Detergent compositions and related methods

Abstract

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

Description

[Technical Field]

[0001] Disclosed herein are one or more subtilisin variants, including one or more subtilisin variants having improved stability and / or stain removal properties compared to one or more reference subtilisins, nucleic acids encoding same, and compositions and methods related to their production and use.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 403,332, filed September 2, 2022, and U.S. Provisional Patent Application No. 63 / 492,619, filed March 28, 2023, which are incorporated by reference in their entireties.

[0003] Reference to an electronically submitted sequence listing The contents of the sequence listing submitted electronically with this application as an XML file (Name: NB42142PCT_SequenceListing; Size: 12,186 bytes; Creation Date: August 22, 2023) constitute part of this application and are incorporated by reference in their entirety herein. [Background technology]

[0004] Proteases (also known as proteinases) are enzymes capable of degrading other proteins. Proteases have the proteolytic ability to initiate protein catabolism by hydrolyzing the peptide bonds linking amino acids in the peptide or polypeptide chain that forms the protein. This activity of proteases as protein-digesting enzymes is called proteolytic activity. Many well-known methods exist for measuring proteolytic activity (Kalisz, "Microbial Proteinases," in: Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology, 1988). For example, proteolytic activity can be confirmed by a comparative assay that analyzes the hydrolysis ability of each protease against a commercially available substrate. Exemplary substrates useful for analyzing protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and keratin Azure (Sigma-Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (see, e.g., WO 99 / 34011 and U.S. Pat. No. 6,376,450, both of which are incorporated herein by reference). Summary of the Invention [Problem to be solved by the invention]

[0005] Serine proteases are enzymes (EC number 3.4.21) that contain an active site serine that initiates hydrolysis of peptide bonds in proteins. Based on their structure, serine proteases comprise a diverse class of enzymes with a wide range of specificities and biological functions, further divided into chymotrypsin-like (trypsin-like) and subtilisin-like enzymes. The prototypic subtilisin (EC number 3.4.21.62) was first isolated from Bacillus subtilis. Subtilisin and its homologs are members of the S8 peptidase family of the MEROPS classification scheme (Rawlings, ND et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and in Nucleic Acids Res 44, D343-D350). Members of family S8 contain a catalytic triad consisting of Asp, His, and Ser in that order. Although many useful variant proteases have been developed for cleaning applications, there remains a need for improved protease variants. [Means for solving the problem]

[0006] One embodiment is a composition comprising a subtilisin variant in combination with 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, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 9, and wherein the at least one additional enzyme is an acyltran. Spherase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase enzymes, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, lysozymes, mannanases, metalloproteases, nucleases (e.g., DNases and / or RNases), oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phospholipases, phosphodiesterases, phytases, polygalacturonans, and any combination or mixture thereof, wherein at least one additional enzyme is selected from the group consisting of: enzymes, polyesterases, proteases, pullulanases, reductases, rhamnogalacturonase, beta-glucanases, tannases, transglutaminase, xanthan lyase, xylan acetyl esterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof, wherein the at least one additional enzyme has increased residual activity compared to a reference enzyme in a detergent composition that does not contain the subtilisin variant.

[0007] In one embodiment, the composition comprises a subtilisin variant in combination with 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, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 9, and ... The enzymes include acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, Hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polygala and any combination or mixture thereof, wherein the at least one additional enzyme is selected from the group consisting of: subtilase, polyesterase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof, and wherein the at least one additional enzyme has increased residual activity compared to a reference enzyme in a detergent composition that does not contain the subtilisin variant.

[0008] In another embodiment, the disclosure provides a composition comprising a subtilisin variant in combination with 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, amino acid positions numbered according to SEQ ID NO: 9, and the subtilisin variant is selected from the group consisting of X9T, X17H, X77N, X78I, X103I, X127T, X165Q, X184Q, X202V, X20 and further comprising one or more amino acid substitutions selected from the group consisting of X203N, X217S, X258P, X3E, X203N, X217S, and X258P, wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 9, and the at least one additional enzyme is selected from the group consisting of acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, The present invention relates to a detergent composition comprising at least one additional enzyme selected from the group consisting of phosphatase, phospholipase, phosphodiesterase, phytase, polygalacturonase, polyesterase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has increased residual activity compared to a reference enzyme in a detergent composition that does not contain the subtilisin variant.

[0009] In another embodiment, the disclosure provides a composition comprising a subtilisin variant in combination with 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, and the subtilisin variant is selected from the group consisting of P009T, Q017H, T077N, T078I, Y103I, G127T, G165Q, N184Q, A202V, and further comprising one or more amino acid substitutions selected from the group consisting of G203E, G203N, N217S, and S258P, wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO:9, and the at least one additional enzyme is selected from the group consisting of acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, The present invention relates to a detergent composition comprising at least one additional enzyme selected from the group consisting of phosphatase, phospholipase, phosphodiesterase, phytase, polygalacturonase, polyesterase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has increased residual activity compared to a reference enzyme in a detergent composition that does not contain the subtilisin variant.

[0010] Still other embodiments are directed to methods of producing subtilisin variants, variants described herein, comprising stably transforming a host cell with an expression vector comprising a polynucleotide encoding one or more subtilisin variants described herein. Still further embodiments are directed to polynucleotides comprising a nucleic acid sequence encoding at least one of the subtilisin variants described herein. Compositions, such as enzyme compositions, comprising the subtilisin variants provided herein are also provided. DETAILED DESCRIPTION OF THE INVENTION

[0011] In one embodiment, the present disclosure provides one or more subtilisin variants comprising one, two, three or more amino acid positions at positions selected from the group consisting of 96, 103, 108, 115, 128, 129 and 181, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the variants provided herein exhibit one or more improved properties, such as improved stability or improved cleaning performance, or both improved stability and improved cleaning performance, when compared to a subtilisin having the amino acid sequence of SEQ ID NO: 9. The subtilisin variants provided herein are used in the preparation of cleaning compositions (e.g., automatic dishwashing compositions or laundry detergent compositions) comprising at least one additional enzyme, wherein the at least one additional enzyme has increased residual activity compared to a reference enzyme in a detergent composition that does not contain a subtilisin variant provided herein. Additionally, the subtilisin variants and compositions provided herein are also used in methods of cleaning (e.g., dishwashing methods or laundry cleaning methods) using such variants or compositions comprising such subtilisin variants.

[0012] Unless otherwise indicated herein, one or more subtilisin variants described herein can be made and used by a variety of techniques used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA fields, and industrial enzyme use and development. Terms and abbreviations not defined should be given their ordinary meanings as used in the art. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any definitions provided herein should be interpreted in the context of the specification as a whole. As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Unless otherwise specified, nucleic acid sequences are written from left to right in 5' to 3' direction, and amino acid sequences are written from left to right in amino to carboxy direction. As used herein, each numerical range includes all narrower numerical ranges that fall within such broader numerical range, as if all such narrower numerical ranges were expressly set forth herein.

[0013] As used herein in connection 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.

[0014] The nomenclature for the amino acid substitutions in one or more subtilisin variants described herein uses one or more of the following: position; position:amino acid substitution; or starting amino acid:position:amino acid substitution. Reference to a "position" (i.e., 5, 8, 17, 22, etc.) encompasses any starting amino acid that may be present at such position and any substitution that may be present at such position. Reference to a "position:amino acid substitution" (i.e., 1S / T / G, 3G, 17T, etc.) encompasses any starting amino acid that may be present at such position and one or more amino acids that may replace such starting amino acid. Reference to a position can be referred to in several ways, for example, position 003 can also be referred to as position 03 or 3. Reference to a starting amino acid or a replacing amino acid can further be represented as several starting or replacing amino acids separated by slashes (" / "). For example, D275S / K indicates a substitution of serine (S) or lysine (K) at position 275, and P / S197K indicates a substitution of lysine (K) for the starting amino acid proline (P) or serine (S) at position 197. Reference to X as an amino acid at a position refers to any amino acid at the referenced position.

[0015] The positions of amino acid residues in a given amino acid sequence are numbered relative to the amino acid sequence of SEQ ID NO: 9. That is, the amino acid sequence of SEQ ID NO: 9 serves as a reference sequence for numbering the positions of the amino acid residues. For example, the amino acid sequences of one or more subtilisin variants described herein are aligned with the amino acid sequence of SEQ ID NO: 9 using an alignment algorithm described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with the amino acid residue of SEQ ID NO: 9 is conveniently numbered with reference to the position number of the corresponding amino acid residue. For example, sequence alignment algorithms such as those described herein identify locations where insertions or deletions have occurred in a subject sequence compared to a query sequence (sometimes referred to as a "reference sequence"). Sequence alignment with other subtilisin amino acid sequences can be determined, for example, using the amino acid alignment shown in Figure 1 of WO 2018 / 118917.

[0016] As used herein, the terms "protease" and "proteinase" refer to enzymes capable of degrading proteins and peptides. Proteases have the ability to "degrade proteins" by hydrolyzing the peptide bonds that link amino acids in the peptide or polypeptide chain that forms the protein. This activity of proteases as protein-digesting enzymes is referred to as "proteolytic activity." There are many well-known procedures for measuring proteolytic activity. For example, proteolytic activity can be confirmed by a comparative assay that analyzes the ability of each protease to hydrolyze a suitable substrate. Exemplary substrates useful for analyzing protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and keratin Azure (Sigma-Aldrich K8500). Colorimetric assays using these substrates are well known in the art (see, e.g., WO 99 / 34011 and U.S. Pat. No. 6,376,450). The pNA peptidyl assay (see, e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) is also used to measure active enzyme concentration. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes a soluble synthetic substrate, such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA). The rate at which yellow color develops from the hydrolysis reaction is measured spectrophotometrically at 405 or 410 nm and is proportional to the concentration of active enzyme. Additionally, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a sample of purified protein. Dividing the activity on the substrate by the protein concentration gives the specific activity of the enzyme.

[0017] As used herein, the term "Bacillus" includes all species within the genus "Bacillus" known to those skilled in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkaloids, B. arginine ... Examples of Bacillus species include B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, B. pumilus, B sp. TY-145, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomic reorganization. Thus, this genus is intended to include organisms such as species that have been reclassified, for example, but not limited to, B. stearothermophilus, now referred to as "Geobacillus stearothermophilus," or B. polymyxa, now Paenibacillus polymyxa.The production of resistant endospores under stressful environmental conditions is considered a defining feature of the genus Bacillus, but this characteristic also applies to the recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0018] The term "vector" refers to a nucleic acid construct used to introduce or transfer a nucleic acid into a target cell or tissue. Vectors are typically used to introduce foreign DNA into cells or tissues. Vectors include plasmids, cloning vectors, bacteriophages, viruses (e.g., viral vectors), cosmids, expression vectors, and shuttle vectors. Vectors typically contain an origin of replication, a multicloning site, and a selection marker. The process of introducing a vector into a target cell is typically referred to as transformation. In some embodiments, the present invention includes vectors comprising a DNA sequence encoding a serine protease polypeptide (e.g., a precursor or mature serine protease polypeptide) operably linked to a suitable prosequence (e.g., a secretory sequence, a signal peptide sequence, etc.) capable of effecting expression of the DNA sequence and folding and translocation of the recombinant polypeptide chain in a suitable host.

[0019] As used herein, the term "introduction" in reference to introducing a nucleic acid sequence into a cell refers to any method suitable for transferring a nucleic acid sequence into a cell. Such introduction methods include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic modification of a cell resulting from the uptake, optional genomic integration, and expression of genetic material (e.g., DNA).

[0020] The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid molecule of the present disclosure. Expression can also refer to the translation of mRNA into a polypeptide. Thus, the term "expression" includes any step involved in producing a polypeptide, such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0021] An "expression cassette" or "expression vector" refers to a recombinantly or synthetically produced nucleic acid construct or vector for the expression of a nucleic acid of interest (e.g., a foreign nucleic acid or a transgene) in a target cell. The nucleic acid of interest usually expresses a protein of interest. An expression vector or expression cassette usually contains a promoter nucleotide sequence that drives or promotes the expression of the foreign nucleic acid. An expression vector or expression cassette usually also contains other specific nucleic acid elements that allow transcription of the specific nucleic acid in the target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Some expression vectors are capable of integrating and expressing heterologous DNA fragments into a host cell or the genome of a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. It is within the knowledge of one of ordinary skill in the art to select an appropriate expression vector for expressing a protein from a nucleic acid sequence incorporated into the expression vector.

[0022] As used herein, a nucleic acid is "operably linked" to another nucleic acid sequence when it is placed into a functional relationship with the other nucleic acid sequence. For example, a promoter or enhancer is operably linked to a nucleotide coding sequence if the promoter affects the transcription of the coding sequence. A ribosome binding site may be operably linked to a coding sequence if it is positioned so as to promote transcription of the coding sequence. Typically, "operably linked" DNA sequences are contiguous. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers can be used in accordance with conventional methods.

[0023] The term "gene" refers to a polynucleotide (e.g., a DNA fragment) that encodes a polypeptide and includes regions preceding and following the coding region. In some instances, genes contain intervening sequences (introns) between individual coding segments (exons).

[0024] The term "recombinant," when used in reference to a cell, typically indicates that the cell has been modified by the introduction of an exogenous nucleic acid sequence or that the cell is derived from a cell so modified. For example, a recombinant cell can contain a gene not found in the same form within the native (non-recombinant) form of the cell, or a recombinant cell can contain a native gene (found in the native form of the cell) that has been modified and reintroduced into the cell. A recombinant cell can contain nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include modifications achieved by gene replacement, site-specific mutagenesis, and related techniques known to those of skill in the art. Recombinant DNA technology includes techniques for producing recombinant DNA in vitro and introducing the recombinant DNA into cells capable of expressing or propagating the recombinant DNA, thereby producing a recombinant polypeptide. "Recombination" or "recombining" of polynucleotides or nucleic acids generally refers to the assembly or combination of two or more nucleic acids or polynucleotide strands or fragments to create a new polynucleotide or nucleic acid.

[0025] A nucleic acid or polynucleotide is said to "encode" a polypeptide if, in its natural state or when manipulated by methods known to those of skill in the art, it can be transcribed and / or translated to produce the polypeptide or a fragment thereof. The antisense strand of such a nucleic acid is also said to encode the sequence.

[0026] The terms "host strain" and "host cell" refer to a suitable host for an expression vector containing a DNA sequence of interest.

[0027] A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. Amino acids are referred to throughout this disclosure using one-letter or three-letter codes defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). The single letter X refers to any of the 20 amino acids. It is also recognized that a polypeptide can be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0028] The term "prosequence" or "propeptide sequence" refers to an amino acid sequence between the signal peptide sequence and the mature protease sequence necessary for proper folding and secretion of the protease; these are sometimes referred to as intramolecular chaperones. Cleavage of the prosequence or propeptide sequence results in the mature, active protease. Bacterial serine proteases are often expressed as proenzymes. Examples of modified propeptides are provided, for example, in WO 2016 / 205710.

[0029] The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that may be involved in the secretion or direct export of a mature or precursor form of a protein. A signal sequence is generally located at the N-terminus of a precursor or mature protein sequence. A signal sequence may be endogenous or exogenous. A signal sequence is usually not present in the mature protein. A signal sequence is generally cleaved from a protein by a signal peptidase after the protein has been exported.

[0030] The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide that does not include signal peptide and propeptide sequences.

[0031] The term "precursor" form of a protein or peptide refers to the mature form of the protein having a prosequence operably linked to the amino- or carbonyl-terminus of the protein. A precursor may also have a "signal" sequence operably linked to the amino-terminus of the prosequence. A precursor may also have additional polypeptides associated with post-translational activity (e.g., polypeptides cleaved from it to leave the mature form of the protein or peptide).

[0032] With respect to polypeptides, the term "wild-type" refers to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion 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 contain an artificial substitution, insertion, or deletion at one or more nucleotides. However, a polynucleotide encoding a wild-type polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes a wild-type or parent polypeptide.

[0033] With respect to a polypeptide, the term "parent" refers to a naturally occurring or wild-type polypeptide or a naturally occurring polypeptide that has been artificially substituted, inserted, or deleted at one or more amino acid positions to serve as a basis for introducing substitutions or additional substitutions to generate a variant enzyme provided herein. With respect to a polypeptide, the term "parent" also includes any polypeptide with protease activity that serves as a starting polypeptide for modifications, such as substitutions, additions, and / or deletions, that result in a variant with one or more modifications compared to the starting polypeptide. That is, a parent or reference polypeptide is not limited to naturally occurring wild-type polypeptides but includes any wild-type, parent, or reference polypeptide. Similarly, with respect to a polynucleotide, the term "parent" refers to a naturally occurring polynucleotide or a polynucleotide that does not contain an artificial substitution, insertion, or deletion at one or more nucleotides. With respect to a polynucleotide, the term "parent" also includes any polynucleotide encoding a polypeptide with protease activity that serves as a starting polynucleotide for modifications that result in a variant protease with modifications, such as substitutions, additions, and / or deletions compared to the starting polynucleotide. That is, a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to naturally occurring polynucleotides but includes any polynucleotide encoding a wild-type, parent, or reference polypeptide. In some embodiments, a parent polypeptide herein comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:9.

[0034] The term "naturally occurring" refers, for example, to a sequence and the residues contained therein (e.g., a polypeptide sequence and the amino acids or nucleotide sequences contained therein and the nucleotides contained therein) that are found in nature. Conversely, the term "non-naturally occurring" refers, for example, to a sequence and the residues contained therein that are not found in nature (e.g., a polypeptide sequence and the amino acids or nucleotide sequences contained therein and the nucleotides contained therein).

[0035] As used herein in reference to an amino acid residue position, "corresponding to," or "corresponding to," or "corresponding" refers to the amino acid residue at the recited position in the protein or peptide, or an amino acid residue similar, homologous, or equivalent to the recited residue in the protein or peptide. As used herein, a "region corresponding to" generally refers to an analogous position in a related or reference protein.

[0036] The terms "derived from" and "obtained from" refer not only to proteins produced or capable of being produced by a strain of organism of interest, but also to proteins encoded by DNA sequences isolated from such a strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to proteins encoded by DNA sequences of synthetic and / or cDNA origin and having characteristics that allow the protein of interest to be identified. To illustrate, a "protease derived from Bacillus" refers to an enzyme with proteolytic activity naturally produced by Bacillus, as well as a serine protease produced by a Bacillus source, but which is produced by other host cells transformed with a nucleic acid encoding the serine protease through the use of genetic engineering techniques.

[0037] The term "identical" in the context of two polynucleotide or polypeptide sequences refers to the nucleic acids or amino acids in the two sequences that are the same when the two sequences are aligned for maximum correspondence, as determined by sequence comparison algorithms or sequence analysis algorithms described below and known in the art.

[0038] The phrases "% identity," "percent identity," or "PID" refer to protein sequence identity. Percent identity can be determined using standard techniques known in the art. The percent amino acid identity shared by sequences of interest can be determined by aligning the sequences and directly comparing sequence information using programs such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described, for example, in Altschul et al., J Mol Biol, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5877 (1993). The percent amino acid sequence identity (%) value is determined by dividing the number of matching identical residues by the total number of residues in the "reference" sequence, including any gaps created by the program for optimal / maximal alignment. The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0039] As used herein, "homologous protein" or "homologous protease" refers to proteins that share clear similarities in primary, secondary, and / or tertiary structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins are aligned. Homology can be determined by amino acid sequence alignment using programs such as BLAST, MUSCLE, or CLUSTAL. Homology searches of protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST with a threshold (E-value cutoff) of 0.001 (Altschul et al., "Gapped BLAST and PSI BLAST: a new generation of protein database search programs," Nucleic Acids Res, Set 1;25(17):3389-402 (1997)). BLAST programs use several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences in terms of 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: neighbor word 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 size = 3; E-value cutoff = 10; scoring matrix = BLOSUM62; gap opening = 11; and gap extension = 1. This information can be used to group protein sequences and / or construct phylogenetic trees from them.Amino acid sequences can be entered into programs such as the Vector NTI Advance suite, and guide trees can be created using the neighbor-joining (NJ) method (Saitou and Nei, Mol Biol Evol, 4:406-425, 1987). Phylogenetic tree construction can be calculated using the Kimura sequence distance correction and ignoring positions containing gaps. Programs such as AlignX can present calculated distance values ​​in parentheses following the molecule names shown on the phylogenetic tree.

[0040] Understanding homology between molecules can reveal information about a molecule's evolutionary history and function. If a newly sequenced protein is homologous to a previously characterized protein, this strongly indicates the biochemical function of the new protein. Two molecules are said to be homologous if they are derived from a common ancestor. Homologous molecules, or homologs, can be classified into two classes: paralogs and orthologs. Paralogs are homologs present within a single species. Paralogs often differ in their detailed biochemical function. Orthologs are homologs present in different species that have very similar or identical functions. Protein superfamilies are the largest groups (clades) of proteins for which a common ancestry can be inferred. This common ancestry is usually based on sequence alignment and mechanistic similarity. Superfamilies usually include several protein families (exhibiting sequence similarity within the family). The term "protein clan" is commonly used to refer to protease superfamilies based on the MEROPS protease classification system. As used herein, the term "subtilisin" includes any member of the S8 serine protease family 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 44:D343-D350).

[0041] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994). Default parameters for the CLUSTAL W algorithm include: gap opening penalty = 10.0; gap extension penalty = 0.05; protein weight matrix = BLOSUM series; DNA weight matrix = IUB; delay divergence sequence % = 40; gap separation distance = 8; DNA transition weight = 0.50; list of hydrophilic residues = GPSNDQEKR; use negative matrix = OFF; toggle residue specific penalty = ON; toggle hydrophilicity penalty = ON; and toggle end gap separation penalty = OFF. The CLUSTAL algorithm includes deletions that occur at either end. For example, a variant in which 5 amino acids are deleted at one end of (or within) a 500 amino acid polypeptide has a percent sequence identity of 99% (495 / 500 identical residues x 100) to the "reference" polypeptide. Such a variant would be encompassed by variants having "at least 99% sequence identity" to the polypeptide.

[0042] Nucleic acids and polynucleotides are "isolated" when they are at least partially or completely separated from other components, including, but not limited to, other proteins, nucleic acids, cells, etc. Similarly, polypeptides, proteins, or peptides are "isolated" when they are at least partially or completely separated from other components, including, but not limited to, other proteins, nucleic acids, cells, etc. On a molar basis, an isolated species is enriched in other species in a composition. For example, an isolated species may comprise at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (on a molar basis) of all macromolecular species present. Preferably, the species of interest is purified to substantial homogeneity (i.e., contaminating species cannot be detected in the composition by conventional detection methods). Purity and homogeneity can be determined using several techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of nucleic acid or protein samples, respectively, followed by visualization immediately after staining. If desired, high-resolution techniques, such as high-performance liquid chromatography (HPLC) or similar means, can be used to purify materials.

[0043] The term "purified," as applied to a nucleic acid or polypeptide, generally refers to a nucleic acid or polypeptide that is substantially free from other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide will form a distinct band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to substantially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, and typically is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% (e.g., percent by weight on a molar basis) or more pure. In this context, a composition is enriched for a molecule if the concentration of that molecule is substantially increased after application of a purification or concentration procedure. The term "enriched" means that a compound, polypeptide, cell, nucleic acid, amino acid, or other particular material or component is present in a composition at a higher relative or absolute concentration than in the resulting composition.

[0044] The term "cleaning activity" refers to the cleaning performance achieved by a serine protease polypeptide, variant, or reference subtilisin under conditions used in the proteolytic, hydrolytic, cleaning, or other processes disclosed. In some embodiments, the cleaning performance of a serine protease or reference subtilisin can be determined by using various assays to clean one or more various enzyme-sensitive item or surface stains (e.g., stains resulting from food, grass, blood, ink, milk, oil, and / or egg proteins). The cleaning performance of one or more subtilisin variants or reference subtilisins described herein can be measured by subjecting the item or surface stain to standard washing conditions and assessing the extent to which the stain is removed using various chromatographic, spectrophotometric, or other quantitative methods. Exemplary cleaning assays and methods are known in the art and include, but are not limited to, those described in WO 99 / 34011 and U.S. Pat. No. 6,605,458, as well as those included in Example 2 below.

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

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

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

[0048] As used herein, cleaning compositions or cleaning formulations, unless otherwise indicated, include granular or powdered all-purpose or heavy-duty cleaning agents, especially cleaning detergents; all-purpose cleaning agents in liquid, granular, gel, solid, tablet, paste or unit dose form, especially so-called heavy-duty liquid (HDL) or heavy-duty dry (HDD) detergents; liquid detergents for fine fabrics; hand or manual dishwashing agents, such as high-foaming detergents; hand or manual dishwashing agents, automatic dishwashing agents or dish or tableware cleaners, such as various tablet, powder, solid, granular, liquid, gel and rinse aid types for domestic or institutional use; liquid cleaning and disinfecting agents for humans and other animals, such as antibacterial hand wash types, wash bars, mouthwashes, denture cleaners, car shampoos, carpet shampoos, bathroom cleaners, hair shampoos and / or hair rinses; shower gels and foam baths and metal cleaners; and cleaning aids, such as bleach additives and "stain sticks" or pre-treats. In some embodiments, the granular composition is in a "compact" form, and in some embodiments, the liquid composition is in a "concentrated" form.

[0049] The terms "detergent composition" or "detergent formulation" are used in reference to compositions intended for use in a wash medium for cleaning soiled or stained objects, including certain fabric and / or non-fabric objects or articles. In some embodiments, detergents of the present disclosure comprise one or more subtilisin variants of the present disclosure and, in addition, one or more surfactants, transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, fluorescent dyes, anti-caking agents, masking agents, enzyme stabilizers, calcium, enzyme activators, antioxidants, and / or solubilizers. In some examples, the builder salts are mixtures of silicates and phosphates, preferably with more silicates (e.g., sodium metasilicate) than phosphates (e.g., sodium tripolyphosphate). Some embodiments are directed to cleaning or detergent compositions that do not contain phosphates (e.g., phosphates or phosphate builders). The detergent composition may also contain biological components, such as one or more microorganisms or microbial extracts (as described in WO 2018 / 060475 and U.S. Pat. No. 10,968,556). Microorganisms can be used as the sole biologically active ingredient, but they can also be used in combination with one or more of the enzymes described herein. For example, a Bacillus strain having deposit accession number PTA-7543 can be used to reduce malodors, as described in WO 2012 / 112718. Other objectives may include the in situ production of desirable biological compounds or inoculating / inhabiting a locus with microorganisms to competitively prevent other undesirable microorganisms from inhabiting the same locus (competitive exclusion).

[0050] The phrases "substantially boron-free composition" or "substantially boron-free detergent" refer to compositions or detergents, respectively, that contain trace amounts of boron, e.g., perhaps less than about 1000 ppm (1 mg / kg or liter equals 1 ppm), less than about 100 ppm, less than about 50 ppm, less than about 10 ppm, or less than about 5 ppm, or less than about 1 ppm of other composition or detergent ingredients.

[0051] The term "bleaching" refers to treating a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient time and / or under appropriate pH and / or temperature conditions to result in brightening (i.e., whitening) and / or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, ClO, HO, peracids, NO, etc. Bleaching agents also include enzymatic bleaches, such as perhydrolases and arylesterases. Another embodiment is directed to a composition comprising one or more subtilisin variants described herein and one or more perhydrolases, such as those described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0052] The term "washing performance" of a protease (e.g., one or more subtilisin variants described herein or recombinant polypeptides or active fragments thereof) refers to the contribution of one or more subtilisin variants described herein to washing, providing additional cleaning performance to a detergent compared to a detergent without the addition of one or more subtilisin variants described herein to the composition. Washing performance is compared under relevant washing conditions. In some test systems, other suitable factors, such as detergent composition, foam concentration, water hardness, wash mechanism, time, pH and / or temperature, can be controlled in such a way that conditions typical for household use in a particular market segment (e.g., hand or manual dishwashing, automatic dishwashing, dishwashing, tableware washing, fabric washing, etc.) are mimicked.

[0053] The phrase "suitable washing conditions" is used herein to refer to the conditions actually used in the domestic hand dishwashing, automatic dishwashing or laundry detergent market segments, in particular washing temperature, time, washing mechanism, foam concentration, detergent type and water hardness.

[0054] The term "dishwashing" refers to both domestic and industrial dishwashing, and relates to both automatic dishwashing (e.g., in a dishwasher) and manual dishwashing (e.g., by hand).

[0055] The term "compact" form of the cleaning composition herein is best reflected in terms of density and composition by the amount of inorganic filler salt. Inorganic filler salts are conventional components of powder-form detergent compositions. In conventional detergent compositions, filler salts are present in significant amounts, typically about 17 to about 35% by weight of the total composition. In contrast, in compact compositions, filler salts are present in amounts less than about 15% by weight of the total composition. In some embodiments, the filler salt is present in an amount of about 10% by weight or less, more preferably about 5% by weight or less, of the composition. In some embodiments, the inorganic filler salt is selected from sulfate and chloride salts of alkali metals and alkaline earth metals. In some embodiments, the filler salt is sodium sulfate.

[0056] Disclosed herein are one or more subtilisin variants useful for cleaning applications and methods, as well as various industrial applications. Also disclosed herein are one or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin variants. In some embodiments, one or more subtilisin variants described herein can be incorporated into cleaning compositions useful in cleaning applications and methods for cleaning articles or surfaces in need thereof, such as laundry or textiles.

[0057] In one embodiment a subtilisin variant is provided, which comprises two, three, four or more amino acid substitutions compared to the parent subtilisin enzyme of SEQ ID NO: 0, said substitutions being 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 to the amino acid sequence of SEQ ID NO: 9.

[0058] In one embodiment a subtilisin variant is provided, wherein the variant comprises one, two, three, four or more amino acid 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, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 9.

[0059] 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 corresponding to the amino acid sequence of SEQ ID NO: 9.

[0060] 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.

[0061] Another embodiment is directed to one or more subtilisin variants described herein, with the proviso that one or more amino acid substitutions are not naturally occurring. Still further embodiments are directed to one or more subtilisin variants described herein, wherein the variants (i) are derived from B. licheniformis subtilisin, (ii) are isolated, (iii) have proteolytic activity, or (iv) comprise a combination of (i)-(iii). Yet another embodiment is directed to one or more subtilisin variants described herein, wherein said variants are derived from a parent or reference polypeptide having (i) 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 9, or (ii) 100% amino acid sequence identity to 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. Yet another embodiment is directed to one or more subtilisin variants described herein, wherein the variants comprise an amino acid sequence having (i) less than 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:9.

[0062] In yet other embodiments, one or more subtilisin variants described herein have one or more improved properties compared to a reference or parent subtilisin, the improved properties being selected from cleaning performance in detergent, stability in detergent or buffer, and aged cleaning performance, and combinations thereof. Aged cleaning performance refers to the difference in stain removal measured on aged test samples (enzyme pre-incubated in detergent at an elevated temperature, such as 37°C, for an extended period of time, e.g., 3-8 weeks) compared to cleaning of "fresh" stains with the same enzyme (no enzyme pre-incubation). Thus, enzymes with improved aged cleaning performance will show a smaller difference between aged and freshly prepared samples compared to the same evaluation performed with the reference / parent enzyme.

[0063] 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 cleaning performance in a detergent (wherein the variant has improved cleaning performance against blood / milk / ink stains or egg stains on woven cotton compared to the parent subtilisin), and / or (ii) improved stability (wherein the variant has greater residual activity compared to the parent or reference subtilisin). In yet another embodiment, cleaning performance in a detergent is measured according to the cleaning performance assay of Example 2, and / or stability is measured according to the stability assay of Example 2.

[0064] In another embodiment, the subtilisin variants provided herein exhibit reduced activity against an additional enzyme in a liquid detergent composition. Thus, the additional enzyme (e.g., an alpha-amylase or mannanase) is degraded less in a liquid detergent composition compared to the same additional enzyme (e.g., an alpha-amylase or mannanase) in a liquid detergent composition containing a subtilisin enzyme that does not have one or more substitutions selected from X96D, X103F, X108Q, X115L, X128K, X129Q, or X181Q. In some embodiments, the additional enzyme (e.g., an alpha-amylase or mannanase) is degraded less in a liquid detergent composition compared to the same additional enzyme (e.g., an alpha-amylase or mannanase) in a liquid detergent composition containing a subtilisin enzyme that does not have 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.

[0065] The term "enhanced stability" or "improved stability" in the context of oxidation-, chelating-, denaturing-, detergent-, heat-, and / or pH-stable proteases refers to a greater retention of proteolytic activity of a subtilisin variant over a longer period of time compared to a wild-type or parent protease, such as a reference or parent subtilisin protease, e.g., SEQ ID NO: 9. Autolysis has been identified as one form of subtilisin activity loss 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.)

[0066] The terms "thermostable" and "thermostable" and "thermostable" with respect to protease variants refer to proteases that retain a greater amount of residual activity compared to a parent or reference protease after exposure to an altered temperature for a given period of time under conditions (or "stress conditions") prevailing during proteolysis, hydrolysis, cleaning, or other processes. Residual activity is the amount of activity remaining after testing compared to the initial activity of the sample and can be reported as a percentage, e.g., % residual activity. An "altered temperature" encompasses an increase or decrease in temperature. In some embodiments, the variant proteases provided herein are incubated at a temperature between 40°C and 80°C for a 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 The variant subtilisins provided herein retain at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% proteolytic activity after exposure for 840 minutes, about 900 minutes, about 960 minutes, about 1020 minutes, about 1080 minutes, about 1140 minutes, or about 1200 minutes. In some embodiments, the variant subtilisins provided herein have a residual activity that is greater than the residual activity of the parent or reference protease using the method described in Example 2. In some embodiments, the variant subtilisins provided herein have at least 10% improved residual activity compared to the parent subtilisin when measured after 20 minutes of incubation in a liquid detergent at 37-63°C.

[0067] The subtilisin variants provided herein can be used in the manufacture of various compositions, such as enzyme compositions and cleaning or detergent compositions. The enzyme compositions comprise the subtilisin variants provided herein. The enzyme compositions can be in any form, such as granules, liquid formulations, or enzyme slurries.

[0068] Enzyme granules can be made by, for example, rotary atomization, wet granulation, dry granulation, spray drying, disk granulation, extrusion, pan coating, spheronization, drum granulation, fluidized bed agglomeration, high shear granulation, fluidized bed spray coating, crystallization, precipitation, emulsion gelation, rotary disk atomization and other casting methods, and prilling granulation. The core of the granule can be the granule itself or can be the inner core of a layered granule.

[0069] The core may comprise one or more water-soluble or water-dispersible materials, such as, but not limited to, sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate, citric acid, sugars (e.g., sucrose, lactose, glucose, granular sucrose, maltodextrin, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives such as hydroxypropylmethylcellulose, carboxymethylcellulose, and hydroxyethylcellulose), phosphates, calcium, protease inhibitors, and combinations thereof. Suitable dispersible materials include, but are not limited to, clays, nonpareils (combinations of sugars and starches, such as starch-sucrose nonpareils-ASNP), talc, silicates, carboxymethylcellulose, starch, and combinations thereof.

[0070] In some embodiments, the core comprises primarily sodium sulfate. In some embodiments, the core consists essentially of sodium sulfate. In certain embodiments, the core consists solely of sodium sulfate.

[0071] In some embodiments, the core comprises a subtilisin variant provided herein. In other embodiments, the core comprises one or more enzymes in addition to a protease. In other embodiments, the core is inactive and does not contain any enzymes.

[0072] In some embodiments, the core is an enzyme powder comprising a UFC containing an enzyme. The enzyme powder can be spray dried and optionally mixed with any of the water-soluble or water-dispersible materials listed herein. The enzyme can be or include a stabilized protease, in which case the enzyme powder will further include a stabilizer.

[0073] In some embodiments, the core is coated with at least one coating layer. In certain embodiments, the core is coated with at least two coating layers. In another particular embodiment, the core is coated with at least three coating layers. Materials used in the coating layers may be suitable for use in cleaning and / or detergent compositions (see, e.g., U.S. Patent Application Publication No. 2010 / 0124586, WO 99 / 32595, and U.S. Patent No. 5,324,649).

[0074] In some embodiments, the coating layer 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 (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, nonpareils (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-agglomerating agents (e.g., talc, clay, amorphous silica, and titanium dioxide), anti-foaming agents (FOAMBLAST 882® and EROL 6000K® and talc. U.S. Patent Application Publication No. 2010 / 0124586, WO 99 / 32595 and U.S. Patent No. 5,324,649 detail suitable ingredients for the coating layer.

[0075] In some embodiments, the coating layer comprises a sugar (e.g., sucrose, lactose, glucose, granular sucrose, maltodextrin, and fructose). In some embodiments, the coating layer comprises a polymer such as polyvinyl alcohol (PVA). PVA suitable for incorporation into the coating layer of the multi-layer granules includes partially hydrolyzed, fully hydrolyzed, and moderately hydrolyzed low to high viscosity. In some embodiments, the coating layer comprises an inorganic salt such as sodium sulfate.

[0076] In some embodiments, at least one coating layer is an enzyme coating layer. In some embodiments, the core is coated with at least two enzyme layers. In other embodiments, the core is coated with at least three or more enzyme layers.

[0077] In some embodiments, the enzyme granules comprise an enzyme selected from the group consisting of acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or R N-ase), oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polygalacturonase, polyesterase, additional protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof. Generally, at least one enzyme coating layer comprises at least one subtilisin variant provided herein.

[0078] The above list of enzymes is merely exemplary and is not meant to be exhaustive. Any enzyme can be used in the granules described herein, including wild-type, recombinant, and mutant enzymes of bacterial, fungal, and yeast origin, as well as acidic, neutral, or alkaline enzymes.

[0079] Another embodiment is directed to a method of cleaning a surface, the method comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition containing one or more subtilisin variants provided herein. In some embodiments, the surface or item in need of cleaning comprises a proteinaceous stain on the surface. In some embodiments, the surface or item in need of cleaning comprises a proteinaceous stain. The term "stain" includes any type of soiling on the surface of an item, such as a hard surface item (e.g., a dish), a medical device, or a textile. In some embodiments, the stain is a proteinaceous stain. As used herein, a "proteinaceous stain" is a stain or soil that contains protein.

[0080] Further embodiments are directed to methods of cleaning proteinaceous stains, comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition containing one or more subtilisin variants provided herein.

[0081] Another embodiment is directed to a method of cleaning egg stains, the method comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition containing one or more subtilisin variants provided herein.

[0082] Another embodiment is directed 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 provided herein or a composition containing one or more subtilisin variants provided herein.

[0083] One or more subtilisin variants described herein can be modified in various ways, such as the insertion, deletion, and / or substitution of one or more conservative or non-conservative amino acids (including cases where such modifications do not substantially alter the enzymatic activity of the variant). Similarly, the nucleic acids herein can be modified in various ways, such as one or more substitutions of one or more nucleotides in one or more codons, such that particular codons encode the same or different amino acids, resulting in silent mutations (e.g., where the nucleotide mutation does not change the encoded amino acid) or non-silent mutations; one or more deletions of one or more nucleotides (or codons) in the sequence; one or more additions or insertions of one or more nucleic acids (or codons) in the sequence; and / or one or more truncations or cleavage of one or more nucleotides (or codons) in the sequence. Many such modifications in nucleic acid sequences will not substantially alter the enzymatic activity of the resulting encoded polypeptide enzyme compared to the polypeptide enzyme encoded by the original nucleic acid sequence. One or more of the nucleic acid sequences described herein can also be modified to include one or more codons that provide for optimal expression in an expression system (e.g., a bacterial expression system), while, if necessary, the one or more codons still encode the same amino acid.

[0084] Described herein are one or more isolated, non-naturally occurring, or recombinant polynucleotides comprising a nucleic acid sequence encoding one or more subtilisin variants described herein, or recombinant polypeptides or active fragments thereof. One or more nucleic acid sequences described herein are useful for recombinantly producing (e.g., expressing) one or more subtilisin variants described herein, typically by expression of a plasmid expression vector comprising a sequence encoding one or more subtilisin variants described herein, or fragments thereof. One embodiment provides a nucleic acid encoding one or more subtilisin variants described herein, wherein the variants are in a mature form that has proteolytic activity. In some embodiments, one or more subtilisin variants described herein are recombinantly expressed using a homologous propeptide sequence. In some embodiments, one or more subtilisin variants described herein are recombinantly expressed using a heterologous or naturally occurring propeptide sequence (e.g., a propeptide sequence from B. licheniformis).

[0085] One or more nucleic acid sequences described herein can be produced using any suitable synthesis, manipulation, and / or isolation method, or a combination thereof. For example, one or more polynucleotides described herein can be made using standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques well known to those of skill in the art. Such techniques typically involve the synthesis of fragments of up to 50 or more nucleic acid bases, which are then joined (e.g., by enzymatic or chemical ligation) to form a substantially desired contiguous nucleic acid sequence. Synthesis of one or more polynucleotides described herein can also be facilitated by any suitable method known in the art, including, but not limited to, the classical phosphoramidite method (see, e.g., Beaucage et al., Tetrahedron Letters 22:1859-69 (1981)) or the method described by Matthes et al., EMBO J. 3:801-805 (1984), as typically practiced in automated synthesis. One or more polynucleotides described herein can also be produced using an automated DNA synthesizer. Customized nucleic acids can be ordered from various 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, for example, in Itakura et al., Ann. Rev. Biochem. 53:323 (1984) and Itakura et al., Science 198:1056 (1984).

[0086] Recombinant DNA techniques useful for modifying nucleic acids are well known in the art, such as restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more polynucleotides described herein can also be obtained by screening a cDNA library with one or more hybridizable oligonucleotide probes, or by PCR-amplifying polynucleotides encoding one or more subtilisin variants described herein, or recombinant polypeptides or active fragments thereof. Procedures for screening and isolating cDNA clones and PCR amplification procedures are well known to those of skill in the art and are described in standard references known to those of skill in the art. One or more polynucleotides described herein can be obtained by modifying a naturally occurring polynucleotide backbone (e.g., encoding one or more subtilisin variants or a reference subtilisin described herein), for example, by known mutagenesis methods (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro genetic recombination). A variety of methods suitable for generating modified polynucleotides encoding one or more subtilisin variants described herein are known in the art, including, but not limited to, site-saturation mutagenesis, systematic mutagenesis, insertional mutagenesis, deletional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, as well as various other recombinant methods.

[0087] Further embodiments are directed to one or more vectors comprising one or more subtilisin variants described herein (e.g., polynucleotides encoding one or more subtilisin variants described herein); one or more 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; one or more 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.

[0088] Some embodiments are directed to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein, which contain one or more nucleic acids or polynucleotides described herein. Some such recombinant cells are transformed or transfected with at least one such vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells include bacterial cells, for example, cells of Bacillus sp., such as, but not limited to, B. subtilis cells or B. licheniformis cells. Other embodiments are directed to recombinant cells (e.g., recombinant host cells) comprising one or more subtilisins described herein.

[0089] In some embodiments, one or more of the vectors described herein are expression vectors or expression cassettes in which one or more of the polynucleotide sequences described herein are operably linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., a promoter operably linked to one or more of the polynucleotide sequences described herein). The vector may also include a transcription terminator and / or a selection gene (e.g., an antibiotic resistance gene) that allows for continuous cultural maintenance of plasmid-infected host cells by growth in antimicrobial-containing medium.

[0090] Expression vectors can be derived from plasmids or viral DNA, or in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (see Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990); suitable replicating plasmids for B. subtilis include those listed on p. 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).

[0091] For expression and production of a protein of interest (e.g., one or more subtilisin variants described herein) in a cell, one or more expression vectors containing one or more copies, and in some cases multiple copies, of a polynucleotide encoding one or more subtilisin variants described herein are transformed into the cell under conditions suitable for expression of the variants. In some embodiments, one or more subtilisin variants described herein (and other sequences contained in the vector) are integrated into the genome of the host cell, while in other embodiments, a plasmid vector containing a polynucleotide sequence encoding one or more subtilisin variants described herein remains as a separate extrachromosomal element within the cell. Some embodiments provide both an extrachromosomal nucleic acid element and an incoming nucleotide sequence that is integrated into the genome of the host cell. The vectors described herein are useful for production of one or more subtilisin variants described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variants described herein is present on an integrating vector, which allows for integration and, optionally, amplification of the polynucleotide into the host chromosome. Examples of integration sites are well known to those of skill in the art. In some embodiments, transcription of a polynucleotide encoding one or more subtilisin variants described herein is by a promoter that is the wild-type promoter for the parent subtilisin, hi some other embodiments, the promoter is heterologous to one or more subtilisin variants described herein but is functional in the host cell.Examples of promoters for use in bacterial host cells include, but are not limited to, the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII, rrnIp2 promoters; the promoters of the B. stearothermophilus maltogenic amylase gene, the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene, the B. pumilus xylosidase gene; the B. thuringiensis cryIIIA; and the B. licheniformis alpha-amylase gene. Additional promoters include, but are not limited to, the A4 promoter, as well as the PR or PL promoters of phage lambda and the lac, trp or tac promoters of E. coli.

[0092] One or more subtilisin variants described herein can be produced in any suitable microbial host cell, including bacteria and fungi. In some embodiments, one or more subtilisin variants described herein can be produced in Gram-positive bacteria. In some embodiments, the host cell is Bacillus spp., Streptomyces spp., Escherichia spp., Aspergillus spp., Trichoderma spp., Pseudomonas spp., Corynebacterium spp., Saccharomyces spp., or Pichia spp. In some embodiments, one or more subtilisin variants described herein are produced by a Bacillus sp. host cell. The Bacillus sp. host cells used to produce one or more subtilisin variants described herein are Examples of Bacillus sp. host cells include, but are not limited to, B. licheniformis, B. gibsonii, B. lentus, B. subtilis, B. amyloliquefaciens, B. brevis, B. stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B. pumilis, B. thuringiensis, B. clausii, and B. megaterium, as well as other organisms within the genus Bacillus. In some embodiments, B. subtilis host cells are used to generate the variants described herein.U.S. Pat. Nos. 5,264,366 and 4,760,025 (Reissue Patent Application No. 34,606) describe various Bacillus host strains that can be used to produce one or more of the subtilisin variants described herein, although other suitable strains can also be used.

[0093] Some bacterial strains that can be used to produce one or more subtilisin variants described herein include non-recombinant (i.e., wild-type) Bacillus sp. strains as well as naturally occurring and / or recombinant strains. In some embodiments, the host strain is a recombinant strain, and a polynucleotide encoding one or more subtilisin variants described herein has been introduced into the host. In some embodiments, the host strain is a B. subtilis host strain, particularly a recombinant B. subtilis host strain. Many B. subtilis strains are known, including, but not limited to, strains 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753-PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211 (see, e.g., Hoch et al., Genetics 73:215-228 (1973); see also U.S. Pat. Nos. 4,450,235, 4,302,544, and EP 0134048). The use of B. subtilis as an expression host cell is well known in the art (see, e.g., Palva et al., Gene 19:81-87 (1982); Fahnestock and Fischer, J. Bacteriol., 165:796-804 (1986); and Wang et al., Gene 69:39-47 (1988)).

[0094] In some embodiments, the Bacillus host cell is a Bacillus sp. that includes a mutation or deletion in at least one of the following genes: degU, degS, degR, and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU(Hy)32 (see, e.g., Msadek et al., J. Bacteriol. 172:824-834 (1990); and Olmos et al., Mol. Gen. Genet. 253:562-567 (1997)). In some embodiments, the Bacillus host comprises a mutation or deletion in scoC4 (see, e.g., Caldwell et al., J. Bacteriol. 183:7329-7340 (2001)); spoIIE (see, e.g., Arigoni et al., Mol. Microbiol. 31:1407-1415 (1999)); and / or oppA or other genes of the opp operon (see, e.g., Perego et al., Mol. Microbiol. 5:173-185 (1991)). Indeed, any mutation in the opp operon that results in the same phenotype as a mutation in the oppA gene is contemplated for use in some embodiments of the altered Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, modified Bacillus host cell strains that can be used to produce one or more subtilisin variants described herein are Bacillus host strains that already contain mutations in one or more of the above genes. Additionally, Bacillus sp. host cells that contain mutations and / or deletions of endogenous protease genes are also used. In some embodiments, the Bacillus host cell contains deletions of the aprE and nprE genes.In other embodiments, the Bacillus sp. host cell comprises a deletion of five protease genes, while in other embodiments, the Bacillus sp. host cell comprises a deletion of nine protease genes (see, e.g., U.S. Patent Application Publication No. 2005 / 0202535).

[0095] Host cells are transformed with nucleic acid sequences encoding one or more of the subtilisin variants described herein by any suitable method known in the art. Methods for introducing nucleic acids (e.g., DNA) into Bacillus cells or E. coli cells using plasmid DNA constructs or vectors and transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmid is then isolated from the E. coli cells and transformed into a Bacillus cell. However, the use of an intermediary microorganism such as E. coli is not required; in some embodiments, the DNA construct or vector is introduced directly into the Bacillus host.

[0096] Exemplary methods for introducing one or more of the nucleic acid sequences described herein into Bacillus cells are described, for example, in Ferrari et al., "Genetics," Harwood et al. [eds.], Bacillus, Plenum Publishing Corp. (1989), pp. 57-72; Saunders et al., J. Bacteriol. 157:718-726 (1984); Hoch et al., J. Bacteriol. 93:1925-1937 (1967); Mann et al., Current Microbiol. 13:131-135 (1986); Holubova, Folia Microbiol. 30:97 (1985); Chang et al., Mol. Gen. Genet. 168:11-115 (1979); Vorobjeva et al., FEMS Microbiol. Lett. 7:261-263 (1980); Smith et al., Appl. Env. Microbiol. 51:634 (1986); Fisher et al., Arch. Microbiol. 139:213-217 (1981); and McDonald, J. Gen. Microbiol. 130:203 (1984)). Indeed, methods such as transformation, including, for example, 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 harboring a partially homologous resident plasmid (see Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol. 154:1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169:1205-1211 (1987)).In this method, an incoming donor plasmid recombines with homologous regions of a resident "helper" plasmid in a process that mimics chromosomal transformation.

[0097] In addition to commonly used methods, in some embodiments, host cells are directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more subtilisin variants described herein (i.e., no intermediate cells are used to amplify or otherwise process the DNA construct or vector before introduction into the host cell). Introduction of the DNA construct or vector described herein into a host cell includes physical and chemical methods known in the art for introducing a nucleic acid sequence (e.g., a DNA sequence) into a host cell without inserting it into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In additional embodiments, the DNA construct or vector is not inserted into a plasmid but is co-transformed with a plasmid. In a further embodiment, the selectable marker is deleted from the modified Bacillus strain using methods known in the art (see Stahl et al., J. Bacteriol. 158:411-418 (1984); and Palmeros et al., Gene 247:255-264 (2000)).

[0098] In some embodiments, the transformed cells are cultured in a conventional nutrient medium. Suitable specific culture conditions, such as temperature and pH, are known to those of skill in the art and are well described in the scientific literature. Some embodiments provide one or more cultures (e.g., cell cultures) comprising one or more subtilisin variants or nucleic acid sequences described herein.

[0099] 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 for expression of the variants, and the resulting variants are then recovered from the culture. In some embodiments, the variants produced by the cells are recovered from the culture medium by conventional procedures, such as, but not limited to, separating the host cells from the culture medium by centrifugation or filtration, precipitating the protein components of the supernatant or filtrate with a salt (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).

[0100] In some embodiments, one or more subtilisin variants produced by recombinant host cells are secreted into the culture medium. A nucleic acid sequence encoding a purification-facilitating domain can be used to facilitate purification of the variants. A vector or DNA construct comprising a polynucleotide sequence encoding a subtilisin variant described herein may further comprise a nucleic acid sequence encoding a purification-facilitating domain that facilitates purification of the variant (see, e.g., Kroll et al., DNA Cell Biol. 12:441-53 (1993)). Examples of such purification-facilitating domains include, but are not limited to, metal-chelating peptides such as histidine-tryptophan modules, which enable purification on immobilized metals (see, e.g., Porath, Protein Expr. Purif. 3:263-281 (1992)), protein A domains, which enable purification on immobilized immunoglobulins, and domains utilized in the FLAGS extension / affinity purification system. The inclusion of a cleavable linker sequence, such as factor XA or enterokinase (e.g., those available from Invitrogen, San Diego, Calif.), between the purification domain and the heterologous protein can also be used to facilitate purification.

[0101] The variant proteins of the present invention can be produced in host cells by, for example, secretion or intracellular expression, using methods well known in the art. Fermentation, separation, and concentration techniques are well known in the art, and conventional methods can be used to prepare concentrated enzyme-containing solutions. The host cells can be further treated, for example, by heating or altering the pH or salt content to release the enzyme or improve cell separation, or by treating with an enzyme, such as hen egg white lysozyme, T4 lysozyme, or an enzyme described in WO 2022 / 047149. To restore production scale, the variant polypeptide can be concentrated or partially purified, generally as described above, by removing cells by flocculation with a polymer. Alternatively, the enzyme can be concentrated or purified by microfiltration, followed by concentration by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme does not need to be concentrated or purified; the whole broth culture can be dissolved and used without further processing. The enzyme can then be processed, for example, into granules.

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

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

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

[0105] Further embodiments are directed to methods of improving the cleaning performance or stability of a subtilisin, comprising modifying the subtilisin to include one or more substitutions or combinations of substitutions provided herein.

[0106] Yet other embodiments are directed to methods for reducing the degradation ability of a subtilisin variant for an accessory enzyme (e.g., alpha-amylase or mannanase) in a detergent composition, comprising modifying the subtilisin to include one or more substitutions or combinations of substitutions provided herein.

[0107] Unless otherwise stated, the level of all ingredients or compositions provided herein is adjusted based on the active level of that ingredient or composition, and does not include impurities that may be present in commercially available sources, such as residual solvents or by-products. The weight of enzyme ingredients is based on total active protein. Unless otherwise stated, all percentages and ratios are calculated by weight. Unless otherwise stated, all percentages and ratios are calculated based on the total composition. The compositions described herein include cleaning compositions, such as detergent compositions. In exemplary detergent compositions, the enzyme level is expressed by pure enzyme relative to the weight of the total composition, and detergent ingredients are expressed by the weight of the total composition, unless otherwise stated. In one embodiment, one or more subtilisin variants described herein are useful in cleaning applications, such as, but not limited to, cleaning dish or tableware products, fabrics, medical devices, and products having hard surfaces (e.g., tables, tabletops, walls, furniture products, floor and ceiling hard surfaces). In other embodiments, one or more subtilisin variants described herein are useful in sanitizing applications, such as, but not limited to, sanitizing automatic dishwashers or washing machines. In one embodiment, the cleaning composition is a cleaning composition comprising one or more subtilisin variants described herein, wherein the cleaning composition is a composition selected from the group consisting of laundry detergents, fabric softeners, dishwashing detergents (e.g., automatic or hand dishwashing detergents), hard surface cleaning detergents, and medical device cleaning compositions.

[0108] Another embodiment is directed to a composition comprising one or more subtilisin variants described herein. In some embodiments, the composition is a cleaning composition. In other embodiments, the composition is a detergent composition. In still other embodiments, the composition is selected from a laundry detergent composition, an automatic dishwashing (ADW) composition, a hand (manual) dishwashing detergent composition, a hard surface cleaning composition, an eyeglass cleaning composition, a medical device cleaning composition, a disinfecting (e.g., malodor or microbial) composition, and a personal care cleaning composition. In still other embodiments, the composition is a laundry detergent composition, an ADW composition, or a hand (manual) dishwashing detergent composition. Still further embodiments are directed to fabric cleaning compositions, while other embodiments are directed to non-fabric cleaning compositions. In some embodiments, the cleaning composition is boron-free. In other embodiments, the cleaning composition is phosphate-free. In still other embodiments, the composition comprises one or more subtilisin variants described herein and one or more excipients, adjuvants, and / or additional enzymes.

[0109] In one embodiment there is provided a composition comprising a subtilisin variant in combination with 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 or X181Q, wherein the positions are numbered according to SEQ ID NO: 9, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 9, and wherein the at least one additional enzyme is an acyltransferase. ferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronanase enzymes such as maltase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polygala and wherein the at least one additional enzyme is selected from the group consisting of: subtilisin, polyesterase, additional protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof, and wherein the at least one additional enzyme has increased residual activity compared to a reference enzyme in a detergent composition that does not contain the subtilisin variant. In some embodiments, such compositions comprise a subtilisin variant selected from the group consisting of SEQ ID NOs: 1-8.

[0110] In another embodiment, the present disclosure provides a detergent composition (e.g., an ADW composition) comprising a surfactant and at least one subtilisin variant provided herein. Such compositions may further comprise one or more of excipients, adjuvants, and / or additional enzymes.

[0111] In still further embodiments, the compositions described herein contain phosphate, are phosphate-free, contain boron, are boron-free, or are a combination thereof. In other embodiments, the compositions are boron-free. In some embodiments, the boron-free compositions are compositions without added borate stabilizers. In another embodiment, the boron-free compositions are compositions with less than 5.5% boron. In yet a further embodiment, the boron-free compositions are compositions with less than 4.5% boron. In yet another embodiment, the boron-free compositions are compositions with less than 3.5% boron. In yet a further embodiment, the boron-free compositions are compositions with less than 2.5% boron. In yet another embodiment, the boron-free compositions are compositions with less than 1.5% boron. In another embodiment, the boron-free compositions are compositions with less than 1.0% boron. In yet a further embodiment, the boron-free compositions are compositions with less than 0.5% boron. In other embodiments, the composition is free or substantially free of enzyme stabilizers or peptide inhibitors.

[0112] In another embodiment, one or more compositions described herein have the form of a gel, tablet, powder, granule, solid, liquid, unit dosage form, and combinations thereof. In yet another embodiment, one or more compositions described herein have a form selected from a low-moisture compact formulation, a low-moisture HDL or unit dose (UD), or a high-moisture formulation or HDL. In some embodiments, the cleaning compositions described herein have a unit dosage form. In other embodiments, the unit dosage form is selected from a pill, tablet, capsule, gel capsule, sachet, pouch, multi-compartment pouch, and pre-measured powder or liquid. In some embodiments, the unit dosage form is designed to control the release of ingredients in the multi-compartment pouch (or other unit dosage form). Suitable unit dosage forms and controlled-release dosage forms are described, for example, in EP 2100949; WO 02 / 102955; U.S. Pat. No. 4,765,916; U.S. Pat. No. 4,972,017; and WO 04 / 111178. In some embodiments, the unit dosage form is a tablet or powder contained in a water-soluble film or pouch.

[0113] Examples of laundry detergent compositions include, but are not limited to, liquid and powder laundry detergent compositions. Examples of hard surface cleaning compositions include, but are not limited to, compositions used to clean hard surfaces such as non-dishware products, non-tableware products, tables, tabletops, furniture products, walls, floors, and ceilings. Examples of hard surface cleaning compositions are described, for example, in U.S. Patent Nos. 6,610,642, 6,376,450, and 6,376,450. Examples of personal care compositions include, but are not limited to, compositions used to clean dentures, teeth, hair, contact lenses, and skin. Examples of ingredients for such oral care compositions include, for example, those described in U.S. Patent No. 6,376,450.

[0114] In some embodiments, one or more subtilisin variants described herein clean at low temperatures. In other embodiments, one or more compositions described herein clean at low temperatures. In other embodiments, one or more compositions described herein comprise an effective amount of one or more subtilisin variants described herein as useful or effective for cleaning surfaces in need of proteinaceous stain removal.

[0115] In some embodiments, auxiliary substances are included to, for example, aid or enhance cleaning performance, treat the substrate to be cleaned, or alter the aesthetics of the cleaning composition, as in the case of fragrances, colorants, dyes, etc. One embodiment is directed to a composition comprising one or more auxiliary substances and one or more subtilisin variants described herein. Another embodiment is a composition comprising one or more auxiliary substances and one or more subtilisin variants described herein, wherein the auxiliary substances are selected from the group consisting of bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilization systems), chelating agents, optical brighteners, soil release polymers, dye transfer inhibitors, dispersants, suds suppressors, dyes, fragrances, colorants, fillers, photoactivators, fluorescent agents, fabric finishes, hydrolyzable surfactants, preservatives, antioxidants, shrinkage inhibitors, anti-wrinkle agents, disinfectants, fungicides , color speckle, silver care agents, tarnish inhibitors, corrosion inhibitors, alkalinity sources, solubilizers, carriers, processing aids, pigments, pH adjusters, surfactants, builders, chelating agents, dye transfer inhibitors, deposition aids, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymeric dispersants, clay stain removal / anti-redeposition agents, structural elasticizers, fabric softeners, carriers, hydrotropes, processing aids, pigments, and combinations thereof. Examples of other adjuncts and concentrations of use can be found in U.S. Patent Nos. 5,576,282, 6,306,812, 6,326,348, 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014, and 5,646,101. In embodiments where one or more cleaning adjuncts and one or more subtilisin variants described herein do not mix well, methods of keeping the adjuncts and variants separated (i.e., not in contact with each other) can be used until the two components are suitable for mixing. Such separation methods include any suitable method known in the art (eg, gelcaps, encapsulation, tablets, physical separation, etc.).

[0116] Some embodiments are directed to one or more cleaning additive products comprising one or more subtilisin variants described herein. In some embodiments, the additive is packaged in a dosage form for addition to a cleaning process. In some embodiments, the additive is packaged in a dosage form for addition to a cleaning process in which a source of peroxide is utilized and enhanced bleaching is desired.

[0117] Examples of fillers or carriers for granular compositions include, but are not limited to, various salts of sulfates, carbonates, and silicates, talc, and clay. Examples of fillers or carriers for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols, such as polyols and diols (e.g., methanol, ethanol, propanol, and isopropanol). In some embodiments, the composition contains from about 5% to about 90% of such fillers or carriers. Such compositions can contain acidic fillers to lower the pH of solutions resulting from cleaning methods or applications.

[0118] In one embodiment, one or more cleaning compositions described herein comprise an effective amount of one or more subtilisin variants described herein, alone or in combination with one or more additional enzymes. Typically, the cleaning composition comprises at least about 0.0001 to about 20%, about 0.0001 to about 10%, about 0.0001 to about 1%, about 0.001 to about 1%, or about 0.01 to about 0.2% by weight 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, from about 0.01 to about 5 mg, from about 0.01 to about 2 mg, from about 0.01 to about 1 mg, from about 0.05 to about 1 mg, from about 0.5 to about 10 mg, from about 0.5 to about 5 mg, from about 0.5 to about 4 mg, from about 0.5 to about 3 mg, from about 0.5 to about 2 mg, from about 0.5 to about 1 mg, from about 0.1 to about 10 mg, from about 0.1 to about 5 mg, from about 0.1 to about 4 mg, from about 0.1 to about 3 mg, from about 0.1 to about 2 mg, from about 0.1 to about 2 mg, from about 0.1 to about 1 mg, or from about 0.1 to about 0.5 mg of one or more subtilisin variants described herein per gram of composition.

[0119] The cleaning compositions herein are typically formulated to provide a wash water pH of about 4.0 to about 11.5, or about 5.0 to about 11.5, or about 5.0 to about 8.0, or about 7.5 to about 10.5 during use in aqueous cleaning operations. Liquid product formulations are typically formulated to have a pH of about 3.0 to about 9.0, or about 3 to about 5. Granular laundry products are typically formulated to have a pH of about 8 to about 11. In some embodiments, the cleaning compositions of the present invention can be formulated to have an alkaline pH under wash conditions, for example, a pH of about 8.0 to about 12.0, or about 8.5 to about 11.0, or about 9.0 to about 11.0. In some embodiments, the cleaning compositions of the present invention can be formulated to have a neutral pH under wash conditions, for example, a pH of about 5.0 to about 8.0, or about 5.5 to about 8.0, or about 6.0 to about 8.0, or about 6.0 to about 7.5. In some embodiments, neutral pH conditions may be measured using a conventional pH meter when the cleaning composition is dissolved 1:100 (wt:wt) in deionized water at 20° C. Techniques for adjusting pH to recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0120] In some embodiments, one or more subtilisin variants described herein are encapsulated to protect them from other components in the composition during storage and / or to control the availability of the variant during cleaning. In some embodiments, encapsulation enhances the performance of the variant and / or additional enzymes. In some embodiments, the encapsulating material typically encapsulates at least a portion of the subtilisin variant described herein. Typically, the encapsulating material is water-soluble and / or water-dispersible. In some embodiments, the encapsulating material has a glass transition temperature (Tg) of 0°C or higher. Examples of encapsulating materials include, but are not limited to, carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin wax, and combinations thereof. When the encapsulating material is a carbohydrate, the encapsulating material is typically selected from monosaccharides, oligosaccharides, and combinations thereof. In some embodiments, the encapsulant is starch (see, e.g., EP 0922499, U.S. Pat. No. 4,977,252, U.S. Pat. No. 5,354,559, and U.S. Pat. No. 5,935,826). In some embodiments, the encapsulant is a microsphere made from a plastic, such as a thermoplastic, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof. Examples of commercially available microspheres include, but are not limited to, EXPANCEL® (Stockviksverken, Sweden); and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (PQCorp., Valley Forge, PA).

[0121] There are a variety of wash conditions to which one or more subtilisin variants described herein may be exposed, including various detergent formulations, wash water volumes, wash water temperatures, and wash time lengths. A low detergent concentration system is directed to wash water containing less than about 800 ppm of detergent components. A medium detergent concentration system is directed to wash water containing from about 800 ppm to about 2000 ppm of detergent components. A high detergent concentration system is directed to wash water containing more than about 2000 ppm of detergent components. In some embodiments, the "cold water wash" of the present invention utilizes a "cold water detergent" suitable for washing at temperatures between about 10°C and about 40°C, between about 20°C and about 30°C, or between about 15°C and about 25°C, as well as all other combinations within the ranges of about 15°C to about 35°C, or between 10°C and 40°C.

[0122] Water hardness varies depending on the region. Hardness is determined by the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) is a measure of the amount of Ca 2+ / Mg 2+ It is stated in grains per gallon (gpg) of the mixture. Most water in the United States is hard, but hardness levels vary. Medium-hard water (60-120 ppm) to hard water (121-181 ppm) has hardness minerals between 60 and 181 ppm (ppm can be converted to grains per U.S. gallon by dividing ppm by 17.1). [Table A]

[0123] Other embodiments are directed to one or more cleaning compositions comprising from about 0.00001% to about 10% by weight of the composition of one or more subtilisin variants described herein and from about 99.999% to about 90.0% by weight of the composition of one or more adjunct substances. In further embodiments, 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%, or from about 99.995% to about 99.5% by weight of the composition of one or more adjunct substances.

[0124] In other embodiments, the compositions described herein comprise one or more subtilisin variants described herein and one or more additional enzymes, such as acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nucleic acid enzyme, or the like. The enzymes are selected from enzymes selected from the group consisting of lyases (e.g., DNase and / or RNase), oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phosphodiesterases, phospholipases, phytases, polygalacturonases, polyesterases, additional proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xanthan lyases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof. Some embodiments are directed to enzyme combinations (i.e., "cocktails") comprising traditional enzymes such as amylases, lipases, cutinases, mannanases, and / or cellulases, together with one or more subtilisin variants described herein and / or one or more additional proteases.

[0125] In another embodiment, one or more compositions described herein comprise one or more subtilisin variants described herein and one or more additional proteases. In one embodiment, the additional protease is a serine protease. In another embodiment, the additional protease is not immunologically related (e.g., the protease does not cross-react in antibody-based tests known in the art for assessing shared immunological epitopes). In another embodiment, the additional protease is a protease with a different net charge (e.g., +2 to -1 relative to the parent protease). In another embodiment, the additional protease is a metalloprotease, a fungal subtilisin, an alkaline microbial protease, or a trypsin-like protease. Suitable additional proteases include those of animal, vegetable, or microbial origin. In some embodiments, the additional protease is a microbial protease. In other embodiments, the additional protease is a chemically modified or genetically engineered variant. In another embodiment, the additional protease is an alkaline microbial protease or a trypsin-like protease, hi other embodiments, the additional protease does not contain cross-reactive epitopes with the subtilisin variant as determined by antibody binding assays or other assays available in the art. Exemplary alkaline proteases include, for example, subtilisins derived from Bacillus (e.g., BPN', Carlsberg, subtilisin 309, subtilisin 147, subtilisin PB92, subtilisin DY, subtilisin 168, B. gibsonii DSM 14391 subtilisin, B. gibsonii AGS 78407 subtilisin, B. pumilus subtilisin, B. lentus DSM 5483, and B. sp. TY-145 subtilisin) or subtilisins of fungal origin, such as those described in U.S. Pat. No. 8,362,222. Examples of additional proteases include, but are not limited to, those described in WO 92 / 21760, WO 95 / 23221, WO 2008 / 010925,WO 09 / 149200, WO 09 / 149144, WO 09 / 149145, WO 10 / 056640, WO 10 / 056653, WO 2010 / 0566356, WO 11 / 072099, WO 2011 / 13022, WO 11 / 140364, WO 12 / 151534, WO 2015 / 038792 FRET, WO 2015 / 089447 pamphlet, WO 2015 / 089441 pamphlet, WO 2017 / 215925 pamphlet, U.S. Patent Application Publication No. 2008 / 0090747 specification, U.S. Patent No. 5,801,039, U.S. Patent No. 5,340,735, U.S. Patent No. 5,500,364, U.S. Patent No. 5,855,625, Reissue Patent Application No. 34,606 specification, U.S. Patent No. 5,955,340, U.S. Patent No. 5,700,676, U.S. Patent No. 6,312, 936, U.S. Patent No. 6,482,628, U.S. Patent No. 8,530,219, U.S. Provisional Patent Application Nos. 62 / 180673 and 62 / 161077, and International Application Nos. PCT / US2015 / 021813, PCT / US2015 / 055900, PCT / US2015 / 057497, PCT / US2015 / 057492, PCT / US2015 / 057512, and PCT / US2015 / 057526 Documents, International Application No. PCT / US2015 / 057520, International Application No. PCT / US2015 / 057502, International Application No. PCT / US2016 / 022282, International Publication No. WO 2016 / 074925, International Publication No. WO 2020 / 178102, International Publication No. WO 2022 / 106400, International Publication No. WO 2016 / 203064, European Patent No. 3380599, International Publication No. WO 2017 / 215925, International Publication No. WO 2019 / 48495,Those described in International Publication Nos. 2020 / 221578, 2016 / 203064, U.S. Patent No. 7,294,499, 2016 / 097354, and International Application No. PCT / US16 / 32514, as well as those described in International Publication Nos. 1999 / 014341, 1999 / 033960, 1999 / 014342, and 1999 / 034003 No. WO 2007 / 044993, WO 2009 / 058303, WO 2009 / 058661, WO 2014 / 071410, WO 2014 / 194032, WO 2014 / 194034, WO 2014 / 194054, and WO 2014 / 194117. Exemplary additional proteases include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and the Fusarium proteases described in WO 89 / 06270. Examples of commercially available proteases include, but are not limited to, MAXATASE®, MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX™, EXCELLASE™, PREFERENZ™ proteases (e.g., P100, P110, P280, P300), EFFECTENZ™ proteases (e.g., P100, P110, P280, P300), and EFFECTENZ™ proteases (e.g., P100, P110, P280, P300). Proteases (e.g., P1000, P1050, P2000), EXCELLENZ™ Proteases (e.g., P1000), ULTIMASE® and PURAFAST™ (DuPont / Danisco / Genencor); ALCALASE®, ALCALASE® ULTRA, BLAZE®, BLAZE® Variants, BLAZE® EVITY®, BLAZE® EVITY® 16L,Examples include CORONASE®, SAVINASE®, SAVINASE® ULTRA, SAVINASE® EVITY®, SAVINASE® EVERIS®, PRIMASE®, DURAZYM™, POLARZYME®, OVOZYME®, KANNASE®, LIQUANASE®, LIQUANASE EVERIS®, NEUTRASE®, PROGRESS UNO®, RELASE®, and ESPERASE® (Novozymes); BLAP® and BLAP® Varian® (Henkel); LAVERGY® PRO 104L (BASF), KAP (B. alkalophilus subtilisin (Kao Corporation)), and BIOTOUCH® (AB Enzymes).

[0126] Another embodiment is directed to a composition comprising one or more subtilisin variants described herein and one or more lipases. In some embodiments, the composition comprises from about 0.00001% to about 10%, from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% lipase by weight of the composition. Exemplary lipases can be chemically modified or genetically engineered variants.Examples of lipases include, but are not limited to, H. lanuginosa lipase (see, e.g., EP 258068 and EP 305216), T. lanuginosus lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009), and Rhizomucor miehei lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009). miehei lipase (see, e.g., EP 238023), Candida lipases such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, e.g., EP 214761), P. alcaligenes and P. pseudoalcaligenes lipases (see, e.g., EP 218 Pseudomonas lipases, such as P. cepacia lipase (see, e.g., European Patent No. 331376), P. stutzeri lipase (see, e.g., British Patent No. 1,372,034), P. fluorescens lipase, Bacillus lipases (e.g., Dartois lipase), Bacillus lipases (e.g., B. subtilis lipase), et al., Biochem. Biophys. Acta 1131:253-260(1993)), B. stearothermophilus lipase (see, for example, JP-A-64-744992), and B. pumilus lipase (see, for example, WO 91 / 16422), for example, those of bacterial or fungal origin.Examples of 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 R. delemar lipase (see Hass et al., Gene 109:117-113 (1991)). al., Biosci. Biotech. Biochem. 56:716-719 (1992)) and various Rhizopus lipases, such as R. oryzae lipase. Other lipolytic enzymes, such as cutinases, may also be used in one or more of the compositions described herein, including, but not limited to, cutinase derived from Pseudomonas mendocina (see WO 88 / 09367) and / or cutinase derived from Fusarium solanipisi (see WO 90 / 09446). Examples of commercially available lipases include, but are not limited to, M1 LIPASE™, LUMA FAST™, LIPOMAX™, and PREFERENZ™ L100 (DuPont); LIPEX®, LIPOCLEAN®, LIPOLASE®, and LIPOLASE® ULTRA (Novozymes), and LIPASE P™ (Amano Pharmaceutical Co. Ltd).

[0127] Still other embodiments are directed to compositions comprising one or more subtilisin variants described herein and one or more amylases. In one embodiment, the composition comprises about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% of the amylase by weight of the composition. Any amylase (e.g., alpha and / or beta) suitable for use in alkaline solutions may be useful for inclusion in such compositions. Exemplary amylases may be chemically modified or genetically engineered variants. Examples of amylases include, but are not limited to, those described in, for example, GB 1,296,839, WO 91 / 00353, WO 94 / 02597, WO 94 / 183314, WO 95 / 10603, WO 95 / 26397, WO 95 / 35382, WO 96 / 05295, WO 96 / 23873, WO 96 / 23874, WO 96 / 30481, WO 97 / 10342, WO 97 / 41213, WO 97 / 43424, WO 98 / 13481, WO 98 / 26078, and the like. FRET, International Publication No. 99 / 02702, International Publication No. 99 / 09183, International Publication No. 99 / 19467, International Publication No. 99 / 23211, International Publication No. 99 / 29876, International Publication No. 99 / 42567, International Publication No. 99 / 43793, International Publication No. 99 / 43794, International Publication No. 99 / 46399, International Publication No. 00 / 29560, International Publication No. 00 / 60058, International Publication No. 00 / 60059, International Publication No. 00 / 60060, International Publication No. 01 / 14532, International Publication No. 01 / 34784, International Publication No. 01 / 64852, International Publication No. 01 / 66712,International Publication No. 01 / 88107, International Publication No. 01 / 96537, International Publication No. 02 / 092797, International Publication No. 02 / 10355, International Publication No. 02 / 31124, International Publication No. 2004 / 055178, International Publication No. 2004 / 113551, International Publication No. 2005 / 001064, International Publication No. 2005 / 003311, International Publication No. 2005 / 018336, International Publication No. 2005 / 019443 , WO 2005 / 066338, WO 2006 / 002643, WO 2006 / 012899, WO 2006 / 012902, WO 2006 / 031554, WO 2006 / 063594, WO 2006 / 066594, WO 2006 / 066596, WO 2006 / 136161, WO 2008 / 000825, WO International Publication No. 2008 / 088493, International Publication No. 2008 / 092919, International Publication No. 2008 / 101894, International Publication No. 2008 / 112459, International Publication No. 2009 / 061380, International Publication No. 2009 / 061381, International Publication No. 2009 / 100102, International Publication No. 2009 / 140504, International Publication No. 2009 / 149419, International Publication No. 2010 / 059413, International Publication No. 2010 / 088 447, International Publication No. 2010 / 091221, International Publication No. 2010 / 104675, International Publication No. 2010 / 115021, International Publication No. 10 / 115028, International Publication No. 2010 / 117511, International Publication No. 2011 / 076123, International Publication No. 2011 / 076897, International Publication No. 2011 / 080352, International Publication No. 2011 / 080353, International Publication No. 2011 / 080354,These include amylases of bacterial or fungal origin, such as those described in WO 2011 / 082425, WO 2011 / 082429, WO 2011 / 087836, WO 2011 / 098531, WO 2013 / 063460, WO 2013 / 184577, WO 2014 / 099523, WO 2014 / 164777, WO 2015 / 077126, WO 2022 / 175435 and WO 2018 / 184004. Examples of commercially available amylases include, but are not limited to, AMPLIFY®, DURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, STAINZYME PLUS®, AMPLIFY PRIME®, STAINZYME ULTRA®, EVITY®, and BAN™ (Novozymes); EFFECTENZ™ S1000, POWERASE™, PREFERENZ™ S100, PREFERENZ™ S110, PREFERENZ™ S210, EXCELLENZ™ S2000, RAPIDASE®, and MAXAMYL® P (DuPont). In some embodiments, the subtilisin variants provided herein may be combined with one or more amylases selected from the group consisting of AA707, AA560, AAI10, BspAmy24, SP722, and CspAmy1, and variants thereof, and combinations thereof.

[0128] Still further embodiments are directed to compositions comprising one or more subtilisin variants described herein and one or more cellulases. In one embodiment, the composition comprises about 0.00001% to about 10%, 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% of cellulase by weight of the composition. Any suitable cellulase can be used in the compositions described herein. Exemplary cellulases can be chemically modified or genetically engineered variants. Examples of cellulases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO 2005 / 054475, WO 2005 / 056787, U.S. Pat. No. 7,449,318, U.S. Pat. No. 7,833,773, U.S. Pat. No. 4,435,307; EP 0495257 and U.S. Provisional Patent Application No. 62 / 296,678. Examples of commercially available cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®, CAREZYME®, ENDOLASE®, RENOZYME®, and CAREZYME® PREMIUM (Novozymes); REVITALENZ™ 100, REVITALENZ™ 200 / 220, and REVITALENZ® 2000 (DuPont); and KAC-500(B)™ (Kao Corporation). In some embodiments, the cellulase is incorporated as a portion or fragment of a mature wild-type or variant cellulase, with a portion of the N-terminus removed (see, e.g., U.S. Pat. No. 5,874,276).

[0129] Still further embodiments are directed to compositions comprising one or more subtilisin variants described herein and one or more mannanases. In one embodiment, the composition comprises about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% of the mannanase by weight of the composition. Exemplary mannanases can be chemically modified or genetically engineered variants. Examples of mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO 2016 / 007929; U.S. Patent Nos. 6,566,114; 6,602,842; and 6,440,991; and U.S. Provisional Patent Applications Nos. 62 / 251516, 62 / 278383, and 62 / 278387. Commercially available mannanases include, but are not limited to, MANNAWAY® (Novozymes), EFFECTENZ™ M1000, EFFECTENZ™ M2000, PREFERENZ™ M100, MANNASTAR®, BIOTOUCH® (AB Enzymes), and PURABRITE™ (DuPont).

[0130] Still further embodiments are directed to compositions comprising one or more subtilisin variants described herein and one or more nucleases, such as DNase or RNase, hi one embodiment, the composition comprises from about 0.00001% to about 10%, from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% of the nuclease by weight of the composition. Examples of nucleases include, but are not limited to, those described in WO 2015 / 181287, WO 2015 / 155350, WO 2016 / 162556, WO 2017 / 162836, WO 2017 / 060475 (e.g., SEQ ID NO: 21), WO 2018 / 184816, WO 2018 / 177936, WO 2018 / 177938, WO 2018 / 185269, WO 2018 / 1 Examples of such compounds include those described in International Publication Nos. 85285, WO 2018 / 177203, WO 2018 / 184817, WO 2019 / 084349, WO 2019 / 084350, WO 2019 / 081721, WO 2018 / 076800, WO 2018 / 185267, WO 2018 / 185280, WO 2018 / 206553 and WO 2020 / 099490.Other nucleases that can be used in combination with the subtilisin variants provided herein in the compositions and methods provided herein include those described in Nijland R, Hall MJ, Burgess JG (2010) Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase. PLoS ONE 5(12) and Whitchurch, CB, Tolker-Nielsen, T., Ragas, PC, Mattick, JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295:1487.

[0131] Still yet other embodiments are directed to compositions comprising one or more subtilisin variants described herein and one or more peroxidase and / or oxidase enzymes. In one embodiment, the composition comprises from about 0.00001% to about 10%, from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% of the peroxidase or oxidase, by weight of the composition. Peroxidases may be used in combination with hydrogen peroxide or a source thereof (e.g., percarbonate, perborate, or persulfate), and oxidases may be used in combination with oxygen. Peroxidases and oxidases are used alone or in combination with enhancers for "solution bleaching" (i.e., to prevent transfer of textile dyes from dyed fabrics to other fabrics when the fabrics are washed together in the wash liquor) (see, e.g., WO 94 / 12621 and WO 95 / 01426). Exemplary peroxidases and / or oxidases can be chemically modified or genetically engineered variants. Examples of peroxidases / oxidases include, but are not limited to, those of plant, bacterial, or fungal origin.

[0132] Another embodiment is directed to a composition comprising one or more subtilisin variants described herein and one or more perhydrolases, e.g., as described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214 and WO 2008 / 106215.

[0133] Another embodiment relates to a composition comprising one or more subtilisin variants described herein and a modified polysaccharide biopolymer having the benefit of improved cleaning / whitening or fabric surface modification.

[0134] In yet another embodiment, one or more of the subtilisin variants described herein and one or more additional enzymes contained in one or more of the compositions described herein may each independently range up to about 10% of the composition, with the remainder of the cleaning composition being one or more adjuncts.

[0135] In some embodiments, one or more compositions described herein are used as detergent additives, and the additives are in solid or liquid form. Such additive products are intended to supplement and / or enhance the performance of conventional detergent compositions and can be added at any stage of the cleaning process. In some embodiments, the laundry detergent compositions have a density ranging from about 400 to about 1200 g / liter, measured at 20°C, while in other embodiments, the density ranges from about 500 to about 950 g / liter.

[0136] Some embodiments are directed to laundry detergent compositions comprising one or more subtilisin variants described herein and one or more adjuncts selected from the group consisting of surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime soap dispersants, soil suspending agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry composition also contains a fabric softener.

[0137] Further embodiments are directed to manual dishwashing compositions comprising one or more subtilisin variants described herein and one or more adjuvants selected from surfactants, organic polymeric compounds, suds boosters, Group II metal ions, solvents, hydrotropes, and additional enzymes.

[0138] Other embodiments are directed to one or more compositions described herein, wherein the composition is a compact granular fabric cleaning composition or a heavy duty liquid (HDL) fabric cleaning composition for use in laundering colored fabrics or providing softness through cleaning performance. Examples of fabric cleaning compositions and / or methods for making the same are described in U.S. Patent Nos. 6,610,642 and 6,376,450. Other examples of cleaning compositions are described in, for example, U.S. Patent Nos. 6,605,458; 6,294,514; 5,929,022; 5,879,584; 5,691,297; 5,565,145; 5,574,005; 5,569,645; 5,565,42 ... ,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.

[0139] In some embodiments, the cleaning composition comprises acidified particles or aminocarboxylic acid builders. Examples of aminocarboxylic acid builders include aminocarboxylic acids, aminocarboxylic acid salts, and derivatives thereof. In some embodiments, the aminocarboxylic acid builders are aminopolycarboxylic acid builders, such as those of the general formula MOOC-CHR-N(CHCOOM)2, where R is C 1~12and M is an alkali metal), glycine-N,N-diacetic acid or derivatives thereof. In some embodiments, the aminocarboxylic acid builder is methylglycine diacetate (MGDA), GLDA (glutamic acid-N,N-diacetic acid), iminodisuccinic acid (IDS), carboxymethyl inulin and salts and derivatives thereof, aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N- The acidifying agent may be (2-sulfoethyl)glutamic acid (SEGL), iminodiacetic acid (IDA), its salts and derivatives, such as N-methyliminodiacetic acid (MIDA), alpha-alanine-N,N-diacetic acid (alpha-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), as well as alkali metal salts and derivatives thereof. In some embodiments, the acidifying particles have a weighted geometric mean particle size of about 400 μm to about 1200 μm and a bulk density of at least 550 g / L. In some embodiments, the acidifying particles comprise at least about 5% builder.

[0140] In some embodiments, the acidified particles can contain any acid, including organic and mineral acids. The organic acid can have one or two carboxyls, and in some cases up to 15 carbon atoms, particularly up to 10 carbon atoms, such as formic acid, acetic acid, propionic acid, capric acid, oxalic acid, succinic acid, adipic acid, maleic acid, fumaric acid, sebacic acid, malic acid, lactic acid, glycolic acid, tartaric acid, and glyoxylic acid. In some embodiments, the acid is citric acid. Mineral acids include hydrochloric acid and sulfuric acid. In some cases, the acidified particles are high-activity particles containing a high concentration of aminocarboxylic acid builder. Sulfuric acid has also been found to further contribute to the stability of the microparticles.

[0141] Further embodiments are directed to cleaning compositions comprising one or more subtilisin variants and one or more surfactants and / or surfactant systems, wherein the surfactants are selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some embodiments, the surfactants are present in a concentration of from about 0.1% to about 60% by weight of the cleaning composition; in alternative embodiments, the concentration is from about 1% to about 50% by weight; and in still further embodiments, the concentration is from about 5% to about 40% by weight.

[0142] In some embodiments, one or more compositions described herein comprise one or more detergent builders or builder systems. In one embodiment, the composition comprises 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. Examples of builders include, but are not limited to, alkali metal, ammonium, and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth metal carbonates, and alkali metal carbonates, aluminosilicates, polycarboxylate compounds, etherhydroxypolycarboxylates, copolymers of maleic anhydride and ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid and carboxymethyloxysuccinic acid, ammonium and substituted ammonium salts of polyacetic acid (e.g., ethylenediaminetetraacetic acid and nitrilotriacetic acid), polycarboxylates (e.g., mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid), and soluble salts thereof. In some such compositions, the builder forms water-soluble hardness ion complexes (e.g., sequestering builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate, sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixtures of sodium tripolyphosphate and potassium tripolyphosphate). Examples of builders are described, for example, in EP 2100949. In some embodiments, the builder includes a phosphate builder and a non-phosphate builder. In some embodiments, the builder is a phosphate builder. In some embodiments, the builder is a non-phosphate builder. In some embodiments, the builder comprises a mixture of a phosphate builder and a non-phosphate builder. Examples of phosphate builders include monophosphates, diphosphates, triphosphates, or oligomeric polyphosphates, such as alkali metal salts of these compounds, e.g., sodium salts. In some embodiments, the builder may be sodium tripolyphosphate (STPP).Additionally, the composition may contain carbonates and / or citrates. Other suitable non-phosphate builders include homopolymers and copolymers of polycarboxylic acids and their partially or fully neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids, and their salts. In some embodiments, salts of the above compounds include ammonium and / or alkali metal salts, i.e., lithium, sodium, and potassium salts, such as sodium salts. Suitable polycarboxylic acids include acyclic, alicyclic, heterocyclic, and aromatic carboxylic acids, which in some embodiments may contain at least two carboxyl groups, each separated from the other by, in some cases, no more than two carbon atoms.

[0143] In some embodiments, one or more compositions described herein include one or more chelating agents. In one embodiment, the composition includes from about 0.1% to about 15% or from about 3% to about 10% chelating agent by weight of the composition. Examples of chelating agents include, but are not limited to, copper, iron, manganese, and mixtures thereof.

[0144] In some embodiments, one or more compositions described herein comprise one or more deposition aids. Examples of deposition aids include, but are not limited to, polyethylene glycol; polypropylene glycol; polycarbonates; soil release polymers such as polyethylene terephthalate; clays such as kaolinite, montmorillonite, attapulgite, illite, bentonite, and halloysite, and mixtures thereof.

[0145] In other embodiments, one or more compositions described herein include one or more anti-redeposition agents or nonionic surfactants that can prevent soil redeposition (see, e.g., EP 2100949). For example, in ADW compositions, nonionic surfactants can be used for surface modification purposes, particularly for spreading, preventing filming and staining, and improving gloss. These nonionic surfactants can also be used to prevent soil redeposition. In some embodiments, the nonionic surfactant can be an ethoxylated nonionic surfactant, an epoxy-capped poly(oxyalkylated) alcohol, or an amine oxide surfactant.

[0146] In some embodiments, one or more compositions described herein comprise one or more dye transfer inhibitors. Examples of polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone, polyvinylimidazole, and mixtures thereof. In one embodiment, the composition comprises from about 0.0001% to about 10%, from about 0.01% to about 5%, or from about 0.1% to about 3% of a dye transfer inhibitor by weight of the composition.

[0147] In some embodiments, one or more compositions described herein comprise one or more silicates. Examples of silicates include, but are not limited to, sodium silicates, such as sodium disilicate, sodium metasilicate, and crystalline phyllosilicates. In some embodiments, the silicate is present at a concentration of about 1% to about 20% or about 5% to about 15% by weight of the composition.

[0148] In some still further embodiments, one or more compositions described herein comprise one or more dispersants. Examples of water-soluble organic materials include, but are not limited to, homopolymeric or copolymeric acids or salts thereof, such as polycarboxylic acids containing at least two carboxyl groups separated from each other by no more than two carbon atoms.

[0149] In some further embodiments, one or more compositions described herein comprise one or more enzyme stabilizers. In some embodiments, the enzyme stabilizer is a water-soluble source of calcium and / or magnesium ions. In some embodiments, enzyme stabilizers include oligosaccharides, polysaccharides, and inorganic divalent metal salts, e.g., alkaline earth metal salts such as calcium salts. In some embodiments, the enzymes used herein are stabilized by the presence in the final composition of a water-soluble source of zinc(II), calcium(II), and / or magnesium(II) ions to provide such ions to the enzyme, as well as other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and oxovanadium(IV)). Chlorides and sulfates are also used in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described, for example, in WO 07 / 145964. In some embodiments, reversible protease inhibitors, such as boron-containing compounds (e.g., borates, 4-formylphenylboronic acid and phenylboronic acid derivatives (e.g., those described in WO 96 / 41859) and / or peptide aldehydes, such as those further described in WO 2009 / 118375 and WO 2013 / 004636, are also used.

[0150] Peptide aldehydes can be used as protease stabilizers in detergent formulations, as previously described (WO 1998 / 13458, WO 2011 / 036153, U.S. Patent Application Publication No. 2014 / 0228274). Examples of peptide aldehyde stabilizers are peptide aldehydes, ketones, or halomethyl ketones, which can be "N-capped" with, for example, a ureido, carbamate, or urea moiety, or "doubly N-capped" with, for example, a carbonyl, ureido, oxamide, thioureido, dithiooxamide, or thiooxamide moiety (EP 2358857B1). The molar ratio of these inhibitors to protease can be 0.1:1 to 100:1, for example, 0.5:1 to 50:1, 1:1 to 25:1, or 2:1 to 10:1. Other examples of protease stabilizers are benzophenone or benzoanilide derivatives, which may contain carboxyl groups (U.S. Pat. No. 7,968,508 B2). The molar ratio of these stabilizers to protease is preferably in the range of 1:1 to 1000:1, in particular 1:1 to 500:1, particularly preferably 1:1 to 100:1, and most particularly preferably 1:1 to 20:1.

[0151] 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. Examples of 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, the inorganic perhydrate salts are present as crystalline solids without additional protection, while in other embodiments, the salts are coated. Bleach activators are typically organic peracid precursors that enhance bleaching activity during cleaning processes at temperatures of 60°C or less. Examples of bleach activators include compounds that, under perhydrolysis conditions, yield aliphatic peroxycarboxylic acids having from about 1 to about 10 carbon atoms or from about 2 to about 4 carbon atoms and / or optionally substituted perbenzoic acids. Examples of bleach activators are described, for example, in EP 2100949. Examples of bleach catalysts include, but are not limited to, manganese triazacyclononane complexes and related complexes, as well as cobalt, copper, manganese, and iron complexes. Further examples of bleach catalysts are described, for example, in U.S. Pat. No. 4,246,612; U.S. Pat. No. 5,227,084; U.S. Pat. No. 4,810,410; WO 99 / 06521; and EP 2100949.

[0152] In some embodiments, one or more compositions described herein contain one or more catalytic metal complexes. In some embodiments, a metal-containing bleach catalyst is used. In some embodiments, the metal bleach catalyst comprises a catalyst system containing a transition metal cation of predetermined bleach catalytic activity (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation), an auxiliary metal cation (e.g., zinc or aluminum cation) with little or no bleach catalytic activity, and a sequestering agent having a predetermined stability constant for the catalyst and auxiliary metal cation, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and water-soluble salts thereof (see, e.g., U.S. Pat. No. 4,430,243). In some embodiments, one or more compositions described herein are catalyzed by a manganese compound. Such compounds and amounts used are described, for example, in U.S. Pat. No. 5,576,282. In further embodiments, a cobalt bleach catalyst is used and included in one or more compositions described herein. Various cobalt bleach catalysts are described, for example, in US Pat. Nos. 5,597,936 and 5,595,967.

[0153] In some further embodiments, one or more compositions described herein comprise a transition metal complex of a macropolycyclic rigid ligand (MRL). As a practical matter, and not by way of limitation, in some embodiments, the compositions and cleaning processes described herein are adjusted to provide an active MRL in the cleaning solution of at least about 0.005 ppm to about 25 ppm, about 0.05 ppm to about 10 ppm, or about 0.1 ppm to about 5 ppm, on the order of parts per billion. Examples of MRLs include, but are not limited to, specialized bridged ultrarigid ligands such as 5,12-diethyl-1,5,8,12-tetraazabicyclo(6.6.2)hexadecane. Examples of metal MRLs are described, for example, in WO 2000 / 32601 and U.S. Pat. No. 6,225,464.

[0154] In another embodiment, one or more compositions described herein include one or more metal care agents. In some embodiments, the composition includes about 0.1% to about 5% by weight of the composition of the metal care agent. Examples of metal care agents include, for example, aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). Additional examples of metal care agents are described, for example, in EP 2100949, WO 94 / 26860, and WO 94 / 26859. In some compositions, the metal care agent is a zinc salt.

[0155] In some embodiments, the cleaning composition is a heavy-duty liquid (HDL) composition comprising one or more subtilisin variants described herein. HDL liquid laundry detergents may comprise a detersive surfactant (10% to 40%), which is selected from the group consisting of anionic detersive surfactants (straight-chain, branched-chain, or random-chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof); and an optional nonionic surfactant (straight-chain, branched-chain, or random-chain, substituted or unsubstituted alkyl alkoxylated alcohols, e.g., C8 to C9). 18 Alkyl ethoxylated alcohol and / or C6-C 12 alkylphenol alkoxylates), optionally with a weight ratio of anionic detersive surfactants (having a hydrophilicity index (HIc) of 6.0 to 9) to nonionic detersive surfactants of greater than 1:1. Suitable detersive surfactants also include cationic detersive surfactants (selected from the group of alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary 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.

[0156] In another embodiment, the cleaning composition may be aqueous, typically a non-unit-dose liquid or gel detergent, containing at least 20% to 95% water by weight, e.g., up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, or up to about 35% water by weight. Other types of liquids, including but not limited to alkanols, amines, diols, ethers, and polyols, may be included in the aqueous liquid or gel. The aqueous liquid or gel detergent may contain 0-30% organic solvent. The liquid or gel detergent may be non-aqueous.

[0157] The composition optionally comprises an amphiphilic alkoxylated grease cleaning polymer selected from the group of branched hydrophilic and hydrophobic alkoxylated polymers, such as an alkoxylated polyalkyleneimine and / or random graft polymer in the range of 0.05% to 10% by weight (typically a hydrophilic backbone comprising monomers selected from the group consisting of unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, saturated polyalcohols such as maleic anhydride, glycerol, and mixtures thereof, and a C4-C6 25 The polymer may comprise a surfactant-enhancing polymer having hydrophobic side chains selected from the group consisting of alkyl groups, polypropylene, polybutylene, vinyl esters of saturated C2-C6 monocarboxylic acids, C1-C6 alkyl esters of acrylic or methacrylic acid, and mixtures thereof.

[0158] The composition may further comprise an additional polymer, such as a soil release polymer (e.g., an anionically end-capped polyester, e.g., SRP1; a polymer comprising at least one monomer unit selected from saccharides, dicarboxylic acids, polyols, and combinations thereof, in a random or block configuration; an ethylene terephthalate-based polymer and copolymers thereof, e.g., Repel-o-tex SF, SF-2, and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325, Marloquest SL); anti-redeposition polymers (0.1% to 10% by weight, for example, carboxylic acid polymers, for example, polymers containing 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; vinylpyrrolidone homopolymer and / or polyethylene glycol, having a molecular weight in the range of 500 to 100,000 Da); cellulose polymers (for example, alkyl celluloses; alkyl alkoxyalkyl celluloses; carboxyalkyl celluloses; alkyl carboxyalkyl celluloses (examples of which include carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, and methylcarboxymethyl cellulose); and mixtures thereof), and polymeric carboxylates (for example, maleate / acrylate random copolymers or polyacrylate homopolymers).

[0159] The composition comprises a saturated or unsaturated fatty acid, preferably a saturated or unsaturated C 12 ~C 24It may further include fatty acids (0-10% by weight); deposition aids (including, for example, polysaccharides, cellulose polymers, polydiallyldimethylammonium halide (DADMAC) and random or block copolymers of DADMAC with vinylpyrrolidone, acrylamide, imidazole, imidazolinium halide, and mixtures thereof); cationic guar gum, cationic cellulose, such as cationic hydroxyethyl cellulose; cationic starch, cationic polyacrylamide; and mixtures thereof.

[0160] The compositions may contain dye transfer inhibitors (examples of which include manganese phthalocyanine, peroxidase, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone and polyvinylimidazole, 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), propylene glycol diisopropyl ether (PG ... The surfactants may further include methylglycine diacetate (MGDA), glutamic acid N,N-diacetic acid (including N,N-dicarboxymethylglutamic acid tetrasodium salt (GLDA), nitrilotriacetic acid (NTA), 4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any salts thereof, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), and derivatives thereof).

[0161] The composition may further comprise silicone or fatty acid based suds suppressors; hue dyes, calcium and magnesium cations, visual signaling components, antifoaming agents (0.001 to about 4.0% by weight) and / or structurants / thickeners (0.01 to 5% by weight) selected from the group consisting of diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulosic materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.

[0162] In some embodiments, the cleaning composition is a heavy duty powder (HDD) composition comprising one or more subtilisin variants described herein. HDD powder laundry detergents are characterized by the presence of detersive surfactants, such as anionic detersive surfactants (selected from linear, branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof), nonionic detersive surfactants (linear, branched or random chain, substituted or unsubstituted C8-C9 18 Alkyl ethoxylate and / or C6-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 sulfo-betaines); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof; builders (phosphate-free builders, e.g., zeolite builders (examples of which include zeolite A, zeolite B, zeolite C, zeolite D, zeolite E, zeolite F, zeolite G, zeolite H, zeolite I ... phosphate builders, such as sodium tripolyphosphate (in the range of 0 to less than 10% by weight), citric acid, citrates, and nitrilotriacetic acid or its salts (in the range of less than 15% by weight); silicates (in the range of 0 to less than 10% by weight of sodium or potassium silicate or sodium metasilicate or layered silicate (SKS-6)); carbonates (in the range of 0 to less than 10% by weight of sodium carbonate and / or sodium bicarbonate); and bleaching agents (photobleaches, e.g., sulfones sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes and mixtures thereof; hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED and nonanoyloxybenzenesulfonate-NOBS, nitrile quats and mixtures thereof); hydrogen peroxide; hydrogen peroxide sources (inorganic perhydrate salts, e.g., peroxides) monohydrate or tetrahydrate of sodium salts of peracid, percarbonate, persulfate, perphosphate or persilicic acid; preformed hydrophilic and / or hydrophobic peracids (selected from percarboxylic acids and salts, percarbonates and salts, perimidic acids and salts, peroxymonosulfates and salts, and mixtures thereof); and / or bleach catalysts (e.g., imine bleach accelerators, such as iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulfonylimines; N-phosphonylimines; N-acylimines; thiadiazole dioxides; perfluoroimines;cyclic sugar ketones and mixtures thereof), metal-containing bleach catalysts (e.g., cations of copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese with auxiliary metal cations such as zinc or aluminum, and sequestering agents such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and water-soluble salts thereof);

[0163] The composition may further comprise additional detergent ingredients such as perfume microcapsules, starch encapsulated perfume accords, hueing agents, additional polymers including fabric integrity and cationic polymers, dielock components, fabric softeners, brighteners (e.g., CI optical brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids, and / or cyclodextrins.

[0164] In some embodiments, the cleaning composition comprising one or more subtilisin variants described herein is a detergent composition selected from the group consisting of laundry detergents, soap bars, fabric softeners, dishwashing detergents, medical device detergents, and hard surface cleaning detergents.

[0165] In some embodiments, the present invention is directed to detergent compositions comprising at least two proteases in combination with one or more additional cleaning composition ingredients, such as, but not limited to, the liquid laundry compositions described in WO 2022 / 106404.

[0166] In some embodiments, one or more subtilisin variants described herein may be selected from the group consisting of, but not limited to, liquid laundry compositions described in U.S. Pat. No. 1,046,919 B2, WO 2021 / 223552, WO 2022 / 167251, WO 2022 / 074037, WO 2021 / 123184, WO 2021 / 037895, WO 2022 / 10372, WO 2020 / 264077, WO 2022 / 106404 and / or WO 2017 / 54983; compact liquid laundry compositions (U.S. Pat. No. 10,683,474 B2); fatty alkyl ethers; The present invention may be part of or added to liquid laundry detergent compositions such as water-soluble unit dose products comprising steralkoxylate nonionic surfactants and alkoxylated alcohol nonionic surfactants (U.S. Patent Application Publication No. 2022 / 0162523A1); liquid laundry detergent compositions comprising improved alkyl benzene sulfonate surfactants (WO 2021 / 108307); liquid laundry detergent compositions comprising benzyl benzoate (WO 2020 / 223959) and / or detergent compositions comprising branched surfactants (WO 2021 / 247801); water-soluble unit dose products comprising amphiphilic graft polymers and polyester terephthalates (WO 2019 / 032257).

[0167] In some embodiments, 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 to provide softening benefits (WO 2013 / 087286).

[0168] 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).

[0169] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition described in US Patent Application Publication No. 2021 / 0317387A1, WO 2021 / 219296, WO 2021 / 127662, WO 2021 / 041685, US Pat. No. 11208619, or US Pat. Appl. No. 2022 / 0186144.

[0170] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition comprising a dispersin variant, such as, but not limited to, the liquid laundry detergent compositions described in U.S. Patent Application Publication No. 2021 / 0317387A1.

[0171] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition comprising a highly alkaline fabric detergent, such as, but not limited to, the liquid laundry detergent compositions described in WO 2021 / 219296.

[0172] In some embodiments, the cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition comprising a low-density unit dose detergent comprising an encapsulated perfume, such as, but not limited to, the detergent compositions described in WO 2021 / 127662.

[0173] 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 compositions described in WO 2021 / 041685.

[0174] 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 compositions described in WO 2021 / 041685.

[0175] In some embodiments, cleaning compositions comprising one or more subtilisin variants described herein are detergent compositions effective against protein stains, such as, but not limited to, those described in U.S. Pat. No. 1,120,8619.

[0176] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a detergent composition containing a soil release polymer, such as, but not limited to, the detergent compositions described in U.S. Patent Application Publication No. 2022 / 0186144.

[0177] Examples of laundry detergent compositions include those provided in the Examples or Tables below. [Table B]

[0178] In some embodiments, the cleaning composition is an ADW detergent composition comprising one or more subtilisin variants described herein. The ADW detergent composition comprises two or more nonionic surfactants selected from ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohols, and amine oxide surfactants, present in an amount of 0-10% by weight; a builder in the range of 5-60% by weight, including a phosphate (monophosphate, diphosphate, tripolyphosphate, or oligomeric polyphosphate), sodium tripolyphosphate -STPP, or a phosphate-free builder (amino acid-based compounds, such as MGDA (methyl-glycine-diacetic acid) and its salts and derivatives, GLDA (glutamic acid-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 their salts); a builder in the range of 0.5-50% by weight, including a polycarboxylic acid and its partially or fully neutralized salt, a monomeric polycarboxylic acid, or a carboxylic acid-based compound in the range of 5-60% by weight. , homopolymers and copolymers of hydroxycarboxylic acids and their salts; sulfonated / carboxylated polymers in the range of about 0.1 to about 50% by weight (which impart dimensional stability to the product); drying aids in the range of about 0.1 to about 10% by weight (polyesters, especially anionic polyesters (optionally with monomers having 3 to 6 functional groups leading to polycondensation, especially with acid, alcohol or ester functional groups), polycarbonate-, polyurethane- and / or polyurea-polyorganosiloxane compounds or reactive cyclic carbonates and ureas). These include: silicates (sodium or potassium silicates, e.g., sodium disilicate, sodium metasilicate, and crystalline phyllosilicates) in the range of about 1 to about 20% by weight; bleaches—inorganic (e.g., perhydrate salts such as perborates, percarbonates, perphosphates, persulfates, and persilicates) and organic (e.g., organic peracids, including diacyl peroxides and tetraacyl peroxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); and about 0.a bleach activator-organic peracid precursor in the range of 1 to about 10% by weight; a bleach catalyst (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn, and Fe bispyridylamine and related complexes, and cobalt(III) pentamine acetate and related complexes); a metal care agent (selected from benzatriazoles, metal salts and complexes, and silicates) in the range of about 0.1 to 5% by weight; an enzyme (acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactosidase ... tannase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, peroxidase, phenoloxidase, phosphatase, phospholipase, phytase, polyesterase, polygalacturonase, additional protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and mixtures thereof; and an enzyme stabilizer component (selected from oligosaccharides, polysaccharides, and inorganic divalent metal salts).

[0179] Exemplary ADW compositions are shown in the table below. [Table C]

[0180] In more embodiments, compositions and methods are also provided for treating fabric (e.g., for desizing woven fabrics) using one or more subtilisin variants described herein. Methods for treating fabric are well known in the art (see, e.g., U.S. Pat. No. 6,077,316). For example, the feel and appearance of fabric can be improved by a method comprising contacting the fabric in a solution with a variant described herein. The fabric can be treated with the solution under pressure.

[0181] One or more subtilisin variants described herein can be applied during or after weaving of fibers, in the desizing stage, or in one or more additional fabric processing steps. During weaving of woven fabrics, yarns are subjected to significant mechanical strain. Before weaving on a loom, warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. One or more subtilisin variants described herein can be applied during or after weaving to remove these sizing starches or starch derivatives. After weaving, the variants can be used to remove the sizing coating before further processing of the fabric to ensure a uniform and wash-resistant result. One or more subtilisin variants described herein can be used as detergent additives, for example, in aqueous compositions, alone or in combination with other desizing agents and / or enzymes, for desizing fabrics, such as cotton-containing fabrics. Amylases can also be used in compositions and methods for creating a stonewashed look on indigo-dyed denim fabrics and garments. To produce garments, fabrics can be cut and sewn into garments or garments, which are then finished. Various enzymatic finishing methods have been developed, particularly for producing denim jeans. Denim garment finishing typically begins with an enzymatic desizing process, during which the garment is subjected to the action of proteolytic enzymes to soften the fabric and make the cotton more accessible to subsequent enzymatic finishing steps. One or more subtilisin variants described herein can be used in denim garment finishing (e.g., "bio-stoning"), enzymatic desizing, and fabric softening and / or finishing processes.

[0182] The present disclosure also provides methods of cleaning the surface of an article, the method comprising contacting the article with at least one subtilisin variant provided herein (or a composition comprising such a subtilisin variant). In some embodiments, the article may have, for example, a proteinaceous stain on its surface. In some embodiments, the proteinaceous stain may include egg or egg-based stains, such as creme brûlée, baked cheese, BMI, or other protein-containing substances.

[0183] Non-limiting examples of the compositions and methods disclosed herein are as follows. 1. A composition comprising a subtilisin variant in combination with at least one additional enzyme, wherein the subtilisin 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, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 9, and wherein the at least one additional enzyme is an acyl Transferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase ronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polygala 1. The detergent composition of claim 1, wherein the at least one additional enzyme is selected from the group consisting of: subtilisin, polyesterase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof, and wherein the at least one additional enzyme has increased residual activity compared to a reference enzyme in a detergent composition that does not contain the subtilisin variant.

[0184] 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 the parent subtilisin, the substitutions being selected from the group consisting of X96D, X103F, X108Q, X115L, X128K, X129Q and X181Q, and the amino acid positions of the variant are numbered according to the amino acid sequence of the parent enzyme of SEQ ID NO: 9, and the variant has at least 75 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 9. % identity, and at least one additional enzyme is an acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemistearate, or the like. enzymes, such as hydroxylase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phenoloxidase, phospholipase, phosphodiesterase, phospholip ... 10. The detergent composition of claim 1, wherein the at least one additional enzyme is selected from the group consisting of: α-glucanase, β ...

[0185] 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.

[0186] 3. Any of the compositions of embodiments 1, 1b and 2, 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 positions are numbered according to SEQ ID NO: 9.

[0187] 4. The composition of embodiment 3, wherein said one or more additional amino acid substitutions in said subtilisin are selected from the group consisting of P009T, Q017H, T077N, T078I, Y103I, G127T, G165Q, N184Q, A202V, G203E, G203N, N217S and S258P.

[0188] 5. The composition of embodiment 1, wherein the subtilisin variant is selected from the group consisting of SEQ ID NOs: 1-8.

[0189] 6. Any of the compositions of embodiments 1, 1b and 2-5, wherein the subtilisin variant comprises an amino acid sequence having less than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 9.

[0190] 7. Any of the compositions of embodiments 1, 1b and 2-6, 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 one combination thereof.

[0191] 8. Any of the compositions of embodiments 1, 1b and 2-7, wherein the at least one additional enzyme is an alpha-amylase.

[0192] 9. Any of the compositions of embodiments 1, 1b and 2-8, wherein the at least one additional enzyme is a mannanase.

[0193] 10. Any of the compositions of embodiments 1, 1b, and 2-9, wherein the composition is a detergent composition selected from the group consisting of laundry detergents, fabric softeners, dishwashing detergents (e.g., automatic or hand dishwashing detergents), medical device detergents, and hard surface cleaning detergents.

[0194] 11. A method of cleaning, comprising contacting a surface or item in need of cleaning with an effective amount of a composition of any one of embodiments 1-10, and optionally further comprising rinsing the surface or item after contacting the surface or item with the composition of any one of embodiments 1-10.

[0195] 12. The method of embodiment 11, wherein the article is tableware, fabric, or a medical device. [Example]

[0196] Stability of coenzymes in laundry detergents in the presence of proteases. The in-detergent enzymatic activity of auxiliary enzymes (amylase and mannanase) was evaluated in the presence of various subtilisin proteases. The effects of the commercial proteases PREFERENZ® P300 (IFF) and Progress UNO® 100L (Novozymes) were compared to the effects of experimental protease samples: variants 1, 2, 3, 4, 5, 6, 7, and 8 (SEQ ID NOs: 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. [Table 1]

[0197] The experimental proteases SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 8 were prepared by concentrating culture supernatants from B. licheniformis strains using ultrafiltration and diafiltration against water.

[0198] The stability of accessory enzymes such as amylase and mannanase variants in the presence of proteases was measured by adding the enzymes to detergents at the following loading levels: 0.06% protease, 0.007% amylase, and 0.003% mannanase by weight based on active protein. Detergent / enzyme samples were incubated in sealed containers at 37°C for the indicated storage times. At each time point, samples were removed from the incubator, mixed (by rotation) at room temperature, and an aliquot was removed for enzyme activity analysis.

[0199] Amylase activity The amylase activity of Stainzyme® 12L Amylase (Novozymes) was tested by measuring the release of reducing sugars due to the action of the amylase on an amylopectin substrate. For the amylopectin assay, the reagent solution used was 50 mM MOPS (pH 7.15), 50 mM NaCl, 2.5 mM CaCl (MOPS buffer), and 0.125% amylopectin (amylopectin stock solution) (Sigma: A-8515) in water. The detergent / enzyme sample was diluted in MOPS buffer and added to a microtiter plate (MTP) containing the amylopectin stock solution. The plate was incubated at 40°C for 30 minutes with agitation. The reducing sugars produced during the reaction were determined using a BCA assay (Pierce: 23225) and measured at 562 nm using a SpectraMax plate reader.

[0200] Amylase activity of Amplify® Prime 100L (Novozymes) amylase was tested using Ceralpha HR substrate (Megazyme: AMHR4). Detergent / enzyme samples were diluted in MOPS buffer and added to the MTP-containing Ceralpha HR substrate. The plate was incubated at room temperature for 2 minutes, and the reaction was quenched by the addition of 200 mM borate (pH 10.2). Amylase activity was measured at 405 nm using a SpectraMax plate reader.

[0201] Mannanase activity The mannanase activity of PREFERENZ® M100 (IFF) was tested by measuring the release of reducing sugars due to the action of mannanase on locust bean gum (LBG) substrate. For the LBG assay, the reagent solution used was 100 mM HEPES (pH 8.2), 0.005% Tween-80 (HEPES buffer), and 0.5% LBG stock solution (Sigma: G0753) in 50 mM Tris-HCl (pH 7.15). The detergent / enzyme sample was diluted in HEPES buffer and added to the MTP-containing LBG stock solution. The plate was incubated at 40°C for 20 minutes with agitation. The reducing sugars produced during the reaction were determined using a BCA assay (Pierce: 23225) and measured at 562 nm using a SpectraMax plate reader.

[0202] Enzyme residual activity The percent residual activity of the amylase and mannanase enzymes in Test Detergent A was determined by comparing the activity at the indicated time points (1 week and 4 weeks) to the activity at time = 0 or no incubation.

[0203] The residual activities of PREFERENZ® M100 mannanase and the indicated proteases are shown in Table 2. [Table 2]

[0204] The residual activity of Stainzyme 12L amylase added to detergents in combination with the indicated proteases is shown in Table 3. [Table 3]

[0205] The residual activity of Amplify Prime 100L amylase added to detergents in combination with the indicated proteases is shown in Table 4. [Table 4]

[0206] The results shown in Tables 2, 3 and 4 demonstrate the benefit of the experimental proteases: variants 1-8 (SEQ ID NOs: 1-8) compared to the commercial proteases PREFERENZ P300 (IFF) and Progress UNO 100L (Novozymes) in increasing the residual activity of commercially relevant amylase and mannanase co-enzymes in liquid detergents over a period of at least 4 weeks.

[0207] A time course study of mannanase stability in various protease combinations was conducted in HDL detergent using test detergent A. The stability of the commercially available mannanase PREFERENZ® M100 and the PspMan138 variant mannanase TL1219 (SEQ ID NO: 10, U.S. Provisional Patent Application No. 63 / 403332, filed September 2, 2022) in the detergent was investigated. The following loading levels, based on active protein, were used in the detergent: 0.06% by weight protease, 0.007% by weight amylase, and 0.003% by weight mannanase. Samples were incubated at 37°C, and after the indicated storage times, the percent remaining activity of the mannanase was determined as described above. Table 5 shows the percent (%) residual activity of mannanase PREFERENZ® M 100 and PspMan138 variant mannanase TL1219 (TL1219) in the presence of various proteases. [Table 5]

[0208] In the presence of PREFERENZ® P 300 protease, mannanase TL1219 has a higher % residual activity compared to PREFERENZ® M 100 mannanase. This indicates that TL1219 mannanase is more stable in the presence of commercially relevant proteases. Mannanase TL1219 also shows a higher % residual activity in the presence of protease variant 2 (SEQ ID NO: 2) compared to protease PREFERENZ® P 300. This result demonstrates the advantage of protease variant 2 over commercially available PREFERENZ® P 300.

Claims

1. 1. A composition comprising a subtilisin variant in combination with at least one additional enzyme, wherein the subtilisin variant comprises one, two or more amino acid substitutions selected from the group consisting of X96D, X103F, X108Q, X115L, X128K, X129Q and X181Q, the amino acid positions being numbered according to SEQ ID NO:9, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO:9, and the at least one additional enzyme is Syltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, protease, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polygalactose 1. The detergent composition of claim 1, wherein the at least one additional enzyme is selected from the group consisting of: subtilisin, polyesterase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof, wherein the at least one additional enzyme has increased residual activity compared to a reference enzyme in a detergent composition not comprising the subtilisin variant.

2. 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. 2. 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. 4. The composition of claim 3, wherein the one or more additional amino acid substitutions in 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. 2. The composition of claim 1, wherein the subtilisin variant comprises an amino acid sequence having less than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:

9.

7. 10. 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. 10. The composition of claim 1, wherein the at least one additional enzyme is an alpha-amylase.

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

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

11. 10. A method of cleaning comprising contacting a surface or item requiring cleaning with an effective amount of the composition of claim 1, and optionally further comprising rinsing the surface or item after contacting the surface or item with the composition of claim 1.

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

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