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] This application claims the benefit of U.S. Provisional Application No. 63 / 482,634, filed February 1, 2023, and incorporates by reference in its entirety.

[0002] Disclosed herein are one or more subtilisin protease variants, nucleic acids encoding the same, and compositions and methods related to the production and use thereof, including one or more subtilisin protease variants having improved stability and / or stain removal compared to one or more reference subtilisin proteases.

[0003] Reference to a sequence listing submitted electronically

[0004] An official copy of the sequence listing was submitted electronically via Patent Center as an XML document named "20240117_NB42193WOPCT_SequenceListing" created on January 2024 January 17 , and the size is 37,708 The sequence listing contained in this XML format file is a part of this specification and is incorporated herein by reference in its entirety. Background Art

[0005] Proteases (also known as prase enzymes) are enzymes that have the ability to break down other proteins. Proteases have the ability to initiate protein breakdown by hydrolyzing the peptide bonds that link amino acids in the peptide or polypeptide chains that form proteins, thus performing proteolysis. This activity of proteases as protein-digesting enzymes is known as proteolytic activity. There are many well-known procedures for measuring proteolytic activity (Kalisz, "Microbial Proteinases," in: Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology [ Biochemical Industry Program / Biotechnology Progress ], (1988). For example, proteolytic activity can be determined by comparative assays analyzing the ability of individual proteases to hydrolyze commercial substrates. Exemplary substrates that can be used to analyze protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and 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 herein by reference).

[0006] Serine proteases are enzymes with an active site serine that initiates the hydrolysis of peptide bonds in proteins (EC number 3.4.21). Serine proteases encompass a diverse group of enzymes with a wide range of specificities and biological functions, further classified based on their structure as chymotrypsin-like (trypsin-like) and subtilisin-like. The prototypical subtilisin (EC number 3.4.21.62) was originally isolated from Bacillus subtilis. Subtilisin and its homologs are members of the S8 peptidase family of the MEROPS classification scheme (Rawlings, ND et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 44, D343-D350). Members of the S8 family possess a catalytic triad in the order Asp, His, and Ser in their amino acid sequences. While a number of variant proteases have been developed for use in cleaning applications, there remains a need for improved protease variants. Summary of the Invention

[0007] One embodiment relates to a subtilisin variant comprising two or more substitutions selected from the group consisting of: X006W, X024K, X055P, X162Q, X183N, X204Q, X206Y, X222Q, X248A or X254A, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NO: 1.

[0008] Another embodiment relates to a subtilisin variant comprising two substitutions selected from the group consisting of: X006W, X024K, X055P, X109Q, X162Q, X182Q, X183N, X204Q, X206Y, X222Q, X248A, or X254A, and further comprising one, two, or more additional substitutions from the group consisting of: X003Q, X022Y, X024Q, X033T, X045V, X053G, X076D, X078N, X087D, X101N, X109Q, X118R, X128A, X128S, X145R, X166Q, X169A, X182Q, X217Q, and X218S, wherein the positions are identified by the alignment with SEQ ID NO: The variants are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, wherein the variants have at least 55% identity with the amino acid sequence of SEQ ID NO: 1.

[0009] Still other embodiments relate to the method for producing variant as herein described, the method comprising stably transforming host cells with an expression vector comprising a polynucleotide encoding one or more subtilisin variants as herein described. Still other embodiments relate to the polynucleotide comprising a nucleotide sequence encoding one or more subtilisin variants as herein described. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A schematic diagram of a box plot depicting the cumulative contribution of substitutions in the BPN' backbone to stability improvement is provided, showing the number of mutations versus the percent (%) residual activity for the variants described in Table 4 (tested in PNB detergent at 40°C).

[0011] Figure 2 A schematic diagram of a box plot depicting the cumulative contribution of substitutions in the BPN' backbone to stability improvement is provided, showing the number of mutations versus the percent (%) of residual activity for the variants described in Table 4 (tested in HDL 1 detergent at 41°C). DETAILED DESCRIPTION

[0012] In one embodiment, the present disclosure provides two or more subtilisin protease variants, the two or more subtilisin protease variants are included in two, three or more amino acid substitutions at the position selected from the group consisting of: 6, 24, 55, 162, 183, 204, 206, 222, 248 and 254, wherein these positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, when compared to the subtilisin protease with the amino acid sequence of SEQ ID NO: 1, the variants provided herein exhibit one or more improved properties, such as improved stability or improved cleaning performance and improved stability. The subtilisin protease variants provided herein can be used to prepare cleaning compositions (e.g., laundry detergent compositions or automatic dishwashing compositions). In addition, the subtilisin protease variants provided herein can also be used in cleaning methods (e.g., laundry washing methods or dishwashing methods) using such variants or compositions comprising such subtilisin protease variants.

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

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

[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. Reference to a "position" (e.g., 5, 8, 17, 22, etc.) encompasses any starting amino acid that may be present at such a position, as well as any substitution that may be present at such a position. Reference to a "position: one or more amino acid substitutions" (e.g., 1S / T / G, 3G, 17T, etc.) encompasses any starting amino acid that may be present at such a position and one or more amino acids that may replace such a starting amino acid. Reference to a position can be given in several forms, for example, position 003 can also be referred to as position 03 or 3. Reference to a starting or substituted amino acid can be further represented as several starting or substituted amino acids separated by a foreslash (" / "). For example, D275S / K indicates that position 275 is substituted with serine (S) or lysine (K), and P / S197K indicates that the starting amino acid proline (P) or serine (S) at position 197 is substituted with lysine (K). Reference to X as the amino acid at a position refers to any amino acid at the listed position.

[0016] The positions of the amino acid residues in a given amino acid sequence are numbered by corresponding to the amino acid sequence of SEQ ID NO: 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 sequence of one or more subtilisin variants described herein is aligned with the amino acid sequence of SEQ ID NO: 1 using an alignment algorithm as described herein, and each amino acid residue in a given amino acid sequence aligned (preferably, optimally aligned) with the amino acid residues in SEQ ID NO: 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 a "reference sequence"), a sequence alignment algorithm such as that described herein will identify one or more positions at which insertions or deletions occur in the subject sequence. For example, as described in PCT Publication No. WO 2018118917, Figure 1 Provided, amino acid alignments can be used to determine sequence alignment 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 hydrolyzing the peptide bonds that link amino acids in the peptide or polypeptide chains that form proteins. 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 each protease to hydrolyze a suitable substrate. Exemplary substrates that can be used to analyze protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and Keratin Azure (Sigma-Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (see, for example, WO 99 / 34011 and US Pat. No. 6,376,450). The pNA peptidyl assay (see, e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) can also be used to determine active enzyme concentration. This assay measures the rate at which p-nitroaniline is released when the enzyme hydrolyzes a soluble synthetic substrate, such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (suc-AAPF-pNA). The rate of yellow color formation from the hydrolysis reaction is measured spectrophotometrically at 405 or 410 nm and is proportional to the active enzyme concentration. Additionally, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a purified protein sample. The activity on substrate divided by the protein concentration gives the specific enzyme activity.

[0018] As used herein, "Bacillus" includes all species within the genus "Bacillus" as known to those of skill in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, Bacillus species TY145, B. patagoniensis, and B. thuringiensis. It should be recognized that the genus Bacillus is continually undergoing taxonomic reorganization. Therefore, this 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 B. polymyxa (now "Paenibacillus polymyxa").The production of resistant endospores under stressful environmental conditions is considered a defining characteristic of the genus Bacillus, although this feature also applies to the more recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, Alkalihalobacillus, Peribacillus, and Cystosporium. Cytobacillus, Mesobacillus, Neobacillus, Metabacillus, Virgibacillus, and the recently proposed genera: Alteribacter, Ectobacillus, Evansella, Ferdinandcohnia, Gottfriedia, Heyndrickxia, Lederbergia, Litchfieldia, Margalitia, Niallia, Priestia, Robertmurraya, Rossellomorea, Schinkia, Siminovitchia, Sutcliffiella, and Weizmannia (Gupta et al., Int. J. Syst. Evol. Microbiol. 2020;70:5753–5798). “Bacillus lentus subtilisin” includes any subtilisin obtained or derived from a Bacillus lentus (e.g., Lederbergia lentus) source, including P29600.

[0019] “Bacillus clade subtilisin” includes any subtilisin obtained or derived from a Bacillus clade source (genus Bacillus sensu stricto) described in: GuptaRS , Patel S, Saini N, Chen S (2020) Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, byphylogenomics and comparative genomic analyses: description of Robertmurrayakyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting itonly to the members of the Subtilis and Cereus clades of species. Int J Syst Evol Microbiol [International Journal of Systematic and Evolutionary Microbiology] 70 :5753-5798, including Bacillus amyloliquefaciens (BPN', WP_013351733.1, CAA24990.1).

[0020] In other embodiments, the present invention provides a "BPN' variant" (or "CAA24990.1 variant" or "WP_013351733.1 variant" or "BPN' subtilisin variant") wherein the mutation is present in the mature BPN' amino acid sequence set forth in SEQ ID NO: 1. In other embodiments, BPN' subtilisin and variants thereof include those polypeptides having an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 1. In some embodiments, the protease is a serine protease. In some embodiments, the protease is a subtilisin. In some embodiments, the protease is a Bacillus clade subtilisin. In some embodiments, the subtilisin is from a Bacillus species. In some embodiments, the protease is from Bacillus amyloliquefaciens (BPN'). In some embodiments, the protease is from B. xiamenensis. In some embodiments, the protease is from B. subtilisin. In some embodiments, the variant comprises an amino acid sequence having at least 55% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 7, 8, or 9.

[0021] In some embodiments, the protease is a serine protease. In some embodiments, the protease is a subtilisin. In some embodiments, the protease is a Bacillus clade subtilisin. In some embodiments, the subtilisin is from a Bacillus species. In some embodiments, the protease is from Bacillus amyloliquefaciens (BPN'). In some embodiments, the protease is from a Bacillus species. In some embodiments, the protease is from Bacillus subtilis. In some embodiments, the protease is from Bacillus licheniformis. In some embodiments, the protease is from Bacillus lentus. In some embodiments, the protease is from Bacillus gibberellins. In some embodiments, the variant comprises an amino acid sequence having at least 55% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 7 to 28.

[0022] 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, vectors are used to introduce exogenous DNA into cells or tissues. Vectors include plasmids, cloning vectors, phages, viruses (e.g., viral vectors), cosmids, expression vectors, shuttle vectors, and the like. 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 referred to as 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 (e.g., a secretion or signal peptide sequence, etc.), which is capable of achieving expression of the DNA sequence and folding and translocation of the recombinant polypeptide chain in a suitable host.

[0023] As used herein, in the context of introducing a nucleic acid sequence into a cell, the term "introduction" refers to any method suitable for transferring a nucleic acid sequence into a cell. Such methods of introduction include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic alteration of a cell resulting from the uptake, optional genomic incorporation, and expression of genetic material (e.g., DNA).

[0024] 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 can also refer to the translation of mRNA into a polypeptide. Thus, the term "expression" encompasses any step involved in the "production of a polypeptide," including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, secretion, and the like.

[0025] The phrases "expression cassette" or "expression vector" refer to a nucleic acid construct or vector that is recombinantly or synthetically produced and used to express a target nucleic acid (e.g., an exogenous nucleic acid or a transgene) in a target cell. Typically, the target nucleic acid expresses a target protein. Typically, the expression vector or expression cassette contains a promoter nucleotide sequence that drives or promotes expression of the exogenous nucleic acid. Typically, the expression vector or expression cassette also includes other designated nucleic acid elements that allow transcription of the specific nucleic acid in the target cell. The 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 heterologous DNA fragments in a host cell or host cell genome. Many prokaryotic and eukaryotic expression vectors are commercially available. It is within the knowledge of those skilled in the art to select an appropriate expression vector for expressing a protein from the nucleic acid sequence incorporated into the expression vector.

[0026] As used herein, a nucleic acid is "operably linked" to another nucleic acid sequence when the nucleic acid is placed in a functional relationship with the other nucleic acid sequence. For example, if a promoter affects the transcription of a coding sequence, then a promoter or enhancer is operably linked to a nucleotide coding sequence. If a ribosome binding site is positioned so as to promote translation of a coding sequence, then the ribosome binding site may be operably linked to a coding sequence. Typically, an "operably linked" DNA sequence is continuous. However, an enhancer does not have to be continuous. Connection is achieved by connecting at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or joint may be used according to conventional practice.

[0027] The term "gene" refers to a polynucleotide (e.g., a DNA segment) that encodes a polypeptide and includes regions preceding and following the coding region. In some cases, a gene includes intervening sequences (introns) between individual coding segments (exons).

[0028] When used with respect to cells, the term "recombinant" typically indicates that the cell has been modified by the introduction of exogenous nucleic acid sequences, or that the cell is derived from a cell that has been so modified. For example, a recombinant cell can contain genes that are not present in the same form in the native (non-recombinant) form of the cell, or a recombinant cell can contain native genes (found in the native form of the cell) that have been modified and reintroduced into the cell. A recombinant cell can contain nucleic acids that are endogenous to the cell that have 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 cells where it can be expressed or propagated, thereby producing recombinant polypeptides. "Recombination" and "recombining" of polynucleotides or nucleic acids generally refer to the assembly or combination of two or more nucleic acid or polynucleotide chains or fragments to produce a new polynucleotide or nucleic acid.

[0029] If a nucleic acid or polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof in its native state or when manipulated by methods known to those skilled in the art, then the nucleic acid or polynucleotide can be said to "encode" the polypeptide. The antisense strand of such a nucleic acid can also be said to encode the sequence.

[0030] The terms "host strain" and "host cell" refer to a suitable host for an expression vector containing a DNA sequence of interest.

[0031] A "protein" or "polypeptide" comprises 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 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) 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.

[0032] The term "prosequence" or "propeptide sequence" refers to the amino acid sequence between the signal peptide sequence and the mature protease sequence that is essential for the proper folding and secretion of the protease; these are sometimes referred to as intramolecular chaperones. Cleavage of the prosequence or propeptide sequence yields the mature, active protease. Bacterial serine proteases are often referred to as proenzymes. For example, WO 2016 / 205710 provides examples of modified propeptides.

[0033] 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 the mature or precursor form of a protein. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. Signal sequences are generally not present in mature proteins. Typically, after protein transport, the signal sequence is cleaved from the protein by a signal peptidase.

[0034] The term "mature" form of a protein, polypeptide or peptide refers to the functional form of the protein, polypeptide or peptide without the signal peptide sequence and the propeptide sequence.

[0035] The term "precursor" form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a "signal" sequence operably linked to the amino terminus of the prosequence. The precursor may also have additional polypeptides involved in post-translational activity (e.g., polypeptides that are cleaved from it to leave the mature form of the protein or peptide).

[0036] With respect to polypeptides, 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 polynucleotides, 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, polynucleotides encoding wild-type polypeptides are not limited to naturally occurring polynucleotides and encompass any polynucleotide encoding a wild-type or parent polypeptide.

[0037] About polypeptide, term " parent " includes the polypeptide of mentioning naturally occurring or wild type, or the naturally occurring polypeptide that wherein carries out artificial substitution, insertion or disappearance at one or more amino acid positions, it is used as the basis of introducing substitution or other substitution to produce variant enzyme provided herein.About polypeptide, term " parent " also includes any polypeptide with protease activity, and this polypeptide serves as the starting polypeptide for changing (as replacing, adding and / or disappearance), to produce the variant with one or more changes compared with this starting polypeptide.That is, parent or reference polypeptide are not limited to naturally occurring wild-type polypeptide, and contain any wild type, parent or reference polypeptide.Similarly, about polynucleotide, term " parent " can refer to naturally occurring polynucleotide or the polynucleotide that is really included in artificial substitution, insertion or disappearance at one or more nucleotide places.About polynucleotide, term " parent " also includes any polynucleotide of coding with polypeptide of protease activity, and this polynucleotide serves as the starting polynucleotide for changing, thereby produces the variant protease with modifications such as replacing, adding and / or disappearance compared with this starting polynucleotide. That is, the polynucleotides encoding wild-type, parental or reference polypeptides are not limited to naturally occurring polynucleotides, and encompass any polynucleotides encoding wild-type, parental or reference polypeptides. In some embodiments, the parent polypeptide herein comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0038] The term "naturally occurring" refers to, for example, sequences found in nature and the residues contained therein (e.g., a polypeptide sequence and the amino acids contained therein or a nucleotide sequence 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., a polypeptide sequence and the amino acids contained therein or a nucleotide sequence and the nucleic acids contained therein).

[0039] As used herein, with respect to amino acid residue positions, "corresponding to," "corresponds to," or "corresponds" 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 identical to the recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related protein or reference protein.

[0040] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by the strain of the organism in question, but also to proteins encoded by a DNA sequence isolated from such a strain and produced in a host organism containing such a DNA sequence. In addition, the term refers to proteins encoded by a DNA sequence of synthetic and / or cDNA origin and having the identifying characteristics of the protein in question. For example, a "protease derived from Bacillus" refers to those enzymes with proteolytic activity that are naturally produced by Bacillus, as well as serine proteases, such as those produced by Bacillus sources but produced by other host cells transformed with nucleic acids encoding serine proteases using genetic engineering techniques.

[0041] The term "identity" in the context of two polynucleotide or polypeptide sequences refers to the nucleotides or amino acids in the two sequences that are the same when aligned for maximum correspondence, as measured using a sequence comparison or analysis algorithm described below and known in the art.

[0042] 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 a sequence 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, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993). Percent (%) amino acid sequence identity values ​​are 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.

[0043] As used herein, "homologous proteins" or "homologous proteases" refer to proteins that have different similarities 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 by, for example, amino acid sequence alignment using programs such as BLAST, MUSCLE, or CLUSTAL. Homology searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST using a threshold (E-value cutoff) of 0.001. (Altschul et al., "Gapped BLAST and PSI BLAST a new generation of protein database search programs," Nucleic Acids Res, Vol. 1; 25(17): 3389-402 (1997)). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences based on biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997 and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: neighborhood word length threshold = 11; E-value cutoff = 10; Scoring Matrix = NUC.3.1 (match = 1, mismatch = -3); Gap opening = 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 opening = 11; and Gap extension = 1. Using this information, protein sequences can be grouped and / or a phylogenetic tree can be constructed therefrom. Amino acid sequences can be input in programs such as the VectorNTI Advance suite, and a guide tree (Saitou and Nei, Mol Biol Evol [Molecular Biology and Evolution], 4:406-425, 1987) can be created using the Neighbor Joining (NJ) method. Tree structures can be calculated using the Kimura correction for sequence distance and ignoring positions with gaps. Programs such as AlignX can display calculated distance values ​​in brackets after the molecular names displayed on the phylogenetic tree.Another useful algorithm for the alignment and comparison of multiple protein sequences is the MUSCLE program (Robert C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucl. Acids Res. (2004) 32 (5): 1792-1797), available from Geneious software (Biomatters Ltd.).

[0044] Understanding the homology between molecules can reveal information about their evolutionary history and their functions. If a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the new protein's biochemical function. Two molecules are said to be homologous if they derive from a common ancestor. Homologous molecules, or homologs, can be divided into two categories: paralogs and orthologs. Paralogs are homologs that exist within a 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 to which a common ancestor can be inferred. This common ancestor is usually based on sequence alignments and mechanistic similarities. A superfamily typically contains several protein families that display sequence similarity within the family. Based on the MEROPS protease classification system, the term "protein lineage" is often 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, ND et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 44, D343-D350).

[0045] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res 22:4673-4680, 1994). The default parameters for the CLUSTAL W algorithm include: Gap Open Penalty = 10.0; Gap Extension Penalty = 0.05; Protein Weight Matrix = BLOSUM Series; DNA Weight Matrix = IUB; Delayed Divergent Sequence % = 40; Gap Separation Distance = 8; DNA Transition Weight = 0.50; List Hydrophilic Residues = GPSNDQEKR; Use Negativity Matrix = Off; Toggle Special Residue Penalty = On; Toggle Hydrophilic Penalty = On; and Toggle End Gap Separation Penalty = Off. In the CLUSTAL algorithm, deletions occurring at either end are included. For example, a variant having five amino acid deletions at either terminus of a 500 amino acid polypeptide (or within a polypeptide) would have a percent sequence identity of 99% (495 / 500 identical residues x 100) relative to a "reference" polypeptide. Such a variant would be encompassed by variants having "at least 99% sequence identity" to that polypeptide.

[0046] 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.). On a molar basis, the isolated species is more abundant in the composition than other species. For example, the isolated species can account for 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). Purity and homogeneity can be determined using a variety of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of nucleic acid or protein samples, respectively, followed by visualization after staining. If necessary, the substance can be purified using high-resolution techniques such as high performance liquid chromatography (HPLC) or similar methods.

[0047] The term "purified" as applied to nucleic acids or polypeptides generally refers to a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percentages by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid or other specific substance or component in a composition at a relative or absolute concentration higher than that in the starting composition.

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

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

[0050] The term "auxiliary material" refers to any liquid, solid or gaseous material, or recombinant polypeptide or its active fragment, included in the cleaning compositions except one or more subtilisin variants as herein described. In certain embodiments, cleaning compositions of the present disclosure include one or more cleaning auxiliary materials. Typically, depending on the specific type and form (for example liquid, granule, powder, stick, paste, spray, tablet, gel, foam or other compositions) of the cleaning compositions, every kind of cleaning auxiliary material is selected. Preferably, every kind of cleaning auxiliary material is compatible with the protease used in the compositions.

[0051] Cleaning compositions and cleaning formulations include any composition suitable for cleaning, bleaching, disinfecting and / or sterilizing any object, article and / or surface. Such compositions and formulations include, but are not limited to, for example, liquid and / or solid compositions, including cleaning compositions or detergent compositions (e.g., liquid, tablet, gel, stick, granular and / or solid laundry cleaning or detergent compositions) and fine fabric detergent compositions; hard surface cleaning compositions and formulations, such as for glass, wood, ceramic and metal countertops and windows; carpet cleaners; oven cleaners; fabric refreshers; fabric softeners; as well as textile and clothing boosting cleaning or detergent compositions, laundry additive cleaning compositions and laundry pre-spotter cleaning compositions; dishwashing compositions, including hand or manual dishwashing compositions (e.g., "hand wash" or "manual" dishwashing detergents) and automatic dishwashing compositions (e.g., "automatic dishwashing detergents"). The present invention may also be used in single dose unit forms, including but not limited to pills, tablets, gelcaps or other single dose units such as pre-measured powders or liquids.

[0052] In some embodiments, the present invention relates to detergent compositions comprising at least two proteases in combination with one or more additional cleaning composition components, such as, but not limited to, the liquid laundry compositions described in WO 2022106404.

[0053] In some embodiments, the present invention relates to detergent compositions comprising at least two proteases in separate compartments, such as, but not limited to, the liquid compositions described in EP 3483250.

[0054] Unless otherwise indicated, cleaning compositions or cleaning formulations as used herein include all-purpose or heavy-duty detergents in granular or powder form, particularly cleaning detergents; all-purpose detergents in liquid, granular, gel, solid, tablet, paste or unit dosage form, particularly the so-called heavy-duty liquid (HDL) detergent or heavy-duty dry cleaning (HDD) detergent types; liquid delicate fabric detergents; hand or manual dishwashing detergents, including those of the high-sudsing types; hand or manual dishwashing detergents, automatic dishwashing detergents, or dishware or tableware detergents, including various tablet, powder, solid, granular, liquid, gel and rinse aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand wash types, cleaning bars, mouthwashes, denture cleaners, car wash shampoos, carpet shampoos, bathroom cleaners; hair shampoos and / or hair rinses for humans and other animals; body washes and bubble baths and metal cleaners; and cleaning aids, such as bleach additives and "stain bars" or pre-treatment types. In some embodiments, the granular composition is in a "compacted" form; in some embodiments, the liquid composition is in a "concentrated" form.

[0055] The terms "detergent composition" or "detergent formulation" are used with respect to compositions intended for use in wash media for cleaning soiled or dirty objects (including specific fabrics and / or non-fabric objects or articles). In some embodiments, the detergents of the present disclosure comprise one or more subtilisin variants as described herein, and further comprise one or more surfactants, one or more transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, sequestering agents, enzyme stabilizers, calcium, enzyme activators, antioxidants, solubilizers, or one or more microorganisms or microbial extracts or microbial spores. Microorganisms can be used as the sole bioactive ingredient, but they can also be used in combination with one or more enzymes as described herein. For example, a Bacillus strain with deposit accession number PTA-7543 can be used to reduce malodors, as described in WO 2012 / 112718. Exemplary commercial microbial products include, but are not limited to, Microvia™ (Novozymes). Other purposes may include the in situ production of a desired biological compound, or inoculation / propagation of a locus with one or more microorganisms to competitively prevent other undesirable microorganisms from propagating the same locus (competitive exclusion).

[0056] In some embodiments, the cleaning composition comprising one or more protease variants described herein is a liquid laundry detergent composition containing an alkyl ether carboxylic acid, a betaine, an anionic surfactant, a nonionic surfactant to provide softening benefits (WO 2013 / 087286).

[0057] In some embodiments, the cleaning composition comprising one or more protease variants described herein is a liquid laundry detergent composition containing a sulfite radical scavenger, a protease stabilizer / inhibitor, or a combination thereof (WO 2022 / 157311).

[0058] In some embodiments, the cleaning composition comprising one or more protease variants described herein is a liquid laundry detergent composition as described in: US 20210317387 A1, WO 2021 / 219296, WO 2021 / 127662, WO 2021 / 041685, US 11208619, US 20220186144, WO 2022 / 043045, WO 2022 / 043138, WO2023 / 117494, WO 2023 / 088776, WO 2023 / 227332, WO 2024 / 002922.

[0059] In some embodiments, the cleaning composition comprising one or more protease variants described herein is a liquid laundry detergent composition comprising a dispersin variant, such as, but not limited to, the liquid laundry detergent compositions described in US 20210317387 A1.

[0060] In some embodiments, the cleaning composition comprising one or more protease variants described herein is a liquid laundry detergent composition that is a high alkaline textile cleaner, such as, but not limited to, the liquid laundry detergent compositions described in WO 2021 / 219296.

[0061] In some embodiments, the cleaning composition comprising one or more protease variants described herein is a liquid laundry detergent composition that is a low-density unit dose detergent encapsulated with a perfume, such as, but not limited to, the detergent compositions described in WO 2021 / 127662.

[0062] In some embodiments, the cleaning composition comprising one or more protease variants described herein is a liquid laundry detergent composition containing polyethylene glycol and an organic acid, such as, but not limited to, the detergent compositions described in WO 2021 / 041685.

[0063] In some embodiments, the cleaning composition comprising one or more protease variants described herein is a detergent composition containing polyethylene glycol and an organic acid, such as, but not limited to, the detergent compositions described in WO 2021 / 041685.

[0064] In some embodiments, the cleaning compositions comprising one or more protease variants described herein are detergent compositions that are active on proteinaceous stains, such as, but not limited to, the detergent compositions described in US 11208619.

[0065] In some embodiments, the cleaning compositions comprising one or more protease variants described herein are detergent compositions containing soil release polymers, such as, but not limited to, the detergent compositions described in US 20220186144.

[0066] In some cases, the builder salt is a mixture of silicate and phosphate, preferably with more silicate (e.g., sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate).Some embodiments relate to cleaning compositions or detergent compositions that do not contain any phosphates (e.g., phosphates or phosphate builders).

[0067] The phrase "substantially boron-free composition(s)" or "substantially boron-free detergent(s)" refers to one or more compositions or one or more detergents, respectively, that contain 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 may come from other compositions or detergent ingredients.

[0068] The term "bleaching" refers to treating a material (e.g., fabric, clothing, paper pulp, etc.) or surface for a sufficient period of time and / or under appropriate pH and / or temperature conditions to achieve whitening (i.e., bleaching) and / or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, ClO 2 , H 2 O 2 , peracids, NO 2 , and the like. Bleaching agents also include enzymatic bleaching agents such as perhydrolases and aryl esterases. Another embodiment relates to a composition comprising one or more subtilisin variants as 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 .

[0069] " washing performance " of term protease (for example, one or more subtilisin enzyme variants as herein described or its recombinant polypeptide or active fragment) refers to that one or more subtilisin enzyme variants as herein described provide the cleaning contribution of other cleaning performance to washing with the detergent that does not add one or more subtilisin enzyme variants as herein described to compositions.Compare washing performance under relevant washing conditions.In some test systems, other relevant factors, such as detergent composition, foam concentration (sud concentration), water hardness, washing mechanics, time, pH and / or temperature can be controlled in the following such manner: imitate in some market segments (for example hand-washing or manual dishwashing, automatic dishwashing, dishwashing cleaning, tableware cleaning, fabric cleaning etc.) for one or more conditions typical of household applications.

[0070] The phrase "relevant washing conditions" is used herein to indicate the conditions actually used in households in the hand dishwashing, automatic dishwashing or laundry detergent segments, in particular wash temperature, time, wash mechanics, foam concentration, detergent type and water hardness.

[0071] The term "dishwashing" refers to both domestic and industrial dishwashing and relates to both automatic dishwashing (eg washing with a dishwashing machine) and manual dishwashing (eg washing by hand).

[0072] The term "tight" form of cleaning compositions herein is preferably reflected by density, and with respect to composition, by the amount of inorganic filler salts. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, filler salts exist with a basic amount, typically about 17% to about 35% by the weight of the total composition. By contrast, in tight compositions, filler salts exist with an amount less than about 15% of the total composition. In certain embodiments, filler salts exist with an amount no more than about 10% or more preferably about 5% by the weight of the composition. In certain embodiments, the inorganic filler salts are selected from alkali salts and alkaline earth metal salts of sulfate and chloride. In certain embodiments, the filler salt is sodium sulfate.

[0073] Disclosed herein are one or more subtilisin protease variants that can be used for cleaning applications and cleaning methods and various industrial applications.Also disclosed herein are one or more separated, recombinant, substantially pure or non-naturally occurring subtilisin protease variants.In certain embodiments, one or more subtilisin protease variants as herein described can be used for cleaning applications and can be incorporated into the cleaning compositions in the method for cleaning articles or surfaces (e.g., clothing items or textiles) in need.

[0074] 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: 6, 24, 55, 162, 183, 204, 206, 222, 248 and 254, wherein positions are numbered according to SEQ ID NO: 1, and wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28.

[0075] 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: X006W, X024K, X055P, X162Q, X183N, X204Q, X206Y, X222Q, X248A and X254A, wherein positions are numbered according to SEQ ID NO: 1, and wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28.

[0076] In some embodiments, the subtilisin variant comprises a combination of substitutions selected from the group consisting of: X006W-X024K, X006W-X055P, X006W-X162Q, X006W-X183N, X006W-X204Q, X006W-X206Y, X006W-X222Q, X006W-X248A, X006W-X254A, X006W-X266Q, 4K-X055P, X024K-X162Q, X024K-X183N, X024K-X204Q, K-X248A, X024K-X254A, X055P-X162Q, X055P-X183N, X055P-X204Q, -X222Q, X055P-X248A, X055P-X254A, X162Q-X183N, X222Q, X162Q-X248A, X162Q-X254A, X183N-X204Q, X183N-X206Y, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28.

[0077] In some embodiments, the combination of substitutions is selected from the group consisting of: Y006W-S024K, Y006W-T055P, Y006W-S162Q, Y006W-S183N, Y006W-S204Q, Y006W-Q206Y, Y006W-M222Q, Y006W-S248A, Y006W-T254A, S024K-T055P, S024K-S162Q, S024K-S183N, S024K-S204Q, S024K-Q206Y, S024K-M222Q, S024K-S248A , S024K-T254A, T055P-S162Q, T055P-S183N, T055P-S204Q, T055P-Q206Y, T055P-M222Q , T055P-S248A, T055P-T254A, S162Q-S183N, S162Q-S204Q, S162Q-Q206Y, S162Q-M222 Q. S162Q-S248A, S162Q-T254A, S183N-S204Q, S183N-Q206Y, S183N-M222Q, S183N-S248 In some embodiments, the variant subtilisin comprises an amino acid sequence having at least 55%, 60%, 65%, 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 NOs: 1 and 7-28.

[0078] In some embodiments, the combination of substitutions is selected from the group consisting of: X006W-X024K-X055P, X006W-X024K-X162Q, X006W-X024K-X183N, X006W-X024K-X204Q, X006W-X024K-X206Y, X006W-X024K-X222Q, X006W-X024K-X248A, X006W-X024K-X254A, X006W-X055P-X162Q, X006W-X055P-X183N, X006W-X024K-X204Q, X006W-X024K-X206Y, 5P-X222Q, X006W-X055P-X248A, X006W-X055P-X254A, , X006W-X162Q-X204Q, X006W-X162Q-X206Y, 162Q-X248A, X006W-X162Q-X254A, X006W-X183N-X204Q, X006W-X183N-X20 6Y, X006W-X183N-X222Q, X006W-X183N-X248A, X204Q-X206Y, X006W-X204Q-X222Q, X006W-X204Q-X248A, X006W-X204Q-X2 54A, X006W-X206Y-X222Q, X006W-X206Y-X248A, W-X222Q-X248A, X006W-X222Q-X254A, X006W-X248A-X254A, X024K-X055P- X162Q, X024K-X055P-X183N, X024K-X055P-X204Q, 4K-X055P-X222Q, X024K-X055P-X248A, X024K-X055P-X254A, X024K-X162Q -X183N, X024K-X162Q-X204Q, X024K-X162Q-X206Y, 024K-X162Q-X248A, X024K-X162Q-X254A, X024K-X183N-X204Q, X024K-X18 3N-X206Y, X024K-X183N-X222Q, X024K-X183N-X248A,X024K-X204Q-X206Y、X024K-X204Q-X222Q、X024K-X204Q-X248A、X024K-X204Q-X254A、X024K-X206Y-X222Q、X024K-X206Y-X248A、X024K-X206Y-X254A、X024K-X222Q-X248A、X024K-X222Q-X254A、X024K-X248A-X254A、X055P-X162Q-X183N、X055P-X162Q-X204Q、X055P-X162Q-X206Y、X055P-X162Q-X222Q、X055P-X162Q-X248A、X055P-X162Q-X254A、X055P-X183N-X204Q、X055P-X183N-X206Y、X055P-X183N-X222Q、X055P-X183N-X248A、X055P-X183N-X254A、X055P-X204Q-X206Y、X055P-X204Q-X222Q、X055P-X204Q-X248A、X055P-X204Q-X254A、X055P-X206Y-X222Q、X055P-X206Y-X248A、X055P-X206Y-X254A、X055P-X222Q-X248A、X055P-X222Q-X254A、X055P-X248A-X254A、X162Q-X183N-X204Q、X162Q-X183N-X206Y、X162Q-X183N-X222Q、X162Q-X183N-X248A、X162Q-X183N-X254A、X162Q-X204Q-X206Y、X162Q-X204Q-X222Q、X162Q-X204Q-X248A、X162Q-X204Q-X254A、X162Q-X206Y-X222Q、X162Q-X206Y-X248A、X162Q-X206Y-X254A、X162Q-X222Q-X248A、X162Q-X222Q-X254A、X162Q-X248A-X254A、X183N-X204Q-X206Y、X183N-X204Q-X222Q、X183N-X204Q-X248A、X183N-X204Q-X254A、X183N-X206Y-X222Q、X183N-X206Y-X248A、X183N-X206Y-X254A、X183N-X222Q-X248A、X183N-X222Q-X254A、X183N-X248A-X254A, X204Q-X206Y-X222Q, X204Q-X206Y-X248A, X204Q-X206Y-X254A, X204Q-X222Q-X248A, X204Q-X222Q-X254A, X204Q-X248A-X254A, X206Y-X222Q-X248A, X206Y-X222Q-X254A, X206Y-X248A-X254A and X222Q-X248A-X254A, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1 and the variant subtilisin comprises the amino acid sequence of SEQ ID NO: 2. 1 and 7-28 have an amino acid sequence with at least 55%, 60%, 65%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity.

[0079] In some embodiments, the combination of substitutions is selected from the group consisting of: Y006W-S024K-T055P, Y006W-S024K-S162Q, Y006W-S024K-S183N, Y006W-S024K-S204Q, Y006W-S024K-Q206Y, Y006W-S024K-M222Q, Y006W-S024K-S248A, Y006W-S024K-T254A, Y006W-T055P-S162Q, Y006W-S055P-S183N, Y006W-S024K-S204Q, Y006W-S024K-Q206Y, 5P-M222Q, Y006W-T055P-S248A, Y006W-T055P-T254A, Y006W-S162Q-S183N , Y006W-S162Q-S204Q, Y006W-S162Q-Q206Y, Y006W-S162Q-M222Q, Y006W-S 162Q-S248A, Y006W-S162Q-T254A, Y006W-S183N-S204Q, Y006W-S183N-Q20 6Y, Y006W-S183N-M222Q, Y006W-S183N-S248A, Y006W-S183N-T254A, Y006W- S204Q-Q206Y, Y006W-S204Q-M222Q, Y006W-S204Q-S248A, Y006W-S204Q-T2 54A, Y006W-Q206Y-M222Q, Y006W-Q206Y-S248A, Y006W-Q206Y-T254A, Y006 W-M222Q-S248A, Y006W-M222Q-T254A, Y006W-S248A-T254A, S024K-T055P- S162Q, S024K-T055P-S183N, S024K-T055P-S204Q, S024K-T055P-Q206Y, S02 4K-T055P-M222Q, S024K-T055P-S248A, S024K-T055P-T254A, S024K-S162Q -S183N, S024K-S162Q-S204Q, S024K-S162Q-Q206Y, S024K-S162Q-M222Q, S 024K-S162Q-S248A, S024K-S162Q-T254A, S024K-S183N-S204Q, S024K-S18 3N-Q206Y, S024K-S183N-M222Q, S024K-S183N-S248A, S024K-S183N-T254A,S024K-S204Q-Q206Y、S024K-S204Q-M222Q、S024K-S204Q-S248A、S024K-S204Q-T254A、S024K-Q206Y-M222Q、S024K-Q206Y-S248A、S024K-Q206Y-T254A、S024K-M222Q-S248A、S024K-M222Q-T254A、S024K-S248A-T254A、T055P-S162Q-S183N、T055P-S162Q-S204Q、T055P-S162Q-Q206Y、T055P-S162Q-M222Q、T055P-S162Q-S248A、T055P-S162Q-T254A、T055P-S183N-S204Q、T055P-S183N-Q206Y、T055P-S183N-M222Q、T055P-S183N-S248A、T055P-S183N-T254A、T055P-S204Q-Q206Y、T055P-S204Q-M222Q、T055P-S204Q-S248A、T055P-S204Q-T254A、T055P-Q206Y-M222Q、T055P-Q206Y-S248A、T055P-Q206Y-T254A、T055P-M222Q-S248A、T055P-M222Q-T254A、T055P-S248A-T254A、S162Q-S183N-S204Q、S162Q-S183N-Q206Y、S162Q-S183N-M222Q、S162Q-S183N-S248A、S162Q-S183N-T254A、S162Q-S204Q-Q206Y、S162Q-S204Q-M222Q、S162Q-S204Q-S248A、S162Q-S204Q-T254A、S162Q-Q206Y-M222Q、S162Q-Q206Y-S248A、S162Q-Q206Y-T254A、S162Q-M222Q-S248A、S162Q-M222Q-T254A、S162Q-S248A-T254A、S183N-S204Q-Q206Y、S183N-S204Q-M222Q、S183N-S204Q-S248A、S183N-S204Q-T254A、S183N-Q206Y-M222Q、S183N-Q206Y-S248A、S183N-Q206Y-T254A、S183N-M222Q-S248A、S183N-M222Q-T254A、S183N-S248A-T254A, S204Q-Q206Y-M222Q, S204Q-Q206Y-S248A, S204Q-Q206Y-T254A, S204Q-M222Q-S248A, S204Q-M222Q-T254A, S204Q-S248A-T254A, Q206Y-M222Q-S248A, Q206Y-M222Q-T254A, Q206Y-S248A-T254A and M222Q-S248A-T254A, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1 and the variant subtilisin comprises the amino acid sequence of SEQ ID NO: 2. 1 and 7-28 have an amino acid sequence with at least 55%, 60%, 65%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity.

[0080] In some embodiments, the combination of substitutions is selected from the group consisting of: X055P-X204Q, X162Q-X204Q, X006W-X024K, X222Q-X254A, X206Y-X222Q, X055P-X222Q, X183N-X204Q, X162Q-X222Q, X006W-X183N, X183N-X222Q, X006W-X204Q, X222Q-X248A, X006W-X254A, X045V-X204Q-X222Q, X101N-X162Q-X222Q, X055P-X109Q-X204Q, X006W-X109Q-X204Q, X006W-X109Q-X222Q 2Q, X109Q-X162Q-X222Q, X055P-X162Q-X204Q, -X182Q-X222Q, X055P-X182Q-X254A, X204Q-X217Q-X222Q, X006W-X183N-X 206Y, X055P-X204Q-X222Q, X109Q-X204Q-X254A, 5P-X162Q-X254A, X055P-X222Q-X254A, X003Q-X162Q-X254A, X078N-X162Q- X254A, X109Q-X204Q-X222Q, X003Q-X222Q-X254A, 24Q-X204Q-X222Q, X022Y-X162Q-X254A, X003Q-X162Q-X222Q, X109Q-X222 Q-X254A, X087D-X162Q-X254A, X076D-X162Q-X254A, X128S-X204Q-X254A, X076D-X204Q-X254A, X182Q-X204Q-X254A, X101N-X20 4Q-X254A, X024Q-X204Q-X254A, X128A-X162Q-X254A, , X128A-X204Q-X254A, X055P-X109Q-X182Q-X204Q, 222Q, X055P-X182Q-X204Q-X222Q, X055P-X109Q-X162Q-X204Q, 2Q-X182Q-X204Q, X006W-X109Q-X183N-X248A,X055P-X162Q-X182Q-X254A、X162Q-X182Q-X248A-X254A、X055P-X162Q-X204Q-X222Q、X006W-X109Q-X182Q-X248A、X055P-X109Q-X162Q-X254A、X055P-X109Q-X162Q-X222Q、X183N-X222Q-X248A-X254A、X055P-X087D-X145R-X254A、X109Q-X182Q-X204Q-X222Q、X162Q-X182Q-X204Q-X222Q、X006W-X162Q-X182Q-X254A、X109Q-X204Q-X222Q-X254A、X055P-X162Q-X204Q-X254A、X006W-X109Q-X162Q-X254A、X055P-X109Q-X182Q-X222Q、X006W-X024K-X183N-X254A、X055P-X182Q-X183N-X254A、X055P-X182Q-X204Q-X254A、X162Q-X182Q-X222Q-X254A、X055P-X182Q-X222Q-X254A、X162Q-X182Q-X183N-X254A、X006W-X024K-X182Q-X254A、X182Q-X204Q-X222Q-X254A、X006W-X183N-X248A-X254A、X182Q-X183N-X204Q-X254A、X006W-X109Q-X204Q-X254A、X109Q-X183N-X204Q-X254A、X006W-X055P-X182Q-X254A、X006W-X182Q-X248A-X254A、X006W-X162Q-X204Q-X254A、X024K-X033T-X166Q-X222Q、X003Q-X076D-X222Q-X254A、X162Q-X182Q-X204Q-X254A、X006W-X128S-X217Q-X254A、X006W-X183N-X204Q-X254A、X024Q-X109Q-X145R-X162Q-X217Q-X222Q、X053G-X076D-X162Q-X182Q-X204Q-X217Q、X003Q-X053G-X101N-X109Q-X183N-X254A、X024Q-X033T-X076D-X182Q-X183N-X254A、X003Q-X053G-X055P-X076D-X128A-X254A, X033T-X076D-X145R-X162Q-X182Q-X254A, 7Q-X222Q-X254A, X024Q-X076D-X162Q-X204Q-X222Q-X254A, 162Q-X182Q-X206Y-X222Q-X254A, X024K-X087D-X145R-X183N-X222Q-X254A, Q-X222Q, X022Y-X087D-X109Q-X183N-X222Q-X254A, 82Q-X204Q-X222Q-X254A, X003Q-X109Q-X128S-X162Q-X166Q-X183N, ,X006W-X024K-X101N-X128A-X169A-X183N, 4Q-X206Y-X254A, X024K-X087D-X169A-X204Q-X218S-X222Q, X006W-X045V-X128A-X145R-X182Q-X254A, X006W-X087D-X109Q-X145R-X162Q-X169A, and X006W-X055P-X182Q-X183N-X204Q-X206Y-X222Q-X248A-X254A, where the positions are determined by comparing with SEQ The variant subtilisin enzymes are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin enzymes comprise an amino acid sequence having at least 55%, 60%, 65%, 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 and 7-28.

[0081] In some embodiments, the combination of substitutions is selected from the group consisting of: T055P-S204Q, S162Q-S204Q, Y006W-S024K, M222Q-T254A, Q206Y-M222Q, T055P-M222Q, S183N-S204Q, S162Q-M222Q, Y006W-S183N, S183N-M222Q, Y006W-S204Q, M222Q-S248A, Y006W-T254A, A045V-S204Q-M222Q, S101N-S162Q-M222Q 2Q, N109Q-S162Q-M222Q, T055P-S162Q-S204Q, S162Q-S182Q-S204Q, S162Q -S182Q-M222Q, T055P-S182Q-T254A, S204Q-Y217Q-M222Q, Y006W-S183N-Q 206Y, T055P-S204Q-M222Q, N109Q-S204Q-T254A, T055P-S182Q-M222Q, T05 5P-S162Q-T254A, T055P-M222Q-T254A, S003Q-S162Q-T254A, S078N-S162Q- T254A, N109Q-S204Q-M222Q, S003Q-M222Q-T254A, S033T-M222Q-T254A, S0 24Q-S204Q-M222Q, T022Y-S162Q-T254A, S003Q-S162Q-M222Q, N109Q-M222 Q-T254A, S087D-S162Q-T254A, N076D-S162Q-T254A, S078N-S204Q-T254A, G128S-S204Q-T254A, N076D-S204Q-T254A, S182Q-S204Q-T254A, S101N-S20 4Q-T254A, S024Q-S204Q-T254A, G128A-S162Q-T254A, S182Q-M222Q-T254A , G128A-S204Q-T254A, T055P-N109Q-S182Q-S204Q, N109Q-S162Q-S182Q-M 222Q, T055P-S182Q-S204Q-M222Q, T055P-N109Q-S162Q-S204Q, T055P-S16 2Q-S182Q-S204Q, Y006W-N109Q-S183N-S248A, T055P-S162Q-S182Q-M222Q,T055P-S162Q-S182Q-T254A、S162Q-S182Q-S248A-T254A、T055P-S162Q-S204Q-M222Q、Y006W-N109Q-S182Q-S248A、T055P-N109Q-S162Q-T254A、T055P-N109Q-S162Q-M222Q、S183N-M222Q-S248A-T254A、T055P-S087D-S145R-T254A、N109Q-S182Q-S204Q-M222Q、S162Q-S182Q-S204Q-M222Q、Y006W-S162Q-S182Q-T254A、N109Q-S204Q-M222Q-T254A、T055P-S162Q-S204Q-T254A、Y006W-N109Q-S162Q-T254A、T055P-N109Q-S182Q-M222Q、Y006W-S024K-S183N-T254A、T055P-S182Q-S183N-T254A、T055P-S182Q-S204Q-T254A、S162Q-S182Q-M222Q-T254A、T055P-S182Q-M222Q-T254A、S162Q-S182Q-S183N-T254A、Y006W-S024K-S182Q-T254A、S182Q-S204Q-M222Q-T254A、Y006W-S183N-S248A-T254A、S182Q-S183N-S204Q-T254A、Y006W-N109Q-S204Q-T254A、N109Q-S183N-S204Q-T254A、Y006W-T055P-S182Q-T254A、Y006W-S182Q-S248A-T254A、Y006W-S162Q-S204Q-T254A、S024K-S033T-G166Q-M222Q、S003Q-N076D-M222Q-T254A、S162Q-S182Q-S204Q-T254A、Y006W-G128S-Y217Q-T254A、Y006W-S183N-S204Q-T254A、S024Q-N109Q-S145R-S162Q-Y217Q-M222Q、S053G-N076D-S162Q-S182Q-S204Q-Y217Q、S003Q-S053G-S101N-N109Q-S183N-T254A、S024Q-S033T-N076D-S182Q-S183N-T254A、S003Q-S053G-T055P-N076D-G128A-T254A, S033T-N076D-S145R-S162Q-S182Q-T254A, Y006W-T055P-G128S-Y21 7Q-M222Q-T254A, S024Q-N076D-S162Q-S204Q-M222Q-T254A, S101N-S145R-S182Q-S183N-S204Q-N218S, S024K-S 162Q-S182Q-Q206Y-M222Q-T254A, S024K-S087D-S145R-S183N-M222Q-T254A, S024Q-S101N-S145R-S182Q-S204 Q-M222Q, T022Y-S087D-N109Q-S183N-M222Q-T254A, S024Q-N118R-G169A-S182Q-S183N-S248A, S024Q-S101N-S1 82Q-S204Q-M222Q-T254A, S003Q-N109Q-G128S-S162Q-G166Q-S183N, N076D-N109Q-S145R-S182Q-M222Q-T254A , Y006W-S024K-S101N-G128A-G169A-S183N, S101N-G166Q-G169A-S182Q-S204Q-Q206Y, N109Q-S182Q-S183N-S20 4Q-Q206Y-T254A, S024K-S087D-G169A-S204Q-N218S-M222Q, Y006W-A045V-G128A-S145R-S182Q-T254A, Y006W-S087D-N109Q-S145R-S162Q-G169A, and Y006W-T055P-S182Q-S183N-S204Q-Q206Y-M222Q-S248A-T254A, where the positions are determined by comparing with SEQ The variant subtilisin enzymes are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin enzymes comprise an amino acid sequence having at least 55%, 60%, 65%, 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 and 7-28.

[0082] In some embodiments, the substituted combinations include those in Tables 1 and 2, such as those selected from the group consisting of: S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-S248A-T254A, S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-S248A-T254A. 3N-Y217Q-S248A-T254A, S003Q-Y006W-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y21 7Q-S248A-T254A, S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-S248 A-T254A, S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254 A. S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-N218S-S248A-T254A, S003Q -T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-N218S-S248A-T254A, S003Q -Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-S248A-T254A,S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-S183N-Y217Q-S248A-T254A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-S182Q-S183N-Y217Q-S248A-T254A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-G169A-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S162Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-N218S-S248A-T254A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-T022Y-S024K-S033T-S053G-N076D-S078N-S101N-N118R-G128S-S182Q-S183N-Y217Q-M222Q-S248A-T254A、S003Q-S024K-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S182Q-S183N-Q206Y-Y217Q-M222Q-T254A、S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128A-S145R-S162Q-S182Q-S183N-Y217Q-M222Q-S248A-T254A、S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128A-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128S-G169A-S182Q-Y217Q-S248A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128A-G169A-S182Q-S183N-S204Q-Q206Y-Y217Q-N218S-T254A、S003Q-Y006W-S024Q-S033T-S053G-N076D-S078N-S101N-G128S-G169A-S182Q-Y217Q-N218S-S248A-T254A、Y006W-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-G128S-S145R-S162Q-S182Q-Y217Q-N218S-T254A、S003Q-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-N118R-G128S-S162Q-S182Q-S183N-Y217Q-T254A、S024Q-S033T-A045V-S053G-N076D-S078N-S101N-G128A-S145R-Y217Q-S248A-T254A、S003Q-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S162Q-G169A-S183N-Y217Q-N218S-T254A、S003Q-Y006W-S024Q-S033T-S053G-N076D-S078N-S101N-N109Q-G128S-S145R-G169A-S183N-Y217Q-N218S-S248A、Y006W-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-G128S-S162Q-S183N-Y217Q-N218S-T254A、S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-G128S-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A、S003Q-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-G128S-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A、Y006W-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-G128S-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-S053G-S078N-S087D-S101N-N118R-G128S-S162Q-S182Q-Y217Q-T254A、S003Q-T022Y-S024K-S033T-A045V-S053G-N076D-S078N-S087D-S101N-N118R-G1 28S-S182Q-S183N-S204Q-Q206Y-Y217Q-M222Q-S248A-T254A, S003Q-T022Y-S024 K-S033T-S053G-N076D-S078N-S101N-G128S-S145R-S162Q-G166Q-S182Q-Q206Y- Y217Q-N218S-M222Q-T254A, S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-T0 55P-N076D-S078N-S087D-S101N-N118R-G128S-S182Q-S183N-S204Q-Q206Y-Y217 Q-N218S-M222Q-S248A-T254A, S003Q-S024Q-S033T-A045V-S053G-T055P-N076D- S078N-S101N-N109Q-G128A-S145R-S162Q-S182Q-S204Q-Y217Q-M222Q-T254A and S033T-S053G-N076D-S078N-S101N-G128S-S145R-S183N-Y217Q-S248A, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin comprises an amino acid sequence having at least 55%, 60%, 65%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequences of SEQ ID NOs: 1 and 7-28.

[0083] In some embodiments, the substituted combinations include those in Tables 4 and 5, such as those selected from the group consisting of: T055P-S204Q, S182Q-M222Q, S162Q-S204Q, Y006W-S024K, M222Q-T254A, Q206Y-M222Q, T055P-M222Q, N109Q-M222Q, S183N-S204Q, S162Q-M222Q, Y006W-S183N, S204Q-M222Q, S183N-M222Q, Y006W-S204Q, M222Q-S248A, S204Q-T254A, Y006W-T254A, N109Q- S162Q-S182Q, N076D-S182Q-M222Q, A045V-S204Q-M222Q, S101N-S162Q-M2 22Q, T055P-N109Q-S204Q, Y006W-S024K-M222Q, N109Q-S162Q-M222Q, T055P -S162Q-S204Q, S162Q-S182Q-S204Q, S162Q-S182Q-M222Q, S101N-S182Q-M 222Q, T055P-S182Q-T254A, S204Q-Y217Q-M222Q, Y006W-S183N-Q206Y, N109 Q-S182Q-M222Q, T055P-S204Q-M222Q, N109Q-S204Q-T254A, T055P-S182Q- M222Q, T055P-S162Q-T254A, T055P-M222Q-T254A, S003Q-S162Q-T254A, S07 8N-S162Q-T254A, N109Q-S204Q-M222Q, S003Q-S182Q-T254A, S003Q-M222Q -T254A, S033T-M222Q-T254A, S024Q-S204Q-M222Q, T022Y-S162Q-T254A, S0 78N-S182Q-T254A, S003Q-S162Q-M222Q, N109Q-M222Q-T254A, T022Y-S182 Q-T254A, S087D-S162Q-T254A, N076D-S182Q-T254A, S204Q-Y217Q-T254A, N 076D-S162Q-T254A, S078N-S204Q-T254A, G128S-S204Q-T254A, N076D-S20 4Q-T254A, S087D-S182Q-T254A, S182Q-S204Q-T254A, S101N-S204Q-T254A,S024Q-S204Q-T254A、G128A-S162Q-T254A、S182Q-M222Q-T254A、G128A-S204Q-T254A、T055P-N109Q-S182Q-S204Q、N109Q-S162Q-S182Q-M222Q、T055P-S182Q-S204Q-M222Q、T055P-N109Q-S162Q-S204Q、T055P-S162Q-S182Q-S204Q、Y006W-N109Q-S183N-S248A、T055P-S162Q-S182Q-M222Q、S024Q-S078N-S182Q-T254A、T055P-S162Q-S182Q-T254A、S162Q-S182Q-S248A-T254A、T055P-S162Q-S204Q-M222Q、Y006W-N109Q-S182Q-S248A、T055P-N109Q-S162Q-T254A、T055P-N109Q-S162Q-M222Q、S183N-M222Q-S248A-T254A、S078N-N109Q-S145R-T254A、T055P-S087D-S145R-T254A、N109Q-S182Q-S204Q-M222Q、S162Q-S182Q-S204Q-M222Q、Y006W-S162Q-S182Q-T254A、N109Q-S204Q-M222Q-T254A、T055P-S162Q-S204Q-T254A、Y006W-N109Q-S162Q-T254A、T055P-N109Q-S182Q-M222Q、Y006W-S024K-S183N-T254A、T055P-S182Q-S183N-T254A、T055P-S182Q-S204Q-T254A、S162Q-S182Q-M222Q-T254A、T055P-S182Q-M222Q-T254A、S162Q-S182Q-S183N-T254A、Y006W-S024K-S182Q-T254A、S182Q-S204Q-M222Q-T254A、Y006W-S183N-S248A-T254A、S182Q-S183N-S204Q-T254A、Y006W-N109Q-S204Q-T254A、N109Q-N118R-G169A-M222Q、N109Q-S183N-S204Q-T254A、Y006W-T055P-S182Q-T254A、Y006W-S182Q-S248A-T254A、Y006W-S162Q-S204Q-T254A、S024K-S033T-G166Q-M222Q、S003Q-N076D-M222Q-T254A、S162Q-S182Q-S204Q-T254A、Y006W-G128S-Y217Q-T254A、Y006W-S183N-S204Q-T254A、S024Q-A045V-T055P-S078N-N109Q-S182Q、S024Q-N109Q-S145R-S162Q-Y217Q-M222Q、S053G-N076D-S162Q-S182Q-S204Q-Y217Q、S003Q-S053G-S101N-N109Q-S183N-T254A、S024Q-S033T-N076D-S182Q-S183N-T254A、S003Q-S053G-T055P-N076D-G128A-T254A、S033T-N076D-S145R-S162Q-S182Q-T254A、Y006W-T055P-G128S-Y217Q-M222Q-T254A、S024Q-N076D-S162Q-S204Q-M222Q-T254A、S101N-S145R-S182Q-S183N-S204Q-N218S、S024K-S162Q-S182Q-Q206Y-M222Q-T254A、S024K-S087D-S145R-S183N-M222Q-T254A、S024Q-S101N-S145R-S182Q-S204Q-M222Q、T022Y-S087D-N109Q-S183N-M222Q-T254A、S024Q-N118R-G169A-S182Q-S183N-S248A、S024Q-S101N-S182Q-S204Q-M222Q-T254A、S003Q-N109Q-G128S-S162Q-G166Q-S183N、N076D-N109Q-S145R-S182Q-M222Q-T254A、Y006W-S024K-S101N-G128A-G169A-S183N、S101N-G166Q-G169A-S182Q-S204Q-Q206Y、N109Q-S182Q-S183N-S204Q-Q206Y-T254A、S024K-S087D-G169A-S204Q-N218S-M222Q、Y006W-A045V-G128A-S145R-S182Q-T254A、Y006W-S087D-N109Q-S145R-S162Q-G169A and Y006W-T055P-S182Q-S183N-S204Q-Q206Y-M222Q-S248A-T254A, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin comprises an amino acid sequence having at least 55%, 60%, 65%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequences of SEQ ID NOs: 1 and 7-28.

[0084] In another embodiment, the subtilisin variant further comprises two or more substitutions selected from the group consisting of X003Q, X006W, X022Y, X024K, X024Q, X033T, X045V, X053G, X055P, X076D, X078N, X087D, X101N, X109Q, X118R, X128A, X128S, X145R, X162Q, X166Q, X169A, X182Q, X183N, X204Q, X206Y, X217Q, X218S, X222Q, X248A, or X254A, wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, wherein the positions are identified by the amino acid sequence of SEQ ID NOs: 1 and 7-28. The variant is numbered corresponding to the amino acid sequence of NO: 1, and wherein the variant does not have 100% sequence identity with the naturally occurring amino acid sequence.

[0085] In yet another embodiment, the subtilisin variant further comprises two or more substitutions selected from the group consisting of S003Q, Y006W, T022Y, S024K, S024Q, S033T, A045V, S053G, T055P, N076D, S078N, S087D, S101N, N109Q, N118R, G128A, G128S, S145R, S162Q, G166Q, G169A, S182Q, S183N, S204Q, Q206Y, Y217Q, N218S, M222Q, S248A, or T254A, wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, wherein the positions are identified by the amino acid sequence of SEQ ID NOs: 1 and 7-28. The variant is numbered corresponding to the amino acid sequence of NO: 1, and wherein the variant does not have 100% sequence identity with the naturally occurring amino acid sequence.

[0086] The disclosure includes subtilisin enzyme variants, and these variants have one or more modifications on the amino acid exposed on the surface.By having the stability of the enzyme in the detergent composition and the lowest performance index of the thermostability of the enzyme for washability, the detergent composition, there is simultaneously at least one in these characteristics improved with respect to the parent subtilisin enzyme, the surface modification of the enzyme variants can be used in the detergent composition.In certain embodiments, surface modification changes the amino acid whose hydrophobicity and / or the electric charge at that position.Hydrophobicity can be determined using technology known in the art, such as those (White, SH and Wimley, WC,. (1999) Annu. Rev. Biophys. Biomol. Struct [biophysics and biomolecular structure annual review] 28:319-65) that White and Wimley describe.

[0087] As used herein, "surface properties" may refer to electrostatic charge, and properties such as hydrophobicity and hydrophilicity exhibited by a protein surface. In even further embodiments, one or more 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.

[0088] In another embodiment, the parent subtilisin comprises the amino acid sequence of SEQ ID NO: 1. In another embodiment, the parent subtilisin is a polypeptide with the amino acid sequence of SEQ ID NO: 1. In yet another embodiment, the improved property is improved stability, wherein the variant has a higher residual activity than the parent or reference subtilisin. In yet another embodiment, the improved stability of the detergent is measured according to the stability assay of Example 2.

[0089] In the context of proteases that are stable to oxidation, chelating agents, denaturing agents, surfactants, heat, and / or pH, the term "enhanced stability" or "improved stability" refers to a subtilisin variant that has a higher retention of proteolytic activity over time compared to a reference subtilisin or parent subtilisin (e.g., a wild-type protease or parent protease, such as SEQ ID NO: 1). 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).

[0090] With respect to protease variants, the terms "thermally stable" and "thermostable" and "thermostability" refer to proteases that retain a greater amount of residual activity than a parent or reference protease after exposure to varying temperatures for a given period of time, under conditions (or "stress conditions") that prevail during proteolysis, hydrolysis, cleaning, or other processes. Residual activity is the amount of activity that remains after testing compared to the initial activity of the sample and can be reported as a percentage, e.g., residual activity %. "Altered temperature" encompasses increases or decreases in temperature. 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 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 certain embodiments, the method shown in use example 2, the residual activity of variant subtilisin provided herein is higher than the residual activity of parent or reference protease.In certain embodiments, when measuring after 20 minutes at 40-80 degrees Celsius in liquid detergent, variant subtilisin provided herein has at least 5% improved residual activity compared with parent subtilisin.In certain embodiments, when measuring after 20 minutes at 40-80 degrees Celsius in liquid detergent, variant subtilisin provided herein has at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% improved residual activity compared with parent subtilisin.

[0091] In yet another embodiment, the improved property is cleaning performance, wherein the variant has a higher performance index compared to the parent reference subtilisin protease.In yet another embodiment, the improved property is cleaning performance as measured according to the cleaning assay of Example 2.

[0092] Subtilisin variants provided herein can be used to produce various compositions, such as enzyme compositions and cleaning compositions or detergent compositions. The enzyme compositions comprise subtilisin variants as provided herein. The enzyme compositions can be in any form, such as liquid preparations, gels, particles, or enzyme slurries.

[0093] Enzyme granules can be prepared by methods such as 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, rotating disk atomization and other casting methods, as well as spheronization. The core of the granule can be the granule itself or the inner core of a layered granule.

[0094] The core may comprise 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, granulated 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, carboxymethyl cellulose and hydroxyethyl cellulose), phosphates, calcium, protease inhibitors and combinations thereof. Suitable dispersants include but are not limited to clays, sphericals (combinations of sugar and starch; e.g., starch-sucrose sphericals-ASNPs), talc, silicates, carboxymethyl cellulose, starch and combinations thereof.

[0095] In some embodiments, the core comprises primarily sodium sulfate. In some embodiments, the core consists essentially of sodium sulfate. In specific embodiments, the core consists only of sodium sulfate.

[0096] In certain embodiments, the core comprises a subtilisin variant as provided herein. In other embodiments, the core comprises one or more enzymes in addition to a protease. In other embodiments, the core is inert and does not comprise an enzyme.

[0097] In some embodiments, the core is an enzyme powder, including a UFC containing an enzyme. The enzyme powder can be spray-dried and optionally blended with any of the water-soluble or water-dispersible agents listed herein. The enzyme can be or include a protease to be stabilized, in which case the enzyme powder should further include a stabilizer.

[0098] In some embodiments, the core is coated with at least one coating. In a specific embodiment, the core is coated with at least two coatings. In another specific embodiment, the core is coated with at least three coatings. The materials used for the one or more coatings can be suitable for use in cleaning compositions and / or detergent compositions (see, for example, US 20100124586, WO 9932595, and US5324649).

[0099] 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, carboxymethyl cellulose, and hydroxyethyl cellulose), clays, sugar pellets (combinations of sugar and starch), silicates, carboxymethyl cellulose, phosphates, starches (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 (e.g., FOAMBLAST 882 ® and EROL 6000K ® ) and talc. US 20100124586, WO9932595 and US 5324649 describe suitable components for the coating in detail.

[0100] In some embodiments, the coating comprises a sugar (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin, and fructose). In some embodiments, the coating comprises a polymer such as polyvinyl alcohol (PVA). Suitable PVA for incorporation into one or more coatings of the multilayered particles include partially hydrolyzed, fully hydrolyzed, and moderately hydrolyzed PVAs having low to high viscosity. In some embodiments, the coating comprises an inorganic salt such as sodium sulfate.

[0101] 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.

[0102] In some embodiments, the enzyme granule comprises a subtilisin variant as provided herein in combination with one or more additional enzymes selected from the group consisting of: acyltransferases, alginate lyases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, β-glucanases, carrageenanases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, dispersins, endo-β-1,4-glucanases, endo-β-mannanases, esterases, exo-mannanases, fructosidases, galactanases, glucoamylases, hemicellulases, hexosaminidase, hyaluronidase, keratinase, lacquer The enzyme coating comprises at least one subtilisin enzyme variant as provided herein.

[0103] The above enzyme list is only an example 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 of bacterial, fungal, and yeast origin, as well as acidic, neutral or alkaline enzymes.

[0104] Another embodiment relates to a method for cleaning a surface, wherein the method comprises contacting a surface or article in need of cleaning with an effective amount of one or more subtilisin variants as provided herein or a composition containing one or more subtilisin variants as provided herein. In certain embodiments, the surface or article in need of cleaning comprises a protein stain on the surface. In certain embodiments, the surface or article in need of cleaning comprises a protein stain. The term "stain" comprises any type of dirt on the surface of an article (for example, a hard surface article (for example, tableware) or a textile). In certain embodiments, the stain is a protein stain. As used herein, a "protein stain" is a stain or dirt containing protein.

[0105] Additional embodiments relate to methods of cleaning protein stains comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants as provided herein or a composition containing one or more subtilisin variants as provided herein.

[0106] Another embodiment relates to a method of cleaning egg stains comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants as provided herein, or a composition containing one or more such subtilisin variants.

[0107] Another embodiment relates to a method of cleaning BMI stains comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants as provided herein, or a composition containing one or more such subtilisin variants.

[0108] One or more subtilisin variants described herein may be subjected to various changes, such as one or more amino acid insertions, deletions, and / or substitutions (conservative or non-conservative), including situations where such changes do not substantially alter the enzymatic activity of the variant. Similarly, the nucleic acids of the present invention may also be subjected 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 different amino acid, thereby producing a silent change (e.g., when the encoded amino acid is not altered by the nucleotide mutation) or a non-silent change; 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 of these changes in the nucleic acid sequence do not substantially alter the enzymatic activity of the resulting encoded polypeptide enzyme compared to the polypeptide enzyme encoded by the original nucleic acid sequence. The nucleic acid sequences described herein may also be modified to include one or more codons that provide optimal expression in an expression system (e.g., a bacterial expression system), while, if desired, the one or more codons still encode the same amino acid(s).

[0109] Described herein are one or more isolated, non-naturally occurring or recombinant polynucleotides comprising nucleic acid sequences encoding one or more subtilisin variants as described herein, or recombinant polypeptides or active fragments thereof. One or more nucleic acid sequences as described herein can be used in the recombinant production (e.g., expression) of one or more subtilisin variants as described herein, typically by expressing a plasmid expression vector comprising a sequence encoding one or more subtilisin variants as described herein or fragments thereof. One embodiment provides nucleic acids encoding one or more subtilisin variants as described herein, wherein the variant is a mature form having proteolytic activity. In some embodiments, one or more subtilisin variants as described herein are recombinantly expressed using homologous propeptide sequences. In other embodiments, one or more subtilisin variants as described herein are recombinantly expressed using heterologous propeptide sequences (e.g., propeptide sequences from Bacillus amyloliquefaciens (SEQ ID NO: 4) or variants thereof).

[0110] One or more nucleotide sequences as herein described can be produced by using any suitable synthesis, operation and / or separation technology or its combination.For example, one or more polynucleotides as herein described can be produced using standard nucleic acid synthesis techniques well known to those skilled in the art such as solid phase synthesis technology.In such technology, typically synthesize fragments up to 50 or more nucleotide bases, then connect (for example by enzyme or chemical connection method) to form basically any desired continuous nucleic acid sequence.Can also promote the synthesis of one or more polynucleotides as herein described by any suitable method known in the art, including but not limited to using the following method of chemical synthesis: classical phosphoramidite method (see for example, Beaucage et al., Tetrahedron Letters [tetrahedron express] 22:1859-69 (1981)), or as typically practiced in automatic synthesis method at Matthes et al., EMBO J. [European Molecular Biology Association magazine] 3:801-805 (1984) method described.One or more polynucleotides as herein described can also be produced by using automatic 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 BV, 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).

[0111] 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 (such as PCR). One or more polynucleotides as described herein can also be obtained by screening a cDNA library using one or more oligonucleotide probes, which can hybridize with the polynucleotides encoding one or more subtilisin variants as described herein, or recombinant polypeptides or their active fragments or by PCR amplification. Programs for screening and separating cDNA clones and PCR amplification programs are well known to those skilled in the art and are described in standard references known to those skilled in the art. One or more polynucleotides as described herein can be obtained by, for example, changing naturally occurring polynucleotide backbones (such as, encoding one or more subtilisin variants as described herein or the polynucleotide backbones of a reference subtilisin) through known mutagenesis procedures (such as, site-directed mutagenesis, site-saturation mutagenesis and in vitro recombination). A variety of methods for producing modified polynucleotides as described herein encoding one or more subtilisin variants are known in the art, including, but not limited to, site-saturation mutagenesis, scanning mutagenesis, insertion mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution and various other recombination methods.

[0112] Additional embodiments relate to one or more vectors comprising one or more subtilisin variants described herein (e.g., polynucleotides encoding one or more subtilisin variants described herein); expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequences described herein; isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequences described herein; isolated or recombinant cells comprising one or more polynucleotide sequences described herein; and compositions comprising one or more such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof.

[0113] Some embodiments relate to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein, comprising one or more nucleic acid or polynucleotide sequences described herein. Some such recombinant cells are transformed or transfected using at least one such vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells comprise bacterial cells, including but not limited to Bacillus species cells, such as Bacillus subtilis cells. Other embodiments relate to recombinant cells (e.g., recombinant host cells) comprising one or more subtilisins described herein.

[0114] In some embodiments, one or more vectors described herein are expression vectors or expression cassettes comprising one or more polynucleotide sequences described herein operably linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., a promoter operably linked to one or more polynucleotide sequences described herein). The vectors may include transcription terminators and / or selection genes (e.g., antibiotic resistance genes) that enable continuous culture maintenance of plasmid-infected host cells by growth in a culture medium containing an antimicrobial agent.

[0115] Expression vectors can be derived from plasmids or viral DNA, or in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (see Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990); suitable replicating plasmids for Bacillus subtilis include those listed on page 92). (See also, Perego, “Integrational Vectors for Genetic Manipulations in Bacillus subtilis”; Sonenshein et al., [eds.]; “Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics”, American Society for Microbiology, Washington, DC (1993), pp. 615-624; and p2JM103BBI).

[0116] To express and produce a protein of interest (e.g., one or more subtilisin variants described herein) in a cell, one or more expression vectors containing one or more copies (and in some cases, multiple copies) of a polynucleotide encoding one or more subtilisin variants described herein are transformed into the cell under conditions suitable for expression of the variant. In some embodiments, the polynucleotide sequence encoding one or more subtilisin variants described herein (as well as other sequences contained in the vector) is integrated into the host cell's genome; however, in other embodiments, a plasmid vector containing a 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 and imported nucleotide sequences that integrate into the host cell genome. 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 integration vector that is capable of integrating the polynucleotide encoding the variant into the host chromosome and optionally amplifying the polynucleotide within the host chromosome. Examples of integration sites are well known to those skilled in the art. In some embodiments, transcription of the polynucleotide encoding one or more subtilisin variants described herein is achieved via a promoter that is the wild-type promoter of the parent subtilisin. In some other embodiments, the promoter is heterologous to one or more of the 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, and 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; the Bacillus thuringiensis cryIIIA; and the Bacillus licheniformis α-amylase gene. Additional promoters include, but are not limited to, the A4 promoter, as well as the bacteriophage lambda PR or PL promoters and the Escherichia coli (E. coli) lac, trp, or tac promoters.

[0117] 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, a Streptomyces species, an Escherichia species, an Aspergillus species, a Trichoderma species, a Pseudomonas species, a Corynebacterium species, a Saccharomyces species, or a Pichia species. In some embodiments, one or more subtilisin variants described herein are produced by a Bacillus species host cell. Examples of Bacillus species host cells that can be used to produce one or more subtilisin variants described herein include, but are not limited to, Bacillus licheniformis, Bacillus gibberellin, Bacillus lentus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus coagulans, Bacillus circulans, Bacillus pumilus, Bacillus thuringiensis, Bacillus clausii, and Bacillus megaterium, as well as other organisms within the genus Bacillus. In some embodiments, Bacillus subtilis host cells are used to produce the variants described herein. USPNs 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus host strains that can be used to produce one or more subtilisin variants described herein, but other suitable strains can be used.

[0118] Several bacterial strains that can be used to produce one or more subtilisin protease variants as herein described include non-recombinant (i.e. wild-type) Bacillus species strains, and variants of naturally occurring bacterial strains and / or recombinant strains. In certain embodiments, the host strain is a recombinant strain, wherein the polynucleotides encoding one or more subtilisin protease variants as herein described have been introduced into the host. In certain embodiments, the host strain is a subtilis host strain, particularly a recombinant subtilis host strain. Many strains of Bacillus subtilis are known, including, but not limited to, e.g., 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 to PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211 strains (see, e.g., Hoch et al., Genetics 73:215–228 (1973); see also, U.S. Pat. No. 4,450,235; U.S. Pat. No. 4,302,544; and EP 0 134 048). The use of Bacillus subtilis as an expression host cell is well known in the art (see, e.g., Palva et al., Gene 19:81-87 (1982); Fahnestock and Fischer, J. Bacteriol. 165:796-804 (1986); and Wang et al., Gene 69:39–47 (1988)).

[0119] In some embodiments, the Bacillus sp. host cell is a Bacillus sp. that comprises a mutation or deletion in at least one of the following genes: degU, degS, degR, and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU(Hy)32 (see, e.g., Msadek et al., J. Bacteriol. 172:824-834 (1990); and Olmos et al., Mol. Gen. Genet. 253:562–567 (1997)). In some embodiments, the Bacillus host comprises a mutation or deletion in: scoC4 (see, e.g., Caldwell et al., J. Bacteriol. 183:7329-7340 (2001)); spoIIE (see, e.g., Arigoni et al., Mol. Microbiol. 31:1407-1415 (1999)); and / or oppA or other genes of the opp operon (see, e.g., Perego et al., Mol. Microbiol. 5:173-185 (1991)). Indeed, it is contemplated that any mutation in the opp operon that results in the same phenotype as a mutation in the oppA gene will find use in some embodiments of the altered Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, the modified Bacillus host cell strain that can be used to produce one or more subtilisin variants described herein is a Bacillus host strain that already contains a mutation in one or more of the above-mentioned genes. Additionally, Bacillus sp. host cells that contain one or more mutations and / or one or more deletions of endogenous protease genes can be used. In some embodiments, the Bacillus host cell contains a deletion of the aprE and nprE genes. In other embodiments, the Bacillus sp. host cell contains a deletion of 5 protease genes, and in other embodiments, the Bacillus host cell contains a deletion of 9 protease genes (see, e.g., US 2005 / 0202535).

[0120] Use any suitable method known in the art to transform host cells with one or more nucleic acid sequences of encoding one or more subtilisin enzyme variants as herein described.Utilize plasmid DNA construct or carrier that nucleic acid (for example DNA) is introduced into bacillus cell or escherichia coli cell and the method that such plasmid DNA construct or carrier are transformed into such cell is well known.In certain embodiments, plasmid is separated and transformed into the bacillus cell from escherichia coli cell subsequently.Yet, use intervention microorganism such as escherichia coli is not essential, and in certain embodiments, DNA construct or carrier are directly introduced into the bacillus host.

[0121] Exemplary methods for introducing one or more nucleic acid sequences described herein into Bacillus cells are described, for example, in Ferrari et al., "Genetics," in Hardwood et al. [eds.], Bacillus, Plenum Publishing Corp. (1989), pp. 57-72; Saunders et al., J. Bacteriol., 157:718-726 (1984); Hoch et al., J. Bacteriol., 93:1925-1937 (1967); Mann et al., Current Microbiol., 13:131-135 (1986); Holubova, Folia Microbiol., 30:97 (1985); Chang et al., Mol. Gen. Genet. 168:11-115 (1979); Vorobjeva et al., FEMS Microbiol. Lett. 7:261-263 (1980); Smith et al., Appl. Env. Microbiol. 51:634 (1986); Fisher et al., Arch. Microbiol. 139:213-217 (1981); and McDonald, J. Gen. Microbiol. 130:203 (1984). Indeed, methods such as transformation, including protoplast transformation and transfection, transduction, and protoplast fusion, are well known and suitable for use herein. Methods known in the art for transforming Bacillus cells include, for example, plasmid marker rescue transformation, which involves uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (see, Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol. 154:1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169:1205-1211 (1987)).In this method, an incoming donor plasmid recombines with homologous regions of a resident "helper" plasmid in a process that mimics chromosomal transformation.

[0122] In addition to commonly used methods, in some embodiments, host cells are directly transformed with DNA constructs or vectors comprising nucleic acids encoding one or more subtilisin variants described herein (i.e., before introduction into the host cells, intermediate cells are not used to amplify or otherwise process the DNA constructs or vectors). Introducing DNA constructs or vectors as described herein into host cells includes those physical and chemical methods known in the art for introducing nucleic acid sequences (e.g., DNA sequences) into host cells without inserting them into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In other embodiments, the DNA construct or vector is co-transformed with a plasmid without inserting a plasmid. In further embodiments, a selection marker is deleted from a modified Bacillus strain by methods known in the art (see, Stahl et al., J. Bacteriol. [Journal of Bacteriology] 158:411-418 (1984); and Palmeros et al., Gene [Gene] 247:255-264 (2000)).

[0123] In some embodiments, the transformed cells are cultured in a conventional nutrient medium. Suitable specific culture conditions, such as temperature, pH, etc., are known to those skilled in the art and are described in detail in the scientific literature. Some embodiments provide cultures (e.g., cell cultures) comprising one or more subtilisin variants or nucleic acid sequences described herein.

[0124] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more subtilisin variants as described herein are cultured in a suitable nutrient medium under conditions permitting expression of the variant, and the resulting variants are thereafter recovered from the culture. In some embodiments, the variants produced by the cells are recovered from the culture medium by conventional procedures including, but not limited to, separation of the host cells from the culture medium by centrifugation or filtration, precipitation of the protein components of the supernatant or filtrate with the aid of salts (e.g., ammonium sulfate), and purification by chromatography (e.g., ion exchange, gel filtration, affinity, etc.).

[0125] In certain embodiments, one or more subtilisin protease variants produced by recombinant host cells are secreted into the substratum. The nucleic acid sequence encoding the purification promotion domain can be used to promote the purification of this variant. The carrier or DNA construct comprising the polynucleotide sequence encoding one or more subtilisin protease variants as herein described can further comprise the nucleic acid sequence encoding the purification promotion domain that promotes the purification of the variant (see, for example, Kroll et al., DNA Cell Biol. [DNA cell biology] 12:441-53 (1993)). Such purification promotion domain includes but is not limited to, for example, metal chelating peptides, such as the histidine-tryptophan module (see Porath, Protein Expr. Purif. [protein expression and purification] 3:263-281

[1992] ) allowed to be purified on immobilized metals, the protein A domain allowed to be purified on immobilized immunoglobulins, and the domain adopted in the FLAGS extension / affinity purification system. It has also been found useful to include a cleavable linker sequence such as Factor XA or enterokinase (eg, sequences available from Invitrogen, San Diego, CA) between the purification domain and the heterologous protein to facilitate purification.

[0126] The variant protein of the present invention can be produced in host cells using methods well known in the art, such as by secretion or intracellular expression. Fermentation, separation and concentration techniques are well known in the art, and conventional methods can be used to prepare concentrated, enzyme-containing solutions. Host cells can be further processed, for example, by heating or by changing pH or salt content or by enzymatic treatment with enzymes including hen egg white lysozyme, T4 lysozyme or WO 2022047149, for example, to release enzymes or to improve cell separation. For production-scale recovery, variant polypeptides can be enriched or partially purified by removing cells via polymer flocculation as 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 full broth culture can be cracked and used without further processing. The enzyme can then be processed into, for example, particles or non-aqueous particles.

[0127] Various methods can be used to determine the production level of one or more mature subtilisin protease variants as described herein in a host cell. Such methods include, but are not limited to, methods such as utilizing polyclonal or monoclonal antibodies specific for 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. [Journal of Experimental Medicine] 158:1211 (1983)).

[0128] Some other embodiments provide methods for preparing or producing one or more mature subtilisin protease variants as herein described.Mature subtilisin protease variants do not include signal peptide or propeptide sequences.Some methods are included in recombinant bacterial host cells (for example, as Bacillus species cells (for example, Bacillus subtilis cells)) and prepare or produce one or more subtilisin protease variants as herein described.Other embodiments provide methods for producing one or more subtilisin protease variants as herein described, wherein the method is included in cultivating a recombinant host cell comprising a recombinant expression vector under the conditions that are conducive to producing this variant, and this recombinant expression vector comprises the nucleotide sequence of encoding one or more subtilisin protease variants as herein described.Some such methods further comprise reclaiming this variant from culture.

[0129] Additional embodiments provide methods for producing one or more subtilisin variants as described herein, wherein the methods include: (a) introducing a recombinant expression vector comprising a nucleic acid encoding the variant into a cell population (e.g., bacterial cells, such as Bacillus subtilis cells); and (b) culturing the cells in a culture medium under conditions conducive to producing the variant encoded by the expression vector. Some such methods further include: (c) isolating the variant from the cells or from the culture medium.

[0130] Additional embodiments relate to methods of improving the cleaning performance or stability of a subtilisin, comprising modifying the subtilisin to comprise one or more substitutions, or combinations of substitutions, as provided herein.

[0131] Unless otherwise noted, all components or composition levels provided herein are given with reference to the activity level of the component or composition, and do not include impurities that may be present in commercially available sources, such as residual solvents or by-products. Enzyme component weight is based on total active protein. Unless otherwise indicated, all percentages and ratios are calculated by weight. Unless otherwise indicated, all percentages and ratios are calculated based on total composition. Compositions as described herein include cleaning compositions, such as detergent compositions. In the detergent compositions of examples, enzyme levels are expressed by pure enzyme by weight of the total composition and, unless otherwise specified, detergent ingredients are expressed by weight of the total composition.

[0132] In one embodiment, one or more subtilisin protease variants as described herein can be used in cleaning applications, such as, but not limited to, cleaning tableware items or tableware items, fabrics, medical devices, and items with hard surfaces (e.g., the hard surfaces of tables, desktops, walls, furniture items, floors, ceilings). In other embodiments, one or more subtilisin protease variants as described herein can be used in disinfecting applications, such as, but not limited to, disinfecting automatic dishwashing machines or washing machines.

[0133] In some embodiments, the cleaning compositions described herein further comprise a surfactant. In some embodiments, the surfactant is selected from nonionic surfactants, amphoteric surfactants, semi-polar surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations and mixtures thereof. In yet other embodiments, the surfactant is selected from anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations thereof. In some embodiments, the cleaning compositions described herein comprise from about 0.1% to about 60%, from about 1% to about 50%, or from about 5% to about 40% of a surfactant by weight of the composition. Exemplary surfactants include, but are not limited to, sodium dodecylbenzenesulfonate, C12-14 pareth-7, C12-15 pareth-7, C12-15 pareth-4, sodium laureth sulfate (e.g., Steol CS-370), sodium hydrogenated coconut oil, C 12Ethoxylates (Alfonic 1012-6, Hetoxol LA7, Hetoxol LA4), sodium alkylbenzene sulfonate (e.g., Nacconol 90G), and combinations and mixtures thereof. Anionic surfactants include, but are not limited to, linear alkylbenzene sulfonate (LAS), α-olefin sulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkane sulfonate (SAS), α-sulfo fatty acid methyl ester, alkyl- or alkenyl succinic acid, or soap. Nonionic surfactants include, but are not limited to, alcohol ethoxylates (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethylamine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides, polyhydroxyalkyl fatty acid amides (e.g., as described in WO 92 / 06154), polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan esters (e.g., TWEEN), polyoxyethylene alcohols, polyoxyethylene isoalcohols, polyoxyethylene ethers (e.g., TRITON and BRIJ), polyoxyethylene esters, polyoxyethylene-p-tert-octylphenol or octylphenyl-ethylene oxide condensates (e.g., NONIDET P40), condensates of ethylene oxide with fatty alcohols (e.g., LUBROL), polyoxyethylene nonylphenols, polyalkylene glycols (SYNPERONIC F108), sugar-based surfactants (e.g., glucopyranosides, thioglucopyranosides), and combinations and mixtures thereof.

[0134] In additional embodiments, the detergent compositions disclosed herein further comprise a surfactant mixture including, but not limited to, 5%-15% anionic surfactants, <5% nonionic surfactants, cationic surfactants, phosphonates, soaps, enzymes, perfumes, butylphenyl methylpropionate, geraniol, zeolites, polycarboxylates, hexyl cinnamaldehyde, limonene, cationic surfactants, citronellol, and benzisothiazolinone.

[0135] The cleaning compositions described herein may additionally comprise one or more detergent builders or builder systems, complexing agents, polymers, bleaching systems, stabilizers, foam builders, foam inhibitors, corrosion inhibitors, soil suspending agents, soil redeposition inhibitors, dyes, bactericides, hydrogen promoters, tarnish inhibitors, optical brighteners, fabric conditioners, and perfumes. The cleaning compositions described herein may also comprise additional enzymes selected from the group consisting of mannanases, amylases, cellulases, lipases, pectin degrading enzymes, xyloglucanases, or additional carboxylic ester hydrolases.

[0136] In some embodiments, the cleaning compositions described herein further comprise from about 1%, from about 3% to about 60%, or even from about 5% to about 40% of a builder, based on the weight of the cleaning composition. Builders can include, but are not limited to, alkali metal, ammonium, and alkanolammonium salts of polyphosphates; alkali metal silicates, alkaline earth metal, and alkali metal carbonates; aluminosilicates; polycarboxylic acid 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; various alkali metal, ammonium, and substituted ammonium salts of polyacetic acid, such as ethylenediaminetetraacetic acid and nitrilotriacetic acid; as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof. In some aspects, oxidized α-1,3-glucan can be included as a co-builder; oxidized α-1,3-glucan compounds for use herein are disclosed in US Patent Application Publication No. 2015 / 0259439.

[0137] In some embodiments, builders form water-soluble hardness ion complexes (e.g., chelating builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixed sodium and potassium tripolyphosphates, etc.). Any suitable builder can be used in the compositions described herein, including those known in the art (see, for example, EP 2100949).

[0138] As indicated herein, in some embodiments, the cleaning compositions described herein further comprise adjunct ingredients including, but not limited to, surfactants, builders, bleaching agents, bleach activators, bleach catalysts, other enzymes, enzyme stabilization systems, chelating agents, optical brighteners, soil release polymers, dye transfer agents, dye transfer inhibitors, catalytic materials, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersants, clay soil removers, structure elasticizers, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, solvents, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-tarnish and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents (see, e.g., U.S. Patent No. 5,237,779). 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 some embodiments, one or more adjunct ingredients are incorporated, for example, to assist or enhance cleaning performance (for treating the substrate to be cleaned) or to modify the aesthetics of the cleaning composition (as with fragrances, colorants, dyes, etc.). Any such adjunct ingredients are in addition to the protease variants described herein. The precise nature of these additional components and their incorporation levels will depend on the physical form of the composition and the nature of the cleaning operation in which the composition will be used.

[0139] In embodiments where one or more adjunct ingredients are incompatible with the protease variant, suitable methods can be employed to keep the cleaning adjunct ingredient and the protease separated (i.e., out of contact with each other) until the combination of the two components is appropriate. Such separation methods include any suitable method known in the art (e.g., capsules, encapsulation, tablets, physical separation, etc.). The specific selection of suitable adjunct ingredients is readily accomplished by considering the surface, article, or fabric to be cleaned, and the desired composition form for the cleaning conditions during use (e.g., throughout detergent washing use).

[0140] The cleaning compositions described herein are advantageously used in, for example, laundry applications, hard surface cleaning, dishwashing applications, and cosmetic applications.In addition, the polypeptides of the present invention can be used in granular compositions and liquid compositions.

[0141] The protease variants described herein can also be used in cleaning additive products. In some embodiments, the additive is encapsulated in a dosage form suitable for addition to a cleaning process. In some embodiments, the additive is encapsulated in a dosage form for addition to a cleaning process where a peroxide source is used and an increased bleaching effect is desired. The present disclosure may use any suitable single unit dosage form, including but not limited to pills, tablets, capsules, or other single unit dosage forms (such as pre-measured powders or liquids). In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such a composition. Suitable fillers or carrier materials include but are not limited to various salts of sulfates, carbonates, and silicates, as well as talc, clay, and the like. Suitable fillers or carrier materials for liquid compositions include but are not limited to water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include but are not limited to methanol, ethanol, propanol, and isopropanol. In some embodiments, the composition contains from about 5% to about 90% of such materials. Acidic fillers can be used to lower the pH. Alternatively, in some embodiments, the cleaning additive includes one or more auxiliary ingredients.

[0142] Another embodiment relates to a composition comprising one or more subtilisin variants as described herein. In some embodiments, the composition is a cleaning composition. In other embodiments, the composition is a detergent composition. In yet other embodiments, the composition is selected from laundry detergent compositions, automatic dishwashing (ADW) compositions, hand-washing (manual) dishwashing detergent compositions, hard surface cleaning compositions, eyeglass cleaning compositions, medical device cleaning compositions, disinfectant (e.g., malodor or microorganism) compositions, and personal care cleaning compositions. In still other embodiments, the composition is a laundry detergent composition, an ADW composition, or a hand-washing (manual) dishwashing detergent composition. Even further embodiments relate to fabric cleaning compositions, while other embodiments relate to non-fabric cleaning compositions. In some embodiments, the cleaning composition does not contain boron. In other embodiments, the cleaning composition does not contain phosphate. In still other embodiments, the composition comprises one or more subtilisin variants as described herein and one or more excipients, auxiliary materials, and / or additional enzymes.

[0143] In another embodiment, the present disclosure provides detergent compositions (e.g., laundry detergent compositions) comprising a surfactant and at least one subtilisin protease variant as provided herein. Such compositions may further comprise one or more excipients, auxiliary materials, and / or additional enzymes.

[0144] In some aspects, in addition to the α-glucan ester derivatives disclosed herein, the detergent composition may further comprise one or more other types of polymers. Examples of other types of polymers that can be used herein include carboxymethylcellulose (CMC), dextran, poly(vinyl pyrrolidone) (PVP), polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers.

[0145] In yet further embodiments, the compositions described herein contain phosphate, do not contain phosphate, contain boron, do not contain boron, or a combination thereof. In other embodiments, the composition is a boron-free composition. In some embodiments, a boron-free composition is a composition to which no borate stabilizer has been added. In another embodiment, a boron-free composition is a composition containing less than 5.5% boron. In yet further embodiments, a boron-free composition is a composition containing less than 4.5% boron. In yet further embodiments, a boron-free composition is a composition containing less than 3.5% boron. In yet further embodiments, a boron-free composition is a composition containing less than 2.5% boron. In even further embodiments, a boron-free composition is a composition containing less than 1.5% boron. In another embodiment, a boron-free composition is a composition containing less than 1.0% boron. In yet further embodiments, a boron-free composition is a composition containing less than 0.5% boron. In other embodiments, the composition is a composition that is free of or substantially free of enzyme stabilizers or peptide inhibitors.

[0146] In another embodiment, one or more compositions described herein are in a form selected from a gel, tablet, powder, granule, solid, liquid, unit dose, and combinations thereof. In yet another embodiment, one or more compositions described herein are in a form selected from a low water compact formula, a low water HDL or unit dose (UD), or a high water formula or HDL. In some embodiments, the cleaning compositions described herein are in unit dosage form. In other embodiments, the unit dosage form is selected from a pill, tablet, capsule, caplet, sachet, pouch, multi-compartment pouch, and pre-measured powder or liquid. In some embodiments, the unit dosage form is designed to provide controlled release of the ingredients within the 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 WO04 / 111178. In some embodiments, the unit dosage form is a tablet or powder contained in a water-soluble film or pouch.

[0147] 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, compositions for example, cleaning the hard surfaces of non-tableware items, non-desktop utensil items, tables, desktops, furniture items, walls, floors, and ceilings. Exemplary hard surface cleaning compositions are described in, for example, USPN 6,610,642, 6,376,450, and 6,376,450. Exemplary personal care compositions include, but are not limited to, compositions for cleaning dentures, teeth, hair, contact lenses, and skin. Exemplary components of such oral care compositions are included in, for example, those described in US 6,376,450.

[0148] In certain embodiments, one or more subtilisin enzyme variants as described herein clean at low temperatures. In other embodiments, one or more compositions as described herein clean at low temperatures. In other embodiments, one or more compositions as described herein comprise an effective amount of one or more subtilisin enzyme variants as described herein, which are useful or effective for cleaning surfaces that need to remove protein stains.

[0149] In some embodiments, adjunct materials are incorporated, for example, to assist or enhance cleaning performance; to treat the substrate to be cleaned; or to modify the aesthetics of the cleaning composition, such as in the case of fragrances, colorants, dyes, and the like. One embodiment relates to a composition comprising one or more adjunct materials as described herein and one or more subtilisin variants. Another embodiment relates to a composition comprising one or more adjunct materials as described herein and one or more subtilisin variants, wherein the adjunct material is selected from the group consisting of: bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilization systems), chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, fillers, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericidal agents, agents, fungicides, color embellishments, silver care agents, anti-tarnish agents, anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, pH control agents, surfactants, builders, chelating agents, dye transfer inhibitors, deposition aids, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersants, clay soil removers / anti-redeposition agents, structure elasticizers, fabric softeners, carriers, hydrotropes, processing aids, pigments, and combinations thereof. Exemplary auxiliary materials and usage levels can be found in USPN 5,576,282, 6,306,812, 6,326,348, 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101. In embodiments where one or more cleaning auxiliary materials are incompatible with one or more subtilisin variants described herein, a method is employed where the auxiliary material and one or more variants are kept separate (i.e., not in contact with each other) until the combination of the two components is suitable. Such separation methods include any suitable method known in the art (e.g., capsules, encapsulation, tablets, physical separation, etc.).

[0150] Some embodiments relate to cleaning additive products comprising one or more subtilisin variants as described herein. In certain embodiments, the additive is encapsulated in a formulation for addition to a cleaning process. In certain embodiments, the additive is encapsulated in a formulation for addition to a cleaning process in which a peroxide source is used and an increased bleaching effect is desired.

[0151] Exemplary fillers or carriers for granular compositions include, but are not limited to, various salts such as sulfates, carbonates, and silicates; talc; and clay. Exemplary fillers or carriers for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols, 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 during cleaning methods or applications.

[0152] In one embodiment, one or more cleaning compositions described herein comprise an effective amount of one or more subtilisin variants described herein, alone or in combination with one or more additional enzymes. Typically, the cleaning compositions comprise at least about 0.0001 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 %, from about 0.001 wt % to about 1 wt %, or from about 0.01 wt % to about 0.2 wt % of one or more subtilisin variants described herein. In another embodiment, one or more cleaning compositions described herein comprise from about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 2 mg, about 0.01 to about 1 mg, about 0.05 to about 1 mg, about 0.5 to about 10 mg, about 0.5 to about 5 mg, about 0.5 to about 4 mg, about 0.5 to about 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 subtilisin variants described herein per gram of the composition.

[0153] The cleaning compositions described herein are typically formulated so that during use in aqueous cleaning operations, the wash water will have a pH of 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 of 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 of 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 wash conditions, such as a pH of 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 wash conditions, such as a pH of 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 a ratio of 1:100 (wt:wt) at 20°C, as measured using a conventional pH meter. Techniques for controlling pH at recommended usage levels include the use of buffers, bases, acids, etc., and are well known to those skilled in the art.

[0154] Suitable low-pH cleaning compositions typically have a neat pH of from about 3.0 to about 5.0, or even from about 3.5 to about 4.5. Low-pH cleaning compositions typically do not contain surfactants that hydrolyze in such pH environments. Such surfactants include sodium alkyl sulfate surfactants containing at least one ethylene oxide moiety or even from about 1 to about 16 moles of ethylene oxide. Such cleaning compositions typically contain a sufficient amount of a pH adjuster (such as sodium hydroxide, monoethanolamine, or hydrochloric acid) to provide such cleaning compositions with a neat pH of from about 3.0 to about 5.0. Such compositions typically contain at least one acid-stable enzyme. In some embodiments, the compositions are liquids, while in other embodiments, they are solids. The pH of such liquid compositions is typically measured neat. The pH of such solid compositions is measured as a 10% solids solution of the composition in distilled water. In these examples, all pH measurements are taken at 20°C unless otherwise stated.

[0155] Suitable high pH cleaning compositions typically have a neat pH of from about 9.0 to about 11.0, or even a neat pH of from 9.5 to 10.5. Such cleaning compositions typically include a sufficient amount of a pH adjusting agent (such as sodium hydroxide, monoethanolamine, or hydrochloric acid) to provide such cleaning compositions with a neat pH of from about 9.0 to about 11.0. Such compositions typically include at least one alkali-stable enzyme. In some embodiments, the composition is a liquid, while in other embodiments, these compositions are solid. The pH of such liquid compositions is typically measured as a neat pH. The pH of such solid compositions is measured as a 10% solids solution of the composition, wherein the solvent is distilled water. In these embodiments, unless otherwise stated, all pH measurements are performed at 20°C.

[0156] In some embodiments, one or more subtilisin variants described herein are encapsulated to protect them from the influence of other components in the composition during storage and / or to control the availability of the variants during cleaning. In some embodiments, encapsulation enhances the performance of the variants and / or other enzymes. In some embodiments, an encapsulating material typically encapsulates at least a portion of the subtilisin variants described herein. Typically, the encapsulating material is water-soluble and / or water-dispersible. In some embodiments, the encapsulating material has a glass transition temperature (Tg) of 0°C or higher. 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 a microsphere made of plastic (such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof). Exemplary commercial microspheres include but are not limited to EXPANCEL ® (Stockviksverken, Sweden); and PM 6545, PM6550, PM 7220, PM 7228, EXTENDOSPHERES ® 、LUXSIL ® 、Q-CEL ® and SPHERICEL ® (PQCorp., Valley Forge, PA).

[0157] In some embodiments, the present invention provides a kind of washing condition of the present invention.There are various washing conditions, comprise the different detergent formulations, wash water volume, wash water temperature and the length of washing time that one or more subtilisin variants as herein described can expose.Low detergent concentration system relates to the washing water that contains less than about 800 ppm detergent component.Medium detergent concentration system relates to the washing water that contains the detergent component of about 800 ppm to about 2000ppm.High detergent concentration system relates to the washing water that contains greater than about 2000 ppm detergent component.In certain embodiments, " cold water washing " of the present invention utilizes and is suitable for " cold water detergent " washing under the temperature of every other combination from about 10 ℃ to about 40 ℃, from about 20 ℃ to about 30 ℃ or from about 15 ℃ to about 25 ℃ and about 15 ℃ to about 35 ℃ or 10 ℃ to 40 ℃ scope.

[0158] Different geographical locations have different water hardness. Hardness is the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) is a measure of the amount of Ca2+ mixed in a container. Usually measured in grains per gallon (gpg) 2+ / Mg 2+ To describe water hardness. Most water in the United States is hard water, but there are different levels of hardness. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has between 60 and 181 ppm (ppm can be converted to grains per US gallon by dividing ppm by 17.1) of hardness minerals.

[0159]

[0160] Other embodiments relate to one or more cleaning compositions comprising from about 0.00001% to about 10% by weight of the composition of one or more subtilisin variants described herein, and from about 99.999% to about 90.0% by weight of the composition of one or more adjunct 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 adjunct materials.

[0161] 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, alginate lyases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, β-glucanases, carrageenanases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, dispersins, endo-β-1, β-glucanase, endo-β-mannanase, esterase, exo-mannanase, fructosidase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, laminarinase, lichenase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease (e.g., DNA enzyme and / or RNA enzyme), oxidase, oxidoreductase, pectate lyase, pectin acetylcholine, thiazolinase ... The invention also provides a method for preparing the enzyme mixture of the present invention and the method for preparing the enzyme mixture of the present invention. The method comprises preparing the enzyme mixture of the present invention and the method for preparing the enzyme mixture of the present invention. The method comprises preparing the enzyme mixture of the present invention and the method for preparing the enzyme mixture of the present invention.

[0162] In another embodiment, one or more compositions as herein described comprise one or more subtilisin variants as herein described and one or more other proteases. In one embodiment, other proteases are serine proteases. In another embodiment, other proteases are metalloproteases, fungal subtilisin or alkaline microbial proteases or trypsin-like proteases. Suitable other proteases include those of animal, plant or microbial origin. In certain embodiments, other proteases are microbial proteases. In other embodiments, other proteases are chemically or genetically modified mutants. In another embodiment, other proteases are alkaline microbial proteases or trypsin-like proteases. In other embodiments, other proteases do not contain an epitope that cross-reacts with the subtilisin variants as measured by antibodies available in the art in conjunction with or other assays. Exemplary alkaline proteases include subtilisins derived from, for example, Bacillus (e.g., B. lentus, B. gibberellin, B. pumilus, TY-145, Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168), or fungal sources, such as those described in U.S. Pat. No. 8,362,222.Exemplary additional proteases include, but are not limited to, WO 92 / 21760, WO 95 / 23221, WO 2008 / 010925, WO 09 / 149200, WO 09 / 149144, WO09 / 149145, WO 10 / 056640, WO 10 / 056653, WO 2010 / 0566356、WO 11 / 072099、WO 2011 / 13022、WO 11 / 140364、WO 12 / 151534、WO 2015 / 038792、WO 2015 / 089447、WO 2015 / 089441、WO 2017 / 215925、WO 2022 / 106400, U.S. Publication No. 2008 / 0090747, US 5,801,039, US 5,340,735, US 5,500,364, US 5,855,625, RE 34,606, US 5,955,340, US 5,700,676, US 6,312,936, US 6,482,628, US 8,530,219, U.S. 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, as well as those described in WO 1999014341, WO 1999033960, WO 1999014342, WO 1999034003, WO 2007044993, WO 2009058303, WO 2009058661, WO2014071410, WO 2014194032, WO 2014194034, WO 2014194054, WO 2014 / 194117, EP3380599, 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, MAXATASE. ® , MAXACAL ™ , MAXAPEM ™ OPTICLEAN® 、OPTIMASE ® 、PROPERASE ® 、PURAFECT ® 、PURAFECT ® OXP, PURAMAX ™ 、EXCELLASE ™ 、PREFERENZ ™ Proteases (e.g., P100, P110, P280, P300), EFFECTENZ ™ Proteases (e.g., P1000, P1050, P2000), EXCELLENZ ™ Proteases (e.g., P1000), ULTIMASE ® , and PURAFAST ™ (International Flavors & Fragrances (IFF)); ALCALASE ® 、ALCALASE ® 、ULTRA、BLAZE ® , BLAZE ® Variant, BLAZE ® EVITY ® , BLAZE ® EVITY ® 16L, CORONASE ® 、SAVINASE ® 、SAVINASE ® ULTRA, SAVINASE ® EVITY ® 、SAVINASE ® EVERIS ® PRIMASE ® 、DURAZYM ™ 、POLARZYME ® 、OVOZYME ® 、KANNASE ® LIQUANASE ® LIQUANASE EVERIS ® 、NEUTRASE ® 、PROGRESS UNO ® 、RELASE ® , and ESPERASE ® (Novozymes); BLAP ™ and BLAP ™ Variant (Henkel); LAVERGY ™PRO 104 L (BASF), KAP (alkalophilic Bacillus subtilisin (Kao), PuriWise 1.0L (Bestzyme), and BIOTOUCH® (AB Enzymes).

[0163] Another embodiment relates to a composition comprising one or more subtilisin variants as described herein and one or more lipases. In certain embodiments, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% lipase by weight of the composition. Exemplary lipases can be chemically or genetically modified mutants. Exemplary lipases include, but are not limited to, those of bacterial or fungal origin, such as, for example, H. lanuginosa lipase (see, e.g., EP 258068 and EP 305216), T. lanuginosa lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase (see, e.g., EP 238023), Candida lipases, such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, e.g., EP 214761), Pseudomonas lipases, such as P. alcaligenes and P. pseudoalcaligenes lipases (see, e.g., EP 238023). 218272), P. cepacia lipase (see, e.g., EP 331376), P. stutzeri lipase (see, e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipases (e.g., B. subtilis lipase (Dartois et al., Biochem. Biophys. 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 cloned lipases 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 R. delemar lipase (see Hass et al., Gene 109:117-113 (1991)), R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 (1992)), and R. oryzae lipase. Other lipolytic enzymes (e.g., cutinases) may also be used in one or more compositions described herein, including, but not limited to, cutinases derived from, for example, Pseudomonas mendocina (see WO 88 / 09367) and / or Fusarium solani pisi (see WO 90 / 09446). Exemplary commercial lipases include, but are not limited to, M1 LIPASE. ™ LUMA FAST ™ 、LIPOMAX ™ and PREFERENZ ™ L100 (International Flavors & Fragrances); LIPEX®, LIPEX® Evity® 200L, LIPOCLEAN ® 、LIPOLASE ® and LIPOLASE ® ULTRA (Novozymes); and LIPASE P ™ (Amano Pharmaceutical Co. Ltd).

[0164] Still other embodiments relate to compositions comprising one or more subtilisin protease variants as herein described 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 (e.g., alpha amylase and / or beta amylase) suitable for use in alkaline solutions can be used to be included 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, as described, for example, in GB 1,296,839, WO 9100353, WO 9402597, WO94183314, WO 9510603, WO 9526397, WO 9535382, WO 9605295, WO 9623873, WO 9623874, WO9630481, WO 9710342, WO 9741213, WO 9743424, WO 9813481, WO 9826078, WO 9902702, WO9909183, WO 9919467, WO 9923211, WO 9929876, WO 9942567, WO 9943793, WO 9943794, WO9946399, WO 0029560, WO 0060058, WO 0060059, WO 0060060, WO 0114532, WO 0134784, WO0164852, WO 0166712, WO 0188107, WO 0196537, WO 02092797, WO 0210355, WO 0231124, WO2004055178, WO 2004113551, WO 2005001064, WO 2005003311, WO 2005018336, WO2005019443, WO 2005066338, WO 2006002643, WO 2006012899, WO 2006012902, WO2006031554, WO 2006063594, WO 2006066594, WO 2006066596, WO 2006136161, WO2008000825, WO 2008088493, WO 2008092919, WO 2008101894, WO 2008 / 112459, WO2009061380, WO 2009061381, WO 2009100102, WO 2009140504, WO 2009149419, WO2010 / 059413, WO 2010088447, WO 2010091221, WO 2010104675, WO 2010115021, WO 10115028, WO2010117511, WO 2011076123, WO 2011076897, WO 2011080352, WO 2011080353, WO2011080354, WO 2011082425, WO 2011082429, WO 2011087836, WO 2011098531, WO2013063460, WO 2013184577, WO 2014099523, WO 2014164777, WO 2015077126, and WO2018184004. Exemplary commercial amylases include, but are not limited to, AMPLIFY®, DURAMYL ® TERMAMYL ® 、FUNGAMYL ® 、STAINZYME ® 、STAINZYME PLUS ® 、STAINZYME PLUS ® 、STAINZYME ULTRA ® EVITY ® , and BAN ™ (Novozymes); EFFECTENZ ™ S 1000, POWERASE ™ 、PREFERENZ ™ S 100, PREFERENZ ™ S 110, PREFERENZ ™ S 210, EXCELLENZ ™ S 2000, RAPIDASE ® and MAXAMYL ® P (International Flavors & Fragrances, Inc.). In some embodiments, the subtilisin variants provided herein can be combined with one or more amylases and variants thereof, and combinations of the one or more amylases and variants thereof, the one or more amylases selected from the group consisting of AA707, AA560, AAI10, BspAmy24, SP722, and CspAmy1.

[0165] Still other embodiments relate to compositions comprising one or more 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 of bacterial or fungal origin, such as those described in WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. Provisional Application No. 62 / 296,678. Exemplary commercial cellulases include, but are not limited to, CELLUCLEAN ® 、CELLUZYME ® 、CAREZYME ® ENDOLASE ® 、RENOZYME ® 、CAREZYME ® Elite 100L and CAREZYME ® PREMIUM (Novozymes); REVITALENZ ™ 100. REVITALENZ ™ 200 / 220, and REVITALENZ ® 2000 (International Flavors & Fragrances); Lavergy C Bright 100 (BASF) and KAC-500(B) ™ (Kao Corporation). In some embodiments, the cellulase is incorporated as a portion or fragment of a mature wild-type or variant cellulase in which a portion of the N-terminus is deleted (see, eg, US 5,874,276).

[0166] Even further embodiments relate to compositions comprising one or more subtilisin variants as 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 of bacterial or fungal origin, such as those described in WO 99 / 64619, WO2016 / 007929; USPN 6,566,114, 6,602,842, and 6,440,991; and U.S. Provisional Application Nos. 62 / 251516, 62 / 278383, and 62 / 278387. Exemplary commercial mannanases include, but are not limited to, MANNAWAY ® (Novozymes), Lavergy M Ace 100L (BASF), and EFFECTENZ ™ M 1000, EFFECTENZ ™ M 2000, PREFERENZ ® M 100, MANNASTAR ® , and PURABRITE ™ (International Flavors & Fragrances, Inc.).

[0167] In some embodiments, the cleaning compositions described herein further comprise a suitable pectin-degrading enzyme. As used herein, "one or more pectin degrading enzymes" encompasses arabinanases (EC 3.2.1.99), galactanases (EC 3.2.1.89), polygalacturonases (EC 3.2.1.15), exo-polygalacturonases (EC 3.2.1.67), exo-poly-α-galacturonosidases (EC 3.2.1.82), pectin lyases (EC 4.2.2.10), pectinesterases (EC 3.1.1.11), pectate lyases (EC 4.2.2.2), exo-polygalacturonate lyases (EC 4.2.2.9), and hemicellulases such as endo-1,3-β-xylosidase (EC 3.2.1.32), xylan-1,4-β-xylosidase (EC 3.2.1.47), and xylan-1,4-β-xylosidase (EC 3.2.1.66). 3.2.1.37), xanthan gum lyase, and α-L-arabinofuranosidase (EC 3.2.1.55). Pectin-degrading enzymes are naturally occurring mixtures of the enzyme activities mentioned above. Thus, pectinases include pectin methylesterase, which hydrolyzes pectin methyl ester bonds; polygalacturonase, which cleaves glycosidic bonds between galacturonic acid molecules; and pectin transeliminase or lyase, which acts on pectic acid to non-hydrolytically cleave α-1,4 glycosidic bonds to form unsaturated derivatives of galacturonic acid.

[0168] Suitable pectin degrading enzymes include those of plant, fungus or microbial origin. In certain embodiments, include chemical or genetically modified mutants. In certain embodiments, the pectin degrading enzyme is an alkaline pectin degrading enzyme, i.e., an enzyme having at least 10%, at least 25% or at least 40% enzymatic activity at a pH of from about 7.0 to about 12 of its maximum activity. In certain other embodiments, the pectin degrading enzyme is an enzyme having at least 7.0% or at least 40% enzymatic activity at a pH of from about 7.0 to about 12 of its maximum activity. The alkaline pectin degrading enzyme is produced by alkaliphilic microorganisms, which are, for example, bacteria, fungi and yeast microorganisms, such as Bacillus species. In certain embodiments, as described in JP 56131376 and JP 56068393, the microorganism is Bacillus firmus, Bacillus circulans and Bacillus subtilis. Alkaline pectinolytic enzymes may include, but are not limited to, galacturonan-1,4-α-galacturonase (EC 3.2.1.67), polygalacturonase activity (EC 3.2.1.15), pectinesterase (EC 3.1.1.11), pectate lyase (EC 4.2.2.2), and isozymes thereof. Alkaline pectinolytic enzymes may be produced by Erwinia species. In some embodiments, alkaline pectinase is produced by E. chrysanthemi, E. carotovora, E. amylovora, E. herbicola, and E. dissolvens, as described in JP 59066588, JP 63042988, and World J. Microbiol. Biotechnol. (8, 2, 115-120) 1992. In certain other embodiments, alkaline pectinase is produced by Bacillus species, as described in JP 73006557 and Agr. Biol. Chem. (1972), 36(2) 285-93. Exemplary commercial xanthan gum lyases include, but are not limited to, XPect® 1000L (Novozymes). In some embodiments, the cleaning compositions described herein further comprise from about 0.00001% to about 10%, from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5%, by weight of the composition, of a pectin degrading enzyme.

[0169] In some other embodiments, the cleaning compositions described herein further comprise suitable beta-glucanases. Suitable beta-glucanases include, but are not limited to, those of plant, fungal or bacterial origin. In some embodiments, chemically or genetically modified mutants are included. As used herein, beta-glucanases refer to endo-beta-1,4-glucanase activity (e.g., endo-1,4^-D-glucanases) that catalyze the hydrolysis of the beta-1,4-bonds connecting two glucosyl residues in beta-glucans. Non-limiting examples of beta-glucanases as defined herein include cellulases (e.g., EC 3.2.1.4, e.g., endo-cellulase activity on the beta-1,4-bonds between D-glucose units) and lichenases (or lichenases) (e.g., EC 3.2.1.73), which hydrolyze the (1,4)-beta-D-glycosidic bonds in beta-D-glucans containing (1,3)- and (1,4)-bonds. For example, β-glucanases (e.g., EC 3.2.1.4) can endohydrolyze (1,4)-β-D-glycosidic bonds in cellulose, lichenin, and cereal β-D-glucans, and can also hydrolyze 1,4-bonds in β-D-glucans containing 1,3-bonds. Examples of useful β-glucanases have been described in Bacillus (e.g., B agaradhaerens, B akibai, B mojavensis, WO 2021148364).

[0170] In some other embodiments, the cleaning compositions described herein further comprise a suitable xyloglucanase. Suitable xyloglucanases include, but are not limited to, those of plant, fungal, or bacterial origin. In certain embodiments, chemically or genetically modified mutants are included. As used herein, "one or more xyloglucanases" encompasses the family of enzymes described by Vincken and Voragen at Wageningen University [Vincken et al. (1994) Plant Physiol. [Plant Physiology], 104, 99-107], and can degrade the xyloglucans described in Hayashi et al. (1989) Annu. Rev. Plant. Physiol. Plant Mol. Biol. [Annual Review of Plant Physiology and Plant Molecular Biology], 40, 139-168. Vincken et al. demonstrated that xyloglucan coatings were removed from the cellulose of isolated apple cell walls by xyloglucanase (endo-IV-glucanase) purified from Trichoderma viride. In some other embodiments, the xyloglucanase for specific applications is an alkaline xyloglucanase, i.e., an enzyme with at least 10%, at least 25% or at least 40% enzymatic activity at a pH range of from 7 to 12. In some other embodiments, the xyloglucanase for specific applications is an alkaline xyloglucanase, i.e., an enzyme with at least 10%, at least 25% or at least 40% enzymatic activity at a pH range of from 7 to 12. In some other embodiments, the xyloglucanase is an enzyme with at least 7.0 to about 12 pH levels.

[0171] Still other embodiments relate to compositions comprising one or more subtilisin variants as described herein and one or more nucleases (e.g., DNA enzyme or RNA enzyme). 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, WO 2015155350, WO 2016162556, WO 2017162836, WO 2017060475 (e.g., SEQ ID NO: 21), WO 2018184816, WO 2018177936, WO 2018177938, WO 2018 / 185269, WO 2018185285, WO 2018177203, WO 2018184817, WO 2019084349, WO 2019084350, WO 2019081721, WO 2018076800, WO 2018185267, WO 2018185280, WO 2018206553 and WO 2020099490. Exemplary commercial nucleases include, but are not limited to, Everis™, Pristine® and Purezyme® (Novozymes). 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 of Biofilms by Secreted, Matrix Degrading, Bacterial DNase. PLoS ONE 5(12) and Whitchurch, CB, Tolker-Nielsen, T., Ragas, PC, Mattick, JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295: 1487.

[0172] In some other embodiments, the cleaning compositions described herein further include suitable peroxidases / oxidases. Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Including chemically modified mutants or protein engineered mutants. Examples of useful peroxidases include peroxidases from Coprinus (e.g., from C. cinereus) and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include Guardzyme™ (Novozymes A / S). Peroxidases are composed of enzyme classification EC 1.11.1.7 as defined by the International Union of Biochemistry and Molecular Biology (IUBMB) Nomenclature Committee or any fragment thereof that exhibits peroxidase activity. Suitable peroxidases include those of plant, bacterial or fungal origin. Including chemically modified mutants or protein engineered mutants. Examples of useful peroxidases include peroxidases from Coprinopsis (e.g., from C. cinerea (EP 179,486)), and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Suitable peroxidases include haloperoxidases, such as chloroperoxidases, bromoperoxidases, and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Suitable oxidases particularly include any laccases comprised by enzyme classification EC 1.10.3.2, or any fragments thereof exhibiting laccase activity, or compounds exhibiting similar activity, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5). Preferred laccases are enzymes of microbial origin. Enzymes may be derived from plants, bacteria, or fungi (including filamentous fungi and yeasts).Suitable examples from fungi include laccases derivable from strains of Aspergillus, Neurospora (e.g., N. crassa), Podospora, Botrytis, Collybia, Pomes, Lentinus, Pleurotus, Trametes (e.g., T. villosa and T. versicolor), Rhizoctonia (e.g., R. solani), Coprinus (e.g., C. cinerea, C. comatus, C. friesii, and C. plicatilis), Psathyrella (e.g., P. condelleana), Panaeolus (e.g., P. papilionaceus), Myceliophthora (e.g., M. thermophila), Schytalidium (e.g., S. thermophilum), Polyporus (e.g., P. pinsitus), Phlebia (e.g., P. radiata (WO 92 / 01046)), or Coriolus (e.g., C. hirsutus (JP 2238885)). Suitable examples of bacterial laccases include laccases derivable from Bacillus strains. Laccases derivable from Coprinus or Myceliophthora are preferred; in particular, laccases derivable from Coprinus cinerea, as disclosed in WO 97 / 08325, or laccases from Myceliophthora thermophila, as disclosed in WO 95 / 33836.

[0173] Another embodiment relates to a composition comprising one or more subtilisin variants described herein and one or more perhydrolases, such as, for example, the perhydrolases described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0174] In yet another embodiment, one or more subtilisin variants described herein and one or more additional enzymes contained in one or more compositions described herein may each independently vary up to about 10% by weight of the composition, with the balance of the cleaning composition being one or more adjunct materials.

[0175] In some embodiments, one or more compositions described herein can be used as detergent additives, wherein the additive is in solid or liquid form. Such additive products are intended to supplement and / or improve the performance of conventional detergent compositions and can be added at any stage of the cleaning process. In some embodiments, the density of the laundry detergent composition ranges from about 400 to about 1200 g / liter, while in other embodiments, it ranges from about 500 to about 950 g / liter of composition measured at 20°C.

[0176] Some embodiments relate to laundry detergent compositions comprising one or more subtilisin variants as described herein and one or more adjunct materials selected from the group consisting of surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime soap dispersants, soil suspending agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry compositions further comprise a softener.

[0177] Additional embodiments relate to hand dishwashing compositions comprising one or more subtilisin variants described herein and one or more adjunct materials selected from the group consisting of surfactants, organic polymeric compounds, suds boosters, Group II metal ions, solvents, hydrotropes, and additional enzymes.

[0178] Other embodiments relate to one or more compositions described herein, wherein the composition is a compact granular fabric cleaning composition for colored fabric laundering or providing softening through wash capacity, or a heavy-duty liquid (HDL) fabric cleaning composition. Exemplary fabric cleaning compositions and / or methods of preparation are described in USPN 6,610,642 and 6,376,450. Other exemplary cleaning compositions are described, for example, in USPNs 6,605,458; 6,294,514; 5,929,022; 5,879,584; 5,691,297; 5,565,145; 5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; and 5,486,303; 4,968,451; 4,597,898; 4,561,998; 4,550,862; 4,537,706; 4,515,707; and 4,515,705.

[0179] 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 builders are aminopolycarboxylic acid builders, such as glycine-N,N-diacetic acid or a builder having the general formula MOOC-CHR-N(CH2COOM)2 (wherein R is C 1-12 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-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL ... In some embodiments, the acidifying particles include glutamic acid (SEGL), IDA (iminodiacetic acid), and salts and derivatives thereof 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), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), and alkali metal salts and derivatives thereof. In some embodiments, the acidifying particles have a weight geometric mean particle size of from about 400 μm to about 1200 μm and a bulk density of at least 550 g / L. In some embodiments, the acidifying particles comprise at least about 5% builder.

[0180] In certain embodiments, the acidifying particle can comprise any acid, including organic acid and mineral acid.Organic acid can have one or two carboxyl groups, and can have up to 15 carbons in some cases, up to 10 carbons in particular, 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 hydrated glyoxylic acid.In certain embodiments, acid is citric acid.Mineral acid includes hydrochloric acid and sulfuric acid.In some cases, the acidifying particle is the highly active particle that comprises high-level aminocarboxylic acid builder.Also been found that sulfuric acid further contributes to the stability of the final particle.

[0181] Additional embodiments relate to cleaning compositions comprising one or more subtilisin variants and one or more surfactants and / or surfactant systems, wherein the surfactant is selected from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some embodiments, the surfactant is present at a level of from about 0.1% to about 60%, while in alternative embodiments, the level is from about 1% to about 50%, and in yet other embodiments, the level is from about 5% to about 40%, based on the weight of the cleaning composition.

[0182] In some embodiments, one or more compositions described herein comprise one or more detergent builders or builder systems. In one embodiment, the composition comprises from 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 and alkanolammonium salts of polyphosphates; alkali metal silicates; alkaline earth and alkali metal carbonates; aluminosilicates; polycarboxylate compounds; ether hydroxypolycarboxylates; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethyloxysuccinic acid; ammonium and substituted ammonium salts of polyacetic acids, such as ethylenediaminetetraacetic acid and nitrilotriacetic acid; polycarboxylates, such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid; and soluble salts thereof. In some such compositions, builders form water-soluble hardness ion complexes (e.g., chelating builders), such as citrates and polyphosphates, for example, sodium tripolyphosphate, sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixed sodium and potassium tripolyphosphates. Exemplary builders are described, for example, in EP 2100949. In some embodiments, builders include 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 alkali metal salts of these compounds, including sodium salts. In some embodiments, the builder can be sodium tripolyphosphate (STPP). In addition, the composition can include carbonates and / or citrates. Other suitable non-phosphate builders include homopolymers and copolymers of polycarboxylic acids and partially or fully neutralized salts thereof, monomeric polycarboxylic acids, and hydroxycarboxylic acids and their salts. In some embodiments, salts of the above compounds include ammonium salts and / or alkali metal salts, i.e., lithium salts, sodium salts, and potassium salts, including sodium salts. Suitable polycarboxylic acids include acyclic, alicyclic, heterocyclic, and aromatic carboxylic acids, which, in some embodiments, may contain at least two carboxyl groups, which are in each case separated from each other, in some cases by no more than two carbon atoms.

[0183] In some embodiments, one or more 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% of a chelating agent by weight of the composition. Exemplary chelating agents include, but are not limited to, copper, iron, manganese, and mixtures thereof.

[0184] In some embodiments, one or more compositions described herein comprise one or more deposition aids. Exemplary deposition aids include, but are not limited to, polyethylene glycol, polypropylene glycol, polycarboxylates, soil release polymers such as, for example, polyethylene terephthalate, clays such as, for example, kaolinite, montmorillonite, attapulgite, illite, bentonite, and halloysite, and mixtures thereof.

[0185] In other embodiments, one or more compositions described herein include one or more anti-redeposition agents or nonionic surfactants (which can prevent the redeposition of soil) (see, for example, EP 2100949). For example, in ADW compositions, nonionic surfactants can be used for surface modification purposes (particularly for thin sheets) to prevent filming and staining and to improve gloss. These nonionic surfactants can also be used to prevent the redeposition of soil. In some embodiments, the nonionic surfactant can be ethoxylated nonionic surfactants, epoxy-terminated poly(alkoxylated) alcohols, and amine oxide surfactants.

[0186] In some embodiments, one or more compositions described herein comprise one or more dye transfer inhibiting agents. Exemplary polymeric dye transfer inhibiting agents include, but are not limited to, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazolidones, polyvinylimidazoles, and mixtures thereof. In one embodiment, the composition comprises from about 0.0001% to about 10%, from about 0.01% to about 5%, or from about 0.1% to about 3% of a dye transfer inhibiting agent, by weight of the composition.

[0187] In some embodiments, one or more compositions described herein comprise one or more silicates. Exemplary silicates include, but are not limited to, sodium silicate, e.g., sodium disilicate, sodium metasilicate, and crystalline phyllosilicates. In some embodiments, the silicate is present at a level of from about 1% to about 20% or from about 5% to about 15% by weight of the composition.

[0188] In some still further embodiments, one or more compositions described herein comprise one or more dispersants.Exemplary water-soluble organic materials include, but are not limited to, for example, homopolymeric or copolymeric acids or their salts, wherein the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

[0189] In some additional embodiments, one or more compositions described herein include one or more enzyme stabilizers. In some embodiments, the enzyme stabilizer is a water-soluble source of calcium and / or magnesium ions. In some embodiments, enzyme stabilizers include 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 water-soluble sources 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 vanadium (IV)) present in the finished composition to provide such ions to the enzyme. Chlorides and sulfates may also be used in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described, for example, in WO 07 / 145964. In some embodiments, reversible protease inhibitors may also be used, for example, in boron-containing compounds (e.g., borate salts, 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 WO 2009 / 118375, WO 2013004636).

[0190] As previously described (WO 199813458, WO 2011036153, US 20140228274, and WO 2022 / 265069), 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-blocked," e.g., with a urea, carbamate, or urea moiety, or "di-N-blocked," e.g., with a carbonyl, urea, oxamide, thiourea, dithiooxamide, or thiooxamide moiety (EP 2358857 B1). In some cases, the protease stabilizer is a bisulfite adduct of the peptide aldehyde compound (WO 2022 / 265069). The molar ratio of these inhibitors to the protease may be 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 carboxyl groups (US Pat. No. 7,968,508 B2). The molar ratio of these stabilizers to the protease is preferably in the range of 1:1 to 1000:1, in particular 1:1 to 500:1, particularly preferably 1:1 to 100:1, and most particularly preferably 1:1 to 20:1.

[0191] In some embodiments, one or more compositions described herein comprise one or more bleaching agents, bleach activators, and / or bleach catalysts. In some embodiments, one or more compositions described herein comprise one or more inorganic and / or organic bleaching compounds. Exemplary inorganic bleaching agents include, but are not limited to, perhydrate salts, such as perborates, percarbonates, perphosphates, persulfates, and persilicates. In some embodiments, the inorganic perhydrate salts are alkali metal salts. In some embodiments, inorganic perhydrate salts are included as crystalline solids without additional protection, but in some other embodiments, the salts are coated. Bleach activators are typically organic peracid precursors that enhance bleaching during cleaning at temperatures of 60°C or below. Exemplary bleach activators include compounds that, under perhydrolysis conditions, yield 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 acids. Exemplary bleach activators are described, for example, in EP 2100949. Exemplary bleach catalysts include, but are not limited to, manganese triazacyclononane and related complexes, and cobalt, copper, manganese, and iron complexes. Additional exemplary bleach catalysts are described in, for example, US 4,246,612; US 5,227,084; US 4,810,410; WO 99 / 06521; and EP 2 100 949.

[0192] In some embodiments, one or more compositions described herein comprise one or more catalytic metal complexes. In some embodiments, metal-containing bleach catalysts may be used. In some embodiments, the metal bleach catalyst comprises a catalytic system comprising: a transition metal cation with defined bleach catalytic activity (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations), an auxiliary metal cation with little or no bleach catalytic activity (e.g., zinc or aluminum cations), and a chelate with defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts (see, e.g., US 4,430,243). In some embodiments, one or more compositions described herein are catalyzed by a manganese compound. Such compounds and usage levels are described, for example, in US 5,576,282. In other embodiments, a cobalt bleach catalyst may be used and included in one or more compositions described herein. Various cobalt bleach catalysts are described, for example, in USPNs 5,597,936 and 5,595,967.

[0193] In some further embodiments, one or more compositions described herein comprise a transition metal complex of a 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 at least one part per hundred million of active MRL in the wash liquor, ranging 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, specialized ultrarigid ligands that are cross-linked and bridged, such as, for example, 5,12-diethyl-1,5,8,12-tetraazabicyclo(6.6.2)hexadecane. Exemplary metal MRLs are described, for example, in WO 2000 / 32601 and US Pat. No. 6,225,464.

[0194] In another embodiment, one or more 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 a 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.

[0195] In some embodiments, the cleaning composition is a heavy-duty liquid (HDL) composition comprising one or more subtilisin variants described herein. The HDL liquid laundry detergent may comprise a detersive surfactant (10%-40%) comprising an anionic detersive surfactant selected from the group consisting of linear or branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof; and optionally a nonionic surfactant selected from the group consisting of linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohols, such as C8-C 18 Alkyl ethoxylated alcohols and / or C6-C 12 Alkylphenol alkoxylate, optionally wherein the weight ratio of anionic detersive surfactant (hydrophilic index (HIc) from 6.0 to 9) to nonionic detersive surfactant is greater than 1: 1. Suitable detersive surfactants also include cationic detersive surfactants (selected from alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and / or mixtures thereof); zwitterionic and / or amphoteric detersive surfactants (selected from alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants; and mixtures thereof.

[0196] In another embodiment, the cleaning composition is a liquid or gel detergent comprising a biopolymer. In some cases, the biopolymer is a glucan. In other cases, the glucan is α-1,6-glucan or an α-glucan derivative. The terms "α-1,6-glucan," "poly-α-1,6-glucan," "α-1,6-glucan polymer," "dextran," and the like herein refer to water-soluble α-glucans comprising glucose monomer units linked together by glycosidic bonds, wherein at least about 40% of the glycosidic bonds are α-1,6. In some aspects, α-1,6-glucans comprise about or at least about 90%, 95%, or 100% α-1,6-glycosidic bonds. Other bonds that may be present in α-1,6-glucans include α-1,2, α-1,3, and / or α-1,4 bonds.

[0197] The dextran herein can be as disclosed (e.g., molecular weight, bond / branching pattern, production method) in, for example, U.S. Patent Application Publication Nos. 2016 / 0122445, 2017 / 0218093, 2018 / 0282385, 2020 / 0165360, or 2019 / 0185893, each of which is incorporated herein by reference. In some aspects, the ester-derivatized dextran can be a dextran produced in a suitable reaction comprising glucosyltransferase (GTF) 0768 (SEQ ID NO: 1 or 2 of US 2016 / 0122445), GTF 8117, GTF 6831, or GTF 5604 (these latter three GTF enzymes are SEQ ID NOs: 30, 32, and 33 of US 2018 / 0282385, respectively), or a GTF comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of GTF 0768, GTF 8117, GTF 6831, or GTF 5604.

[0198] Dextran herein can have for example α-1,2, α-1,3 and / or α-1,4 branch.In some respects, about, at least about or less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45% or 50% of all glycosidic bonds of branched dextran ester are α-1,2, α-1,3 and / or α-1,4 glycosidic branch bonds.The length of such branch is typically mostly (> 90% or> 95%) or all (100%) are single glucose monomers. In some aspects, dextran with α-1,2-branching can be enzymatically produced according to the procedures in U.S. Patent Application Publication Nos. 2017 / 0218093 or 2018 / 0282385 (both of which are incorporated herein by reference), wherein, for example, an α-1,2-branching enzyme such as GTFJ18T1 or GTF9905 can be added during or after dextran production. In some aspects, any other enzyme known to produce α-1,2-branching can be used. Dextran with α-1,3-branching can be prepared, for example, as disclosed in Vuillemin et al. (2016, J. Biol Chem. [Journal of Biological Chemistry] 291:7687-7702) or International Patent Application Publication No. WO 2021 / 007264 (which are incorporated herein by reference).

[0199] As used herein, the terminology regarding "esters" (e.g., α-glucan ester derivatives) may be as disclosed, for example, in U.S. Patent Application Publication Nos. 2014 / 0187767, 2018 / 0155455, or 2020 / 0308371, or International Patent Application Publication No. WO 2021 / 252575 (each of which is incorporated herein by reference). The terms "α-glucan ester derivative," "α-glucan ester compound," "α-glucan ester," and the like are used interchangeably herein. The α-glucan ester derivative herein is an α-glucan that has been esterified with one or more organic groups (e.g., a hydrophobic organic group) such that the derivative has a degree of substitution (DoS) of up to about 3.0 with the one or more organic groups. The α-glucan ester derivative is herein referred to as such due to the inclusion of the substructure C G OCOC is called "ester", where "C G ” refers to a carbon atom of a monomeric unit (e.g., glucose) of an α-glucan ester derivative (wherein such a carbon atom is bonded to a hydroxyl group [OH] in the α-glucan precursor of the ester), and wherein “COC” is included in the acyl group. An example of an α-glucan ester derivative herein is benzoyl α-glucan.

[0200] The hydrophobic acyl group of the α-glucan ester derivative herein can be as disclosed in, for example, U.S. Patent Application Publication Nos. 2014 / 0187767, 2018 / 0155455, or 2020 / 0308371, or International Patent Application Publication No. WO 2021 / 252575 (each of which is incorporated herein by reference).

[0201] As used herein, the term "ether" (e.g., α-glucan ether derivative) may be as disclosed in, for example, U.S. Patent Application Publication Nos. 2016 / 0311935, 2018 / 0237816, or 2020 / 0002646, or International Patent Application Publication Nos. WO 2021 / 257786 or WO 2021 / 252569 (each of which is incorporated herein by reference). The terms "α-glucan ether derivative," "α-glucan ether compound," "α-glucan ether," and the like are used interchangeably herein. The α-glucan ether derivative herein is an α-glucan that has been etherified with one or more organic groups (e.g., charged organic groups, such as cationic groups) such that the derivative has a DoS of up to about 3.0 achieved with the one or more organic groups. α-glucan ether derivatives herein are etherified by the inclusion of the substructure -C G -OC- is called "ether", where "-C G "-" represents a carbon atom of a monomeric unit (typically glucose) of an α-glucan ether derivative (wherein such carbon atom is bonded to a hydroxyl group [-OH] in the α-glucan precursor of the ether), and wherein "-C-" is a carbon atom of an organic group.

[0202] The ether derivatives of α-glucans disclosed herein can be substituted with at least one positively charged organic group described herein that is attached to the α-glucan ether. The positively charged organic group can be, for example, any of those disclosed in U.S. Patent Application Publication Nos. 2016 / 0311935, 2018 / 0237816, or 2020 / 0002646, or International Patent Application Publication No. WO 2021 / 257786 (which are incorporated herein by reference). For example, the positively charged organic group can include a substituted ammonium group. Examples of substituted ammonium groups are primary, secondary, tertiary, and quaternary ammonium groups. In some aspects, one or more positively charged organic groups can include a trimethylammonium hydroxypropyl group.

[0203] In some aspects, the α-glucan derivatives (e.g., esters or ethers) and / or products comprising such derivatives are biodegradable. For example, such biodegradability can be about, at least about, or at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 5%-60%, 5%-80%, 5%-60%, 5%-80%, 5%-90%, 6%-80%, 7%-90%, 8%-80%, 9%-90%, 10%-15 ... -90%, 40%-70%, 50%-70%, 60%-70%, 40%-75%, 50%-75%, 60%-75%, 70%-75%, 40%-80%, 50%-80%, 60%-80%, 70%-80%, 40%-85%, 50%-85%, 60%-85%, 70%-85%, 40%-90%, 50%-90%, 60%-90%, or 70%-90%, or any value between 5% and 90%.

[0204] In another embodiment, the cleaning composition is a liquid or gel detergent (which is not in unit dose). The liquid or gel detergent can be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70% water by weight, up to about 65% water by weight, up to about 55% water by weight, up to about 45% water by weight, or up to about 35% water by weight. Other types of liquids (including but not limited to alkanols, amines, glycols, ethers, and polyols) can be included in the aqueous liquid or gel. The aqueous liquid or gel detergent can contain from 0 to 30% organic solvent. The liquid or gel detergent can be non-aqueous.

[0205] The detergent gel composition may comprise at least one fatty alcohol ethoxylate, wherein the detergent gel composition is contained in a water-soluble container, such as in WO 2022 / 253728.

[0206] The composition may optionally comprise a surfactant enhancing polymer consisting of: amphiphilic alkoxylated grease cleaning polymers 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% to 10 wt%); and / or random grafted polymers typically comprising a hydrophilic backbone comprising monomers selected from the group consisting of unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyols (e.g., glycerol), and mixtures thereof; and one or more hydrophobic side chains selected from the group consisting of: C4-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyols (e.g., glycerol), and mixtures thereof.25 Alkyl groups, polypropylene, polybutene, vinyl esters of saturated C2-C6 monocarboxylic acids, C1-C6 alkyl esters of acrylic or methacrylic acid, and mixtures thereof.

[0207] The composition may comprise additional polymers such as soil release polymers including, for example, anionic terminated polyesters such as SRP1; polymers comprising at least one monomer unit selected from the group consisting of saccharides, dicarboxylic acids, polyols, and combinations thereof in random or block configurations; ethylene terephthalate-based polymers and copolymers thereof in random or block configurations such as Repel-o-tex SF, SF-2, and SRP6; Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325; Marloquest SL; anti-redeposition polymers (0.1 wt % to 10 wt %). wt %, including, for example, carboxylate polymers, such as polymers comprising at least one monomer selected from the group consisting of acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixtures thereof; vinyl pyrrolidone homopolymer; and / or polyethylene glycol having a molecular weight in the range of 500 to 100,000 Da); cellulosic polymers (including, for example, alkyl celluloses; alkyl alkoxyalkyl celluloses; carboxyalkyl celluloses; alkyl carboxyalkyl celluloses, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose; and mixtures thereof); and polymeric carboxylates (such as, for example, maleate / acrylate random copolymers or polyacrylate homopolymers).

[0208] 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 in random or block configuration (including, for example, polysaccharides, cellulosic polymers, polydiallyldimethylammonium halide (DADMAC) and copolymers of DADMAC and vinylpyrrolidone, acrylamide, imidazole, halogenated imidazoline, and mixtures thereof; cationic guar gum; cationic cellulose, such as cationic hydroxyethyl cellulose; cationic starch; cationic polyacrylamide; and mixtures thereof.

[0209] The composition may further comprise dye transfer inhibitors, examples of which include manganese phthalocyanine, peroxidase, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazolidones and polyvinylimidazoles and / or mixtures thereof; chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA); diethylenetriaminepentamethylenephosphonic acid (DTPMP); hydroxyethanediphosphonic acid (HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methylglycine diacetic acid (MGDA); diethylenetriaminepentaacetic acid (DTPA); propylenediaminetetraacetic acid (PDTMP); A); 2-hydroxypyridine-N-oxide (HPNO); or methylglycine diacetic acid (MGDA); glutamic acid N,N-diacetic acid (N,N-dicarboxymethylglutamate tetrasodium salt (GLDA); nitrilotriacetic acid (NTA); 4,5-dihydroxym-benzenedisulfonic acid; citric acid and any salts thereof; N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and their derivatives.

[0210] The composition may further comprise a silicone-based or fatty acid-based foam suppressor; an enzyme stabilizer; a hueing dye, calcium and magnesium cations, a visual signaling ingredient, an anti-foaming agent (0.001 wt% to about 4.0 wt%) and / or a structurant / thickener (0.01 wt%-5 wt%) selected from the group consisting of diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based materials, microcellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.

[0211] In some embodiments, the cleaning composition is a heavy duty powder (HDD) composition comprising one or more subtilisin variants described herein. The HDD powder laundry detergent may comprise a detersive surfactant, including an anionic detersive surfactant (selected from a linear or branched or random chain, substituted or unsubstituted alkyl sulfate, alkyl sulfonate, alkyl alkoxylated sulfate, alkyl phosphate, alkyl phosphonate, alkyl carboxylate and / or mixtures thereof); a nonionic detersive surfactant (selected from a linear or branched or random chain, substituted or unsubstituted C8-C 18 Alkyl ethoxylates and / or C6-C 12alkylphenol alkoxylates); cationic detersive surfactants (selected from alkylpyridinium compounds, alkylquaternary ammonium compounds, alkylquaternary phosphonium compounds, alkyltertiary sulfonium compounds and mixtures thereof); zwitterionic and / or amphoteric detersive 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, citrates 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 silicate or potassium silicate or sodium metasilicate or layered silicate (SKS-6) in the range of 0 wt % to less than 10 wt %); wt% range of sodium carbonate and / or sodium bicarbonate); and bleaching agents (photobleaches 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 its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED, and nonanoyloxybenzenesulfonate-NOBS, nitrile quaternary ammonium salts (nitrile quats), and mixtures thereof); hydrogen peroxide; a source of hydrogen peroxide (an inorganic perhydrate salt, for example, the mono- or tetrahydrated sodium salt of perborate, percarbonate, persulfate, perphosphate or persilicate); a preformed hydrophilic and / or hydrophobic peracid (selected from percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, and mixtures thereof); and / or a bleach catalyst (for example, imine bleach boosters, such as iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulfonylimines; N-phosphonylimines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones, and mixtures thereof); a metal-containing bleach catalyst (for example, copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations and auxiliary metal cations (for example, zinc or aluminum) and chelates (for example, ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and water-soluble salts thereof).

[0212] The composition may further comprise additional detergent ingredients including perfume microcapsules, starch encapsulated perfume coordinators, enzyme stabilizers, hueing agents, additional polymers (including fabric integrity and cationic polymers), dye locking ingredients, fabric softeners, brighteners (e.g., CI fluorescent brighteners), flocculants, chelants, alkoxylated polyamines, fabric deposition aids and / or cyclodextrins.

[0213] 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-terminated poly(alkoxylated) alcohols, and amine oxide surfactants, present in an amount of 0-10% by weight; a builder 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 beta-alanine diacetic acid (B-ADA) and its salts), homopolymers and copolymers of polycarboxylic acids and their partially or fully neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts (which are in the range of 0.5%-50% by weight); sulfonated / carboxylated polymers (which provide dimensional stability to the product); % to about 50% by weight; a drying aid in the range of about 0.1% to about 10% by weight (selected from polyesters, in particular anionic polyesters, optionally together with additional monomers having 3 to 6 functional groups (in particular acid, alcohol or ester functional groups) which facilitate polycondensation, polycarbonate-, polyurethane- and / or polyurea-polyorganosiloxane compounds or their reactive cyclic carbonate and urea type precursor compounds); a silicate in the range of from about 1% to about 20% by weight (sodium or potassium silicate, for example sodium disilicate, sodium metasilicate and crystalline silicate); silicates); inorganic bleaching agents (e.g., perhydrate salts such as perborates, percarbonates, perphosphates, persulfates, and persilicates) and organic bleaching agents (e.g., organic peroxyacids, including diacyl and tetraacyl peroxides, especially diperoxydodecanedioic acid, diperoxytetradecandioic acid, and diperoxyhexadecanedioic acid); bleach activator - an organic peracid precursor, which ranges from about 0.1% to about 10% by weight; a bleach catalyst (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn, and Fe bispyridylamine and related complexes, and cobalt(III) pentamineacetate and related complexes); a metal care agent (selected from benzotriazoles, metal salts and complexes, and silicates) in the range of about 0.1% to 5% by weight; a bleach activator - an organic peracid precursor, which ranges from about 0.1% to about 10% by weight; a bleach catalyst (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn, and Fe bispyridylamine and related complexes, and cobalt(III) pentamineacetate and related complexes); a metal care agent (selected from benzotriazoles, metal salts and complexes, and silicates) in the range of about 0.01% to about 5.0 mg active enzyme / g ADW detergent composition range of enzymes (acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, β-glucanase, carrageenanase, 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) , lipoxygenases, mannanases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phosphodiesterases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonases, β-glucanases, tannases, transglutaminases, xanthan gum lyases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, and mixtures thereof); and an enzyme stabilizer component selected from the group consisting of oligosaccharides, polysaccharides, and inorganic divalent metal salts.

[0214] Exemplary ADW compositions include those provided in the table below.

[0215] Exemplary ADW Compositions

[0216]

[0217] Further embodiments relate to compositions and methods for treating fabrics (e.g., desizing textiles) using one or more of the subtilisin variants described herein. Fabric treatment methods are well known in the art (see, for example, US 6,077,316). For example, the feel and appearance of a fabric can be improved by a method comprising contacting the fabric with a variant described herein in a solution. The fabric can be treated with the solution under pressure.

[0218] One or more subtilisin enzyme variants as described herein can be applied during or after the weaving of a textile, during the desizing stage, or in one or more additional fabric processing steps. During the weaving of a textile, the threads are exposed to considerable mechanical strain. Before weaving on a mechanical loom, the warp yarns are typically coated with sizing starch or starch derivatives to increase their tensile strength and prevent disconnection. One or more subtilisin enzyme variants as described herein can be applied during or after weaving to remove sizing starch or starch derivatives. After weaving, the variants can be used to remove the sizing coating before further processing the fabric to ensure uniform and wash-resistant results. One or more subtilisin enzyme variants as described herein can be used alone or in combination with other desizing chemicals and / or desizing enzymes to desize fabrics (including cotton-containing fabrics) as detergent additives (e.g., in aqueous compositions). Amylases can also be used in combination with subtilisin enzyme variants in compositions and methods for producing a stonewashed appearance on indigo-dyed denim fabrics and clothing. For clothing production, the fabric can be cut and sewn into clothing or garments, which are subsequently finished. In particular, different enzymatic finishing methods have been developed for the production of denim. The finishing process of denim garments typically begins with an enzymatic desizing step, wherein the garments are subjected to the action of proteolytic enzymes to provide softness to the fabric and make the cotton more susceptible to subsequent enzymatic finishing steps. One or more subtilisin variants described herein can be used in methods for finishing denim garments (e.g., the "biosanding process"), enzymatic desizing, and providing softness and / or finishing to fabrics.

[0219] The present disclosure also provides methods for cleaning surfaces of articles, comprising contacting the article with at least one subtilisin variant provided herein (or a composition comprising such a subtilisin variant). In certain embodiments, the article may have, for example, a protein stain on its surface. In certain embodiments, the protein stain may comprise egg or an egg-based stain, such as crème brûlée, baked cheese, BMI, or other protein-containing substances.

[0220] 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.

[0221] Paragraph 1. A subtilisin variant comprising two or more substitutions selected from the group consisting of: X006W, X024K, X055P, X162Q, X183N, X204Q, X206Y, X222Q, X248A or X254A, wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant does not have 100% sequence identity to the naturally occurring polypeptide sequence.

[0222] Paragraph 2. The subtilisin variant of paragraph 1, wherein the substitution is Y006W, S024K, T055P, S162Q, S183N, S204Q, Q206Y, M222Q, S248A, or T254A, and wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant does not have 100% sequence identity to the naturally occurring polypeptide sequence.

[0223] Paragraph 3. The subtilisin protease variant of paragraph 1 or 2, wherein the variant comprises one, two or more additional substitutions from the group consisting of X003Q, X022Y, X024Q, X033T, X045V, X053G, X076D, X078N, X087D, X101N, X109Q, X118R, X128A, X128S, X145R, X166Q, X169A, X182Q, X217Q or X218S, wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, and wherein the variant does not have 100% sequence identity to the naturally occurring polypeptide sequence.

[0224] Paragraph 4. The subtilisin variant of any preceding paragraph, wherein the substitution is S003Q, T022Y, S024Q, S033T, A045V, S053G, N076D, S078N, S087D, S101N, N109Q, N118R, G128A, G128S, S145R, G166Q, G169A, S182Q, Y217Q, or N218S, and wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, and wherein the variant does not have 100% sequence identity to the naturally occurring polypeptide sequence.

[0225] Paragraph 5. The subtilisin variant of paragraph 1 or 2, comprising two mutations selected from the group consisting of: X006W-X024K, X006W-X055P, X006W-X162Q, X006W-X183N, X006W-X204Q, X006W-X206Y, X006W-X222Q, X006W-X248A, X006W-X254 A. X024K-X055P, X024K-X162Q, X024K-X183N, X024K-X204Q, X024K-X248A, X024K-X254A, X055P-X162Q, X055P-X183N, X055P-X204Q, 55P-X222Q, X055P-X248A, X055P-X254A, X162Q-X183N, X162Q-X204Q, Q-X222Q, X162Q-X248A, X162Q-X254A, X183N-X204Q, X183N-X206Y, X248A, X183N-X254A, X204Q-X206Y, X204Q-X222Q, X204Q-X248A, X204Q-X254A, X206Y-X222Q, X206Y-X248A, X206Y-X254A, X222Q-X248A, X222Q-X254A, and X248A-X254A, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, wherein the variant does not have 100% sequence identity to the naturally occurring amino acid sequence, and wherein the variant has at least 55% identity to the subtilisin having the amino acid sequence of SEQ ID NOs: 1 and 7-28.

[0226] Paragraph 6. The subtilisin variant of paragraph 1 or 2, comprising three mutations selected from the group consisting of: X006W-X024K-X055P, X006W-X024K-X162Q, X006W-X024K-X183N, X006W-X024K-X204Q, X006W-X024K-X206Y, X006W-X024K-X222Q, X006W-X024K-X248A, X006W-X024K-X254A, X006W-X055P-X162Q, X006W-X024K-X183N, X006W-X024K-X204Q, X006W-X024K-X206Y 5P-X206Y, X006W-X055P-X222Q, X006W-X055P-X248A, , X006W-X162Q-X183N, X006W-X162Q-X204Q, 162Q-X222Q, X006W-X162Q-X248A, X006W-X162Q-X254A, 4Q, X006W-X183N-X206Y, X006W-X183N-X222Q, X183N-X254A, X006W-X204Q-X206Y, X006W-X204Q-X222Q, X006W-X204Q-X2 48A, X006W-X204Q-X254A, X006W-X206Y-X222Q, W-X206Y-X254A, X006W-X222Q-X248A, X006W-X222Q-X254A, X006W-X248A- X254A, X024K-X055P-X162Q, X024K-X055P-X183N, 4K-X055P-X206Y, X024K-X055P-X222Q, X024K-X055P-X248A, X024K-X055P -X254A, X024K-X162Q-X183N, X024K-X162Q-X204Q, 024K-X162Q-X222Q, X024K-X162Q-X248A, X024K-X162Q-X254A, X024K-X18 3N-X204Q, X024K-X183N-X206Y, X024K-X183N-X222Q,X024K-X183N-X254A、X024K-X204Q-X206Y、X024K-X204Q-X222Q、X024K-X204Q-X248A、X024K-X204Q-X254A、X024K-X206Y-X222Q、X024K-X206Y-X248A、X024K-X206Y-X254A、X024K-X222Q-X248A、X024K-X222Q-X254A、X024K-X248A-X254A、X055P-X162Q-X183N、X055P-X162Q-X204Q、X055P-X162Q-X206Y、X055P-X162Q-X222Q、X055P-X162Q-X248A、X055P-X162Q-X254A、X055P-X183N-X204Q、X055P-X183N-X206Y、X055P-X183N-X222Q、X055P-X183N-X248A、X055P-X183N-X254A、X055P-X204Q-X206Y、X055P-X204Q-X222Q、X055P-X204Q-X248A、X055P-X204Q-X254A、X055P-X206Y-X222Q、X055P-X206Y-X248A、X055P-X206Y-X254A、X055P-X222Q-X248A、X055P-X222Q-X254A、X055P-X248A-X254A、X162Q-X183N-X204Q、X162Q-X183N-X206Y、X162Q-X183N-X222Q、X162Q-X183N-X248A、X162Q-X183N-X254A、X162Q-X204Q-X206Y、X162Q-X204Q-X222Q、X162Q-X204Q-X248A、X162Q-X204Q-X254A、X162Q-X206Y-X222Q、X162Q-X206Y-X248A、X162Q-X206Y-X254A、X162Q-X222Q-X248A、X162Q-X222Q-X254A、X162Q-X248A-X254A、X183N-X204Q-X206Y、X183N-X204Q-X222Q、X183N-X204Q-X248A、X183N-X204Q-X254A、X183N-X206Y-X222Q、X183N-X206Y-X248A、X183N-X206Y-X254A、X183N-X222Q-X248A、X183N-X222Q-X254A, X183N-X248A-X254A, X204Q-X206Y-X222Q, X204Q-X206Y-X248A, X204Q-X206Y-X254A, X204Q-X222Q-X248A, X204Q-X222Q-X254A, X204Q-X248A-X254A, X206Y-X222Q-X248A, X206Y-X222Q-X254A, X206Y-X248A-X254A, and X222Q-X248A-X254A, wherein the positions are indicated by the alignment with SEQ ID NO:1, wherein the variant does not have 100% sequence identity with the naturally occurring amino acid sequence, and wherein the variant has at least 55% identity with the subtilisin having the amino acid sequence of SEQ ID NO: 1 and 7-28.

[0227] Paragraph 7. The subtilisin variant of paragraph 2, comprising two mutations selected from the group consisting of: Y006W-S024K, Y006W-T055P, Y006W-S162Q, Y006W-S183N, Y006W-S204Q, Y006W-Q206Y, Y006W-M222Q, Y006W-S248A, Y006W-T254A, S024K-T055P, S024K-S162Q, S024K-S183N, S024K-S204Q, S024K-Q206Y, S024K-M222Q, S 024K-S248A, S024K-T254A, T055P-S162Q, T055P-S183N, T055P-S204Q, T055P-Q206Y, T05 5P-M222Q, T055P-S248A, T055P-T254A, S162Q-S183N, S162Q-S204Q, S162Q-Q206Y, S162 Q-M222Q, S162Q-S248A, S162Q-T254A, S183N-S204Q, S183N-Q206Y, S183N-M222Q, S183N- S248A, S183N-T254A, S204Q-Q206Y, S204Q-M222Q, S204Q-S248A, S204Q-T254A, Q206Y-M222Q, Q206Y-S248A, Q206Y-T254A, M222Q-S248A, M222Q-T254A, and S248A-T254A, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, wherein the variant does not have 100% sequence identity to the naturally occurring amino acid sequence, and wherein the variant has at least 55% identity to the subtilisin having the amino acid sequence of SEQ ID NOs: 1 and 7-28.

[0228] Paragraph 8. The subtilisin variant of paragraph 2, comprising three mutations selected from the group consisting of: Y006W-S024K-T055P, Y006W-S024K-S162Q, Y006W-S024K-S183N, Y006W-S024K-S204Q, Y006W-S024K-Q206Y, Y006W-S024K-M222Q, Y006W-S024K-S248A, Y006W-S024K-T254A, Y006W-T055P-S162Q, Y006W-S024K-S183N, Y006W-S024K-S204Q, Y006W-S024K-Q206Y -Q206Y, Y006W-T055P-M222Q, Y006W-T055P-S248A, Y006W-T055P-T254A, Y 006W-S162Q-S183N, Y006W-S162Q-S204Q, Y006W-S162Q-Q206Y, Y006W-S16 2Q-M222Q, Y006W-S162Q-S248A, Y006W-S162Q-T254A, Y006W-S183N-S204Q , Y006W-S183N-Q206Y, Y006W-S183N-M222Q, Y006W-S183N-S248A, Y006W-S 183N-T254A, Y006W-S204Q-Q206Y, Y006W-S204Q-M222Q, Y006W-S204Q-S24 8A, Y006W-S204Q-T254A, Y006W-Q206Y-M222Q, Y006W-Q206Y-S248A, Y006W -Q206Y-T254A, Y006W-M222Q-S248A, Y006W-M222Q-T254A, Y006W-S248A-T 254A, S024K-T055P-S162Q, S024K-T055P-S183N, S024K-T055P-S204Q, S02 4K-T055P-Q206Y, S024K-T055P-M222Q, S024K-T055P-S248A, S024K-T055P -T254A, S024K-S162Q-S183N, S024K-S162Q-S204Q, S024K-S162Q-Q206Y, S 024K-S162Q-M222Q, S024K-S162Q-S248A, S024K-S162Q-T254A, S024K-S18 3N-S204Q, S024K-S183N-Q206Y, S024K-S183N-M222Q, S024K-S183N-S248A,S024K-S183N-T254A、S024K-S204Q-Q206Y、S024K-S204Q-M222Q、S024K-S204Q-S248A、S024K-S204Q-T254A、S024K-Q206Y-M222Q、S024K-Q206Y-S248A、S024K-Q206Y-T254A、S024K-M222Q-S248A、S024K-M222Q-T254A、S024K-S248A-T254A、T055P-S162Q-S183N、T055P-S162Q-S204Q、T055P-S162Q-Q206Y、T055P-S162Q-M222Q、T055P-S162Q-S248A、T055P-S162Q-T254A、T055P-S183N-S204Q、T055P-S183N-Q206Y、T055P-S183N-M222Q、T055P-S183N-S248A、T055P-S183N-T254A、T055P-S204Q-Q206Y、T055P-S204Q-M222Q、T055P-S204Q-S248A、T055P-S204Q-T254A、T055P-Q206Y-M222Q、T055P-Q206Y-S248A、T055P-Q206Y-T254A、T055P-M222Q-S248A、T055P-M222Q-T254A、T055P-S248A-T254A、S162Q-S183N-S204Q、S162Q-S183N-Q206Y、S162Q-S183N-M222Q、S162Q-S183N-S248A、S162Q-S183N-T254A、S162Q-S204Q-Q206Y、S162Q-S204Q-M222Q、S162Q-S204Q-S248A、S162Q-S204Q-T254A、S162Q-Q206Y-M222Q、S162Q-Q206Y-S248A、S162Q-Q206Y-T254A、S162Q-M222Q-S248A、S162Q-M222Q-T254A、S162Q-S248A-T254A、S183N-S204Q-Q206Y、S183N-S204Q-M222Q、S183N-S204Q-S248A、S183N-S204Q-T254A、S183N-Q206Y-M222Q、S183N-Q206Y-S248A、S183N-Q206Y-T254A、S183N-M222Q-S248A、S183N-M222Q-T254A, S183N-S248A-T254A, S204Q-Q206Y-M222Q, S204Q-Q206Y-S248A, S204Q-Q206Y-T254A, S204Q-M222Q-S248A, S204Q-M222Q-T254A, S204Q-S248A-T254A, Q206Y-M222Q-S248A, Q206Y-M222Q-T254A, Q206Y-S248A-T254A, and M222Q-S248A-T254A, wherein the positions are indicated by the alignment with SEQ ID NO:1, wherein the variant does not have 100% sequence identity with the naturally occurring amino acid sequence, and wherein the variant has at least 55% identity with the subtilisin having the amino acid sequence of SEQ ID NO: 1 and 7-28.

[0229] Paragraph 9. A subtilisin variant as described in any of the preceding paragraphs, wherein the variant is derived from a parent or reference polypeptide having 50%, 55%, 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 sequences of SEQ ID NOs: 1 and 7-28.

[0230] Paragraph 10. The subtilisin variant of any of the preceding paragraphs, wherein the variant comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0231] Paragraph 11. The subtilisin variant of any preceding paragraph, wherein the variant has improved stability or improved cleaning performance when compared to a reference subtilisin lacking the two, three, four or more substitutions.

[0232] Paragraph 12. The subtilisin protease variant of paragraph 11, wherein i) the improved stability is measured by a residual activity (fraction based on 1) greater than 0.2 compared to the corresponding parent in a 10% detergent solution after 20 minutes at 54 or 60 degrees Celsius as determined in Example 2, or ii) the improved stability is measured by a residual activity percentage ≥ 20% compared to the corresponding parent in a 10% detergent solution after 20 minutes at 40 or 41 degrees Celsius as determined in Example 2.

[0233] Paragraph 13. The subtilisin variant of Paragraph 11, wherein the cleaning performance is compared to the corresponding parent with a performance index ≥ 1.1 on blood, milk, ink, or egg stains in a 10% detergent solution according to the cleaning assay described in Example 2.

[0234] Paragraph 14. The subtilisin variant of any preceding paragraph, wherein the subtilisin variant has protease activity.

[0235] Paragraph 15. A polynucleotide comprising a nucleotide sequence encoding the subtilisin variant of any of paragraphs 1-14, wherein the polynucleotide is optionally isolated.

[0236] Paragraph 16. An expression vector or expression cassette comprising the polynucleotide of paragraph 15.

[0237] Paragraph 17. The expression vector or expression cassette of paragraph 16, wherein the polynucleotide is operably linked to a promoter.

[0238] Paragraph 18. A recombinant host cell comprising the vector or cassette of paragraph 16 or 17.

[0239] Paragraph 19. A composition comprising one or more subtilisin variants as described in any preceding paragraph.

[0240] Paragraph 20. The composition of paragraph 20, wherein the composition is selected from an enzyme composition and a detergent composition.

[0241] Paragraph 21. The composition of paragraph 20, wherein the detergent composition is selected from the group consisting of laundry detergents, fabric softener detergents, dishwashing detergents, and hard surface cleaning products.

[0242] Paragraph 22. The composition of any of paragraphs 19-21, wherein the composition further comprises one or more ions selected from calcium and / or zinc; one or more enzyme stabilizers; from about 0.001% to about 1.0% by weight of the subtilisin variant; one or more bleaching agents; one or more auxiliary materials; one or more microorganisms and / or one or more additional enzymes or enzyme derivatives selected from the group consisting of: acyltransferases, alginate lyases, α-amylases, β-amylases, β-glucanases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, DNase, dispersin, endo-β-1, β-Glucanase, endo-β-mannanase, esterase, exo-mannanase, fructosidase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, laminarinase, lichenase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease (e.g., DNA enzyme and / or RNA enzyme), oxidase, oxidoreductase, pectate lyase , pectin acetylesterase, pectin methylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phosphodiesterase, phospholipase, phytase, polyesterase, polygalacturonase, additional protease, pullulanase, RNase, reductase, rhamnogalacturonase, tannase, transglutaminase, xanthanase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and combinations thereof.

[0243] Paragraph 23. The composition of any of paragraphs 19-22, wherein the composition contains phosphate or is phosphate-free and / or contains boron or is boron-free.

[0244] Paragraph 24. The composition of any of paragraphs 19-22, wherein the composition is free of a protease stabilizer.

[0245] Paragraph 25. The composition of any of paragraphs 19-24, wherein the composition is a granule, a regular or compact powder, a solid, a stick, a regular or compact or concentrated liquid, a uniform tablet, a tablet having two or more layers, a pouch having one or more compartments, a gel, a paste, or a unit dose composition.

[0246] Paragraph 26. A method of cleaning comprising contacting a surface or item in need of cleaning with a subtilisin variant as described in any of paragraphs 1-14 or a composition as described in any of paragraphs 19-25; and optionally further comprising the step of rinsing the surface or item after contacting the surface or item with the variant or composition, wherein optionally the item is dishware or fabric.

[0247] Paragraph 27. A composition comprising the subtilisin variant of any of paragraphs 1-14, wherein the composition is a disinfectant composition, an industrial or institutional cleaning composition, a medical device cleaning composition, a contact lens cleaning composition, a wound cleaning composition, or a textile processing composition.

[0248] Paragraph 28. The variant of any of paragraphs 1-14, wherein the variant does not have the same amino acid sequence as the naturally occurring molecule.

[0249] Paragraph 29. The composition of paragraph 25, wherein the enzyme composition is an enzyme in a liquid medium.

[0250] Example 1

[0251] Generation of enzyme variants

[0252] Bacillus amyloliquefaciens (BPN') wild-type subtilisin and its variants were produced as described below. The amino acid sequence of the mature BPN' parent enzyme is shown in SEQ ID NO: 1. All BPN' subtilisin variants were expressed using a DNA fragment comprising, in order: a 5' AprE flanking region containing a variant of the Bacillus subtilis rrnIp2 promoter sequence (SEQ ID NO: 2) (the B. 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 B. amyloliquefaciens propeptide (SEQ ID NO: 4); a sequence corresponding to the gene encoding the mature BPN' subtilisin; a BPN' terminator (SEQ ID NO: 5); and a 3' AprE flanking sequence including a kanamycin gene expression cassette (SEQ ID NO: 6). This DNA fragment was assembled using standard molecular biology techniques. The linear DNA of the expression cassette is used to transform competent Bacillus subtilis cells of an appropriate strain.A library of BPN' subtilisin variants was generated by the method described above.

[0253] 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.

[0254] For protein expression experiments, transformed cells were grown in 96-well microtiter plates (MTP) in a medium (an enriched, semi-defined medium based on MOPS buffer, with urea as the main nitrogen source, glucose as the main carbon source, supplemented with 1% soytone for robust cell growth, and containing antibiotic selection) in a shaking 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.

[0255] Example 2

[0256] Enzyme assay

[0257] Protein concentration determination: Protein concentration quantification was performed using an Agilent Infinity II 1290 UHPLC equipped with an Agilent 300 SB-C3 RRHD (1.8 μm, 2.1 x 50 mm) column. The column temperature was 65°C, and samples were eluted from the column using a gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.07% TFA in acetonitrile. Absorbance was measured at 220 nm, and peaks were integrated using OpenLab software (Agilent Technologies, USA). The protein concentration of the samples was calculated based on a standard curve of the parent protease.

[0258] Protease activity: The protease activity of the parent subtilisin and its variants was tested by measuring the hydrolysis of the N-suc-AAPF-pNA substrate. For the AAPF assay, the reagent solution used was: 100 mM Tris pH 8.6, 0.005% Tween®-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. Enzyme samples were added to a microtiter plate (MTP) containing 1.6 mM suc-AAPF-pNA working solution, and activity was determined kinetically by measuring absorbance at 405 nm over 3-5 minutes at room temperature using a SpectraMax microplate reader. Protease activity is expressed as mOD / min.

[0259] Cleaning performance determination:Liquid laundry detergents used for cleaning performance testing included Persil Small & Mighty Non-Bio liquid detergent "Persil Non-Bio" (PNB, Unilever) (purchased from a UK supermarket on September 26, 2014) and standard HDL detergent Formula 1 (composition shown in Table 3). For cleaning performance testing, the detergents were diluted to 2.7 g / L in 5 mM HEPES (pH 8.2) in deionized water with a water hardness of 12 gpg (3Ca:1Mg). These detergents are considered boron-free because they contain ≤ 5 mg / kg of boron when tested for elemental boron content. The protease variants were tested for cleaning performance relative to the parent (BPN') on the technical stains C-05 (blood / milk / ink on woven cotton) and CS-39 (aged whole egg carbon with carbon black on woven cotton), both purchased from the Center for Testmaterials BV in Vlaardingen, the Netherlands. Soil is punched into small circular swatches and distributed into Costar 9017 or Greiner 655101 microtiter plates (MTPs). The MTP containing the microswatches is first filled with detergent. Then, a defined amount of the parent enzyme and variant is added to a final volume of 200 μL. The assay is performed at 25°C with gentle shaking for 25 minutes. After the incubation period, 100–150 μL of the supernatant is transferred to a fresh MTP and the absorbance is read at 600 nm for BMI swatches or 405 nm for whole egg swatches using a SpectraMax microplate reader. Absorbance results are obtained by subtracting the blank (no enzyme) value from each sample value. For each condition and subtilisin variant, the cleaning performance index (PI) is calculated by dividing the blank-subtracted absorbance of the variant by the absorbance of the parent protease at the same concentration. Blank-subtracted absorbance values ​​of the parent protease at corresponding concentrations of the variants were determined using a standard curve of the parent protease included in the assay and generated using a Langmuir fit or a Hill sigmoidal fit, as appropriate.

[0260] General sample setup for stability determination:The stability of subtilisin was tested in 10% (v / v) solutions of PNB detergent and standard HDL detergent formula 1. The BPN' parent (wild-type) and BPN' variants were tested at 40°C, 41°C, 54°C, or 60°C. The elevated temperature was set to allow for the identification of residual activity in stressed versus unstressed samples within a 20-minute incubation time, within a range suitable for distinguishing differences between the variant enzyme and its parent or reference variant. The exact temperature required to achieve the desired stability range may depend on the equipment manufacturer and setup used. Incubating samples under a temperature gradient can help determine this temperature. Using an appropriate curve fit to the residual activity under the temperature gradient, the relative stability of the samples can be compared at any temperature within the tested range. Enzyme samples were mixed with diluted detergent, and protease activity on the AAPF substrate was immediately measured to provide the unstressed value. The samples were then placed in a PCR plate, sealed, and incubated at elevated temperature for 20 minutes using a thermal cycler, after which AAPF activity was measured to obtain the stressed value. Residual activity was calculated by taking the ratio of stressed to unstressed activity. For these assays, all enzyme samples were assayed in triplicate. A protein expression cutoff of 200 ppm was applied, and data with a CV (coefficient of variation) of 20% or less were analyzed.

[0261] Example 3

[0262] Variant subtilisins with increased stability in the presence of detergents

[0263] As described in Example 2, variants of subtilisin BPN' were tested against the parent enzyme or a reference variant to determine the relative improvement in stability when measured in detergent at 54°C or 60°C for 20 minutes and reported as the fraction of stressed enzyme activity to unstressed enzyme activity. Tables 1 and 2 show the results of testing a series of BPN' variants with significant stability enhancement relative to wild-type BPN' (SEQ ID NO: 1). Stability results are reported as a fraction based on 1.0 (100% residual activity), with a result of 0.0 corresponding to zero residual activity.

[0264] The subtilisin variants in this example generally exhibited robust cleaning performance.

[0265]

[0266]

[0267]

[0268]

[0269]

[0270] Example 4

[0271] Detergent stability of additional BPN' variants

[0272] As described in Example 2, additional variants of subtilisin BPN' were tested against the parent enzyme or reference variant to determine the relative improvement in stability when measured, with results reported as the percentage (%) of residual activity after stress. Stability was tested at 40°C in PNB detergent and at 41°C in Standard HDL Detergent Formula 1 (the composition of Standard HDL Detergent Formula 1 (HDL 1) is shown in Table 3 below).

[0273]

[0274] Table 4 shows the results of testing a series of BPN' variants having two to nine substitutions relative to the wild-type parent enzyme and having significantly enhanced stability relative to wild-type BPN' (SEQ ID NO: 1). ND means no data.

[0275]

[0276]

[0277]

[0278]

[0279] The cumulative contribution of substitutions under each detergent and temperature stress was visualized by plotting the number of mutations versus the percentage (%) of residual activity for the variants described in Table 4. Data for variants tested in PNB detergent at 40°C are shown in Figure 1 Data for the variants tested in HDL 1 at 41°C are shown in Figure 2 middle.

[0280] Example 5

[0281] Cleaning performance evaluation of BPN' variants

[0282] The cleaning performance of the BPN' variants evaluated in Example 4 was tested under the conditions described in Example 2. Testing was performed in standard HDL detergent formula 1 (HDL 1) at pH 8 and 25°C using technical stains C-05 and CS-39. Table 5 shows the cleaning performance results, expressed as the performance index (PI) of the variants compared to the wild-type parent enzyme. ND indicates no data.

[0283]

[0284]

[0285]

[0286]

[0287] Although the present disclosure has been described in conjunction with the specific embodiments thereof, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, the present disclosure is intended to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0288] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present disclosure. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. A subtilisin protease variant comprising two or more substitutions selected from the group consisting of: X006W, X024K, X055P, X162Q, X183N, X204Q, X206Y, X222Q, X248A, or X254A, wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant does not have 100% sequence identity to the naturally occurring polypeptide sequence.

2. The subtilisin protease variant of claim 1 , wherein the substitution is Y006W, S024K, T055P, S162Q, S183N, S204Q, Q206Y, M222Q, S248A, or T254A, and wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant does not have 100% sequence identity to the naturally occurring polypeptide sequence.

3. The subtilisin protease variant of claim 1 or 2, wherein the variant comprises one, two or more additional substitutions from the group consisting of X003Q, X022Y, X024Q, X033T, X045V, X053G, X076D, X078N, X087D, X101N, X109Q, X118R, X128A, X128S, X145R, X166Q, X169A, X182Q, X217Q or X218S, wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, and wherein the variant does not have 100% sequence identity to the naturally occurring polypeptide sequence.

4. The subtilisin protease variant of any preceding claim, wherein the substitution is S003Q, T022Y, S024Q, S033T, A045V, S053G, N076D, S078N, S087D, S101N, N109Q, N118R, G128A, G128S, S145R, G166Q, G169A, S182Q, Y217Q, or N218S, and wherein the variant has at least 55% identity to the amino acid sequence of SEQ ID NOs: 1 and 7-28, and wherein the variant does not have 100% sequence identity to the naturally occurring polypeptide sequence.

5. The subtilisin variant of claim 1 or 2, comprising two mutations selected from the group consisting of: X006W-X024K, X006W-X055P, X006W-X162Q, X006W-X183N, X006W-X204Q, X006W-X206Y, X006W-X222Q, X006W-X248A, X006W-X254 A. X024K-X055P, X024K-X162Q, X024K-X183N, X024K-X204Q, X024K-X248A, X024K-X254A, X055P-X162Q, X055P-X183N, X055P-X204Q, 55P-X222Q, X055P-X248A, X055P-X254A, X162Q-X183N, X162Q-X204Q, Q-X222Q, X162Q-X248A, X162Q-X254A, X183N-X204Q, X183N-X206Y, X248A, X183N-X254A, X204Q-X206Y, X204Q-X222Q, X204Q-X248A, X204Q-X254A, X206Y-X222Q, X206Y-X248A, X206Y-X254A, X222Q-X248A, X222Q-X254A, and X248A-X254A, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, wherein the variant does not have 100% sequence identity to the naturally occurring amino acid sequence, and wherein the variant has at least 55% identity to the subtilisin having the amino acid sequence of SEQ ID NOs: 1 and 7-28.

6. The subtilisin variant of claim 1 or 2, comprising three mutations selected from the group consisting of: X006W-X024K-X055P, X006W-X024K-X162Q, X006W-X024K-X183N, X006W-X024K-X204Q, X006W-X024K-X206Y, X006W-X024K-X222Q, X006W-X024K-X248A, X006W-X024K-X254A, X006W-X055P-X162Q, X006W-X055P-X183N, X006W-X024K-X204Q, X006W-X055 5P-X206Y, X006W-X055P-X222Q, X006W-X055P-X248A, , X006W-X162Q-X183N, X006W-X162Q-X204Q, 162Q-X222Q, X006W-X162Q-X248A, X006W-X162Q-X254A, 4Q, X006W-X183N-X206Y, X006W-X183N-X222Q, X183N-X254A, X006W-X204Q-X206Y, X006W-X204Q-X222Q, X006W-X204Q-X2 48A, X006W-X204Q-X254A, X006W-X206Y-X222Q, W-X206Y-X254A, X006W-X222Q-X248A, X006W-X222Q-X254A, X006W-X248A- X254A, X024K-X055P-X162Q, X024K-X055P-X183N, 4K-X055P-X206Y, X024K-X055P-X222Q, X024K-X055P-X248A, X024K-X055P -X254A, X024K-X162Q-X183N, X024K-X162Q-X204Q, 024K-X162Q-X222Q, X024K-X162Q-X248A, X024K-X162Q-X254A, X024K-X18 3N-X204Q, X024K-X183N-X206Y, X024K-X183N-X222Q,X024K-X183N-X254A、X024K-X204Q-X206Y、X024K-X204Q-X222Q、X024K-X204Q-X248A、X024K-X204Q-X254A、X024K-X206Y-X222Q、X024K-X206Y-X248A、X024K-X206Y-X254A、X024K-X222Q-X248A、X024K-X222Q-X254A、X024K-X248A-X254A、X055P-X162Q-X183N、X055P-X162Q-X204Q、X055P-X162Q-X206Y、X055P-X162Q-X222Q、X055P-X162Q-X248A、X055P-X162Q-X254A、X055P-X183N-X204Q、X055P-X183N-X206Y、X055P-X183N-X222Q、X055P-X183N-X248A、X055P-X183N-X254A、X055P-X204Q-X206Y、X055P-X204Q-X222Q、X055P-X204Q-X248A、X055P-X204Q-X254A、X055P-X206Y-X222Q、X055P-X206Y-X248A、X055P-X206Y-X254A、X055P-X222Q-X248A、X055P-X222Q-X254A、X055P-X248A-X254A、X162Q-X183N-X204Q、X162Q-X183N-X206Y、X162Q-X183N-X222Q、X162Q-X183N-X248A、X162Q-X183N-X254A、X162Q-X204Q-X206Y、X162Q-X204Q-X222Q、X162Q-X204Q-X248A、X162Q-X204Q-X254A、X162Q-X206Y-X222Q、X162Q-X206Y-X248A、X162Q-X206Y-X254A、X162Q-X222Q-X248A、X162Q-X222Q-X254A、X162Q-X248A-X254A、X183N-X204Q-X206Y、X183N-X204Q-X222Q、X183N-X204Q-X248A、X183N-X204Q-X254A、X183N-X206Y-X222Q、X183N-X206Y-X248A、X183N-X206Y-X254A、X183N-X222Q-X248A、X183N-X222Q-X254A, X183N-X248A-X254A, X204Q-X206Y-X222Q, X204Q-X206Y-X248A, X204Q-X206Y-X254A, X204Q-X222Q-X248A, X204Q-X222Q-X254A, X204Q-X248A-X254A, X206Y-X222Q-X248A, X206Y-X222Q-X254A, X206Y-X248A-X254A, and X222Q-X248A-X254A, wherein the positions are indicated by the alignment with SEQ ID NO:1, wherein the variant does not have 100% sequence identity with the naturally occurring amino acid sequence, and wherein the variant has at least 55% identity with the subtilisin having the amino acid sequence of SEQ ID NO: 1 and 7-28.

7. The subtilisin protease variant of any of the preceding claims, wherein the variant comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:

1.

8. A subtilisin variant according to any preceding claim, wherein the variant has improved stability or improved cleaning performance when compared to a reference subtilisin lacking the two, three, four or more substitutions.

9. The subtilisin protease variant of claim 8, wherein i) the improved stability is measured by a residual activity (based on a score of 1) greater than 0.2 in a 10% detergent solution after 20 minutes at 54 or 60 degrees Celsius, compared to the corresponding parent, according to the assay described in Example 2, or ii) the improved stability is measured by a residual activity percentage of ≥ 20% in a 10% detergent solution after 20 minutes at 40 or 41 degrees Celsius, compared to the corresponding parent, according to the assay described in Example 2.

10. The subtilisin protease variant of claim 9, wherein the cleaning performance is compared to the corresponding parent with a performance index of ≥ 1.1 on blood, milk, ink, or egg stains in a 10% detergent solution according to the cleaning assay described in Example 2.

11. A composition comprising one or more subtilisin variants according to any preceding claim.

12. The composition of claim 11, wherein the composition is selected from the group consisting of an enzyme composition and a detergent composition.

13. The composition of claim 12, wherein the detergent composition is selected from the group consisting of laundry detergents, fabric softener detergents, dishwashing detergents and hard surface cleaning products.

14. The composition of any one of claims 11-13, wherein the composition further comprises one or more ions selected from calcium and / or zinc; one or more enzyme stabilizers; from about 0.001% to about 1.0% by weight of the subtilisin variant; one or more bleaching agents; one or more auxiliary materials; one or more microorganisms and / or one or more additional enzymes or enzyme derivatives selected from the group consisting of: acyltransferases, alginate lyases, α-amylases, β-amylases, β-glucanases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, DNase, dispersin, endo-β-1, β-Glucanase, endo-β-mannanase, esterase, exo-mannanase, fructosidase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, laminarinase, lichenase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease (e.g., DNA enzyme and / or RNA enzyme), oxidase, oxidoreductase, pectate lyase , pectin acetylesterase, pectin methylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phosphodiesterase, phospholipase, phytase, polyesterase, polygalacturonase, additional protease, pullulanase, RNase, reductase, rhamnogalacturonase, tannase, transglutaminase, xanthanase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and combinations thereof.

15. The composition of any one of claims 11 to 14, wherein the composition is a granule, a regular or compact powder, a solid, a stick, a regular or compact or concentrated liquid, a uniform tablet, a tablet having two or more layers, a pouch having one or more compartments, a gel, a paste or a unit dose composition.

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