Detergent compositions and methods related thereto

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] 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.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 403,332 filed on September 2, 2022 and U.S. Provisional Patent Application Serial No. 63 / 492,619 filed on March 28, 2023, which are incorporated by reference in their entirety.

[0004] Reference to a sequence listing submitted electronically

[0005] The contents of the sequence listing submitted electronically with this application as an XML file (file name: NB42142PCT_sequencelisting; size: 12,186 bytes; created on: August 22, 2023) constitute a part of this application and are hereby incorporated by reference in their entirety. Background Art

[0006] Protease (also referred to as protease) refers to an enzyme with the ability to decompose other proteins. Protease has the ability to start protein catabolism to perform proteolysis by hydrolysis of the peptide bonds that connect amino acids together in the peptide or polypeptide chain forming the protein. This activity of protease as a protein digestive enzyme is referred to as proteolytic activity. There are many well-known programs for measuring proteolytic activity (Kalisz, "Microbial Proteinases [microbial protease]," in: Fiechter (editor), Advances in Biochemical Engineering / Biotechnology [biochemical engineering / biotechnology progress], (1988)). For example, proteolytic activity can be determined by comparing the ability of each protease to hydrolyze commercial substrates. Exemplary substrates that can be used to analyze protease or proteolytic activity include but are not limited to dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625) and keratin Azure (Keratin Azure) (Sigma-Aldrich K8500). Colorimetric assays using these substrates are well known in the art (see, eg, WO 99 / 34011 and US Pat. No. 6,376,450, both of which are incorporated herein by reference).

[0007] Serine proteases are enzymes with an active site serine that initiates hydrolysis of protein peptide bonds (EC number 3.4.21). Serine proteases include a wide variety of enzymes with broad specificity and biological functions, which are further divided into chymotrypsin-like (trypsin-like) and subtilisin-like based on their structures. The prototype subtilisin (EC number 3.4.21.62) was originally obtained 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 [Twenty years of MEROPS database of proteolytic enzymes and their substrates and inhibitors]. Nucleic Acids Res [Nucleic Acids Research] 44, D343-D350). Members of the S8 family have a catalytic triad in their amino acid sequence with the order of Asp, His and Ser. Although a number of variant proteases have been developed that are useful in cleaning applications, there remains a need for improved protease variants. Summary of the invention

[0008] One embodiment relates to a composition comprising a subtilisin variant and a combination of at least one additional enzyme, wherein the subtilisin variant comprises one, two or more substitutions selected from the group consisting of: X96D, X103F, X108Q, X115L, X128K, X129Q and X181Q, wherein the amino acid positions are numbered according to SEQ ID NO: 9, wherein the variant is identical to SEQ ID NO: 9. NO:9 has at least 75% identity with the amino acid sequence of, and wherein the at least one additional enzyme is selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, lipoxygenase, lysozyme, mannanase, metalloproteinase, The invention relates to a detergent composition comprising the following: a protease, a nuclease (e.g., a DNase and / or an RNase), an oxidase, an oxidoreductase, a pectate lyase, a pectin acetylesterase, a pectinase, a pentosanase, a perhydrolase, a peroxidase, a phenoloxidase, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, a protease, a pullulanase, a reductase, a rhamnogalacturonase, a β-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in the detergent composition lacking the subtilisin variant.

[0009] In one embodiment, the composition is a composition comprising a subtilisin variant and a combination of at least one additional enzyme, wherein the subtilisin variant comprises one, two or more amino acid substitutions selected from the group consisting of: N096D, Y103F, S108Q, T115L, A128K, S129Q and S181Q, wherein the amino acid positions are numbered according to SEQ ID NO: 9, wherein the variant is identical to SEQ ID NO: 9. NO:9 has at least 75% identity with the amino acid sequence of, and wherein the at least one additional enzyme is selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, lipoxygenase, lysozyme, mannanase, metalloproteinase, The invention relates to a detergent composition comprising the following: a protease, a nuclease (e.g., a DNase and / or an RNase), an oxidase, an oxidoreductase, a pectate lyase, a pectin acetylesterase, a pectinase, a pentosanase, a perhydrolase, a peroxidase, a phenoloxidase, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, a protease, a pullulanase, a reductase, a rhamnogalacturonase, a β-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in the detergent composition lacking the subtilisin variant.

[0010] In another embodiment, the disclosure relates to a composition comprising a combination of a subtilisin variant and at least one additional enzyme, wherein the subtilisin variant comprises at least one, two, three, four or more amino acid substitutions selected from the group consisting of: X96D, X103F, X108Q, X115L, X128K, X129Q and X181Q, wherein the amino acid positions are numbered according to SEQ ID NO:9, wherein the subtilisin variant further comprises one or more additional amino acid substitutions selected from the group consisting of: X9T, X17H, X77N, X78I, X103I, X127T, X165Q, X184Q, X202V, X203E, X203N, X217S and X258P, wherein the variant is identical to SEQ ID NO:9. NO:9 has at least 75% identity with the amino acid sequence of, and wherein the at least one additional enzyme is selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, lipoxygenase, lysozyme, mannanase, metalloproteinase, The invention relates to a detergent composition comprising the following: a protease, a nuclease (e.g., a DNase and / or an RNase), an oxidase, an oxidoreductase, a pectate lyase, a pectin acetylesterase, a pectinase, a pentosanase, a perhydrolase, a peroxidase, a phenoloxidase, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, a protease, a pullulanase, a reductase, a rhamnogalacturonase, a β-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in the detergent composition lacking the subtilisin variant.

[0011] In another embodiment, the disclosure relates to a composition comprising a combination of a subtilisin variant and at least one additional enzyme, wherein the subtilisin variant comprises at least one, two, three, four or more amino acid substitutions selected from the group consisting of: X96D, X103F, X108Q, X115L, X128K, X129Q and X181Q, wherein the amino acid positions are numbered according to SEQ ID NO:9, wherein the subtilisin variant further comprises one or more additional amino acid substitutions selected from the group consisting of: P009T, Q017H, T077N, T078I, Y103I, G127T, G165Q, N184Q, A202V, G203E, G203N, N217S and S258P, wherein the variant is identical to SEQ ID NO:9. NO:9 has at least 75% identity with the amino acid sequence, and wherein the at least one additional enzyme is selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, lipoxygenase, lysozyme, mannanase, The invention relates to a detergent composition comprising the following: a metalloproteinase, a nuclease (e.g., a DNase and / or an RNase), an oxidase, an oxidoreductase, a pectate lyase, a pectin acetylesterase, a pectinase, a pentosanase, a perhydrolase, a peroxidase, a phenoloxidase, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, a protease, a pullulanase, a reductase, a rhamnogalacturonase, a β-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination thereof, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in the detergent composition lacking the subtilisin variant.

[0012] Still other embodiments relate to subtilisin variants, methods for producing variants as described herein, the method comprising stably transforming a host cell with an expression vector comprising a polynucleotide encoding one or more subtilisin variants as described herein. Still other embodiments relate to polynucleotides comprising a nucleotide sequence encoding one or more subtilisin variants as described herein. Compositions comprising subtilisin variants provided herein, such as enzyme compositions, are also provided. DETAILED DESCRIPTION

[0013] In one embodiment, the disclosure provides one or more subtilisin variants, the one or more subtilisin variants are included in one, two, three or more amino acid substitutions at a position selected from the group consisting of: 96, 103, 108, 115, 128, 129 and 181, wherein these amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:9. In some embodiments, when compared with the subtilisin with the amino acid sequence of SEQ ID NO:9, the variants provided herein exhibit one or more improved properties, such as improved stability, or improved cleaning performance, or improved stability and improved cleaning performance. The subtilisin variants provided herein can be used to prepare a cleaning composition (such as an automatic dishwashing composition or a laundry detergent composition) with at least one additional enzyme, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in a detergent composition without the subtilisin variants provided herein. In addition, the subtilisin variants and compositions provided herein can also be used in a cleaning method (such as a dishwashing method or a laundry washing method) using such variants or a composition comprising such subtilisin variants.

[0014] Unless otherwise indicated herein, one or more subtilisin variants described herein can be prepared and used by various techniques used and developed for molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA fields and industrial enzymes. 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 those of ordinary skill in the art generally understand. Any definition provided herein will be interpreted as a whole in the context of the specification. Unless otherwise clearly indicated by the context, as used herein, the singular "a / an" and "the" include plural. Unless otherwise indicated, the nucleic acid sequence is written from left to right in the 5' to 3' direction; and the amino acid sequence is written from left to right in the amino to carboxyl direction. Each numerical range used herein includes each narrower numerical range falling within such a wider numerical range, as if such a narrower numerical range is all clearly written in this article.

[0015] 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.

[0016] The nomenclature of the amino acid substitution 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 "position" (e.g., 5, 8, 17, 22, etc.) covers any starting amino acid that may be present at such a position, and any substitution that may be present at such a position. Reference to "position: one or more amino acid substitutions" (e.g., 1S / T / G, 3G, 17T, etc.) covers any starting amino acid that may be present at such a position and one or more amino acids that may replace such starting amino acids. Reference to position can list several forms, for example, position 003 can also be referred to as position 03 or 3. Reference to starting or substituted amino acids can be further represented as several starting or substituted amino acids separated by a foreslash (" / "). For example, D275S / K means that position 275 is substituted with serine (S) or lysine (K), and P / S197K means that the starting amino acid proline (P) or serine (S) at position 197 is substituted with lysine (K). References to X as an amino acid at a position refer to any amino acid at the listed position.

[0017] The position of the amino acid residue in a given amino acid sequence is numbered by corresponding to the amino acid sequence of SEQ ID NO:9. That is, the amino acid sequence of SEQ ID NO:9 is used as a reference sequence for numbering the position of the amino acid residue. For example, the amino acid sequence of one or more subtilisin variants as described herein is compared with the amino acid sequence of SEQ ID NO:9 using an alignment algorithm as described herein, and each amino acid residue in a given amino acid sequence of the amino acid residue alignment (preferably, optimal alignment) in SEQ ID NO:9 is conveniently numbered by reference to the numerical position of the corresponding amino acid residue. When compared with a query sequence (sometimes also referred to as "reference sequence"), one or more positions where insertion or deletion occurs in the subject sequence will be identified as a sequence alignment algorithm as described herein. For example, as provided in Fig. 1 of PCT Publication No. WO 2018118917, amino acid alignment can be used to determine the sequence alignment with other subtilisin amino acid sequences.

[0018] The terms "protease" and "proteinase" refer to enzymes with the ability to decompose proteins and peptides. Proteases have the ability to "proteolyze" by hydrolyzing the peptide bonds that connect amino acids together in the peptide or polypeptide chain that forms a protein. This activity of proteases as protein digestive 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 determination of the ability of each protease to hydrolyze a suitable substrate. Exemplary substrates that can be used to analyze proteases or proteolytic activity include, but are not limited to, dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625) and Keratin Azure (Sigma-Aldrich K8500). Colorimetric determination using these substrates is well known in the art (see, for example, WO 99 / 34011 and US 6,376,450). The pNA peptide base assay (see, e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) can also be used to determine the active enzyme concentration. The assay measures the rate at which p-nitroaniline is released when the enzyme hydrolyzes a soluble synthetic substrate such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (suc-AAPF-pNA). The rate of generation of the yellow color from the hydrolysis reaction is measured at 405 or 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a purified protein sample. The activity of the substrate divided by the protein concentration gives the enzyme specific activity.

[0019] 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, B. pumilus, Bacillus species TY-145, and B. thuringiensis. It will be recognized that the genus Bacillus is continually undergoing taxonomic reorganization. Thus, the genus is intended to include species that have been reclassified, including, but not limited to, organisms such as Bacillus stearothermophilus (now named "Geobacillus stearothermophilus") or B. polymyxa (now "Paenibacillus polymyxa"). The production of resistant endospores under stressful environmental conditions is considered a defining characteristic of the genus Bacillus, although this feature also applies to the more recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0020] The term "vector" refers to a nucleic acid construct for introducing or transferring one or more nucleic acids into a target cell or target tissue. Typically, a vector is used to introduce exogenous DNA into a cell or tissue. Vectors include plasmids, cloning vectors, phages, viruses (e.g., viral vectors), cosmids, expression vectors, shuttle vectors, etc. Typically, vectors include an origin of replication, a multiple cloning site, and a selective marker. Typically, the process of inserting a vector into a target cell is referred to as transformation. In certain embodiments, the present invention includes a vector comprising a DNA sequence of a serine protease polypeptide (e.g., a precursor or mature serine protease polypeptide) operably connected to a suitable presequence (e.g., secretion, a signal peptide sequence, etc.), the vector being able to achieve expression of the DNA sequence in a suitable host, and folding and translocation of the recombinant polypeptide chain.

[0021] As used herein, in the context of introducing a nucleic acid sequence into a cell, the term "introducing" refers to any method suitable for transferring a nucleic acid sequence into a cell. Such methods of introduction include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to a genetic change in a cell caused by uptake, optional genomic incorporation, and expression of genetic material (e.g., DNA).

[0022] The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecules of the present disclosure. Expression can also refer to the translation of mRNA into polypeptides. Therefore, the term "expression" includes any step involved in the "production of polypeptides", including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0023] The phrase "expression cassette" or "expression vector" refers to a nucleic acid construct or vector that is recombinantly or synthetically produced for expressing a target nucleic acid (e.g., an exogenous nucleic acid or a transgenic) in a target cell. Typically, the target nucleic acid expresses a target protein. Typically, an expression vector or expression cassette comprises a promoter nucleotide sequence that drives or promotes the expression of the exogenous nucleic acid. Typically, an expression vector or expression cassette also comprises other designated nucleic acid elements that allow a specific nucleic acid to be transcribed in a 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 a host cell genome. Many prokaryotic and eukaryotic expression vectors are commercially available. It is within the knowledge of those skilled in the art to select an appropriate expression vector for expressing a protein from the nucleic acid sequence incorporated into the expression vector.

[0024] As used herein, a nucleic acid is "operably linked" to another nucleic acid sequence when it is placed in a functional relationship with the other nucleic acid sequence. For example, if a promoter affects the transcription of a coding sequence, a promoter or enhancer is operably linked to a nucleotide coding sequence. If a ribosome binding site is positioned so as to promote the translation of a coding sequence, the ribosome binding site may be operably linked to a coding sequence. Typically, an "operably linked" DNA sequence is continuous. However, an enhancer need not 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.

[0025] The term "gene" refers to a polynucleotide (eg, 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).

[0026] When used with respect to a cell, the term "recombinant" typically indicates that the cell has been modified by the introduction of an exogenous nucleic acid sequence, or that the cell is derived from a cell that has been so modified. For example, a recombinant cell may contain genes that do not exist in the same form in the natural (non-recombinant) form of the cell, or a recombinant cell may contain native genes (found in the natural form of the cell) that have been modified and reintroduced into the cell. A recombinant cell may contain nucleic acids that are endogenous to a cell that has been modified but not removed from the cell; such modifications include those obtained by gene replacement, site-specific mutations, and related techniques known to those of ordinary skill in the art. Recombinant DNA technology includes techniques for producing recombinant DNA in vitro and transferring the recombinant DNA into cells where it can be expressed or propagated to produce recombinant polypeptides. "Recombination" and "recombining" of polynucleotides or nucleic acids generally refers to assembling or combining two or more nucleic acids or polynucleotide chains or fragments to produce new polynucleotides or nucleic acids.

[0027] If a nucleic acid or polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof in the natural 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 a sequence.

[0028] The terms "host strain" and "host cell" refer to a suitable host for an expression vector comprising a DNA sequence of interest.

[0029] "Protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. Single-letter and 3-letter codes for amino acids defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure. The single letter X refers to any one of the twenty amino acids. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0030] The term "prosequence" or "propeptide sequence" refers to an amino acid sequence between the signal peptide sequence and the mature protease sequence that is necessary for the correct folding and secretion of the protease; they are sometimes referred to as intramolecular chaperones. Cleavage of the prosequence or propeptide sequence produces a mature active protease. Bacterial serine proteases are often denoted as proenzymes. Examples of modified propeptides are provided, for example, in WO 2016 / 205710.

[0031] The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can participate in the secretion or directional transport of a mature or precursor form of a protein. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. Signal sequences are generally not present in mature proteins. Typically, after protein transport, the signal sequence is cut from the protein by a signal peptidase.

[0032] 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.

[0033] The term "precursor" form of a protein or peptide refers to a mature form of a protein having a presequence 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 presequence. The precursor may also have additional polypeptides involved in post-translational activity (e.g., polypeptides from which it is cleaved to leave the mature form of the protein or peptide).

[0034] 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 polynucleotides encoding wild-type or parent polypeptides.

[0035] About polypeptide, term " parent " includes the polypeptide of naturally occurring or wild type, or wherein carries out artificial substitution, insertion or deletion naturally occurring polypeptide 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 (such as replacement, addition and / or deletion), to produce the variant with one or more changes compared with this starting polypeptide.That is, parent or reference polypeptide is not limited to naturally occurring wild-type polypeptide, and encompasses any wild type, parent or reference polypeptide.Similarly, about polynucleotide, term " parent " can refer to naturally occurring polynucleotide or really include artificial substitution, insertion or deletion polynucleotide at one or more nucleotides.About polynucleotide, term " parent " also includes any polynucleotide encoding the polypeptide with protease activity, and this polynucleotide serves as the starting polynucleotide for changing, thereby produces the variant protease with modification such as replacement, addition and / or deletion 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 parental polypeptide herein comprises a polypeptide having the amino acid sequence shown in SEQ ID NO:9.

[0036] The term "naturally occurring" refers to, for example, sequences found in nature and residues contained therein (e.g., polypeptide sequences and amino acids contained therein or nucleotide sequences and nucleotides contained therein). In contrast, the term "non-naturally occurring" refers to, for example, sequences not found in nature and residues contained therein (e.g., polypeptide sequences and amino acids contained therein or nucleotide sequences and nucleic acids contained therein).

[0037] As used herein, with respect to amino acid residue positions, "corresponding to or 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.

[0038] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by a 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, "proteases derived from Bacillus" refer 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.

[0039] 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.

[0040] The phrase "% identity" or "percent identity" or "PID" refers to protein sequence identity. Percent identity can be determined using standard techniques known in the art. The percent amino acid identity shared by the target sequence 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 in, for example, Altschul et al., J Mol Biol [Journal of Molecular Biology], 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 90:5873-5787 (1993). The percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues in the "reference" sequence (including any gaps created by the program for optimal / maximal alignment). The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0041] As used herein, "homologous proteins" or "homologous proteases" refer to proteins with different similarities in primary, secondary and / or tertiary structures. When comparing proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology can be determined by, 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 newgeneration of protein database search programs", Nucleic Acids Res, Group 1; 25(17): 3389-402 (1997)). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences by 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 phylogenetic trees constructed therefrom. The amino acid sequence can be entered in programs such as the Vector NTI Advance suite, and a guide tree can be created using the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol [Molecular Biology and Evolution], 4: 406-425, 1987). The tree structure can be calculated using the Kimura correction for sequence distances and ignoring positions with gaps. Programs such as AlignX can display the calculated distance values ​​in brackets after the molecule name displayed on the phylogenetic tree.

[0042] Understanding the homology between molecules can reveal the evolutionary history of molecules as well as information about their functions; if a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the biochemical function of the new protein. If two molecules are derived from a common ancestor, they are called homologous. Homologous molecules or homologs can be divided into two categories: paralogs and orthologs. Paralogs are homologs present in one species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs present in different species and have very similar or identical functions. The protein superfamily is the largest grouping (branch) of proteins from which a common ancestor can be inferred. Usually this common ancestor is based on sequence alignments and mechanical similarities. Typically, a superfamily contains several protein families that show sequence similarity within the family. Based on the MEROPS protease classification system, the term "protein clan" is often used for the protease superfamily. 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).

[0043] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res 22:4673-4680, 1994). The default parameters of the CLUSTAL W algorithm include: Gap Open Penalty = 10.0; Gap Extension Penalty = 0.05; Protein Weight Matrix = BLOSUM Series; DNA Weight Matrix = IUB; Delayed Divergent Sequence % = 40; Gap Separation Distance = 8; DNA Transition Weight = 0.50; List Hydrophilic Residues = GPSNDQEKR; Use Negativity Matrix = Off; Switch Special Residue Penalty = On; Switch Hydrophilic Penalty = On; and Switch End Gap Separation Penalty = Off. In the CLUSTAL algorithm, deletions occurring at either end are included. For example, a variant having five amino acid deletions at either end of a 500 amino acid polypeptide (or within a polypeptide) would have a percent sequence identity of 99% (495 / 500 identical residues x 100) relative to a "reference" polypeptide. Such variants would be encompassed by variants having "at least 99% sequence identity" to the polypeptide.

[0044] When nucleic acid or polynucleotide is separated from other components (including but not limited to, for example, other proteins, nucleic acids, cells, etc.) at least in part or completely, the nucleic acid or polynucleotide is "separated". Similarly, when polypeptide, protein or peptide is separated from other components (including but not limited to, for example, other proteins, nucleic acids, cells, etc.) at least in part or completely, the polypeptide, protein or peptide is "separated". In mole, the species separated in the composition is more abundant than other species. For example, the species separated can 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% (in mole) of all macromolecular species present. Preferably, the target species is purified to basic homogeneity (that is, 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, high resolution techniques such as high performance liquid chromatography (HPLC) or similar methods can be used to purify the material.

[0045] The term "purified" as applied to nucleic acids or polypeptides generally refers to a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percentages by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of that molecule after application of a purification or enrichment technique. The term "enriched" refers to the presence in a composition of a compound, polypeptide, cell, nucleic acid, amino acid or other specific substance or component at a relative or absolute concentration greater than that in the starting composition.

[0046] The term "cleaning activity" refers to the cleaning performance achieved by serine protease polypeptides, variants or reference subtilisin under the conditions mainly during the disclosed proteolysis, hydrolysis, cleaning or other processes. 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 (e.g., 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 or reference subtilisin as described herein can be determined by subjecting the stain on articles or surfaces to one or more standard washing 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 example 2 provided below.

[0047] The term "effective amount" of one or more subtilisin variants or reference subtilisin 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 used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, tablet, stick) composition is desired, etc.

[0048] The term "auxiliary material" refers to any liquid, solid or gaseous material, or recombinant polypeptide or its active fragment contained in the cleaning compositions except one or more subtilisin variants as described herein. In certain embodiments, the cleaning compositions of the present disclosure include one or more cleaning auxiliary materials. Typically, depending on the specific type and form (such as liquid, granule, powder, rod-shaped, 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.

[0049] Cleaning compositions and cleaning formulations include any composition suitable for cleaning, bleaching, disinfecting and / or sterilizing any object, article and / or surface. Such compositions and formulations include, but are not limited to, for example, liquid and / or solid compositions, including cleaning or detergent compositions (e.g., liquid, tablet, gel, stick, granular and / or solid laundry cleaning or detergent compositions) and fine fabric detergent compositions; medical device cleaning 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; and textiles, clothing synergistic cleaning or detergent compositions, clothing additive cleaning compositions and clothing 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 can also use single dosage unit forms, including but not limited to pills, tablets, gelcaps or other single dosage units such as pre-measured powders or liquids.

[0050] Unless otherwise indicated, cleaning compositions or cleaning formulations as used herein include general-purpose or heavy-duty detergents in granular or powder form, especially cleaning detergents; general-purpose detergents in liquid, granular, gel, solid, tablet, paste or unit dosage form, especially the so-called heavy-duty liquid (HDL) detergent or heavy-duty dry cleaning (HDD) detergent types; liquid fine fabric detergents; hand or manual dishwashing detergents, including those of the high-sudsing type; hand or manual dishwashing detergents, automatic dishwashing detergents, or tableware or tableware detergents, including various tablet, powder, solid, granular, liquid, gel and rinse aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand washing 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, particulate compositions are in a "compacted" form; in some embodiments, liquid compositions are in a "concentrated" form.

[0051] About the composition used in the washing medium intended for cleaning contaminated or dirty objects (including specific fabrics and / or non-fabric objects or articles), the term "detergent composition" or "detergent preparation" is used. In certain embodiments, the detergent of the present disclosure includes one or more subtilisin variants described herein, and further includes one or more surfactants, one or more transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme stabilizers, calcium, enzyme activators, antioxidants and / or solubilizing agents. In some cases, the builder salt is a mixture of silicate and phosphate, preferably with more silicate (e.g., sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate). Some embodiments relate to a cleaning composition or a detergent composition not containing any phosphate (e.g., phosphate or phosphate builder). The detergent composition can also contain biological components, such as one or more microorganisms (microorganism / microbe) or microbial extracts (as described in WO 2018060475 and US10968556). Microorganisms can be used as the sole bioactive ingredient, but they can also be used in combination with one or more enzymes 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. Other purposes may include in situ production of desired biological compounds, or inoculating / propagating a locus with one or more microorganisms to competitively prevent other undesirable microorganisms from propagating the same locus (competitive exclusion).

[0052] The phrase "one or more compositions that are substantially free of boron" or "one or more detergents that are substantially free of boron" refers to one or more compositions or 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.

[0053] The term "bleaching" refers to treating a material (e.g., fabric, clothing, pulp, etc.) or a surface for a sufficient period of time and / or under appropriate pH and / or temperature conditions to achieve whitening (i.e., whitening) and / or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, for example, ClO 2 , H 2 O 2 , peracid, NO 2Etc. Bleaching agents also include enzymatic bleaching agents such as perhydrolases and arylesterases. 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.

[0054] The " washing performance " of the term protease (for example, one or more subtilisin variants as described herein, or its recombinant polypeptide or active fragment) refers to the cleaning contribution that one or more subtilisin variants as described herein provide other cleaning performance to washing compared with the detergent that does not add one or more subtilisin variants as described herein to the composition.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 such a way as follows: simulate one or more conditions typical for household applications in certain market segments (for example hand washing or manual dishwashing, automatic dishwashing, dishwashing cleaning, tableware cleaning, fabric cleaning, etc.).

[0055] 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.

[0056] 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).

[0057] The term "compact" form of cleaning compositions herein is best reflected by density, and in terms of composition, by the amount of inorganic filler salts. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, filler salts are present in a basic amount, typically about 17% to about 35% by weight of the total composition. In contrast, in compact compositions, filler salts are present in an amount less than about 15% of the total composition. In certain embodiments, filler salts are present in an amount not exceeding about 10%, or more preferably about 5%, by weight of the composition. In certain embodiments, inorganic filler salts are selected from alkali salts and alkaline earth metal salts of sulfates and chlorides. In certain embodiments, filler salts are sodium sulfate.

[0058] 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 described herein 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 articles or textiles) that are needed.

[0059] In one embodiment, a subtilisin variant is provided, wherein the variant comprises two, three, four or more amino acid substitutions compared to the parent subtilisin of SEQ ID NO:0, wherein the substitutions are at positions selected from the group consisting of: 96, 103, 108, 115, 128, 129 and 181, wherein the positions are numbered according to SEQ ID NO:9, and wherein the variant has at least 75% identity with the amino acid sequence of SEQ ID NO:9.

[0060] In one embodiment, a subtilisin variant is provided, wherein the variant comprises one, two, three or four or more amino acid substitutions selected from the group consisting of: X96D, X103F, X108Q, X115L, X128K, X129Q or X181Q, wherein these positions are numbered according to SEQ ID NO:9, and wherein the variant has at least 75% identity with the amino acid sequence of SEQ ID NO:9.

[0061] In some embodiments, the subtilisin variant further comprises one or more additional substitutions selected from the group consisting of X9T, X17H, X77N, X78I, X103I, X127T, X165Q, X184Q, X202V, X203N, X203E, X217S, and X258P, wherein positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:9.

[0062] In one embodiment, a subtilisin variant is provided, wherein the variant is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0063] Another embodiment relates to one or more subtilisin variants described herein, provided that one or more amino acid substitutions are non-naturally occurring. Yet even further embodiments relate to one or more subtilisin variants described herein, wherein the variant (i) is derived from Bacillus licheniformis subtilisin; (ii) is isolated; (iii) has proteolytic activity; or (iv) comprises a combination of (i) to (iii). Yet another embodiment relates to one or more subtilisin variants described herein, wherein the variant is derived from a parent or reference polypeptide that (i) has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 9; or (ii) has 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 9. In yet another embodiment, the parent comprises the amino acid sequence of SEQ ID NO: 9. Even further embodiments relate to one or more subtilisin variants described herein, wherein the variant comprises an amino acid sequence having (i) 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0064] 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 properties are selected from improved detergent cleaning performance, improved detergent or buffer stability and improved aged cleaning performance, and combinations thereof. Aged cleaning performance refers to the difference in stain removal measured compared to the "fresh" stain cleaning of the same enzyme (without pre-incubation) for samples of aged test samples (wherein the enzyme is pre-incubated in a detergent for an extended period of time, such as 3-8 weeks at an elevated temperature of, for example, 37°C). Therefore, when compared to the same evaluation performed with a reference / parent enzyme, the enzyme with improved aged cleaning performance shows a smaller difference between aged and freshly prepared samples.

[0065] In another embodiment, the parent subtilisin comprises the amino acid sequence of SEQ ID NO: 9. In yet another embodiment, the improved property is (i) improved detergent cleaning performance, wherein the variant has improved cleaning performance on blood / milk / ink stains or egg stains on woven cotton compared to the parent subtilisin; and / or (ii) improved stability, wherein the variant has higher residual activity compared to the parent or reference subtilisin. In yet another embodiment, the detergent cleaning performance is measured according to the cleaning performance assay of Example 2; and / or the stability is measured according to the stability assay of Example 2.

[0066] In another embodiment, the subtilisin variants provided herein exhibit reduced activity against additional enzymes in liquid detergent compositions. Thus, the additional enzymes (e.g., alpha-amylases or mannanases) exhibit reduced degradation in liquid detergent compositions compared to the same additional enzymes (e.g., alpha-amylases or mannanases) in a liquid detergent composition containing subtilisin without one or more of the substitutions selected from X96D, X103F, X108Q, X115L, X128K, X129Q, or X181Q. In some embodiments, the additional enzyme (e.g., α-amylase or mannanase) exhibits reduced degradation in the liquid detergent composition compared to the same additional enzyme (e.g., α-amylase or mannanase) in a liquid detergent composition containing a subtilisin without one or more of the subtilisin variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0067] In the context of oxidation, chelating agent, denaturing agent, surfactant, heat and / or pH stable proteases, the term "enhanced stability" or "improved stability" refers to a subtilisin variant that has a higher retained proteolytic activity over time compared to a reference or parent subtilisin (e.g., a wild-type protease or a parent protease, such as SEQ ID NO: 9). 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).

[0068] With regard 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 being exposed to a varying temperature over a given period of time under conditions (or "stress conditions") that are prevalent during proteolysis, hydrolysis, cleaning, or other processes. Residual activity is the amount of activity that is residual compared to the initial activity of the sample after testing, and can be reported in percentage form, such as residual activity%. "Changed temperature" encompasses temperature increases or decreases. In some embodiments, variant proteases provided herein are exposed to 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 800 minutes, about 810 minutes, about 820 minutes, about 830 minutes, about 840 minutes, about 860 minutes, about 870 minutes, about 880 minutes, about 890 minutes, about 900 minutes, about 910 minutes, about 920 minutes, about 930 minutes, about 940 minutes, about 950 minutes, about 960 minutes, about 970 minutes, about 980 minutes, about 990 minutes, about 1000 minutes, about 1010 minutes, about 1020 minutes, about 1030 minutes, about 1040 minutes, about 1050 minutes, about 1060 minutes, about 1070 minutes, about 1080 minutes, about 1090 minutes, about 1110 minutes, about 1130 minutes, about 114 In certain embodiments, the variant subtilisin enzyme provided herein is a proteolytic enzyme that is present in an amount of at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity. In certain embodiments, the variant subtilisin enzyme provided herein is a proteolytic enzyme that is present in an amount of at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity. In certain embodiments, the variant subtilisin enzyme provided herein is a proteolytic enzyme that is present in an amount of at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity. In certain embodiments, the variant subtilisin enzyme provided herein is a proteolytic enzyme that is present in an amount of at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity.

[0069] Subtilisin variants provided herein can be used to produce various compositions, such as enzyme compositions and cleaning compositions or detergent compositions. The enzyme composition comprises a subtilisin variant as provided herein. The enzyme composition can be in any form, such as a granule, a liquid preparation, or an enzyme slurry.

[0070] 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 and spheronization processes. The core of the granule can be the granule itself or the inner core of a layered granule.

[0071] 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, sugar spheres (a combination of sugar and starch; e.g., starch-sucrose sugar spheres-ASNP), talc, silicates, carboxymethyl cellulose, starch and combinations thereof.

[0072] 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.

[0073] In some 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.

[0074] In some embodiments, the core is an enzyme powder, including UFC containing an enzyme. The enzyme powder can be spray dried and can be optionally blended with any water-soluble or water-dispersible agent listed herein. The enzyme can be or can include a protease to be stabilized, in which case the enzyme powder should further include a stabilizer.

[0075] In certain 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 material for one or more coatings can be suitable for use in cleaning compositions and / or detergent compositions (see, for example, US20100124586, WO 9932595 and US5324649).

[0076] 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 sugars and starches), 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 and EROL ) and talc. US20100124586, WO9932595 and US 5324649 describe suitable components for coating in detail.

[0077] 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 multilayer 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.

[0078] 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.

[0079] In some embodiments, the enzyme granules comprise a subtilisin variant as provided herein in combination with one or more additional enzymes selected from the group consisting of acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, Laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease (such as DNA enzyme and / or RNA enzyme), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polygalacturonase, polyesterase, other protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof. Typically, at least one enzyme coating comprises at least one subtilisin variant as provided herein.

[0080] 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, yeast origin, and acidic, neutral or alkaline enzymes.

[0081] Another embodiment relates to a method for cleaning surfaces, wherein the method comprises contacting a surface or article that needs to be cleaned 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 that needs to be cleaned comprises a protein stain on the surface. In certain embodiments, the surface or article that needs to be cleaned comprises a protein stain. The term "stain" comprises any type of dirt on the surface of articles (such as hard surface articles (such as tableware), medical equipment or textiles). In certain embodiments, the stain is a protein stain. As used herein, a "protein stain" is a stain or dirt containing protein.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] One or more subtilisin variants described herein may undergo 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 change the enzymatic activity of the variant. Similarly, the nucleic acids of the present invention may also undergo various changes, such as one or more substitutions of one or more nucleotides in one or more codons, so that specific codons encode identical or different amino acids, thereby producing silent changes (e.g., when the encoded amino acids are not changed by nucleotide mutations) or non-silent changes; one or more deletions of one or more nucleotides (or codons) in the sequence; one or more additions or insertions of one or more nucleotides (or codons) in the sequence; and / or cutting or one or more truncations of one or more nucleotides (or codons) in the sequence. Compared to the polypeptide enzyme encoded by the original nucleic acid sequence, many such changes in the nucleic acid sequence do not substantially change the enzymatic activity of the resulting encoded polypeptide enzyme. 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 one or more identical amino acids.

[0086] One or more separated, non-naturally occurring or reorganized polynucleotides are described herein, and the polynucleotides include nucleic acid sequences encoding one or more subtilisin variants as described herein, or recombinant polypeptides or their active fragments. One or more nucleic acid sequences as described herein can be used for 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 its fragments. One embodiment provides nucleic acids encoding one or more subtilisin variants as described herein, wherein the variant is a mature form with proteolytic activity. In certain embodiments, one or more subtilisin variants as described herein are recombinantly expressed with homologous propeptide sequences. In other embodiments, one or more subtilisin variants as described herein are recombinantly expressed with heterologous or natural propeptide sequences (e.g., propeptide sequences from bacillus licheniformis).

[0087] One or more nucleotide sequences described herein can be produced by using any suitable synthesis, operation and / or separation technology or its combination.For example, one or more polynucleotides described herein can be produced using standard nucleic acid synthesis techniques well known to those skilled in the art such as solid phase synthesis techniques.In such a technology, typically a fragment of up to 50 or more nucleotide bases is synthesized, and then connected (for example, by enzyme or chemical connection method) to form any desired continuous nucleic acid sequence.The synthesis of one or more polynucleotides described herein can also be promoted by any suitable method known in the art, including but not limited to the chemical synthesis using the following method: classical phosphoramidite method (see, for example, Beaucage et al., Tetrahedron Letters [tetrahedron express] 22: 1859-69 (1981)), or as typically practiced in automatic synthesis method in Matthes et al., EMBO J. [European Molecular Biology Association Journal] 3: 801-805 (1984) described in the method.One or more polynucleotides described herein can also be produced by using an automatic DNA synthesizer. Custom nucleic acids can be ordered from a variety of commercial sources (e.g., 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).

[0088] Recombinant DNA techniques for modifying nucleic acids are well known in the art, such as restriction endonuclease digestion, connection, reverse transcription and cDNA production and polymerase chain reaction (such as PCR). One or more polynucleotides described herein can also be obtained by screening cDNA libraries using one or more oligonucleotide probes, which can be hybridized with polynucleotides encoding one or more subtilisin variants described herein, or recombinant polypeptides or their active fragments or amplified by PCR. The procedures for screening and separating cDNA clones and PCR amplification procedures are well known to those skilled in the art and are described in standard references known to those skilled in the art. One or more polynucleotides described herein can be obtained by, for example, changing naturally occurring polynucleotide backbones (for example, polynucleotide backbones encoding one or more subtilisin variants described herein or reference subtilisin) through known mutagenesis procedures (such as, site-directed mutagenesis, site-saturation mutagenesis and in vitro recombination). A variety of methods suitable for producing modified polynucleotides described herein encoding one or more subtilisin variants described herein are known in the art, including but not limited to, for example, site-saturation mutagenesis, scanning mutagenesis, insertion mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution, and various other recombination methods.

[0089] 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.

[0090] Some embodiments relate to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) as described herein, comprising one or more nucleic acids or polynucleotide sequences as described herein. Some such recombinant cells are transformed or transfected using at least one such vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells comprise bacterial cells, including but not limited to Bacillus cells, such as Bacillus subtilis or Bacillus licheniformis cells. Other embodiments relate to recombinant cells (e.g., recombinant host cells) comprising one or more subtilisins as described herein.

[0091] 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., operably linked to a promoter of one or more polynucleotide sequences described herein). The vector may include a transcription terminator and / or a selection gene (e.g., an antibiotic resistance gene) that enables continuous culture maintenance of plasmid-infected host cells by growth in a culture medium containing an antimicrobial agent.

[0092] Expression vectors may be derived from plasmids or viral DNA, or in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (see Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus [molecular biological methods for bacillus], John Wiley & Sons [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).

[0093] In order to express and produce target protein (for example, one or more subtilisin variants described herein) in cells, one or more expression vectors comprising one or more copies (and in some cases comprising multiple copies) of polynucleotides encoding one or more subtilisin variants described herein are transformed into cells under conditions suitable for variant expression. In some embodiments, polynucleotide sequences encoding one or more subtilisin variants described herein (and other sequences contained in the vector) are integrated into the genome of the host cell; however, in other embodiments, plasmid vectors comprising polynucleotide sequences encoding one or more subtilisin variants described herein are maintained as autonomous extrachromosomal elements in the cell. Some embodiments provide extrachromosomal nucleic acid elements and imported nucleotide sequences integrated into the host cell genome. Carriers described herein can be used to produce one or more subtilisin variants described herein. In some embodiments, polynucleotide constructs encoding one or more subtilisin variants described herein are present on integration vectors, which can integrate the polynucleotides encoding the variants into the host chromosome and optionally amplify in the host chromosome. Examples of integration sites are well known to those skilled in the art. In some embodiments, the transcription of the polynucleotide encoding one or more subtilisin variants described herein is achieved by the following promoter, which is the wild-type promoter of the parent subtilisin. In some other embodiments, the promoter is heterologous to one or more subtilisin variants described herein, but has function in the host cell. Exemplary promoters for bacterial host cells include but are not limited to amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII, rrnIp2 promoters; promoters of Bacillus stearothermophilus maltogenic amylase genes; Bacillus amyloliquefaciens (BAN) amylase genes; Bacillus subtilis alkaline protease genes; Bacillus clausii alkaline protease genes; Bacillus pumilus xylosidase genes; Bacillus thuringiensis cryIIIA; and Bacillus licheniformis alpha-amylase genes. Other promoters include but are not limited to A4 promoters, as well as bacteriophage λPR or PL promoters and Escherichia coli (E.coli) lac, trp or tac promoters.

[0094] One or more subtilisin variants as described herein can be produced in the host cell of any suitable microorganism (including bacteria and fungi).In certain embodiments, one or more subtilisin variants as described herein can be produced in gram-positive bacteria.In certain embodiments, host cell is bacillus species, streptomyces (Streptomyces) species, Escherichia (Escherichia) species, Aspergillus (Aspergillus) species, Trichoderma (Trichoderma) species, Pseudomonas (Pseudomonas) species, Corynebacterium (Corynebacterium) species, Saccharomyces (Saccharomyces) species or Pichia (Pichia) species.In certain embodiments, one or more subtilisin variants as described herein are produced by bacillus species host cell. Examples of Bacillus host cells that can be used for the production of one or more subtilisin variants described herein include, but are not limited to: Bacillus licheniformis, Bacillus gibsonii, Bacillus lentus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus stearothermophilus, alkaliphilic Bacillus, Bacillus coagulans, Bacillus circulans, Bacillus brevis, Bacillus thuringiensis, Bacillus clausii and Bacillus megaterium, and other organisms within the genus Bacillus. In certain embodiments, Bacillus subtilis host cells are used to produce variants described herein. USPN 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus host strains that can be used to produce one or more subtilisin variants described herein, but other suitable strains can be used.

[0095] Several bacterial strains that can be used to produce one or more subtilisin variants as described herein include non-recombinant (i.e. wild-type) Bacillus strains, and naturally occurring bacterial strains and / or recombinant strain variants. In certain embodiments, the host strain is a recombinant strain, wherein the polynucleotides encoding one or more subtilisin variants as described herein have been introduced into the host. In certain embodiments, the host strain is a subtilis host strain, particularly a recombinant subtilis host strain. Many strains of Bacillus subtilis are known, including, but not limited to, e.g., 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 to PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP211 strains (see, e.g., Hoch et al., Genetics 73:215–228 (1973); see also, U.S. Pat. No. 4,450,235; U.S. Pat. No. 4,302,544; and EP 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)).

[0096] In some embodiments, the Bacillus host cell is a Bacillus comprising a mutation or deletion in at least one of the following genes: degU, degS, degR, and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU(Hy)32 (see, e.g., Msadek et al., J. Bacteriol. 172:824-834 (1990); and Olmos et al., Mol. Gen. Genet. 253:562–567 (1997)). In some embodiments, the Bacillus host comprises a mutation or deletion in: scoC4 (see, e.g., Caldwell et al., J. Bacteriol. 183:7329-7340 (2001)); spoIIE (see, e.g., Arigoni et al., Mol. Microbiol. 31:1407-1415 (1999)); and / or oppA or other genes of the opp operon (see, e.g., Perego et al., Mol. Microbiol. 5:173-185 (1991)). Indeed, it is contemplated that any mutation in the opp operon that causes the same phenotype as a mutation in the oppA gene will be useful in some embodiments of the altered Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, there is a combination of mutations. In certain 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 has already included a mutation of one or more of the above-mentioned genes. In addition, Bacillus host cells comprising one or more mutations and / or one or more deletions of endogenous protease genes can be used. In certain embodiments, the Bacillus host cell comprises the deletion of aprE and nprE genes. In other embodiments, the Bacillus host cell comprises the deletion of 5 protease genes, and in other embodiments, the Bacillus host cell comprises the deletion of 9 protease genes (see, e.g., US2005 / 0202535).

[0097] Use any suitable method known in the art to transform host cells with one or more nucleic acid sequences encoding one or more subtilisin variants as herein described. Utilize plasmid DNA construct or vector that nucleic acid (for example DNA) is introduced into bacillus cell or Escherichia coli cell and the method that such plasmid DNA construct or vector is transformed into such cell is well known. In certain embodiments, plasmid is separated from Escherichia coli cell subsequently and is transformed into bacillus cell. However, use intervention microorganism such as Escherichia coli is not essential, and in certain embodiments, DNA construct or vector are directly introduced into bacillus host.

[0098] 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. [Furria Microbiology], 30:97 (1985); Chang et al., Mol. Gen. Genet. [Molecular and General Genetics] 168:11-115 (1979); Vorobjeva et al., FEMS Microbiol. Lett. [FEMS Microbiology Letters] 7:261-263 (1980); Smith et al., Appl. Env. Microbiol [Application and Environmental Microbiology] 51:634 (1986); Fisher et al., Arch. Microbiol. [Archives of Microbiology], 139:213-217 (1981); and McDonald, J. Gen. Microbiol [Journal of Genetic Microbiology] 130:203 (1984)). In practice, 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 methods such as plasmid marker rescue transformation, which involves uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (see, Contente et al., Plasmid [Plasmid] 2: 555-571 (1979); Haima et al., Mol. Gen. Genet. [Molecular and General Genetics] 223: 185-191 (1990); Weinrauch et al., J. Bacteriol. [Journal of Bacteriology], 154: 1077-1087 (1983); and Weinrauch et al., J. Bacteriol. [Journal of Bacteriology], 169: 1205-1211 (1987)). In this method, the input donor plasmid recombines with homologous regions of a resident "helper" plasmid in a process that simulates chromosomal transformation.

[0099] In addition to commonly used methods, in some embodiments, a DNA construct or vector comprising a nucleic acid encoding one or more subtilisin variants described herein is used to directly transform a host cell (i.e., before introduction into the host cell, the intermediate cell is not used to amplify or otherwise process the DNA construct or vector). Introducing a DNA construct or vector as described herein into a host cell includes those physical and chemical methods known in the art for introducing a nucleic acid sequence (e.g., a DNA sequence) into a host cell without inserting it into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In another embodiment, a DNA construct or vector is co-transformed with a plasmid without inserting a plasmid. In a further embodiment, a selection marker is deleted from a modified bacillus strain by methods known in the art (see, Stahl et al., J. Bacteriol. [Journal of Bacteriology] 158: 411-418 (1984); and Palmeros et al., Gene [Gene] 247: 255-264 (2000)).

[0100] In certain embodiments, the transformed cells are cultured in a conventional nutrient medium. Suitable specific culture conditions, such as temperature, pH, etc. are known to those skilled in the art and are described in detail in the scientific literature. Some embodiments provide a culture (e.g., a cell culture) comprising one or more subtilisin variants or nucleic acid sequences as described herein.

[0101] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more subtilisin variants described herein are cultured in a suitable nutrient medium under conditions that allow expression of the variant, and the resulting variant is thereafter recovered from the culture. In some embodiments, variants produced by the cells are recovered from the culture medium by conventional procedures, including, but not limited to, for example, separation of host cells from the culture medium by centrifugation or filtration, precipitation of protein components of the supernatant or filtrate with the aid of salts (e.g., ammonium sulfate), and chromatography purification (e.g., ion exchange, gel filtration, affinity, etc.).

[0102] In certain embodiments, one or more subtilisin variants produced by recombinant host cells are secreted into the culture medium. The nucleic acid sequence encoding the purification promotion domain can be used to promote the purification of the variant. The vector or DNA construct comprising the polynucleotide sequence encoding one or more subtilisin variants as described herein 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 domains include but are not limited to, for example, metal chelating peptides, such as histidine-tryptophan modules (see Porath, Protein Expr. Purif. [protein expression and purification] 3: 263-281

[1992] ) that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulins, and domains used in FLAGS extension / affinity purification systems. 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.

[0103] Variant proteins 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, such as to release enzymes or to improve cell separation, by heating or by changing pH or salt content or by using enzymes described in egg white lysozyme, T4 lysozyme or WO 2022047149. For production-scale recovery, it is possible to enrich or partially purify variant polypeptides by removing cells via polymer flocculation as described above in general. 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 treatment. The enzyme can then be processed into, for example, particles.

[0104] Various methods can be used to determine the production level of one or more mature subtilisin variants described herein in a host cell. Such methods include, but are not limited to, methods such as using polyclonal or monoclonal antibodies specific for proteases. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescence activated cell sorting (FACS). These and other assays are well known in the art (see, for example, Maddox et al., J. Exp. Med. [Journal of Experimental Medicine] 158: 1211 (1983)).

[0105] Some other embodiments provide methods for preparing or producing one or more mature subtilisin protease variants as described herein.Ripe subtilisin protease variants do not include signal peptide or propeptide sequences.Some methods are included in recombinant bacterial host cells (such as Bacillus cells (such as, subtilis cells)) to prepare or produce one or more subtilisin protease variants as described herein.Other embodiments provide methods for producing one or more subtilisin protease variants described herein, wherein the method is included in cultivating a recombinant host cell comprising a recombinant expression vector under conditions conducive to producing the variant, and the recombinant expression vector comprises a nucleotide sequence encoding one or more subtilisin protease variants described herein.Some such methods further include reclaiming the variant from the culture.

[0106] Other embodiments provide methods for producing one or more subtilisin variants as described herein, wherein the method comprises: (a) introducing a recombinant expression vector comprising a nucleic acid encoding the variant into a cell population (e.g., a bacterial cell, such as a subtilis cell); and (b) culturing cells in a culture medium under conditions conducive to producing a variant encoded by the expression vector. Some such methods further comprise: (c) separating the variant from a cell or from a culture medium.

[0107] 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.

[0108] Still further embodiments relate to methods of reducing the degradation potential of a subtilisin variant towards a coenzyme (eg, α-amylase or mannanase) in a detergent composition comprising modifying the subtilisin to include one or more substitutions or a combination of substitutions as provided herein.

[0109] Unless otherwise noted, all components or composition levels provided herein are given with reference to the activity level of the components or compositions, 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 described herein include cleaning compositions, such as detergent compositions. In the detergent compositions of examples, enzyme levels are expressed by pure enzymes by weight of the total composition and, unless otherwise specified, detergent ingredients are expressed by weight of the total composition.

[0110] In one embodiment, one or more subtilisin 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., hard surfaces of tables, desktops, walls, furniture items, floors, ceilings). In other embodiments, one or more subtilisin variants as described herein can be used in disinfection applications, such as, but not limited to, disinfection automatic dishwashing machines or washing machines. In one embodiment, a cleaning composition is a cleaning composition comprising one or more subtilisin variants as described herein, wherein the cleaning composition is a composition selected from the group consisting of: laundry detergents, fabric softening detergents, dishwashing detergents (e.g., automatic or hand dishwashing detergents), hard surface cleaning detergents and medical device cleaning compositions.

[0111] Another embodiment relates to a composition comprising one or more subtilisin variants as described herein. In certain embodiments, the composition is a cleaning composition. In other embodiments, the composition is a detergent composition. In other embodiments, the composition is selected from laundry detergent compositions, automatic dishwashing (ADW) compositions, hand-washing (manual) dishwashing detergent compositions, hard surface cleaning compositions, glasses cleaning compositions, medical device cleaning compositions, disinfectants (e.g., malodors or microorganisms) compositions, and personal care cleaning compositions. In still other embodiments, the composition is a laundry detergent composition, an ADW composition, or a hand-washing (manual) dishwashing detergent composition. Even further embodiments still relate to fabric cleaning compositions, while other embodiments relate to non-fabric cleaning compositions. In certain embodiments, the cleaning composition does not contain boron. In other embodiments, the cleaning composition does not contain phosphate. In still other embodiments, the composition comprises one or more subtilisin variants as described herein and one or more excipients, auxiliary materials, and / or other enzymes.

[0112] In one embodiment, a composition comprising a combination of a subtilisin variant and at least one additional enzyme is provided, wherein the subtilisin variant comprises one, two or more substitutions selected from the group consisting of: X96D, X103F, X108Q, X115L, X128K, X129Q or X181Q, wherein the positions are numbered according to SEQ ID NO: 9, wherein the variant is identical to SEQ ID NO: NO:9 has at least 75% identity with the amino acid sequence of amino acid sequence, and wherein the at least one additional enzyme is selected from the group consisting of acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, lipoxygenase, lysozyme, mannanase, metalloproteinase, The invention relates to a detergent composition comprising the following: a subtilisin protease variant, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, an additional protease, a pullulanase, a reductase, a rhamnogalacturonase, a β-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in the detergent composition lacking the subtilisin protease variant.

[0113] In some embodiments, such compositions comprise a subtilisin variant selected from the group consisting of SEQ ID NOs: 1-8.

[0114] In another embodiment, the present disclosure provides detergent compositions (e.g., ADW compositions) comprising a surfactant and at least one subtilisin variant as provided herein. Such compositions may further comprise one or more of an excipient, an auxiliary material, and / or an additional enzyme.

[0115] In still 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 a borate stabilizer is not added. In another embodiment, a boron-free composition is a composition containing less than 5.5% boron. In still further embodiments, a boron-free composition is a composition containing less than 4.5% boron. In still further embodiments, a boron-free composition is a composition containing less than 3.5% boron. In still further embodiments, a boron-free composition is a composition containing less than 2.5% boron. In even further embodiments, a boron-free composition is a composition containing less than 1.5% boron. In another embodiment, a boron-free composition is a composition containing less than 1.0% boron. In still further embodiments, a boron-free composition is a composition containing less than 0.5% boron. In other embodiments, the composition is a composition free of or substantially free of enzyme stabilizers or peptide inhibitors.

[0116] In another embodiment, one or more compositions described herein are in a form selected from gel, tablet, powder, granule, solid, liquid, unit dose and combination thereof. In another embodiment, one or more compositions described herein are in a form selected from low water compact formula, low water HDL or unit dose (UD) or high water formula or HDL. In some embodiments, cleaning compositions described herein are in unit dosage form. In other embodiments, the unit dosage form is selected from pills, tablets, capsules, capsules, sachets, pouches, multi-compartment pouches and pre-measured powders or liquids. In some embodiments, the unit dosage form is designed to provide controlled release of ingredients in multi-compartment pouches (or other unit dosage forms). Suitable unit doses and controlled release forms are described in, for example, EP 2100949, WO 02 / 102955, US 4,765,916, US 4,972,017 and WO 04 / 111178. In some embodiments, the unit dosage form is a tablet or powder contained in a water-soluble film or pouch.

[0117] Exemplary laundry detergent compositions include, but are not limited to, for example, liquid and powder laundry detergent compositions. Exemplary hard surface cleaning compositions include, but are not limited to, for example, compositions for cleaning hard surfaces of non-tableware items, non-desktop utensil items, tables, desktops, furniture items, walls, floors, and ceilings. Exemplary hard surface cleaning compositions are described in, for example, USPN 6,610,642, 6,376,450, and 6,376,450. Exemplary personal care compositions include, but are not limited to, compositions for cleaning dentures, teeth, hair, contact lenses, and skin. Exemplary components of such oral care compositions include, for example, those described in US 6,376,450.

[0118] In certain embodiments, one or more subtilisin variants described herein clean at low temperatures. In other embodiments, one or more compositions described herein clean at low temperatures. In other embodiments, one or more compositions described herein comprise an effective amount of one or more subtilisin variants described herein, which are useful or effective for cleaning surfaces that require protein stain removal.

[0119] In some embodiments, adjunct materials are incorporated, for example, to assist or enhance cleaning performance; to treat substrates to be cleaned; or to modify the aesthetics of the cleaning composition, such as in the case of perfumes, colorants, dyes, and the like. One embodiment relates to a composition comprising one or more adjunct materials described herein and one or more subtilisin variants. Another embodiment relates to a composition comprising one or more adjunct materials described herein and one or more subtilisin variants, wherein the adjunct material is selected from: bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilization systems), chelants, brighteners, soil release polymers, dye transfer agents, dispersants, foam inhibitors, dyes, perfumes, colorants, fillers, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides , fungicides, color embellishments, silver care agents, anti-tarnish agents, anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, pH control agents, surfactants, builders, chelating agents, dye transfer inhibitors, deposition aids, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymer dispersants, clay soil removers / anti-redeposition agents, structure elasticizers, fabric softeners, carriers, hydrotropes, processing aids, pigments, and combinations thereof. Exemplary auxiliary materials and use levels can be found in USPN 5,576,282, 6,306,812, 6,326,348, 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101. In embodiments where one or more cleaning auxiliary materials are incompatible with one or more subtilisin variants described herein, a method is used in which the auxiliary materials 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.).

[0120] Some embodiments relate to a cleaning additive product comprising one or more subtilisin variants as described herein. In certain embodiments, the additive is encapsulated in a formulation for adding to a cleaning process. In certain embodiments, the additive is encapsulated in a formulation for adding to a cleaning process, in which a peroxide source is used and an increased bleaching effect is desired.

[0121] 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, for example, 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 about 5% to about 90% of such fillers or carriers. Acidic fillers may be included in such compositions to reduce the pH of the resulting solution in the cleaning method or application.

[0122] In one embodiment, one or more cleaning compositions described herein include an effective amount of one or more subtilisin variants described herein, which are alone or in combination with one or more other enzymes. Typically, the cleaning compositions include at least about 0.0001wt% to about 20wt%, from about 0.0001wt% to about 10wt%, from about 0.0001wt% to about 1wt%, from about 0.001wt% to about 1wt%, from about 0.001wt% to about 1wt%, or from about 0.01wt% to about 0.2wt% 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 composition.

[0123] Typically the cleaning compositions described herein are formulated so that during use in aqueous cleaning operations, the wash water will have a pH from about 4.0 to about 11.5, or even from about 5.0 to about 11.5, or even from about 5.0 to about 8.0, or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from about 3.0 to about 9.0, or even from about 3 to about 5. Granular laundry products are typically formulated to have a pH from about 8 to about 11. In some embodiments, the cleaning compositions of the present invention may be formulated to have an alkaline pH under washing conditions, such as a pH from about 8.0 to about 12.0, or from about 8.5 to about 11.0, or from about 9.0 to about 11.0. In some embodiments, the cleaning compositions of the present invention may be formulated to have a neutral pH under washing conditions, such as a pH from about 5.0 to about 8.0, or from about 5.5 to about 8.0, or from about 6.0 to about 8.0, or from about 6.0 to about 7.5. In some embodiments, neutral pH conditions can be measured when the cleaning composition is dissolved in deionized water at 1:100 (wt:wt) at 20° C., using a conventional pH meter. Techniques for controlling pH at recommended usage levels include the use of buffers, bases, acids, etc., and are well known to those skilled in the art.

[0124] In certain 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 variants during cleaning. In certain embodiments, encapsulation enhances the performance of variants and / or other enzymes. In certain embodiments, encapsulation materials typically encapsulate at least a portion of subtilisin variants described herein. Typically, encapsulation materials are water-soluble and / or water-dispersible. In certain embodiments, encapsulation materials have a glass transition temperature (Tg) of 0°C or higher. Exemplary encapsulation materials include, but are not limited to: carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin and combinations thereof. When encapsulation material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides and combinations thereof. In certain embodiments, encapsulation 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 a plastic such as a thermoplastic, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof. Exemplary commercial microspheres include, but are not limited to (Stockviksverken, Sweden); and PM6545, PM 6550, PM 7220, PM 7228, and (PQ Corp., Valley Forge, PA).

[0125] In some embodiments, " cold water washing " of the present invention is suitable for washing at a temperature of from about 10 ℃ to about 40 ℃, from about 20 ℃ to about 30 ℃, or from about 15 ℃ to about 25 ℃, and at a temperature of from about 15 ℃ to about 35 ℃ or 10 ℃ to 40 ℃.

[0126] Different geographical locations have different water hardness. Hardness is the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) is a measure of the amount of Ca mixed in the mixture. Usually measured in particles per gallon (gpg) 2+ / Mg 2+ To describe water hardness. In the United States, most water is hard water, but there are different levels of hardness. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 ppm (ppm can be converted to particles / US gallon by dividing ppm by 17.1) of hardness minerals.

[0127] water Particles / Gallon Parts per million soft Less than 1.0 Less than 17 Slightly hard 1.0 to 3.5 17 to 60 Medium hard 3.5 to 7.0 60 to 120 hard 7.0 to 10.5 120 to 180 Very hard Greater than 10.5 More than 180

[0128] Other embodiments relate to one or more cleaning compositions comprising from about 0.00001% to about 10% by weight of the composition of one or more subtilisin variants described herein, and from about 99.999% to about 90.0% by weight of the composition of one or more auxiliary materials. In another embodiment, the cleaning composition comprises from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% by weight of the composition of one or more subtilisin variants, and from about 99.9999% to about 90.0%, from about 99.999% to about 98%, from about 99.995% to about 99.5% by weight of the composition of one or more auxiliary materials.

[0129] In other embodiments, the compositions described herein comprise one or more subtilisin variants described herein and one or more additional enzymes. The one or more additional enzymes are selected from acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, The invention relates to a combination of enzymes (i.e., a "mixture") comprising conventional enzymes (such as amylases, lipases, cutinases, mannanases, and / or cellulases) in combination with one or more subtilisin variants and / or one or more additional proteases as described herein.

[0130] In another embodiment, one or more compositions as described herein comprise one or more subtilisin variants as described herein and one or more additional proteases. In one embodiment, additional proteases are serine proteases. In another embodiment, additional proteases are not immunologically relevant (e.g., proteases do not cross-react in antibody-based tests known in the art to assess shared immune epitopes). In another embodiment, additional proteases are proteases with different net charges (e.g., +2 to -1 relative to the parent protease). In another embodiment, additional proteases are metalloproteases, fungal subtilisin or alkaline microbial proteases or trypsin-like proteases. Suitable additional proteases include those of animal, plant or microbial origin. In certain embodiments, additional proteases are microbial proteases. In other embodiments, additional proteases are chemically or genetically modified mutants. In another embodiment, additional proteases are alkaline microbial proteases or trypsin-like proteases. In other embodiments, as measured by antibodies available in the art in combination or other assays, additional proteases do not contain epitopes that cross-react with subtilisin variants. Exemplary alkaline proteases include subtilisins derived from, for example, Bacillus (e.g., BPN', Carlsberg, subtilisin 309, subtilisin 147, subtilisin PB92, subtilisin DY, subtilisin 168, Bacillus gibsonii DSM 14391 subtilisin, Bacillus gibsonii AGS78407 subtilisin, Bacillus pumilus subtilisin, Bacillus lentus DSM 5483, and Bacillus sp. TY-145 subtilisin) or fungal sources, such as, for example, 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, WO 09 / 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 / 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, WO 2016074925, WO2020178102, WO 2022106400, WO 2016203064, EP3380599, WO 2017215925, WO 201948495, WO 2020221578 and WO 2016203064, US7294499, WO 2016 / 097354 and PCT / US16 / 32514, and those described in WO 1999014341, WO 1999033960, WO 1999014342, WO 1999034003, WO2007044993, WO 2009058303, WO 2009058661, WO 2014071410, WO 2014194032, WO2014194034, WO 2014194054 and WO Metalloproteases described in 2014 / 194117. Exemplary additional proteases include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO 89 / 06270.Exemplary commercial proteases include, but are not limited to. MAXACAL TM , MAXAPEM TM , OXP、PURAMAX TM 、EXCELLASE TM 、PREFERENZ TM Proteases (e.g. P100, P110, P280, P300), EFFECTENZ TM Proteases (e.g. P1000, P1050, P2000), EXCELLENZ TM Proteases (e.g. P1000), and PURAFAST TM (DuPont / Danisco / Genencor); ULTRA, Variant, 16L, ULTRA, DURAZYM TM , LIQUANASE PROGRESS and (Novozymes); BLAP TM and BLAP TM Variant (Henkel); LAVERGY TM PRO 104L (BASF), KAP (alkaliphilic Bacillus subtilisin (Kao)) and (AB Enzymes).

[0131] 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, e.g., bacterial or fungal origin, such as, for example, H. lanuginosa lipase (see, e.g., EP 258 068 and EP 305 216), T. lanuginosa lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase (see, e.g., EP 238 023), Candida lipases such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, e.g., EP 238 023). 214761), Pseudomonas lipases such as P. alcaligenes and P. pseudoalcaligenes lipases (see, e.g., EP 218272), P. cepacia lipase (see, e.g., EP 331376), P. stutzeri lipase (see, e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipases (e.g., Bacillus subtilis lipase (Dartois et al., Biochem. Biophys. Acta [Biochemistry and Biophysics] 1131:253-260 (1993)), Bacillus stearothermophilus lipase (see, e.g., JP 64 / 744992), and Bacillus pumilus lipase (see, e.g., WO 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, for example, cutinases derived from Pseudomonas mendocina (see WO 88 / 09367) and / or Fusarium solanipisi (see WO 90 / 09446). Exemplary commercial lipases include, but are not limited to, M1 LIPASE. TM 、LUMAFAST TM 、LIPOMAX TM and PREFERENZ TM L100 (DuPont); and ULTRA (Novozymes); and LIPASE P TM (Amano Pharmaceutical Co. Ltd).

[0132] Still other embodiments relate to compositions comprising one or more subtilisin variants as described herein and one or more amylases. In one embodiment, the composition comprises by composition weight from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% amylase. Any amylase (e.g., alpha amylase and / or beta amylase) suitable for use in alkaline solutions may be used to be included in such compositions. Exemplary amylases may be chemically or genetically modified mutants. Exemplary amylases include, but are not limited to, those of bacterial or fungal origin, such as described in GB 1,296,839, WO 9100353, WO9402597, WO 94183314, WO 9510603, WO 9526397, WO 9535382, WO 9605295, WO 9623873, WO9623874, WO 9630481, WO 9710342, WO 9741213, WO 9743424, WO 9813481, WO 9826078, WO 9902702, WO 9909183, WO 9919467, WO 9923211, WO 9929876, WO 9942567, WO 9943793, WO9943794, WO 9946399, WO 0029560, WO 0060058, WO 0060059, WO 0060060, WO 0114532, WO0134784, WO 0164852, WO 0166712, WO 0188107, WO 0196537, WO 02092797, WO 0210355, WO0231124, WO 2004055178, WO 2004113551, WO 2005001064, WO 2005003311, WO2005018336, WO 2005019443, WO 2005066338, WO 2006002643, WO 2006012899, WO2006012902, WO 2006031554, WO 2006063594, WO 2006066594, WO 2006066596, WO2006136161, WO 2008000825, WO 2008088493, WO 2008092919, WO 2008101894, WO 2008 / 112459, WO 2009061380, WO 2009061381, WO 2009100102, WO 2009140504, WO 2009149419、WO2010 / 059413、WO 2010088447、WO 2010091221、WO 2010104675、WO 2010115021、WO10115028、WO 2010117511、WO 2011076123、WO 2011076897、WO 2011080352, WO2011080353, WO 2011080354, WO 2011082425, WO 2011082429, WO 2011087836, WO2011098531, WO 2013063460, WO 2013184577, WO 2014099523、WO 2014164777, WO2015077126, WO 2022 / 175435 and WO 2018184004. Exemplary commercial amylases include, but are not limited to STAINZYME STAINZYME AMPLIFY STAINZYME and BAN TM (Novozymes); EFFECTENZ TM S1000、POWERASE TM 、PREFERENZ TM S100, PREFERENZ TM S110, PREFERENZ TM S210、EXCELLENZ TM S2000, and P (DuPont). In some embodiments, the subtilisin variants provided herein can be combined with one or more amylases and 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.

[0133] 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, US4,435,307; EP 0495257; and U.S. Provisional Application No. 62 / 296,678. Exemplary commercial cellulases include, but are not limited to and PREMIUM (Novozymes); REVITALENZ TM 100. REVITALENZ TM 200 / 220, and 2000 (DuPont); and KAC-500 (B) TM (Kao Corporation) In some embodiments, the cellulase is incorporated as a portion or fragment of a mature wild-type or variant cellulase in which a portion of the N-terminus is deleted (see, e.g., US 5,874,276).

[0134] Even further embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more mannanases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% mannanase by weight of the composition. Exemplary mannanases may be chemically or genetically modified mutants. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in 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 (Novozymes) and EFFECTENZ TM M 1000, EFFECTENZTM M 2000, M 100, (AB Enzymes) and PURABRITE TM (DuPont).

[0135] Still other embodiments relate to compositions comprising one or more subtilisin variants as described herein and one or more nucleases (e.g., DNA enzymes or RNA enzymes). 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. Other nucleases that can be used in the compositions and methods provided herein in combination with the subtilisin variants provided herein include those described in: Nijland R, Hall MJ, Burgess JG (2010) Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase. PLoS ONE [Public Library of Science: Comprehensive] 5 (12) and Whitchurch, CB, Tolker-Nielsen, T., Ragas, PC, Mattick, JS (2002) Extracellular DNA required for bacterial biofilm formation [Extracellular DNA required for bacterial biofilm formation]. Science [Science] 295: 1487.

[0136] Still even other embodiment relates to the composition comprising one or more subtilisin variants as herein described and one or more peroxidases and / or oxidases.In one embodiment, composition comprises by composition weight from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2% or about 0.005% to about 0.5% peroxidase or oxidase.Peroxidase can be used in combination with hydrogen peroxide or its source (such as percarbonate, perborate or persulfate), and oxidase can be used in combination with oxygen.Peroxidase and oxidase combined with single or synergist are used for " solution bleaching " (i.e., preventing textile dye from transferring to another fabric from a dyed fabric when fabric is washed together in washing liquid) (see, for example, WO 94 / 12621 and WO95 / 01426).Exemplary peroxidase and / or oxidase can be chemical or genetically modified mutants. Exemplary peroxidases / oxidases include, but are not limited to, those of plant, bacterial, or fungal origin.

[0137] 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.

[0138] Another embodiment relates to a composition comprising one or more subtilisin variants described herein and an engineered polysaccharide biopolymer having cleaning / whitening improvement or textile surface modification benefits.

[0139] 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.

[0140] 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.

[0141] Some embodiments relate to laundry detergent compositions comprising one or more subtilisin variants described herein and one or more adjunct materials selected from the group consisting of surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaching agents, additional enzymes, foam suppressors, dispersants, lime soap dispersants, soil suspension agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry composition further comprises a softener.

[0142] Additional embodiments relate to manual dishwashing compositions comprising one or more subtilisin variants described herein and one or more adjunct materials selected from the group consisting of surfactants, organic polymeric compounds, suds boosters, Group II metal ions, solvents, hydrotropes, and additional enzymes.

[0143] 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.

[0144] In some embodiments, the cleaning composition comprises acidified particles or aminocarboxylic acid builders. Examples of aminocarboxylic acid builders include aminocarboxylic acids, salts thereof, and derivatives thereof. In some embodiments, the aminocarboxylic acid builders are aminopolycarboxylic acid builders, such as glycine-N,N-diacetic acid or aminocarboxylic acid builders having the general formula MOOC-CHR-N(CH 2 COOM) 2 (where R is C 1-12alkyl and M is an alkali metal). In some embodiments, the aminocarboxylic acid builder can be methylglycine diacetic acid (MGDA), GLDA (glutamic acid-N,N-diacetic acid), iminodisuccinic acid (IDS), carboxymethyl inulin and its salts and derivatives, aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodiacetic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)aspartic acid (SMGL), N-(2-sulfoethyl)aspartic acid (SMGL), N-(2-sulfoethyl)aspartic acid (SMGL), N-(2-sulfoethyl)aspartic acid (SMGL), N-(2-sulfoethyl)aspartic acid (SMGL), N-(2-sulfoethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SMAS), N-(2-sulfomethyl)glutam ...AS), N-(2-sulfoethyl)aspartic acid (SMAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SMAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)aspartic In some embodiments, the acidifying particles include 1,2-diamino-1,2-diamino-2,2-diamino-3,4-diamino-4,4-diamino-5,4-diamino-6,4-diamino-7,4-diamino-8,4-diamino-9,4-diamino-10,4-diamino-11,4-diamino-2,4-diamino-12,4-diamino-13,4-diamino-2,4-diamino-13,4-diamino-14,4-diamino-2,4-diamino-13,4-diamino-2 ...

[0145] In certain embodiments, the acidified particles can comprise any acid, including organic acids and mineral acids. The 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 acids include hydrochloric acid and sulfuric acid. In some cases, the acidified particles are high-activity particles that comprise high-level aminocarboxylic acid builders. It has also been found that sulfuric acid further contributes to the stability of the final particles.

[0146] Additional embodiments relate to cleaning compositions comprising one or more subtilisin variants and one or more surfactants and / or surfactant systems, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some embodiments, the surfactant is present at a level of from about 0.1% to about 60%, while in alternative embodiments, the level is from about 1% to about 50%, and in yet further embodiments, the level is from about 5% to about 40%, based on the weight of the cleaning composition.

[0147] 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, such as sodium tripolyphosphate, sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixed sodium and potassium tripolyphosphates. Exemplary builders are described in, for example, EP 2100949. In certain embodiments, builders include phosphate builders and non-phosphate builders. In certain embodiments, the builders are phosphate builders. In certain embodiments, the builders are non-phosphate builders. In certain embodiments, the builders include a mixture of phosphate and non-phosphate builders. Exemplary phosphate builders include, but are not limited to, monophosphate, diphosphate, tripolyphosphate or oligophosphate, including alkali metal salts of these compounds, including sodium salts. In certain embodiments, the builders may be sodium tripolyphosphate (STPP). In addition, the composition may include carbonate and / or citrate. Other suitable non-phosphate builders include homopolymers and copolymers of polycarboxylic acids and partially or completely neutralized salts thereof, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts. In certain embodiments, the salts of the above-mentioned compounds include ammonium salts and / or alkali metal salts, i.e., lithium salts, sodium salts and potassium salts, including sodium salts. Suitable polycarboxylic acids include acyclic, cycloaliphatic, heterocyclic and aromatic carboxylic acids, wherein in some embodiments they may contain at least two carboxyl groups, which are in each case separated from one another, in some cases by no more than two carbon atoms.

[0148] In some embodiments, one or more compositions described herein include one or more chelating agents. In one embodiment, the composition includes from about 0.1% to about 15% or about 3% to about 10% of a chelating agent by weight of the composition. Exemplary chelating agents include, but are not limited to, for example, copper, iron, manganese, and mixtures thereof.

[0149] In some embodiments, one or more compositions described herein comprise one or more deposition aids. Exemplary deposition aids include, but are not limited to, for example, polyethylene glycol; polypropylene glycol; polycarboxylates; soil release polymers, such as, for example, polyethylene terephthalate; clays, such as, for example, kaolinite, montmorillonite, attapulgite, illite, bentonite, and halloysite; and mixtures thereof.

[0150] In other embodiments, one or more compositions described herein include one or more anti-redeposition agents or nonionic surfactants (which can prevent the redeposition of soil) (see, e.g., EP 2100949). For example, in ADW compositions, nonionic surfactants can be used for surface modification purposes (especially for thin sheets) to avoid filming and staining and to improve gloss. These nonionic surfactants can also be used to prevent the redeposition of soil. In some embodiments, the nonionic surfactant can be an ethoxylated nonionic surfactant, an epoxy-terminated poly(alkoxylated) alcohol, and an amine oxide surfactant.

[0151] In some embodiments, one or more compositions described herein include one or more dye transfer inhibitors. Exemplary polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones, polyvinylimidazoles, and mixtures thereof. In one embodiment, the composition includes from about 0.0001% to about 10%, about 0.01% to about 5%, or about 0.1% to about 3% of a dye transfer inhibitor by weight of the composition.

[0152] 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 phyllosilicate. 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.

[0153] 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.

[0154] In some other embodiments, one or more compositions described herein include one or more enzyme stabilizers. In certain embodiments, enzyme stabilizers are water-soluble sources of calcium and / or magnesium ions. In certain embodiments, enzyme stabilizers include oligosaccharides, polysaccharides and inorganic divalent metal salts (including alkaline earth metal salts, such as calcium salts). In certain embodiments, the enzyme used herein is stabilized by being present in a water-soluble source of zinc (II), calcium (II) and / or magnesium (II) ions in the finished product composition providing the following ions for the enzyme, and other metal ions (for example, barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II) and vanadium (IV)). Chloride and sulfate may also be used in some embodiments. Exemplary oligosaccharides and polysaccharides (for example, dextrin) are described in, for example, WO 07 / 145964. In some embodiments, reversible protease inhibitors may also be used, for example, in boron-containing compounds (e.g., borates, 4-formylphenylboronic acid, and phenylboronic acid derivatives (e.g., those described in WO 96 / 41859)) and / or peptide aldehydes (e.g., as further described in WO2009 / 118375 and WO2013004636).

[0155] As previously described (WO 199813458, WO 2011036153, US20140228274), peptide aldehydes can be used as protease stabilizers in detergent formulations. Examples of peptide aldehyde stabilizers are peptide aldehydes, ketones or halomethyl ketones, and may be "N-terminated", for example with urea, carbamate or urea moieties, or "double N-terminated", for example with carbonyl, urea, oxamide, thiourea, dithiooxamide or thiooxamide moieties (EP 2358857 B1). The molar ratio of these inhibitors to proteases 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 7,968,508 B2). The molar ratio of these stabilizers to proteases is preferably in the range from 1:1 to 1000:1, in particular 1:1 to 500:1, particularly preferably from 1:1 to 100:1, most particularly preferably from 1:1 to 20:1.

[0156] In some embodiments, one or more compositions described herein comprise one or more bleaching agents, bleach activators, and / or bleach catalysts. In some embodiments, one or more compositions described herein comprise one or more inorganic and / or organic bleaching compounds. Exemplary inorganic bleaching agents include, but are not limited to, perhydrate salts, such as perborates, percarbonates, perphosphates, persulfates, and persilicates. In some embodiments, the inorganic perhydrate salts are alkali metal salts. In some embodiments, inorganic perhydrate salts that are crystalline solids but without additional protection are included, but in some other embodiments, the salts are coated. Bleach activators are typically organic peracid precursors that enhance bleaching during cleaning at temperatures of 60° C. and below. Exemplary bleach activators include compounds that give, under perhydrolysis conditions, aliphatic peroxycarboxylic acids having from about 1 to about 10 carbon atoms or from about 2 to about 4 carbon atoms, and / or optionally substituted peroxybenzoic acid. 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 EP2100949.

[0157] In some embodiments, one or more compositions described herein include one or more catalytic metal complexes. In some embodiments, metal-containing bleaching catalysts can be used. In some embodiments, metal bleaching catalysts include a catalytic system, which includes: transition metal cations (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations) with limited bleaching catalytic activity, auxiliary metal cations (e.g., zinc or aluminum cations) with little or no bleaching catalytic activity, and chelates with limited stability constants for catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetrakis (methylenephosphonic acid) and its water-soluble salts (see, e.g., US 4,430,243). In some embodiments, one or more compositions described herein are catalyzed by means of manganese compounds. Such compounds and usage levels are described in, e.g., US 5,576,282. In other embodiments, cobalt bleaching catalysts can be used for and are included in one or more compositions described herein. Various cobalt bleaching catalysts are described in, e.g., USPN 5,597,936 and 5,595,967.

[0158] In some further embodiments, one or more compositions described herein comprise transition metal complexes of large polycyclic rigid ligands (MRLs). As a practical matter and not by way of limitation, in some embodiments, the compositions and cleaning methods described herein are adjusted to provide at least one part per hundred million, about 0.005 ppm to about 25 ppm, about 0.05 ppm to about 10 ppm, or about 0.1 ppm to about 5 ppm of active MRL in the washing liquid. Exemplary MRLs include, but are not limited to, special ultra-rigid ligands cross-linked and bridged, such as, for example, 5,12-diethyl-1,5,8,12-tetraazabicyclo (6.6.2) hexadecane. Exemplary metal MRLs are described, for example, in WO 2000 / 32601 and US 6,225,464.

[0159] In another embodiment, one or more compositions described herein include one or more metal care agents. In some embodiments, the composition includes from about 0.1% to about 5% of a metal care agent by weight of the composition. Exemplary metal care agents include, for example, aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). 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.

[0160] 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 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; and optionally a nonionic surfactant selected from the group consisting of a linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohol, such as C 8 -C 18 Alkyl ethoxylated alcohol and / or C 6 -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.

[0161] In another embodiment, the cleaning composition is a liquid or gel detergent (which is not unit dosed), which can be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70% water by weight, up to about 65% water by weight, up to about 55% water by weight, up to about 45% water by weight, or up to about 35% water by weight. Other types of liquids (including but not limited to alkanols, amines, glycols, ethers, and polyols) can be included in the aqueous liquid or gel. The aqueous liquid or gel detergent can contain from 0 to 30% of an organic solvent. The liquid or gel detergent can be non-aqueous.

[0162] 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 containing monomers selected from the group consisting of: unsaturated C 1 -C 6 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: C 4 -C 25 Alkyl groups, polypropylene, polybutene, saturated C 2 -C 6 Vinyl esters of monocarboxylic acids, acrylic acid or methacrylic acid 1 -C 6 Alkyl esters and mixtures thereof.

[0163] The composition may contain additional polymers such as soil release polymers including, for example, anionically terminated polyesters such as SRP1; polymers comprising at least one monomer unit in a random or block configuration selected from the group consisting of saccharides, dicarboxylic acids, polyols, and combinations thereof; ethylene terephthalate-based polymers and copolymers thereof in a random or block configuration such as Repel-o-tex SF, SF-2, and SRP6; Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325; Marloquest SL; anti-redeposition polymers (0.1 wt % to 10 wt %, including, for example, carboxylate polymers, such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof; vinyl pyrrolidone homopolymer; and / or polyethylene glycol having a molecular weight in the range of 500 to 100,000 Da); cellulosic polymers (including, for example, alkyl cellulose; alkyl alkoxyalkyl cellulose; carboxyalkyl cellulose; alkyl carboxyalkyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose; and mixtures thereof); and polymeric carboxylates (such as, for example, maleate / acrylate random copolymers or polyacrylate homopolymers).

[0164] 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 configurations (including, for example, polysaccharides, cellulosic polymers, polydiallyldimethylammonium halides (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.

[0165] The composition may further comprise dye transfer inhibiting agents, examples of which include manganese phthalocyanine, peroxidase, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazolidones and polyvinylimidazoles and / or mixtures thereof; chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA); diethylenetriaminepentamethylenephosphonic acid (DTPMP); hydroxyethanediphosphonic acid (HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methylglycine diacetic acid (MGDA); diethylenetriaminepentaacetic acid (DTPA); propylenediaminetetraacetic acid (PDT A); 2-hydroxypyridine-N-oxide (HPNO); or methylglycine diacetic acid (MGDA); glutamic acid N,N-diacetic acid (N,N-dicarboxymethylglutamate tetrasodium salt (GLDA); nitrilotriacetic acid (NTA); 4,5-dihydroxyisobenzene disulfonic acid; citric acid and any salt thereof; N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and its derivatives.

[0166] The composition may further comprise silicone-based or fatty acid-based foam inhibitors; enzyme stabilizers; hueing dyes, calcium and magnesium cations, visual signaling ingredients, defoamers (0.001 wt% to about 4.0 wt%) and / or structurants / thickeners (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.

[0167] 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 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); a nonionic detersive surfactant (selected from linear or branched or random chain, substituted or unsubstituted C 8 -C 18 Alkyl ethoxylate and / or C 6 -C 12alkylphenol alkoxylates); cationic detersive surfactants (selected from alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds and mixtures thereof); zwitterionic and / or amphoteric 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 sub-nitrogen in the range of less than 15 wt %. triacetic acid or its salt; silicate (sodium silicate or potassium silicate or sodium metasilicate or layered silicate (SKS-6) in the range of 0 wt % to less than 10 wt %); carbonate (sodium carbonate and / or sodium bicarbonate in the range of 0 wt % to less than 10 wt %); and bleach (photobleach, 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 salt, 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 (such as zinc or aluminum) and chelates (such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and water-soluble salts thereof).

[0168] 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.

[0169] In some embodiments, the cleaning compositions comprising one or more subtilisin variants described herein are detergent compositions selected from the group consisting of laundry detergents, soap bars, fabric softening detergents, dishwashing detergents, medical device detergents, and hard surface cleaning detergents.

[0170] 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.

[0171] In some embodiments, one or more subtilisin variants described herein can be part of or added to a liquid laundry detergent composition, such as, but not limited to, liquid laundry compositions described in US11046919B2, WO2021 / 223552, WO 2022 / 167251, WO 2022 / 074037, WO 2021 / 123184, WO 2021 / 037895, WO2022 / 10372, WO 2020 / 264077, WO 2022 / 106404 and / or WO 2017 / 54983; concentrated liquid laundry compositions (US10683474B2); water-soluble unit dose preparations comprising fatty alkyl ester alkoxylated nonionic surfactants and alkoxylated alcohol nonionic surfactants (US20220162523A1); liquid laundry detergent compositions comprising improved alkylbenzene sulfonate surfactants (WO 2021 / 108307); a liquid laundry detergent composition comprising benzyl benzoate (WO 2020 / 223959); and / or a detergent composition containing a branched surfactant (WO2021 / 247801), a water-soluble unit dose preparation comprising an amphiphilic graft polymer and a polyester terephthalate (WO2019 / 032257).

[0172] In some embodiments, the cleaning compositions comprising one or more subtilisin variants described herein are liquid laundry detergent compositions containing alkyl ether carboxylic acids, betaines, anionic surfactants, nonionic surfactants for providing softening benefits (WO 2013 / 087286).

[0173] In some embodiments, the cleaning composition comprising one or more subtilisin 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).

[0174] In some embodiments, the cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition as described in US20210317387A1, WO 2021 / 219296, WO 2021 / 127662, WO 2021 / 041685, US11208619, US20220186144.

[0175] In some embodiments, the cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition comprising a dispersoid variant, such as, but not limited to, the liquid laundry detergent compositions described in US20210317387A1.

[0176] In some embodiments, the cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition that is a high alkaline textile detergent, such as, but not limited to, the liquid laundry detergent composition described in WO 2021 / 219296.

[0177] In some embodiments, the cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition that is a low density unit dose detergent with encapsulated fragrance, such as but not limited to the detergent composition described in WO 2021 / 127662.

[0178] In some embodiments, the cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition containing polyethylene glycol and an organic acid, such as, but not limited to, the detergent composition described in WO 2021 / 041685.

[0179] In some embodiments, the cleaning composition comprising one or more subtilisin variants described herein is a detergent composition containing polyethylene glycol and an organic acid, such as, but not limited to, the detergent composition described in WO 2021 / 041685.

[0180] In some embodiments, the cleaning compositions comprising one or more subtilisin variants described herein are detergent compositions that are active on protein stains, such as, but not limited to, the detergent compositions described in US11208619.

[0181] In some embodiments, the cleaning compositions comprising one or more subtilisin variants described herein are detergent compositions containing soil release polymers, such as, but not limited to, the detergent compositions described in US20220186144.

[0182] Examples of laundry detergent compositions include those provided in the following examples or in the following tables:

[0183]

[0184]

[0185] 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; builders in the range of 5%-60% by weight, including phosphate builders (monophosphate, diphosphate, tripolyphosphate, or oligophosphate), sodium tripolyphosphate-STPP or phosphate-free builders (amino acid-based compounds such as MGDA (methyl-glycine-diacetic acid) and its salts and derivatives, GLDA (glutamic acid-N,N-diacetic acid) and its salts and derivatives, IDS (iminodisuccinic acid) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives and mixtures thereof, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and B-alaninediacetic acid (B-ADA) and its salts), homopolymers and copolymers of polycarboxylic acids and their partially or fully neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts (which are in the range of 0.5%-50% by weight); sulfonated / carboxylated polymers (providing dimensional stability to the product), which are in the range of about 0.1% to about 50% by weight; 0.1% to about 10% of drying aids (selected from polyesters, in particular anionic polyesters, optionally together with additional monomers having 3 to 6 functional groups (in particular acid, alcohol or ester functions) which facilitate polycondensation, polycarbonate-, polyurethane- and / or polyurea-polyorganosiloxane compounds or their reactive cyclic carbonate and urea type precursor compounds); silicates (sodium or potassium silicates, for example sodium disilicate, sodium metasilicate and crystalline phyllosilicates) in the range of from about 1% to about 20% by weight; inorganic bleaches (for example, 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 activators - organic peracid precursors, which range from about 0.1% to about 10% by weight; bleach catalysts (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn and Fe bipyridylamine and related complexes, and pentaamine cobalt (III) acetate and related complexes); metal care agents (selected from benzotriazoles, metal salts and complexes, and silicates) in the range of about 0.1%-5% by weight;0 mg active enzyme / gram ADW detergent composition range of enzymes (acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, phospholipases, phytases, polyesterases, polygalacturonase, additional proteases, pullulanases, reductases, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and mixtures thereof); and an enzyme stabilizer component selected from the group consisting of oligosaccharides, polysaccharides, and inorganic divalent metal salts.

[0186] Exemplary ADW compositions are provided in the table below.

[0187]

[0188]

[0189] More embodiments relate to compositions and methods for treating fabrics (e.g., desizing textiles) using one or more subtilisin variants as described herein. Fabric treatment methods are well known in the art (see, e.g., US 6,077,316). For example, the feel and appearance of fabrics can be improved by methods including contacting fabrics with variants as described herein in a solution. Fabrics can be treated with a solution under pressure.

[0190] One or more subtilisin variants described herein can be applied during or after the weaving of textiles, during the desizing stage or in one or more additional fabric processing steps. During the weaving of textiles, the lines are exposed to considerable mechanical strain. Before weaving on a mechanical loom, the warp yarns are usually coated with sizing starch or starch derivatives to increase their tensile strength and prevent disconnection. One or more subtilisin variants described herein can be applied during or after weaving to remove sizing starch or starch derivatives. After weaving, variants can be used to remove the sizing coating before further processing the fabric to ensure uniform and washable results. One or more subtilisin variants described herein can be used alone or together with other desizing chemicals and / or desizing enzymes to desize fabrics (including cotton-containing fabrics) as detergent additives (e.g., in aqueous compositions). Amylases can also be used in combination with subtilisin variants in the composition and method of producing a stone-ground appearance on indigo-dyed denim fabrics and clothing. For clothing production, fabrics can be cut and sewn into clothes or clothing, which are subsequently finished (finish). In particular, for the production of denim, different enzymatic finishing methods have been developed. The finishing process of denim garments usually begins with an enzymatic desizing step, wherein the garments are subjected to the action of proteolytic enzymes to provide softness to the fabric and to make the cotton more susceptible to subsequent enzymatic finishing steps. One or more subtilisin variants described herein can be used in the following methods: finishing denim garments (e.g., "biosanding process"), enzymatic desizing and providing softness and / or finishing processes to fabrics.

[0191] The present disclosure also provides methods for cleaning article surfaces, which methods include contacting articles with at least one subtilisin variant provided herein (or a composition comprising such subtilisin variant). In certain embodiments, articles may have protein stains on, for example, their surfaces. In certain embodiments, protein stains may include eggs or stains based on eggs, such as crème brûlée, baked cheese, BMI or other materials containing protein.

[0192] Non-limiting examples of the compositions and methods disclosed herein are as follows:

[0193] 1. A composition comprising a combination of a subtilisin protease variant and at least one additional enzyme, wherein the subtilisin protease variant comprises one, two or more amino acid substitutions selected from the group consisting of: X96D, X103F, X108Q, X115L, X128K, X129Q and X181Q, wherein the amino acid positions are numbered according to SEQ ID NO: 9, wherein the variant is identical to SEQ ID NO: 1 NO:9 has at least 75% identity with the amino acid sequence of NO:9, and wherein the at least one additional enzyme is selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, lipoxygenase, lysozyme, mannanase, metalloproteinase, The invention relates to a detergent composition comprising the following: a protease, a nuclease (e.g., a DNase and / or an RNase), an oxidase, an oxidoreductase, a pectate lyase, a pectin acetylesterase, a pectinase, a pentosanase, a perhydrolase, a peroxidase, a phenoloxidase, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, a protease, a pullulanase, a reductase, a rhamnogalacturonase, a β-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in the detergent composition lacking the subtilisin protease variant.

[0194] 1b. A composition comprising a combination of a subtilisin variant and at least one additional enzyme, wherein the subtilisin variant comprises one, two or more amino acid substitutions compared to a parent subtilisin, wherein the substitutions are selected from the group consisting of: X96D, X103F, X108Q, X115L, X128K, X129Q and X181Q, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of the parent enzyme of SEQ ID NO: 9, wherein the variant is numbered with respect to SEQ ID NO: NO:9 has at least 75% identity with the amino acid sequence of NO:9, and wherein the at least one additional enzyme is selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, lipoxygenase, lysozyme, mannanase, metalloproteinase, The invention relates to a detergent composition comprising the following: a protease, a nuclease (e.g., a DNase and / or an RNase), an oxidase, an oxidoreductase, a pectate lyase, a pectin acetylesterase, a pectinase, a pentosanase, a perhydrolase, a peroxidase, a phenoloxidase, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, a protease, a pullulanase, a reductase, a rhamnogalacturonase, a β-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in the detergent composition lacking the subtilisin protease variant.

[0195] 2. The composition of embodiment 1 or 1b, wherein the subtilisin variant comprises one, two or more amino acid substitutions selected from the group consisting of N096D, Y103F, S108Q, T115L, A128K, S129Q and S181Q.

[0196] 3. A composition as described in any of the preceding claims, wherein the subtilisin variant further comprises one or more additional amino acid substitutions selected from the group consisting of: X9T, X17H, X77N, X78I, X103I, X127T, X165Q, X184Q, X202V, X203E, X203N, X217S and X258P, wherein the positions are numbered according to SEQ ID NO:9.

[0197] 4. The composition of embodiment 3, wherein the one or more additional amino acid substitutions of the subtilisin are selected from the group consisting of P009T, Q017H, T077N, T078I, Y103I, G127T, G165Q, N184Q, A202V, G203E, G203N, N217S, and S258P.

[0198] 5. The composition of embodiment 1, wherein the subtilisin variant is selected from the group consisting of SEQ ID NOs: 1-8.

[0199] 6. A composition as described in any of the preceding claims, wherein the subtilisin variant comprises an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less than 100% amino acid sequence identity to SEQ ID NO:9.

[0200] 7. The composition of any one of the preceding claims, wherein the at least one additional enzyme is selected from the group consisting of alpha-amylase, mannanase, cellulase, nuclease, polyesterase, protease, xanthan lyase, and any combination thereof.

[0201] 8. The composition of any one of the preceding claims, wherein the at least one additional enzyme is an alpha-amylase.

[0202] 9. The composition of any one of the preceding claims, wherein the at least one additional enzyme is a mannanase.

[0203] 10. The composition of any of the preceding claims, wherein the composition is a detergent composition selected from the group consisting of a laundry detergent, a fabric softener detergent, a dishwashing detergent (e.g., an automatic or hand dishwashing detergent), a medical device detergent, and a hard surface cleaning detergent.

[0204] 11. A method of cleaning, comprising contacting a surface or article in need of cleaning with an effective amount of a composition as described in Examples 1-10; and optionally further comprising the step of: rinsing the surface or article after contacting the surface or article with the composition as described in Examples 1-10.

[0205] 12. The method of embodiment 11, wherein the article is tableware, fabric, or a medical device.

[0206] Example 1

[0207] Stability of coenzymes in laundry detergents in the presence of proteases

[0208] The in-detergent enzymatic activity of the coenzymes (amylase and mannanase) was evaluated in the presence of various subtilisin proteases. P300 (International Flavors & Fragrances (IFF)) and Progress The effect of 100L (Novozymes) was compared with the effect of the experimental protease samples: variants 1, 2, 3, 4, 5, 6, 7 and 8 (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and 8, respectively). The liquid laundry detergent used in the stability assay was Test Detergent A. The composition of Test Detergent A is shown in Table 1.

[0209]

[0210] The experimental proteases SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 8 were prepared by concentrating the culture supernatant of Bacillus licheniformis strains by ultrafiltration and diafiltration with water.

[0211] The stability of coenzymes (e.g., amylase and mannanase variants) in the presence of one or more proteases was measured by adding the enzymes to the detergent at the following inclusion levels: 0.06 wt% protease, 0.007 wt% amylase, and 0.003 wt% mannanase based on active protein. The detergent / enzyme samples were incubated in sealed containers at 37°C for the specified storage time. At each time point, the samples were removed from the incubator, mixed (by rotation) at room temperature, and a small amount was removed for enzyme activity analysis.

[0212] Amylase activity

[0213] The release of reducing sugars by amylase on amylopectin substrate is measured Amylase activity of 12L amylase (Novozymes). For pullulan determination, the reagent solution used was: 50 mM MOPS pH 7.15, 50 mM NaCl, 2.5 mM CaCl in water 2 (MOPS buffer) and 0.125% pullulan (pullulan stock solution) (Sigma: A-8515). The detergent / enzyme samples were diluted in MOPS buffer and added to a microtiter plate (MTP) containing the pullulan stock solution. The plate was incubated at 40°C with agitation for 30 minutes. The reducing sugars generated during the reaction were determined using the BCA assay (Pierce: 23225) and measured at 562nm using a SpectraMax plate reader.

[0214] By using Ceralpha HR substrate (Megazyme: R-AMHR4) Amylase activity of Prime 100L (Novozymes) amylase. The detergent / enzyme samples were diluted in MOPS buffer and added to MTP containing Ceralpha HR substrate. The plate was incubated at room temperature for 2 minutes and the reaction was quenched by adding 200 mM borate (pH 10.2). Amylase activity was measured at 405 nm using a SpectraMax plate reader.

[0215] Mannanase activity

[0216] The release of reducing sugars was tested by measuring the release of reducing sugars by mannanase on locust bean gum (LBG) substrate. M100 (International Flavors & Fragrances, Inc., USA) mannanase activity. For the LBG assay, the reagent solution used was: 100mM HEPES pH 8.2, 0.005% Tween-80 (HEPES buffer) and 0.5% LBG stock solution (Sigma: G0753) in 50mM Tris HCl pH 7.15. The detergent / enzyme samples were diluted in HEPES buffer and added to the MTP containing the LBG stock solution. The plate was incubated at 40°C with stirring for 20 minutes. The reducing sugars generated during the reaction were determined using the BCA assay (Pierce: 23225) and measured at 562nm using a SpectraMax plate reader.

[0217] Residual enzyme activity

[0218] The percent residual activity of amylase and mannanase in Test Detergent A was determined by comparing the activity at the designated time points (weeks 1 and 4) to the activity at time = 0 or no incubation.

[0219] The residual activities of M100 mannanase and the indicated proteases were as shown in

[0220] As shown in Table 2.

[0221]

[0222] The residual activity of Stainzyme 12L amylase added to detergent in combination with the indicated proteases is shown in Table 3.

[0223]

[0224]

[0225] The residual activity of Amplify Prime 100L amylase added to detergent in combination with the indicated proteases is shown in Table 4.

[0226]

[0227] The results shown in Tables 2, 3 and 4 illustrate the benefits of the experimental proteases: Variants 1-8 (SEQ ID NOs: 1-8) increased the residual activity of commercially relevant amylase and mannanase coenzymes in liquid detergents over a period of at least 4 weeks compared to the commercial proteases PREFERENZ P300 (International Flavors & Fragrances Inc.) and Progress UNO 100L (Novozymes).

[0228] Time course study of mannanase stability in different protease combinations in HDL detergent using test detergent A. Commercial mannanase was examined Detergent stability of M100 and PspMan138 variant mannanase TL1219 (SEQ ID NO: 10, U.S. Provisional Application 63 / 403332, filed on September 2, 2022). The following inclusion levels based on active protein were used in the detergent: 0.06 wt% protease, 0.007 wt% amylase, and 0.003 wt% mannanase. The samples were incubated at 37°C and after the specified storage time, the percentage of residual mannanase activity was determined as described above.

[0229] Table 5 shows the activity of mannanase in the presence of various proteases. Residual activity percentage (%) of M100 and PspMan138 variant mannanase TL1219 (TL1219).

[0230]

[0231] exist In the presence of P 300 protease, Compared with M 100 mannanase, mannanase TL1219 had a higher residual activity %. This indicates that TL1219 mannanase is more stable in the presence of commercially relevant proteases. Compared with the presence of P 300, mannanase TL1219 also showed higher residual activity % in the presence of protease variant 2 (SEQ ID NO: 2). This result shows that protease variant 2 is more active than commercial Benefits compared to P 300.

Claims

1. A composition comprising a combination of a subtilisin protease variant and at least one additional enzyme, wherein the subtilisin protease variant comprises one, two or more amino acid substitutions selected from the group consisting of: X96D, X103F, X108Q, X115L, X128K, X129Q and X181Q, wherein the amino acid positions are numbered according to SEQ ID NO: 9, wherein the variant is identical to SEQ ID NO: 1 NO:9 has at least 75% identity with the amino acid sequence of NO:9, and wherein the at least one additional enzyme is selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, 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, lipoxygenase, lysozyme, mannanase, metalloproteinase, The invention relates to a detergent composition comprising the following: a protease, a nuclease (e.g., a DNase and / or an RNase), an oxidase, an oxidoreductase, a pectate lyase, a pectin acetylesterase, a pectinase, a pentosanase, a perhydrolase, a peroxidase, a phenoloxidase, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, a protease, a pullulanase, a reductase, a rhamnogalacturonase, a β-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has an increased residual activity compared to a reference enzyme in the detergent composition lacking the subtilisin protease variant.

2. The composition of claim 1, wherein the subtilisin variant comprises one, two or more amino acid substitutions selected from the group consisting of N096D, Y103F, S108Q, T115L, A128K, S129Q and S181Q.

3. The composition of claim 1, wherein the subtilisin variant further comprises one or more additional amino acid substitutions selected from the group consisting of: X9T, X17H, X77N, X78I, X103I, X127T, X165Q, X184Q, X202V, X203E, X203N, X217S, and X258P, wherein the positions are numbered according to SEQ ID NO:

9.

4. The composition of claim 3, wherein the one or more additional amino acid substitutions of the subtilisin are selected from the group consisting of P009T, Q017H, T077N, T078I, Y103I, G127T, G165Q, N184Q, A202V, G203E, G203N, N217S, and S258P.

5. The composition of claim 1, wherein the subtilisin variant is selected from the group consisting of SEQ ID NOs: 1-8.

6. The composition of claim 1, wherein the subtilisin variant comprises an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less than 100% amino acid sequence identity to SEQ ID NO:

9.

7. The composition of claim 1, wherein the at least one additional enzyme is selected from the group consisting of alpha-amylase, mannanase, cellulase, nuclease, polyesterase, protease, xanthan lyase, and any combination thereof.

8. The composition of claim 1, wherein the at least one additional enzyme is an alpha-amylase.

9. The composition of claim 1, wherein the at least one additional enzyme is a mannanase.

10. The composition of claim 1, wherein the composition is a detergent composition selected from the group consisting of: a laundry detergent, a fabric softener detergent, a dishwashing detergent (e.g., an automatic or hand dishwashing detergent), a medical device detergent, and a hard surface cleaning detergent.

11. A cleaning method, comprising contacting a surface or item in need of cleaning with an effective amount of the composition of claim 1; and the method optionally further comprising the steps of: After contacting the surface or article with the composition of claim 1, rinsing the surface or article.

12. The method of claim 11, wherein the article is tableware, fabric, or a medical device.

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