Enzyme-containing detergent compositions

Abstract

A cleaning composition, particularly for laundry, can comprise one or more subtilisin variants and a cleaning adjunct. A method of treating a surface, particularly a fabric, can include contacting the surface with an aqueous wash liquor comprising the cleaning composition. The subtilisin variants can have improved stability and / or soil removal compared to one or more reference subtilisins.

Description

[Technical Field]

[0001] (Reference to sequence listing) The present specification contains a Sequence Listing in computer readable form. The contents of the electronic Sequence Listing in computer readable form (CM05530M_Sequence_listing.xml, 15,692 bytes, created February 1, 2024) are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to cleaning compositions comprising subtilisin variants that are effective in cold water and have improved stability and / or stain / soil removal compared to compositions comprising a reference subtilisin. The present invention also relates to methods of making and using such cleaning compositions. The present invention is particularly useful for cleaning laundry. [Background technology]

[0003] Detergent manufacturers incorporate proteases into their products to provide good cleaning of stains (such as blood). Providing improved cleaning efficacy is always a challenge. Improved cleaning efficacy is achieved by providing a cleaning composition comprising a protease that has good proteolytic activity and preferably also has good stability. Examples of stability include, for example, stability over time in the cleaning composition, such that the protease provides a good concentration of proteolytic cleaning, even after addition to other ingredients in the cleaning composition, and / or under storage and / or transport conditions of the cleaning composition, and / or under conditions in the wash liquor.

[0004] Many well-known procedures 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 comparative assays that analyze the ability of each protease to hydrolyze a commercially available substrate. Exemplary substrates useful in 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).

[0005] Serine proteases are enzymes (EC number 3.4.21) that contain an active-site serine that initiates hydrolysis of peptide bonds in proteins. Serine proteases comprise a diverse class of enzymes with a wide range of specificities and biological functions, which are further divided into chymotrypsin-like (trypsin-like) and subtilisin-like enzymes based on their structure. The prototypical subtilisin (EC number 3.4.21.62) was first isolated from Bacillus subtilis. Subtilisins and their homologs are members of the S8 peptidase family of the MEROPS classification scheme (Rawlings, ND et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates, and inhibitors. Nucleic Acids Res 44, D343-D350). Family S8 members contain a catalytic triad in their amino acid sequence, in the order Asp, His, and Ser. Although many useful protease variants have been developed for cleaning applications, there remains a need for cleaning compositions that provide good levels of proteolytic cleaning, even with the increasing use of lower wash temperatures (e.g., cold water) and shorter wash cycles, which tend to reduce the stain / soil removal effectiveness of detergent compositions. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 99 / 34011 [Patent Document 2] U.S. Patent No. 6,376,450 [Non-patent literature]

[0007] [Non-Patent Document 1] Kalisz, “Microbial Proteinases”, In: Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology, (1988) [Non-patent document 2] Rawlings,NDet al(2016)Twenty years of the MEROPS database of proteolytic enzymes,their substrates and inhibitors.Nucleic Acids Res 44,D343-D350 Summary of the Invention [Problem to be solved by the invention]

[0008] It is therefore an object of the present invention to provide cleaning compositions, especially for laundry, comprising one or more protease enzymes that can be used in the washing process to provide good cleaning, soil / stain removal even at low temperatures and short wash times. [Means for solving the problem]

[0009] The present invention provides cleaning compositions, particularly for laundry, comprising: (a) a subtilisin variant comprising two or more substitutions selected from the group consisting of X006W, X024K, X055P, X109Q, X162Q, X182Q, X183N, X204Q, X206Y, X222Q, X248A, or X254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1; and (b) a cleaning adjunct.

[0010] The invention also provides a method of treating a surface, the method comprising contacting the surface with an aqueous cleaning liquor, the aqueous cleaning liquor comprising a subtilisin variant comprising two or more substitutions selected from the group consisting of X006W, X024K, X055P, X109Q, X162Q, X182Q, X183N, X204Q, X206Y, X222Q, X248A, or X254A, wherein positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1; and a cleaning adjunct. Preferably, the surface is a fabric surface.

[0011] Preferably, the subtilisin variant comprises two substitutions selected from the group consisting of X006W, X024K, X055P, X109Q, X162Q, X182Q, X183N, X204Q, X206Y, X222Q, X248A, or X254A, and one of X003Q, X022Y, X024Q, X033T, X045V, X053G, X076D, X078N, and further comprising one, two or more additional substitutions from the group consisting of X087D, X101N, X118R, X128A, X128S, X145R, X166Q, X169A, X217Q, and X218S, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1.

[0012] The subtilisin variants herein exhibit one or more improved properties, such as improved stability, or both improved cleaning performance and improved stability, when compared to a subtilisin having the amino acid sequence of SEQ ID NO:1.

[0013] The present invention also provides cleaning compositions comprising one or more subtilisin variants, which can be one or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin variants. Preferably, the subtilisin variants comprise two, three, four, or more amino acid substitutions at positions selected from the group consisting of 6, 24, 55, 109, 162, 182, 183, 204, 206, 222, 248, and 254, wherein the positions are numbered according to SEQ ID NO:1, and wherein the variants have at least 75% identity to the amino acid sequence of SEQ ID NO:1.

[0014] Preferably, the parent subtilisin comprises the amino acid sequence of SEQ ID NO: 1, or the parent subtilisin may comprise a polypeptide having the amino acid sequence of SEQ ID NO: 1. The cleaning composition preferably comprises an improved property which is improved stability, and the subtilisin variant has greater residual activity compared to the parent or reference subtilisin. Improved stability can be measured according to the stability assay of Example 2. In yet another embodiment, the improved property is cleaning performance, and the variant has a greater performance index compared to the parent or reference subtilisin. In yet another embodiment, the improved property is cleaning performance as measured according to the stability assay of Example 2.

[0015] The compositions of the present invention comprise one or more subtilisin variants as described herein and preferably have one or more improved properties when compared to a reference or parent subtilisin, the improved properties being selected from improved cleaning performance, improved stability in detergents, and combinations thereof.

[0016] The term "enhanced stability" or "improved stability" in the context of oxidation-, chelating-, denaturing-, detergent-, heat-, and / or pH-stable proteases refers to greater retention of proteolytic activity of a subtilisin variant over time compared to a reference or parent subtilisin protease, e.g., a wild-type protease or parent protease (such as SEQ ID NO: 1). Autolysis has been identified as one mode 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). [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a box plot showing the cumulative contribution of stability-improving substitutions in the BPN′ backbone as a plot of number of mutations versus percent (%) residual activity for the variants listed in Table 6 tested in Detergent C at 51° C. [Figure 2] 1 is a box plot showing the cumulative contribution of stability-improving substitutions in the BPN′ backbone as a plot of number of mutations versus percent (%) residual activity for the variants listed in Table 6 tested in detergent B at 53° C. [Figure 3] 1 is a box plot showing the cumulative contribution of stability-improving substitutions in the BPN′ backbone as a plot of number of mutations versus percent (%) residual activity for the variants listed in Table 6 tested in PNB detergent at 40° C. DETAILED DESCRIPTION OF THE INVENTION

[0018] definition 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. Unless otherwise indicated, nucleic acid sequences are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation. As used herein, each numerical range includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0019] 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" (e.g., 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" (e.g., 1S / T / G, 3G, 17T, etc.) encompasses any starting amino acid that may be present at such position, and one or more amino acids to which such starting amino acid may be substituted. Reference to positions may be recited in several forms, for example, position 003 may also be referred to as position 03 or position 3. Reference to a starting amino acid or a substituting amino acid may further be represented as several starting or substituting amino acids separated by forward 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 recited position.

[0020] The positions of amino acid residues in a given amino acid sequence are numbered corresponding to the amino acid sequence of SEQ ID NO: 1. That is, the amino acid sequence of SEQ ID NO: 1 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: 1 using an alignment algorithm described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with an amino acid residue in SEQ ID NO: 1 is conveniently numbered by reference to the numerical position of its corresponding amino acid residue. For example, a sequence alignment algorithm such as that described herein identifies one or more positions at which insertions or deletions occur in a subject sequence when 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 an amino acid alignment such as that provided in Figure 1 of PCT Publication WO 2018118917.

[0021] The terms "protease" and "proteinase" refer to enzymes capable of degrading proteins and peptides. Proteases have the ability to perform "proteolysis" by hydrolyzing the peptide bonds that link amino acids together in the peptide or polypeptide chains that form proteins. This activity of proteases as protein-digesting enzymes is referred to as "proteolytic activity." Many well-known procedures exist for measuring proteolytic activity. For example, proteolytic activity can be confirmed by comparative assays that analyze the ability of each protease to hydrolyze a suitable substrate. Exemplary substrates useful in 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). The pNA peptidyl assay (see, e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) is also used to determine active enzyme concentration. This assay measures the rate at which p-nitroaniline is released when the enzyme hydrolyzes a soluble synthetic substrate, such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA). The rate of yellow color production from the hydrolysis reaction is measured spectrophotometrically at 405 or 410 nm and is proportional to the active enzyme concentration. Additionally, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a sample of purified protein. Dividing the activity on the substrate by the protein concentration yields the enzyme specific activity.

[0022] As used herein, the term "Bacillus" includes Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus gibsonii, Bacillus spp. ... The term "Bacillus" includes all species within the genus "Bacillus" known to those of skill in the art, including, but not limited to, B. gibsonii, B. sp. TY145, B. patagonienis, B. pumilus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomic reorganization. Thus, the genus is intended to include reclassified species, including, but not limited to, organisms such as B. stearothermophilus, now referred to as "Geobacillus stearothermophilus," or B. polymyxa, now referred to as "Paenibacillus polymyxa."The production of resistant endospores under stressful environmental conditions is considered a defining characteristic of the genus Bacillus, but this characteristic has also been observed in the recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, and Gracilibacillus. acillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, Alkalihalobacillus, Peribacillus, Cytobacillus, Mesobacillus, Neobacillus The genera Neobacillus, Metabacillus, Virgibacillus, as well as the more recently proposed genera Alteribacter, Ectobacillus, Evansella, Ferdinandcohnia, Gottfriedia, Heyndrickxia, Lederbergia, and This also applies to the genera Schinkia, Siminovitchia, Sutcliffiella, Weizmannia, Litchfieldia, Margalitia, Niallia, Priestia, Robertmurraya, Rossellomorea, Schinkia, Siminovitchia, Sutcliffiella and Weizmannia.

[0023] "Bacillus clade subtilisins" includes any subtilisins obtained or derived from a subtilis clade source (strictly the genus Bacillus) as described in Gupta RS, Patel S, Saini N, Chen S (2020) Robust demarcation of 17 distinct Bacillus species clades, which, through phylogenomic and comparative genomic analysis, proposes a new species in the Bacillaceae family: description of Robertmurraya kyonggiensis sp. nov., and proposal for a revised genus Bacillus that restricts the genus to only members of the subtilis and Cereus clades. Int J Syst Evol Microbiol 70:5753-5798, including Bacillus amyloliquefaciens (BPN', WP_013351733.1, CAA24990.1).

[0024] The present invention provides cleaning compositions comprising a "BPN' variant" (or "CAA24990.1 variant" or "WP_013351733.1 variant" or "BPN' subtilisin variant"), wherein the variant is in the mature BPN' amino acid sequence set forth in SEQ ID NO: 1, and / or BPN' subtilisin and variants thereof include polypeptides having amino acids with at least 75% sequence identity to SEQ ID NO: 1. The protease is preferably a serine protease. The protease is preferably a subtilisin. The protease is preferably a subtilisin of the Bacillus clade. Preferably, the subtilisin is from a species of the genus Bacillus. In some embodiments, the protease is from B. amyloliquefaciens (BPN'). In some embodiments, the protease is from B. xiamenensis. In some embodiments, the protease is derived from B. subtilis. Preferably, the variant comprises an amino acid sequence having at least 75%, or at least 80%, or at least 85%, or at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 7, 8, or 9.

[0025] 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, shuttle vectors, and the like. Vectors typically include an origin of replication, a multiple cloning site, and a selectable marker. The process of inserting a vector into a target cell is typically referred to as transformation. The present disclosure includes vectors comprising a DNA sequence encoding a serine protease polypeptide (e.g., a precursor or mature serine protease polypeptide) operably linked to an appropriate prosequence (e.g., secretory, signal peptide sequence, etc.) capable of effecting expression of the DNA sequence in a suitable host, as well as folding and translocation of the recombinant polypeptide chain.

[0026] As used herein in the context of introducing a nucleic acid sequence into a cell, the term "introduced" 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).

[0027] 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," including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0028] The phrase "expression cassette" or "expression vector" refers to a nucleic acid construct or vector produced recombinantly or synthetically 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 typically expresses a protein of interest. An expression vector or expression cassette typically includes a promoter nucleotide sequence that drives or promotes the expression of the foreign nucleic acid. An expression vector or cassette also typically includes other specific nucleic acid elements that enable 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 have the ability to integrate and express heterologous DNA fragments into a host cell or the genome of a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of an appropriate expression vector for the expression of a protein from a nucleic acid sequence incorporated into the expression vector is within the knowledge of one of ordinary skill in the art.

[0029] 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 facilitate translation 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 may be used in accordance with conventional practice.

[0030] The term "gene" refers to a polynucleotide (e.g., a DNA segment) that encodes a polypeptide and includes regions before and after the coding region. In some cases, a gene contains intervening sequences (introns) between individual coding segments (exons).

[0031] The term "recombinant," when used with respect to cells, typically indicates that the cell has been modified by the introduction of a foreign nucleic acid sequence, or that the cell is derived from a cell so modified. For example, a recombinant cell can contain a gene that is not found in the same form within the native (non-recombinant) form of the cell, or a recombinant cell can contain a native gene (as found in the cell's native form) 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 obtained 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 for introducing recombinant DNA into cells where it may be expressed or propagated, thereby producing a recombinant polypeptide. "Recombination" and "recombining" of polynucleotides or nucleic acids generally refer to the assembly or combination of two or more nucleic acids or polynucleotide chains or fragments to generate a new polynucleotide or nucleic acid.

[0032] A nucleic acid or polynucleotide is said to "encode" a polypeptide if, in its native 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.

[0033] The terms "host strain" and "host cell" refer to a suitable host for an expression vector containing a DNA sequence of interest.

[0034] A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. The one-letter and three-letter codes for amino acids as defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure. The single letter X refers to any of the 20 amino acids. It is also understood that a polypeptide can be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0035] The term "prosequence" or "propeptide sequence" refers to an amino acid sequence between the signal peptide sequence and the mature protease sequence that is necessary for proper folding and secretion of the protease. They are sometimes called intramolecular chaperones. Cleavage of the prosequence or propeptide sequence yields the mature active protease. Bacterial serine proteases are often expressed as proenzymes. Examples of modified propeptides are provided, for example, in WO 2016 / 205710.

[0036] The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can be involved in the secretion or direct export of a mature or precursor form of a protein. A signal sequence is typically located at the N-terminus of a precursor or mature protein sequence. A signal sequence can be endogenous or exogenous. A signal sequence is usually absent from a mature protein. A signal sequence is typically cleaved from a protein by a signal peptidase after the protein has been exported.

[0037] The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide without the signal peptide and propeptide sequences.

[0038] 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 involved in post-translational activity (e.g., polypeptides that are cleaved from the precursor to leave the mature form of the protein or peptide).

[0039] The term "wild-type" with respect to a polypeptide refers to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. Similarly, the term "wild-type" with respect to a polynucleotide refers to a naturally occurring polynucleotide that does not contain an artificial substitution, insertion, or deletion at one or more nucleotides. However, a polynucleotide that encodes a wild-type polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes a wild-type or parent polypeptide.

[0040] With respect to polypeptides, the term "parent" includes reference to a naturally occurring or wild-type polypeptide, or a naturally occurring polypeptide into which an artificial substitution, insertion, or deletion has been made at one or more amino acid positions, which serves as a basis for introducing substitutions or further substitutions to produce the mutant enzymes provided herein. With respect to polypeptides, the term "parent" also includes any polypeptide having protease activity that serves as a starting polypeptide for modification, such as substitutions, additions, and / or deletions, to result in a variant having one or more modifications compared to the starting polypeptide. That is, a parent polypeptide, or reference polypeptide, is not limited to naturally occurring wild-type polypeptides, but encompasses any wild-type polypeptide, parent polypeptide, or reference polypeptide. Similarly, with respect to polynucleotides, the term "parent" can refer to a naturally occurring polynucleotide or a polynucleotide that contains an artificial substitution, insertion, or deletion at one or more nucleotides. With respect to polynucleotides, the term "parent" also includes any polynucleotide encoding a polypeptide having protease activity that serves as a starting polynucleotide for modification to result in a variant protease having modifications, such as substitutions, additions, and / or deletions, compared to the starting polynucleotide. That is, a polynucleotide encoding a wild-type polypeptide, a parent polypeptide, or a reference polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes a wild-type polypeptide, a parent polypeptide, or a reference polypeptide. For example, a parent polypeptide herein may include a polypeptide having the amino acid sequence set forth in SEQ ID NO:1.

[0041] The term "naturally occurring" refers, for example, to a sequence and the residues contained therein that are found in nature (e.g., a polypeptide sequence and the amino acid or nucleotide sequence contained therein and the nucleotides contained therein). 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 acid or nucleotide sequence contained therein and the nucleic acid contained therein).

[0042] As used herein with respect to an amino acid residue position, "corresponding to" or "corresponds to" or "corresponds to" refers to the amino acid residue at the recited position in the protein or peptide, or an amino acid residue that is similar, homologous, or equivalent to the recited residue in the protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related or reference protein.

[0043] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by the strain of organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in host organisms containing such DNA sequences. Furthermore, the terms refer to proteins encoded by synthetic and / or cDNA-derived DNA sequences and having the identifying characteristics of the protein in question. By way of example, a "Bacillus-derived protease" 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.

[0044] The term "identical" in the context of two polynucleotide or polypeptide sequences refers to those nucleotides or amino acids in the two sequences that are the same when aligned for maximum correspondence, as determined using sequence comparison or analysis algorithms described below and known in the art.

[0045] The phrase "% identity" or "percent identity" or "PID" refers to protein sequence identity. Percent identity can be determined using standard techniques known in the art. The percent amino acid identity shared by sequences of interest can be determined by aligning sequences to directly compare sequence information, for example, by using programs such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described, for example, in Altschul et al., J Mol Biol, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993). 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.

[0046] 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, for example, using programs such as BLAST, MUSCLE, or CLUSTAL. Homology searches of protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST at 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 fewer than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997 and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). 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 therefrom.Amino acid sequences can be entered into programs such as the Vector NTI Advance suite, and a guide tree can be created using the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol, 4:406-425, 1987). Tree construction can be calculated using Kimura's correction for sequence distances, ignoring positions with gaps. Programs such as AlignX can display calculated distance values ​​in parentheses following the molecule names displayed on the phylogenetic tree. Another useful algorithm for aligning and comparing multiple protein sequences is the MUSCLE program (Robert C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucl. Acids Res. (2004) 32(5):1792-1797), available from Geneious software (Biomatters Ltd.).

[0047] Understanding the homology between molecules can reveal information about the evolutionary history of molecules and their function. If a newly sequenced protein is homologous to a previously characterized protein, this strongly suggests the biochemical function of the new protein. Two molecules are said to be homologous if they are derived from a common ancestor. Homologous molecules, or homologs, can be divided into two classes: paralogs and orthologs. Paralogs are homologs that exist within a single species. Paralogs often differ in their detailed biochemical function. Orthologs are homologs that exist in different species and have very similar or identical functions. Protein superfamilies are the largest groupings (clades) of proteins for which a common ancestor can be inferred. This common ancestor is usually based on sequence alignment and mechanistic similarity. Superfamilies typically contain several protein families, which show 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 MEROPS - The Peptidase Database (Rawlings, ND, et al (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 44, D343-D350).

[0048] 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, delayed divergent sequence % = 40, gap separation distance = 8, DNA transition weight = 0.50, list of hydrophilic residues = GPSNDQEKR, use negative matrix = OFF, toggle residue-specific penalties = ON, toggle hydrophilicity penalties = ON, and toggle terminal gap separation penalties = OFF. The CLUSTAL algorithm includes deletions that occur at either end. For example, a variant with five amino acid deletions at either end of (or within) a 500-amino acid polypeptide has a percent sequence identity of 99% (495 / 500 identical residues × 100) to the "reference" polypeptide. Such variants are encompassed by variants having "at least 99% sequence identity" to the polypeptide.

[0049] A nucleic acid or polynucleotide is "isolated" if it is at least partially or completely separated from other components, including, but not limited to, other proteins, nucleic acids, cells, etc. Similarly, a polypeptide, protein, or peptide is "isolated" if it is 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 more abundant than other species in a composition. For example, an isolated species can comprise at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% (on a molar basis) of all macromolecular species present. Preferably, the species of interest is purified to essential homogeneity (i.e., contaminating species cannot be detected in the composition by conventional detection methods). Purity and homogeneity can be determined using a number of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of nucleic acid or protein samples, respectively, followed by visualization by staining. If desired, high resolution techniques, such as high performance liquid chromatography (HPLC) or similar means, can be used to purify the material.

[0050] The term "purified" as applied to a nucleic acid or polypeptide generally refers to a nucleic acid or polypeptide that is essentially free from other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, and usually 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% or more pure (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule if the concentration of the molecule is significantly increased after application of a purification or concentration technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other particular substance or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0051] The term "cleaning activity" refers to the cleaning performance achieved by a serine protease polypeptide, variant, or reference subtilisin under conditions prevailing during proteolytic, hydrolytic, cleaning, or other processes of the present disclosure. In some embodiments, the cleaning performance of a serine protease or reference subtilisin can be determined by using various assays for cleaning one or more enzyme-sensitive stains on an item or surface (e.g., stains caused by 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 determined by subjecting the stain on the item or surface to standard cleaning conditions and assessing the extent to which the stain is removed by using various chromatographic, spectrophotometric, or other quantitative methodologies. Exemplary cleaning assays and methods are known in the art and include, but are not limited to, those described in WO 99 / 34011 and U.S. Pat. No. 6,605,458, as well as the cleaning assays and methods included in the Examples provided below.

[0052] 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.

[0053] 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 cleaning contribution of one or more subtilisin variants described herein, which provides additional cleaning performance to a detergent compared to a detergent that does not have one or more subtilisin variants described herein added to the composition. Washing performance is compared under relevant washing conditions. In some test systems, other relevant factors such as detergent composition, foam concentration, water hardness, wash mechanism, time, pH, and / or temperature can be controlled to mimic conditions typical for household use in a particular market segment (e.g., hand or manual dishwashing, automatic dishwashing, dishwashing, tableware washing, fabric washing, etc.).

[0054] The phrase "relevant washing conditions" is used herein to indicate the conditions actually used in households in the manual dishwashing, automatic dishwashing or laundry detergent market segments, in particular washing temperature, time, washing mechanism, foam concentration, detergent type and water hardness.

[0055] The term "dishwashing" refers to both domestic and industrial dishwashing, and relates to both automatic dishwashing (eg, in a dishwasher) and manual dishwashing (eg, by hand).

[0056] The term "variant" refers to a polypeptide having enzymatic activity, which includes modifications / mutations, i.e., substitutions, insertions, and / or deletions, at one or more (e.g., several) positions relative to the parent. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid, a deletion refers to the removal of an amino acid occupying a position, and an insertion refers to the addition of 1-3 amino acids adjacent to and immediately following the amino acid occupying a position.

[0057] The term "wild-type" enzyme refers to an enzyme expressed by a naturally occurring microorganism, such as a bacterium, algae, yeast, or filamentous fungus, as found in nature.

[0058] Cleaning Composition The detergent / cleaning compositions of the present invention are cleaning compositions for cleaning hard surfaces such as glass, wood, ceramic and metal counters, windows, ovens, or for dishwashing, including hand or manual dishwashing compositions (e.g., "hand" or "manual" dishwashing detergents) and automatic dishwashing compositions (e.g., "automatic dishwashing detergents"), or for cleaning fabrics such as fabric fresheners, softeners, and for fabrics, laundry booster cleaning or detergent compositions, laundry additive cleaning compositions, and laundry pre-spotter cleaning compositions, or any other cleaning for consumer or institutional use and for hand or machine cleaning (automatic dish or laundry cleaning). The cleaning compositions of the present invention do not include compositions for cleaning contact lenses, or ultrafiltration membranes, or compositions for wound healing, or compositions for the medical treatment of skin conditions.

[0059] Preferably, the composition is a laundry detergent composition or a dishwashing detergent composition, most preferably a laundry detergent, most preferably an automatic laundry detergent. The compositions of the present invention may be solid, liquid, and / or unit-dose detergent compositions, such as solids and / or liquids encapsulated in a water-soluble pouch. Particularly preferred are liquid laundry detergent compositions, optionally encapsulated in a water-soluble material, in the form of a pouch, optionally a multi-component pouch. The term liquid includes gels, solutions, dispersions, pastes, or mixtures thereof. The solid may be a powder, i.e., a solid particulate, or may be a single homogeneous solid or compressed particles. When liquid, the cleaning composition typically contains less than 15% or less than 12% water by weight of the composition. Liquid cleaning compositions may contain a non-aqueous solvent selected from 1,2-propanediol, dipropylene glycol, tripropylene glycol, glycerol, sorbitol, polyethylene glycol, or a mixture thereof, e.g., 10% to 40% or 15% to 30% by weight of the liquid laundry detergent composition is non-aqueous solvent.

[0060] The cleaning composition comprises: (a) a subtilisin variant comprising two or more substitutions selected from the group consisting of X006W, X024K, X055P, X109Q, X162Q, X182Q, X183N, X204Q, X206Y, X222Q, X248A, or X254A, which has at least 75% identity to the amino acid sequence of SEQ ID NO: 1, and wherein positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1; and (b) a cleaning adjunct.

[0061] The detergent compositions of the present invention are preferably laundry detergents. The compositions may be in the form of compositions for use in the main wash step, or as pre-treatment or rinse-added cleaning compositions for consumer or institutional use.

[0062] Subtilisin mutants The cleaning composition comprises one or more subtilisin variants. Preferably, the one or more subtilisin variants are present in the composition in an amount of 0.0001 to 15 mg of active protein, more preferably 0.0005 to 10% by weight of active protein, or 0.001 to 7% by weight of active protein. The subtilisin variants comprise two, three, four, five, or more amino acid substitutions at positions selected from the group consisting of X006W, X024K, X055P, X109Q, X162Q, X182Q, X183N, X204Q, X206Y, X222Q, X248A, and X254A, wherein the positions are numbered according to SEQ ID NO: 1, and the variants have at least 75% identity to the amino acid sequence of SEQ ID NO: 1.

[0063] Preferably, the subtilisin variants are X006W-X024K, X006W-X055P, X006W-X109Q, X006W-X162Q, X006W-X183N, X006W-X182Q, X006W-X204Q, X006W-X206Y, X006W-X222Q, X006W-X248A, X006W-X254A, X024K-X055P, X024K-X109Q, X024K-X162Q, X024K-X183N, X024K-X182Q, X024K-X204Q, X024K-X206Y 4K-X206Y, X024K-X222Q, X024K-X248A, X024K-X254A, X055P-X109Q, X055P-X162Q, 206Y, X055P-X222Q, X055P-X248A, X055P-X254A, X109Q-X162Q, X109Q-X183N, X109Q-X182Q, X109Q-X204Q, X109Q-X206Y, X109Q-X222Q , X109Q-X248A, X109Q-X254A, X162Q-X183N, X162Q-X182Q, X162Q-X204Q, X162Q-X206Y, 3N-X182Q, X183N-X204Q, X183N-X206Y, X183N-X222Q, X183N-X248A, X183N-X254A, X248A, X182Q-X254A, X204Q-X206Y, X204Q-X222Q, X204Q-X248A, X204Q-X254A, X206Y-X222Q, X206Y-X248A, X206Y-X254A, X222Q-X248A, X222Q-X254A, X248A-X254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1.

[0064] The mutants are preferably Y006W-S024K, Y006W-T055P, Y006W-N109Q, Y006W-S162Q, Y006W-S183N, Y006W-S182Q, Y006W-S204Q, Y006W-Q206Y, Y006W-M222Q, Y006W-S248A, Y006W-T254A, S024K-T055P, S024K-N109Q, S024K-S162Q, S024K-S183N, S024K-S182Q, S024K-S204Q, S024K-Q206Y, S024K-M222Q, S024K-S248A , S024K-T254A, T055P-N109Q, T055P-S162Q, T055P-S183N, T055P-S182Q, T 055P-S204Q, T055P-Q206Y, T055P-M222Q, T055P-S248A, T055P-T254A, N109 Q-S162Q, N109Q-S183N, N109Q-S182Q, N109Q-S204Q, N109Q-Q206Y, N109Q- M222Q, N109Q-S248A, N109Q-T254A, S162Q-S183N, S162Q-S182Q, S162Q-S20 4Q, S162Q-Q206Y, S162Q-M222Q, S162Q-S248A, S162Q-T254A, S183N-S182Q , S183N-S204Q, S183N-Q206Y, S183N-M222Q, S183N-S248A, S183N-T254A, S1 82Q-S204Q, S182Q-Q206Y, S182Q-M222Q, S182Q-S248A, S182Q-T254A, S204 Q-Q206Y, S204Q-M222Q, S204Q-S248A, S204Q-T254A, Q206Y-M222Q, Q206Y-S 248A, Q206Y-T254A, M222Q-S248A, M222Q-T254A, S248A-T254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin has a homology of at least 75%, or at least 80%, or at least 81%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, to the amino acid sequence of SEQ ID NO: 1,or an amino acid sequence having at least 97%, or at least 98% amino acid sequence identity.

[0065] In some embodiments, the substitution combinations are X006W-X024K-X055P, X006W-X024K-X109Q, X006W-X024K-X162Q, X006W-X024K-X183N, X006W-X024K-X182Q, X006W-X024K-X204Q, X006W-X024K-X206Y, X006W-X024K-X222Q, X006W-X024K-X248A, X006W-X024K-X254A, X006W-X055P-X109Q, X006W-X055P-X162Q, X006W-X055 P-X183N, X006W-X055P-X182Q, X006W-X055P-X204Q, X006W-X055P-X206Y, X006W-X055P-X222Q, X006W-X055P-X248A, X006W-X055P-X254A, X006W-X1 09Q-X162Q, X006W-X109Q-X183N, X006W-X109Q-X182Q, X006W-X109Q-X204 Q, X006W-X109Q-X206Y, X006W-X109Q-X222Q, X006W-X109Q-X248A, X006W-X 109Q-X254A, X006W-X162Q-X183N, X006W-X162Q-X182Q, X006W-X162Q-X20 4Q, X006W-X162Q-X206Y, X006W-X162Q-X222Q, X006W-X162Q-X248A, X006W -X162Q-X254A, X006W-X183N-X182Q, X006W-X183N-X204Q, X006W-X183N-X 206Y, X006W-X183N-X222Q, X006W-X183N-X248A, X006W-X183N-X254A, X00 6W-X182Q-X204Q, X006W-X182Q-X206Y, X006W-X182Q-X222Q, X006W-X182Q -X248A, X006W-X182Q-X254A, X006W-X204Q-X206Y, X006W-X204Q-X222Q, X 006W-X204Q-X248A, X006W-X204Q-X254A, X006W-X206Y-X222Q, X006W-X20 6Y-X248A, X006W-X206Y-X254A, X006W-X222Q-X248A, X006W-X222Q-X254A,X006W-X248A-X254A、X024K-X055P-X109Q、X024K-X055P-X162Q、X024K-X055P-X183N、X024K-X055P-X182Q、X024K-X055P-X204Q、X024K-X055P-X206Y、X024K-X055P-X222Q、X024K-X055P-X248A、X024K-X055P-X254A、X024K-X109Q-X162Q、X024K-X109Q-X183N、X024K-X109Q-X182Q、X024K-X109Q-X204Q、X024K-X109Q-X206Y、X024K-X109Q-X222Q、X024K-X109Q-X248A、X024K-X109Q-X254A、X024K-X162Q-X183N、X024K-X162Q-X182Q、X024K-X162Q-X204Q、X024K-X162Q-X206Y、X024K-X162Q-X222Q、X024K-X162Q-X248A、X024K-X162Q-X254A、X024K-X183N-X182Q、X024K-X183N-X204Q、X024K-X183N-X206Y、X024K-X183N-X222Q、X024K-X183N-X248A、X024K-X183N-X254A、X024K-X182Q-X204Q、X024K-X182Q-X206Y、X024K-X182Q-X222Q、X024K-X182Q-X248A、X024K-X182Q-X254A、X024K-X204Q-X206Y、X024K-X204Q-X222Q、X024K-X204Q-X248A、X024K-X204Q-X254A、X024K-X206Y-X222Q、X024K-X206Y-X248A、X024K-X206Y-X254A、X024K-X222Q-X248A、X024K-X222Q-X254A、X024K-X248A-X254A、X055P-X109Q-X162Q、X055P-X109Q-X183N、X055P-X109Q-X182Q、X055P-X109Q-X204Q、X055P-X109Q-X206Y、X055P-X109Q-X222Q、X055P-X109Q-X248A、X055P-X109Q-X254A、X055P-X162Q-X183N、X055P-X162Q-X182Q、X055P-X162Q-X204Q、X055P-X162Q-X206Y、X055P-X162Q-X222Q、X055P-X162Q-X248A、X055P-X162Q-X254A、X055P-X183N-X182Q、X055P-X183N-X204Q、X055P-X183N-X206Y、X055P-X183N-X222Q、X055P-X183N-X248A、X055P-X183N-X254A、X055P-X182Q-X204Q、X055P-X182Q-X206Y、X055P-X182Q-X222Q、X055P-X182Q-X248A、X055P-X182Q-X254A、X055P-X204Q-X206Y、X055P-X204Q-X222Q、X055P-X204Q-X248A、X055P-X204Q-X254A、X055P-X206Y-X222Q、X055P-X206Y-X248A、X055P-X206Y-X254A、X055P-X222Q-X248A、X055P-X222Q-X254A、X055P-X248A-X254A、X109Q-X162Q-X183N、X109Q-X162Q-X182Q、X109Q-X162Q-X204Q、X109Q-X162Q-X206Y、X109Q-X162Q-X222Q、X109Q-X162Q-X248A、X109Q-X162Q-X254A、X109Q-X183N-X182Q、X109Q-X183N-X204Q、X109Q-X183N-X206Y、X109Q-X183N-X222Q、X109Q-X183N-X248A、X109Q-X183N-X254A、X109Q-X182Q-X204Q、X109Q-X182Q-X206Y、X109Q-X182Q-X222Q、X109Q-X182Q-X248A、X109Q-X182Q-X254A、X109Q-X204Q-X206Y、X109Q-X204Q-X222Q、X109Q-X204Q-X248A、X109Q-X204Q-X254A、X109Q-X206Y-X222Q、X109Q-X206Y-X248A、X109Q-X206Y-X254A、X109Q-X222Q-X248A、X109Q-X222Q-X254A、X109Q-X248A-X254A、X162Q-X183N-X182Q、X162Q-X183N-X204Q、X162Q-X183N-X206Y、X162Q-X183N-X222Q、X162Q-X183N-X248A、X162Q-X183N-X254A、X162Q-X182Q-X204Q、X162Q-X182Q-X206Y、X162Q-X182Q-X222Q、X162Q-X182Q-X248A、X162Q-X182Q-X254A、X162Q-X204Q-X206Y、X162Q-X204Q-X222Q、X162Q-X204Q-X248A、X162Q-X204Q-X254A、X162Q-X206Y-X222Q、X162Q-X206Y-X248A、X162Q-X206Y-X254A、X162Q-X222Q-X248A、X162Q-X222Q-X254A、X162Q-X248A-X254A、X183N-X182Q-X204Q、X183N-X182Q-X206Y、X183N-X182Q-X222Q、X183N-X182Q-X248A、X183N-X182Q-X254A、X183N-X204Q-X206Y、X183N-X204Q-X222Q、X183N-X204Q-X248A、X183N-X204Q-X254A、X183N-X206Y-X222Q、X183N-X206Y-X248A、X183N-X206Y-X254A、X183N-X222Q-X248A、X183N-X222Q-X254A、X183N-X248A-X254A、X182Q-X204Q-X206Y、X182Q-X204Q-X222Q、X182Q-X204Q-X248A、X182Q-X204Q-X254A、X182Q-X206Y-X222Q、X182Q-X206Y-X248A、X182Q-X206Y-X254A、X182Q-X222Q-X248A、X182Q-X222Q-X254A、X182Q-X248A-X254A、X204Q-X206Y-X222Q、X204Q-X206Y-X248A、X204Q-X206Y-X254A、X204Q-X222Q-X248A、X204Q-X222Q-X254A、X204Q-X248A-X254A、X206Y-X222Q-X248A、X206Y-X222Q-X254A、X206Y-X248A-X254A、and X222Q-X248A-X254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0066] In some embodiments, the substitution combinations are Y006W-S024K-T055P, Y006W-S024K-N109Q, Y006W-S024K-S162Q, Y006W-S024K-S183N, Y006W-S024K-S182Q, Y006W-S024K-S204Q, Y006W-S024K-Q206Y, Y006W-S024K-M222Q, Y006W-S024K-S248A, Y006W-S024K-T254A, Y006W-T055P-N109Q, Y006W-T055P-S162Q, Y006W-T055 P-S183N, Y006W-T055P-S182Q, Y006W-T055P-S204Q, Y006W-T055P-Q206Y, Y006W-T055P-M222Q, Y006W-T055P-S248A, Y006W-T055P-T254A, Y006W-N1 09Q-S162Q, Y006W-N109Q-S183N, Y006W-N109Q-S182Q, Y006W-N109Q-S204 Q, Y006W-N109Q-Q206Y, Y006W-N109Q-M222Q, Y006W-N109Q-S248A, Y006W-N 109Q-T254A, Y006W-S162Q-S183N, Y006W-S162Q-S182Q, Y006W-S162Q-S20 4Q, Y006W-S162Q-Q206Y, Y006W-S162Q-M222Q, Y006W-S162Q-S248A, Y006W -S162Q-T254A, Y006W-S183N-S182Q, Y006W-S183N-S204Q, Y006W-S183N-Q 206Y, Y006W-S183N-M222Q, Y006W-S183N-S248A, Y006W-S183N-T254A, Y00 6W-S182Q-S204Q, Y006W-S182Q-Q206Y, Y006W-S182Q-M222Q, Y006W-S182Q -S248A, Y006W-S182Q-T254A, Y006W-S204Q-Q206Y, Y006W-S204Q-M222Q, Y 006W-S204Q-S248A, Y006W-S204Q-T254A, Y006W-Q206Y-M222Q, Y006W-Q20 6Y-S248A, Y006W-Q206Y-T254A, Y006W-M222Q-S248A, Y006W-M222Q-T254A,Y006W-S248A-T254A、S024K-T055P-N109Q、S024K-T055P-S162Q、S024K-T055P-S183N、S024K-T055P-S182Q、S024K-T055P-S204Q、S024K-T055P-Q206Y、S024K-T055P-M222Q、S024K-T055P-S248A、S024K-T055P-T254A、S024K-N109Q-S162Q、S024K-N109Q-S183N、S024K-N109Q-S182Q、S024K-N109Q-S204Q、S024K-N109Q-Q206Y、S024K-N109Q-M222Q、S024K-N109Q-S248A、S024K-N109Q-T254A、S024K-S162Q-S183N、S024K-S162Q-S182Q、S024K-S162Q-S204Q、S024K-S162Q-Q206Y、S024K-S162Q-M222Q、S024K-S162Q-S248A、S024K-S162Q-T254A、S024K-S183N-S182Q、S024K-S183N-S204Q、S024K-S183N-Q206Y、S024K-S183N-M222Q、S024K-S183N-S248A、S024K-S183N-T254A、S024K-S182Q-S204Q、S024K-S182Q-Q206Y、S024K-S182Q-M222Q、S024K-S182Q-S248A、S024K-S182Q-T254A、S024K-S204Q-Q206Y、S024K-S204Q-M222Q、S024K-S204Q-S248A、S024K-S204Q-T254A、S024K-Q206Y-M222Q、S024K-Q206Y-S248A、S024K-Q206Y-T254A、S024K-M222Q-S248A、S024K-M222Q-T254A、S024K-S248A-T254A、T055P-N109Q-S162Q、T055P-N109Q-S183N、T055P-N109Q-S182Q、T055P-N109Q-S204Q、T055P-N109Q-Q206Y、T055P-N109Q-M222Q、T055P-N109Q-S248A、T055P-N109Q-T254A、T055P-S162Q-S183N、T055P-S162Q-S182Q、T055P-S162Q-S204Q、T055P-S162Q-Q206Y、T055P-S162Q-M222Q、T055P-S162Q-S248A、T055P-S162Q-T254A、T055P-S183N-S182Q、T055P-S183N-S204Q、T055P-S183N-Q206Y、T055P-S183N-M222Q、T055P-S183N-S248A、T055P-S183N-T254A、T055P-S182Q-S204Q、T055P-S182Q-Q206Y、T055P-S182Q-M222Q、T055P-S182Q-S248A、T055P-S182Q-T254A、T055P-S204Q-Q206Y、T055P-S204Q-M222Q、T055P-S204Q-S248A、T055P-S204Q-T254A、T055P-Q206Y-M222Q、T055P-Q206Y-S248A、T055P-Q206Y-T254A、T055P-M222Q-S248A、T055P-M222Q-T254A、T055P-S248A-T254A、N109Q-S162Q-S183N、N109Q-S162Q-S182Q、N109Q-S162Q-S204Q、N109Q-S162Q-Q206Y、N109Q-S162Q-M222Q、N109Q-S162Q-S248A、N109Q-S162Q-T254A、N109Q-S183N-S182Q、N109Q-S183N-S204Q、N109Q-S183N-Q206Y、N109Q-S183N-M222Q、N109Q-S183N-S248A、N109Q-S183N-T254A、N109Q-S182Q-S204Q、N109Q-S182Q-Q206Y、N109Q-S182Q-M222Q、N109Q-S182Q-S248A、N109Q-S182Q-T254A、N109Q-S204Q-Q206Y、N109Q-S204Q-M222Q、N109Q-S204Q-S248A、N109Q-S204Q-T254A、N109Q-Q206Y-M222Q、N109Q-Q206Y-S248A、N109Q-Q206Y-T254A、N109Q-M222Q-S248A、N109Q-M222Q-T254A、N109Q-S248A-T254A、S162Q-S183N-S182Q、S162Q-S183N-S204Q、S162Q-S183N-Q206Y、S162Q-S183N-M222Q、S162Q-S183N-S248A、S162Q-S183N-T254A、S162Q-S182Q-S204Q、S162Q-S182Q-Q206Y、S162Q-S182Q-M222Q、S162Q-S182Q-S248A、S162Q-S182Q-T254A、S162Q-S204Q-Q206Y、S162Q-S204Q-M222Q、S162Q-S204Q-S248A、S162Q-S204Q-T254A、S162Q-Q206Y-M222Q、S162Q-Q206Y-S248A、S162Q-Q206Y-T254A、S162Q-M222Q-S248A、S162Q-M222Q-T254A、S162Q-S248A-T254A、S183N-S182Q-S204Q、S183N-S182Q-Q206Y、S183N-S182Q-M222Q、S183N-S182Q-S248A、S183N-S182Q-T254A、S183N-S204Q-Q206Y、S183N-S204Q-M222Q、S183N-S204Q-S248A、S183N-S204Q-T254A、S183N-Q206Y-M222Q、S183N-Q206Y-S248A、S183N-Q206Y-T254A、S183N-M222Q-S248A、S183N-M222Q-T254A、S183N-S248A-T254A、S182Q-S204Q-Q206Y、S182Q-S204Q-M222Q、S182Q-S204Q-S248A、S182Q-S204Q-T254A、S182Q-Q206Y-M222Q、S182Q-Q206Y-S248A、S182Q-Q206Y-T254A、S182Q-M222Q-S248A、S182Q-M222Q-T254A、S182Q-S248A-T254A、S204Q-Q206Y-M222Q、S204Q-Q206Y-S248A、S204Q-Q206Y-T254A、S204Q-M222Q-S248A、S204Q-M222Q-T254A、S204Q-S248A-T254A、Q206Y-M222Q-S248A、Q206Y-M222Q-T254A、Q206Y-S248A-T254A、and M222Q-S248A-T254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0067] In another embodiment, the subtilisin variants are X003Q, X006W, X022Y, X024K, X024Q, X033T, X045V, X053G, X055P, X076D, X078N, X087D, X101N, X109Q, X118R, X128A, X128S, X145R, X162Q, X166Q, X169A, X182Q, X183N, X204Q, X20 and further comprising two or more substitutions selected from the group consisting of 6Y, X217Q, X218S, X222Q, X248A, or X254A, wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1, and the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1(BPN'), and the variant does not have 100% sequence identity to the naturally occurring amino acid sequence.

[0068] In yet another embodiment, the subtilisin variants are S003Q, Y006W, T022Y, S024K, S024Q, S033T, A045V, S053G, T055P, N076D, S078N, S087D, S101N, N109Q, N118R, G128A, G128S, S145R, S162Q, G166Q, G169A, S182Q, S183N, S204Q, Q2 and further comprising two or more substitutions selected from the group consisting of 06Y, Y217Q, N218S, M222Q, S248A, or T254A, wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1, and the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1(BPN'), and the variant does not have 100% sequence identity to the naturally occurring amino acid sequence.

[0069] Preferably, the variants are S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-S248A-T254A, S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R- S162Q-S183N-Y217Q-S248A-T254A, S003Q-Y006W-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R- G128S-S145R-S162Q-S183N-Y217Q-S248A-T254A, S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N- N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-S248A-T254A, S003Q-S024Q-S033T-A045V-S053G-N076D-S078N- S101N-N109Q-N118R-G128S-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A, S003Q-S024Q-S033T-A045V-S053G- N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-N218S-S248A-T254A, S003Q-T022Y-S024Q- S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-N218S-S248A-T254A,S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-S248A-T254A,S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-S183N-Y217Q-S248A-T254A,S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-S182Q-S183N-Y217Q-S248A-T254A,S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-G169A-S183N-Y217Q-S248A-T254A,S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S162Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A,S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A,S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A,S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-N218S-S248A-T254A,S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S183N-Y217Q-S248A-T254A,S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S183N-Y217Q-N218S-S248A-T254A,S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-S182Q-S183N-Y217Q-N218S-S248A-T254A,S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-S248A-T254A,S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S145R-S162Q-G166Q-G169A-S182Q-S183N-Y217Q-N218S-S248A-T254A、S003Q-T022Y-S024K-S033T-S053G-N076D-S078N-S101N-N118R-G128S-S182Q-S183N-Y217Q-M222Q-S248A-T254A、S003Q-S024K-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S182Q-S183N-Q206Y-Y217Q-M222Q-T254A、S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128A-S145R-S162Q-S182Q-S183N-Y217Q-M222Q-S248A-T254A、S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128A-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128S-G169A-S182Q-Y217Q-S248A、S003Q-T022Y-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-N109Q-G128A-G169A-S182Q-S183N-S204Q-Q206Y-Y217Q-N218S-T254A、S003Q-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-N118R-G128S-S182Q-Y217Q-N218S-T254A、S003Q-Y006W-S024Q-S033T-S053G-N076D-S078N-S101N-G128S-G169A-S182Q-Y217Q-N218S-S248A-T254A、Y006W-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-G128S-S145R-S162Q-S182Q-Y217Q-N218S-T254A、S003Q-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-N118R-G128S-S162Q-S182Q-S183N-Y217Q-T254A、S024Q-S033T-A045V-S053G-N076D-S078N-S101N-G128A-S145R-Y217Q-S248A-T254A、S024Q-S033T-S053G-N076D-S078N-S101N-N109Q-G128S-S145R-S162Q-G169A-Y217Q-N218S、S003Q-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-N109Q-N118R-G128S-S162Q-G169A-S183N-Y217Q-N218S-T254A、S003Q-Y006W-S024Q-S033T-S053G-N076D-S078N-S101N-N109Q-G128S-S145R-G169A-S183N-Y217Q-N218S-S248A、Y006W-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-G128S-S162Q-S183N-Y217Q-N218S-T254A、T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-N118R-G128S-S145R-S162Q-S182Q-Y217Q-S248A-T254A、S003Q-S024Q-S033T-A045V-S053G-N076D-S078N-S101N-G128S-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A、S003Q-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-G128S-S145R-S162Q-S182Q-S183N-Y217Q-S248A-T254A、Y006W-T022Y-S024Q-S033T-S053G-N076D-S078N-S101N-G128S-S183N-Y217Q-S248A-T254A、S003Q-Y006W-T022Y-S024Q-S033T-S053G-S078N-S087D-S101N-N118R-G128S-S162Q-S182Q-Y217Q-T254A、S003Q-T022Y-S024K-S033T-A045V-S053G-N076D-S078N-S087D-S101N-N118R-G128S-S182Q-S1 83N-S204Q-Q206Y-Y217Q-M222Q-S248A-T254A, S003Q-T022Y-S024K-S033T-S053G-N076D-S07, 8N-S101N-G128S-S145R-S162Q-G166Q-S182Q-Q206Y-Y217Q-N218S-M222Q-T254A, S003Q-Y0 06W-T022Y-S024Q-S033T-A045V-S053G-T055P-N076D-S078N-S087D-S101N-N118R-G128S-S 182Q-S183N-S204Q-Q206Y-Y217Q-N218S-M222Q-S248A-T254A, S003Q-S024Q-S033T-A045V- S053G-T055P-N076D-S078N-S101N-N109Q-G128A-S145R-S162Q-S182Q-S204Q-Y217Q-M222Q- and S033T-S053G-N076D-S078N-S101N-G128S-S145R-S183N-Y217Q-S248A, and S033T-S053G-N076D-S078N-S101N-G128S-S145R-S183N-Y217Q-S248A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin comprises an amino acid sequence having at least 75%, or at least 80%, or at least 81%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0070] In some embodiments, substitution combinations are X055P-X204Q, X182Q-X222Q, X162Q-X204Q, X006W-X024K, X222Q-X254A, X206Y-X222Q, X055P-X222Q, X109Q-X222Q, X183N-X204Q, X162Q-X222Q, X006W-X183N, X204Q-X222Q, X183N-X222Q, X006W-X204Q, X222Q-X248A, X204Q-X254A, X006W-X254A, X109Q-X162Q-X182Q, X076D- X182Q-X222Q, X045V-X204Q-X222Q, X101N-X162Q-X222Q, X055P-X109Q-X2 04Q, X006W-X024K-X222Q, X109Q-X162Q-X222Q, X055P-X162Q-X204Q, X162Q -X182Q-X204Q, X162Q-X182Q-X222Q, X101N-X182Q-X222Q, X055P-X182Q-X 254A, X204Q-X217Q-X222Q, X006W-X183N-X206Y, X109Q-X182Q-X222Q, X055 P-X204Q-X222Q, X109Q-X204Q-X254A, X055P-X182Q-X222Q, X055P-X162Q- X254A, X055P-X222Q-X254A, X003Q-X162Q-X254A, X078N-X162Q-X254A, X10 9Q-X204Q-X222Q, X003Q-X182Q-X254A, X003Q-X222Q-X254A, X033T-X222Q -X254A, X024Q-X204Q-X222Q, X022Y-X162Q-X254A, X078N-X182Q-X254A, X0 03Q-X162Q-X222Q, X109Q-X222Q-X254A, X022Y-X182Q-X254A, X087D-X162 Q-X254A, X076D-X182Q-X254A, X204Q-X217Q-X254A, X076D-X162Q-X254A, X 078N-X204Q-X254A, X128S-X204Q-X254A, X076D-X204Q-X254A, X087D-X18 2Q-X254A, X182Q-X204Q-X254A, X101N-X204Q-X254A, X024Q-X204Q-X254A,X128A-X162Q-X254A、X182Q-X222Q-X254A、X128A-X204Q-X254A、X055P-X109Q-X182Q-X204Q、X109Q-X162Q-X182Q-X222Q、X055P-X182Q-X204Q-X222Q、X055P-X109Q-X162Q-X204Q、X055P-X162Q-X182Q-X204Q、X006W-X109Q-X183N-X248A、X055P-X162Q-X182Q-X222Q、X024Q-X078N-X182Q-X254A、X055P-X162Q-X182Q-X254A、X162Q-X182Q-X248A-X254A、X055P-X162Q-X204Q-X222Q、X006W-X109Q-X182Q-X248A、X055P-X109Q-X162Q-X254A、X055P-X109Q-X162Q-X222Q、X183N-X222Q-X248A-X254A、X078N-X109Q-X145R-X254A、X055P-X087D-X145R-X254A、X109Q-X182Q-X204Q-X222Q、X162Q-X182Q-X204Q-X222Q、X006W-X162Q-X182Q-X254A、X109Q-X204Q-X222Q-X254A、X055P-X162Q-X204Q-X254A、X006W-X109Q-X162Q-X254A、X055P-X109Q-X182Q-X222Q、X006W-X024K-X183N-X254A、X055P-X182Q-X183N-X254A、X055P-X182Q-X204Q-X254A、X162Q-X182Q-X222Q-X254A、X055P-X182Q-X222Q-X254A、X162Q-X182Q-X183N-X254A、X006W-X024K-X182Q-X254A、X182Q-X204Q-X222Q-X254A、X006W-X183N-X248A-X254A、X182Q-X183N-X204Q-X254A、X006W-X109Q-X204Q-X254A、X109Q-X118R-X169A-X222Q、X109Q-X183N-X204Q-X254A、X006W-X055P-X182Q-X254A、X006W-X182Q-X248A-X254A、X006W-X162Q-X204Q-X254A、X024K-X033T-X166Q-X222Q、X003Q-X076D-X222Q-X254A、X162Q-X182Q-X204Q-X254A、X006W-X128S-X217Q-X254A、X006W-X183N-X204Q-X254A、X024Q-X045V-X055P-X078N-X109Q-X182Q、X024Q-X109Q-X145R-X162Q-X217Q-X222Q、X053G-X076D-X162Q-X182Q-X204Q-X217Q、X003Q-X053G-X101N-X109Q-X183N-X254A、X024Q-X033T-X076D-X182Q-X183N-X254A、X003Q-X053G-X055P-X076D-X128A-X254A、X033T-X076D-X145R-X162Q-X182Q-X254A、X006W-X055P-X128S-X217Q-X222Q-X254A、X024Q-X076D-X162Q-X204Q-X222Q-X254A、X101N-X145R-X182Q-X183N-X204Q-X218S、X024K-X162Q-X182Q-X206Y-X222Q-X254A、X024K-X087D-X145R-X183N-X222Q-X254A、X024Q-X101N-X145R-X182Q-X204Q-X222Q、X022Y-X087D-X109Q-X183N-X222Q-X254A、X024Q-X118R-X169A-X182Q-X183N-X248A、X024Q-X101N-X182Q-X204Q-X222Q-X254A、X003Q-X109Q-X128S-X162Q-X166Q-X183N、X076D-X109Q-X145R-X182Q-X222Q-X254A、X006W-X024K-X101N-X128A-X169A-X183N、X101N-X166Q-X169A-X182Q-X204Q-X206Y、X109Q-X182Q-X183N-X204Q-X206Y-X254A、X024K-X087D-X169A-X204Q-X218S-X222Q、X006W-X045V-X128A-X145R-X182Q-X254A、X006W-X087D-X109Q-X145R-X162Q-X169A, X006W-X055P-X182Q-X183N-X204Q-X206Y-X222Q-X248A-X254Axxx, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0071] In some embodiments, substitution combinations are T055P-S204Q, S182Q-M222Q, S162Q-S204Q, Y006W-S024K, M222Q-T254A, Q206Y-M222Q, T055P-M222Q, N109Q-M222Q, S183N-S204Q, S162Q-M222Q, Y006W-S183N, S204Q-M222Q, S183N-M222Q, Y006W-S204Q, M222Q-S248A, S204Q-T254A, Y006W-T254A, N109Q-S162Q-S182Q, N076D- S182Q-M222Q, A045V-S204Q-M222Q, S101N-S162Q-M222Q, T055P-N109Q-S2 04Q, Y006W-S024K-M222Q, N109Q-S162Q-M222Q, T055P-S162Q-S204Q, S162Q -S182Q-S204Q, S162Q-S182Q-M222Q, S101N-S182Q-M222Q, T055P-S182Q-T 254A, S204Q-Y217Q-M222Q, Y006W-S183N-Q206Y, N109Q-S182Q-M222Q, T055 P-S204Q-M222Q, N109Q-S204Q-T254A, T055P-S182Q-M222Q, T055P-S162Q- T254A, T055P-M222Q-T254A, S003Q-S162Q-T254A, S078N-S162Q-T254A, N10 9Q-S204Q-M222Q, S003Q-S182Q-T254A, S003Q-M222Q-T254A, S033T-M222Q -T254A, S024Q-S204Q-M222Q, T022Y-S162Q-T254A, S078N-S182Q-T254A, S0 03Q-S162Q-M222Q, N109Q-M222Q-T254A, T022Y-S182Q-T254A, S087D-S162 Q-T254A, N076D-S182Q-T254A, S204Q-Y217Q-T254A, N076D-S162Q-T254A, S 078N-S204Q-T254A, G128S-S204Q-T254A, N076D-S204Q-T254A, S087D-S18 2Q-T254A, S182Q-S204Q-T254A, S101N-S204Q-T254A, S024Q-S204Q-T254A,G128A-S162Q-T254A、S182Q-M222Q-T254A、G128A-S204Q-T254A、T055P-N109Q-S182Q-S204Q、N109Q-S162Q-S182Q-M222Q、T055P-S182Q-S204Q-M222Q、T055P-N109Q-S162Q-S204Q、T055P-S162Q-S182Q-S204Q、Y006W-N109Q-S183N-S248A、T055P-S162Q-S182Q-M222Q、S024Q-S078N-S182Q-T254A、T055P-S162Q-S182Q-T254A、S162Q-S182Q-S248A-T254A、T055P-S162Q-S204Q-M222Q、Y006W-N109Q-S182Q-S248A、T055P-N109Q-S162Q-T254A、T055P-N109Q-S162Q-M222Q、S183N-M222Q-S248A-T254A、S078N-N109Q-S145R-T254A、T055P-S087D-S145R-T254A、N109Q-S182Q-S204Q-M222Q、S162Q-S182Q-S204Q-M222Q、Y006W-S162Q-S182Q-T254A、N109Q-S204Q-M222Q-T254A、T055P-S162Q-S204Q-T254A、Y006W-N109Q-S162Q-T254A、T055P-N109Q-S182Q-M222Q、Y006W-S024K-S183N-T254A、T055P-S182Q-S183N-T254A、T055P-S182Q-S204Q-T254A、S162Q-S182Q-M222Q-T254A、T055P-S182Q-M222Q-T254A、S162Q-S182Q-S183N-T254A、Y006W-S024K-S182Q-T254A、S182Q-S204Q-M222Q-T254A、Y006W-S183N-S248A-T254A、S182Q-S183N-S204Q-T254A、Y006W-N109Q-S204Q-T254A、N109Q-N118R-G169A-M222Q、N109Q-S183N-S204Q-T254A、Y006W-T055P-S182Q-T254A、Y006W-S182Q-S248A-T254A、Y006W-S162Q-S204Q-T254A、S024K-S033T-G166Q-M222Q、S003Q-N076D-M222Q-T254A、S162Q-S182Q-S204Q-T254A、Y006W-G128S-Y217Q-T254A、Y006W-S183N-S204Q-T254A、S024Q-A045V-T055P-S078N-N109Q-S182Q、S024Q-N109Q-S145R-S162Q-Y217Q-M222Q、S053G-N076D-S162Q-S182Q-S204Q-Y217Q、S003Q-S053G-S101N-N109Q-S183N-T254A、S024Q-S033T-N076D-S182Q-S183N-T254A、S003Q-S053G-T055P-N076D-G128A-T254A、S033T-N076D-S145R-S162Q-S182Q-T254A、Y006W-T055P-G128S-Y217Q-M222Q-T254A、S024Q-N076D-S162Q-S204Q-M222Q-T254A、S101N-S145R-S182Q-S183N-S204Q-N218S、S024K-S162Q-S182Q-Q206Y-M222Q-T254A、S024K-S087D-S145R-S183N-M222Q-T254A、S024Q-S101N-S145R-S182Q-S204Q-M222Q、T022Y-S087D-N109Q-S183N-M222Q-T254A、S024Q-N118R-G169A-S182Q-S183N-S248A、S024Q-S101N-S182Q-S204Q-M222Q-T254A、S003Q-N109Q-G128S-S162Q-G166Q-S183N、N076D-N109Q-S145R-S182Q-M222Q-T254A、Y006W-S024K-S101N-G128A-G169A-S183N、S101N-G166Q-G169A-S182Q-S204Q-Q206Y、N109Q-S182Q-S183N-S204Q-Q206Y-T254A、S024K-S087D-G169A-S204Q-N218S-M222Q、Y006W-A045V-G128A-S145R-S182Q-T254A、Y006W-S087D-N109Q-S145R-S162Q-G169A, Y006W-T055P-S182Q-S183N-S204Q-Q206Y-M222Q-S248A-T254Axxx, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1, and the variant subtilisin comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0072] Typically, the cleaning composition comprises at least about 0.0001 to about 20% by weight, about 0.0001 to about 10% by weight, about 0.0001 to about 1% by weight, about 0.001 to about 1% by weight, or about 0.01 to about 0.2% by weight of one or more subtilisin variants described herein. In another embodiment, one or more cleaning compositions described herein comprise, per gram of composition, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 2 mg, about 0.01 to about 1 mg, about 0.05 to about 1 mg, about 0.5 to about 10 mg, about 0.5 to about 5 mg, about 0.5 to about 4 mg, about 0.5 to about 3 mg, about 0.5 to about 2 mg, about 0.5 to about 1 mg, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 4 mg, about 0.1 to about 3 mg, about 0.1 to about 2 mg, about 0.1 to about 2 mg, about 0.1 to about 1 mg, or about 0.1 to about 0.5 mg of one or more subtilisin variants described herein.

[0073] The subtilisin variants preferably contain one or more modifications to surface-exposed amino acids to alter the surface properties of the variants. The surface modifications in the enzyme variants may be useful in detergent compositions, with at least one of these properties being improved over the parent subtilisin enzyme, while still maintaining a minimum figure of merit for cleaning performance, enzyme stability in detergent compositions, and enzyme thermostability. In some embodiments, the surface modification alters the hydrophobicity and / or charge of the amino acid at that position. Hydrophobicity can be determined using techniques known in the art, such as those described in White and Wimley (White, SH and Wimley, WC,. (1999) Annu. Rev. Biophys. Biomol. Struct. 28:319-65).

[0074] The "surface property" that is modified or changed may be electrostatic charge or the hydrophobicity, hydrophilicity, etc., properties exhibited by the surface of the protein. Preferably, one or more of the subtilisin variants described herein have one or more improved properties when compared to a reference or parent subtilisin, the improved properties being selected from improved cleaning performance, improved stability in detergents, and combinations thereof.

[0075] The parent subtilisin may comprise the amino acid sequence of SEQ ID NO: 1, or a polypeptide having the amino acid sequence of SEQ ID NO: 1. Preferably, the variant has improved properties such as improved stability, and the variant has greater residual activity compared to the parent or reference subtilisin. Improved stability in detergent may be measured according to the stability assay of Example 2.

[0076] The term "enhanced stability" or "improved stability" in the context of oxidation-, chelating-, denaturing-, detergent-, heat-, and / or pH-stable proteases refers to greater retention of proteolytic activity of a subtilisin variant over time compared to a reference or parent subtilisin protease, e.g., a wild-type protease or parent protease (such as SEQ ID NO: 1). Autolysis has been identified as one mode 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).

[0077] The terms "thermostable" and "thermostable" and "thermostable," with respect to protease variants, refer to a protease that retains a greater amount of residual activity when compared to a parent or reference protease after exposure to altered temperatures for a given period of time under conditions prevailing during proteolysis, hydrolysis, cleaning, or other processes (or "stress conditions"). Residual activity is the amount of activity remaining after testing compared to the initial activity of the sample and can be reported as a percentage, e.g., % residual activity. "Altered temperature" encompasses elevated or decreased temperatures. In some embodiments, the protease variants 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 subtilisin variants 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 subtilisin variants provided herein have a residual activity that is greater than that of the parent or reference protease using the method described in Example 2. In some embodiments, the subtilisin variants provided herein have at least 5% improved residual activity compared to the parent subtilisin when measured after 20 minutes at 40-80°C in a liquid detergent. In some embodiments, the subtilisin variants provided herein have at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% improved residual activity compared to the parent subtilisin when measured after 20 minutes at 40-80°C in a liquid detergent.

[0078] The subtilisin variants described herein may be subject to various changes, such as the insertion, deletion, and / or substitution (conservative or non-conservative) of one or more amino acids, including cases where such changes do not substantially alter the enzymatic activity of the variant. Similarly, the nucleic acids of the present invention may also be subject to various changes, such as one or more substitutions of one or more nucleotides in one or more codons, such that a particular codon encodes the same or a different amino acid, resulting in either a silent mutation (e.g., where the encoded amino acid is not altered by the nucleotide mutation) or a non-silent mutation, one or more deletions of one or more nucleotides (or codons) in the sequence, one or more additions or insertions of one or more nucleotides (or codons) in the sequence, and / or one or more truncations of one or more nucleotides (or codons) in the sequence. Many such changes in a nucleic acid sequence may not substantially alter the enzymatic activity of the resulting encoded polypeptide enzyme compared to the polypeptide enzyme encoded by the original nucleic acid sequence. The nucleic acid sequences described herein can also be modified to include one or more codons that provide for optimal expression in an expression system (e.g., a bacterial expression system), while said one or more codons still encode the same amino acid, if desired.

[0079] 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 recombinant production (e.g., expression) of 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. In one embodiment, a nucleic acid encoding one or more subtilisin variants described herein is provided, wherein the variants are mature forms that have proteolytic activity. In some embodiments, one or more subtilisin variants described herein are recombinantly expressed using a homologous propeptide sequence. In other embodiments, one or more subtilisin variants described herein are recombinantly expressed using a heterologous propeptide sequence (e.g., a propeptide sequence from Bacillus amyloliquefaciens (SEQ ID NO: 4) or a variant thereof).

[0080] One or more nucleic acid sequences described herein can be generated by using any suitable synthesis, manipulation, and / or isolation technique, or a combination thereof. For example, one or more polynucleotides described herein can be produced using standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques, well known to those of skill in the art. In such techniques, fragments of up to 50 or more nucleotide bases are typically synthesized and then joined (e.g., by enzymatic or chemical ligation) to form essentially any 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, chemical synthesis using the classical phosphoramidite method (see, e.g., Beaucage et al., Tetrahedron Letters 22:1859-69 (1981)) or the method described in Matthes et al., EMBO J. 3:801-805 (1984), as typically implemented in automated synthesis methods. One or more polynucleotides described herein can also be produced using an automated DNA synthesizer. Customized nucleic acids can be ordered from a variety of commercial sources (e.g., ATUM (DNA 2.0), Newark, CA, USA; Life Tech (GeneArt), Carlsbad, CA, USA; GenScript, Ontario, Canada; Base Clear BV, Leiden, Netherlands; Integrated DNA Technologies, Skokie, IL, USA; Ginkgo Bioworks (Gen9), Boston, MA, USA; and Twist Bioscience, San Francisco, CA, USA). Other techniques and related principles for synthesizing nucleic acids are described, for example, in Itakura et al., Ann. Rev. Biochem. 53:323 (1984) and Itakura et al., Science 198:1056 (1984).

[0081] 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 using one or more oligonucleotide probes that can hybridize to or PCR amplify a polynucleotide encoding one or more subtilisin variants described herein, or a recombinant polypeptide or active fragment 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 procedures (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro recombination). A variety of methods suitable for generating the modified polynucleotides described herein that encode one or more subtilisin variants described herein are known in the art, including, but not limited to, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-specific mutagenesis, and directed evolution, as well as various other recombinant approaches.

[0082] Further embodiments disclosed herein 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), expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequences described herein, isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequences described herein, isolated or recombinant cells comprising one or more polynucleotide sequences described herein, and compositions comprising one or more of such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof.

[0083] Some embodiments are directed to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein that contain one or more nucleic acid or polynucleotide sequences described herein. Some such recombinant cells are transformed or transfected with at least one such vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells include bacterial cells, including, but not limited to, cells of Bacillus species, such as B. subtilis cells. Other embodiments are directed to recombinant cells (e.g., recombinant host cells) that comprise one or more subtilisins described herein.

[0084] In some embodiments, one or more of the vectors described herein are expression vectors or expression cassettes (e.g., a promoter operably linked to one or more of the polynucleotide sequences described herein) that are operably linked to one or more additional nucleic acid segments required for efficient gene expression. The vectors may include a transcription terminator and / or a selection gene (e.g., an antibiotic resistance gene) that allows for continuous culture maintenance of plasmid-infected host cells by growth in antimicrobial-containing medium.

[0085] Expression vectors can be derived from plasmid or viral DNA, or in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (see Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990); suitable 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).

[0086] 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, the polynucleotide sequence encoding one or more subtilisin variants described herein (as well as other sequences contained in the vector) is integrated into the genome of the host cell, while in other embodiments, a plasmid vector containing the polynucleotide sequence encoding one or more subtilisin variants described herein remains within the cell as an autonomous extrachromosomal element. Some embodiments provide both an extrachromosomal nucleic acid element and an input nucleotide sequence that is integrated into the host cell genome. The vectors described herein are useful for producing 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 that allows for integration and optional amplification of the polynucleotide encoding the variant into the host chromosome. Exemplary sites for integration 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 achieved by a promoter that is the wild-type promoter of the parent subtilisin, hi some other embodiments, the promoter is heterologous to one or more subtilisin variants described herein but is functional in the host cell.Exemplary promoters for use in bacterial host cells include, but are not limited to, the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters; the promoter of the B. stearothermophilus maltogenic amylase gene; the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene; the B. pumilis xylosidase gene; the B. thuringiensis cryIIIA gene; and the B. licheniformis α-amylase gene. Additional promoters include, but are not limited to, the A4 promoter, as well as the phage lambda PR or PL promoters and the E. coli lac, trp or tac promoters.

[0087] The 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 a 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 host cell of a Bacillus species. Exemplary host cells of Bacillus species for use in producing one or more of the subtilisin variants described herein include 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. Examples of host cells include Bacillus thuringiensis, B. clausii, and B. megaterium. In some embodiments, B. subtilis host cells are used to produce the variants described herein.U.S. Pat. Nos. 5,264,366 and 4,760,025 (RE 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 be used.

[0088] Some bacterial strains that can be used to produce one or more subtilisin variants described herein include non-recombinant (i.e., wild-type) Bacillus species strains, as well as variants of naturally occurring and / or recombinant strains. In some embodiments, the host strain is a recombinant strain in which 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. Numerous 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. No. 4,450,235, U.S. Pat. No. 4,302,544, and European Patent No. 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)).

[0089] The Bacillus host cell may be a species of the genus Bacillus that includes a mutation or deletion in at least one of the degU, degS, degR, and degQ genes. 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 contains 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 causes 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-mentioned genes. Additionally, Bacillus spp. host cells that contain mutations and / or deletions of endogenous protease genes are used. In some embodiments, the Bacillus spp. host cells contain deletions of the aprE and nprE genes. In other embodiments, the Bacillus spp. host cells contain deletions of five protease genes, while in other embodiments, the Bacillus spp. host cells contain deletions of nine protease genes (see, e.g., U.S. Patent No. 2005 / 0202535).

[0090] Host cells can be transformed with one or more nucleic acid sequences encoding one or more subtilisin variants described herein using any suitable method known in the art. Methods for introducing nucleic acids (e.g., DNA) into Bacillus or E. coli cells using plasmid DNA constructs or vectors, and for transforming such cells with such plasmid DNA constructs or vectors, are well known. In some embodiments, the plasmid is then isolated from the E. coli cells and transformed into Bacillus cells. 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.

[0091] Exemplary methods for introducing one or more nucleic acid sequences described herein into Bacillus cells are described, for example, in Ferrari et al., "Genetics," in 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 protoplast transformation and transfection, transduction, and transformation, including 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 input donor plasmid is recombined with a homologous region of a resident "helper" plasmid in a process that mimics chromosomal transformation.

[0092] In addition to commonly used methods, host cells can also be directly transformed with a DNA construct or vector containing a nucleic acid encoding one or more subtilisin variants described herein (i.e., no intermediate cells are used to amplify or otherwise manipulate the DNA construct or vector prior to introduction into a host cell). Introduction of a DNA construct or vector described herein into a host cell includes physical and chemical methods known in the art for introducing a nucleic acid sequence (e.g., a DNA sequence) into a host cell without insertion into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In a further embodiment, the DNA construct or vector is co-transformed with a plasmid without being inserted into the plasmid. In a further embodiment, a selectable marker is deleted from the modified Bacillus strain by methods known in the art (see, e.g., Stahl et al., J. Bacteriol. 158:411-418 (1984); and Palmeros et al., Gene 247:255-264 (2000)).

[0093] The transformed cells may be cultured in conventional nutrient media. Suitable specific culture conditions, e.g., temperature, pH, etc., are known to those of skill in the art and are well described in the scientific literature. Some embodiments provide cultures (e.g., cell cultures) comprising one or more subtilisin variants or nucleic acid sequences described herein.

[0094] Host cells transformed with one or more polynucleotide sequences encoding one or more subtilisin variants described herein can be cultured in an appropriate nutrient medium under conditions that allow for expression of the variants, and the resulting variants are then recovered from the culture. The variants produced by the cells can be recovered from the culture medium by conventional procedures, including, but not limited to, separating the host cells from the medium by centrifugation or filtration, precipitating the protein components of the supernatant or filtrate with salts (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).

[0095] One or more subtilisin variants produced by recombinant host cells can be secreted into the culture medium. Nucleic acid sequences encoding purification-facilitating domains can be used to facilitate purification of the variants. Vectors or DNA constructs containing polynucleotide sequences encoding one or more subtilisin variants described herein can further include a nucleic acid sequence encoding a purification-facilitating domain to facilitate purification of the variants (see, e.g., Kroll et al., DNA Cell Biol. 12:441-53 (1993)). 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 (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 is also used to facilitate purification.

[0096] The mutant protein can be produced in host cells, for example, by secretion or intracellular expression, using methods well known in the art. Fermentation, separation, and concentration techniques are well known in the art, and concentrated enzyme-containing solutions can be prepared using conventional methods. The host cells can be further treated to release the enzyme or improve cell separation, for example, by heating, altering the pH or salt content, or treating with enzymes, including hen egg white lysozyme, T4 lysozyme, or enzymes described in WO2022047149. For production-scale recovery, the mutant polypeptide can be concentrated or partially purified as generally described above by removing cells by flocculation with a polymer. Alternatively, the enzyme can be concentrated or purified by microfiltration, followed by ultrafiltration using available membranes and equipment. However, in some applications, it is not necessary to concentrate or purify the enzyme; the entire broth culture can be dissolved and used without further processing. The enzyme can then be processed, for example, into granules or non-aqueous particles.

[0097] A variety of methods can be used to determine the production concentration of one or more mature subtilisin variants described herein in a host cell. Such methods include, but are not limited to, methods utilizing either polyclonal or monoclonal antibodies specific for the protease. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), and fluorescent 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)).

[0098] Also disclosed herein are methods of making or producing one or more mature subtilisin variants described herein. Mature subtilisin variants do not contain 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 cell of a Bacillus species (e.g., a B. subtilis cell). Other embodiments provide methods of producing one or more subtilisin variants described herein, the methods 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 conducive to production of the variants. Some such methods further comprise recovering the variants from the culture.

[0099] Also disclosed herein are methods for producing one or more of the subtilisin variants described herein, the methods comprising: (a) introducing a recombinant expression vector containing a nucleic acid encoding the variant into a population of cells (e.g., bacterial cells, such as B. subtilis cells); (b) culturing the cells in a culture medium under conditions conducive to production of the variant encoded by the expression vector; Some such methods further include (c) isolating the mutant from the cell or from the culture medium.

[0100] Further embodiments are directed to methods for improving the cleaning performance or stability of a subtilisin, the methods comprising modifying a subtilisin to include one or more substitutions, or a combination of substitutions, as provided herein. Subtilisin variants suitable for use 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 suitable for incorporation into cleaning compositions, such as a liquid formulation, a gel, granules, or an enzyme slurry.

[0101] 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 approaches, as well as the prilling process. The core of the granule can be the granule itself or the inner core of a layered granule.

[0102] The core may contain one or more water-soluble or dispersible agents, including, but not limited to, sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate, citric acid, sugars (e.g., sucrose, lactose, glucose, granular sucrose, maltodextrin, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives such as hydroxyl-propyl-methylcellulose, carboxy-methylcellulose, and hydroxyl-ethylcellulose), phosphates, calcium, protease inhibitors, and combinations thereof. Suitable dispersible agents include, but are not limited to, clays, nonpareils (combinations of sugars and starches, e.g., starch-sucrose-nonpareils-ASNP), talc, silicates, carboxymethylcellulose, starch, and combinations thereof.

[0103] 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.

[0104] 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 an enzyme.

[0105] In some embodiments, the core is an enzyme powder comprising a UFC containing an enzyme. The enzyme powder may be spray dried and optionally mixed with any of the water-soluble or dispersible agents listed herein. The enzyme may be or include a protease that needs to be stabilized, in which case the enzyme powder should further include a stabilizer.

[0106] 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 other certain embodiments, 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 No. 20100124586, WO 9932595, and U.S. Patent No. 5324649).

[0107] 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, sugars (e.g., sucrose, lactose, glucose, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives such as hydroxyl-propyl-methylcellulose, carboxy-methylcellulose, and hydroxyl-ethylcellulose), clays, nonpareils (combinations of sugars and starches), silicates, carboxymethylcellulose, 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 (e.g., FOAMBLAST 882® and EROL 6000K®, and talc. US 20100124586, WO 9932595, and US 5324649 detail suitable ingredients for the coating layer.

[0108] In some embodiments, the coating layer comprises a sugar (e.g., sucrose, lactose, glucose, granulated 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 PVAs, and have low to high viscosities. In some embodiments, the coating layer comprises an inorganic salt, such as sodium sulfate.

[0109] 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.

[0110] In some embodiments, the enzyme granules comprise the subtilisin variants provided herein in combination with wild-type, recombinant, and mutant enzymes of bacterial, fungal, or yeast origin, as well as one or more additional enzymes, which may be acidic, neutral, or alkaline enzymes. Additional enzymes include acyltransferase, alginate lyase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, β-glucanase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, fructosidase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, laminarinase, lichenase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloproteinase, and the like. The enzyme may be selected from the group consisting of proteases, nucleases (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, rhamnogalacturonase, β-glucanases, tannases, transglutaminases, xanthan lyases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof. Generally, at least one enzyme coating layer comprises at least one subtilisin variant provided herein.

[0111] Auxiliary substances In addition to the subtilisin variant protease enzyme, the cleaning composition comprises a cleaning / detergent adjunct, preferably a mixture of cleaning / detergent adjuncts. The terms cleaning adjunct and cleaning adjunct are used interchangeably herein. Each cleaning adjunct is typically selected depending on the purpose and form of the cleaning composition (e.g., liquid, granules, powder, bar, paste, spray, tablet, gel, foam, or other composition). The term "adjunct" refers to any liquid, solid, or gaseous substance contained in the cleaning composition other than one or more subtilisin variants described herein, or recombinant polypeptides or active fragments thereof. The cleaning adjunct is typically selected depending on the particular type and form of the cleaning composition (e.g., liquid, granules, powder, bar, paste, spray, tablet, gel, foam, or other composition). Where possible, preferred adjuncts are bio-based or contain at least a portion of bio-based material, e.g., more than 10% by weight, or more than 25% by weight, or more than 40% by weight, or more than 50% by weight, or more than 60% by weight, or more than 70% by weight, or more than 80% by weight, or more than 90% by weight. Preferably, the cleaning adjunct, or most of the cleaning adjunct, is compatible with the protease enzyme used in the composition. If one or more adjunct components are incompatible with the protease variant, suitable methods can be used to reduce contact and / or keep the cleaning adjunct and the protease separate (i.e., not in contact with each other) until the two components are suitable for combination. Such separation methods include any suitable method known in the art (e.g., gel caps, encapsulation, tablets, physical separation, etc.).

[0112] Typically, the cleaning adjunct is present in the composition in an amount of 1 to 98.9% by weight, more typically 5 to 90% by weight of the cleaning adjunct. Suitable cleaning adjuncts include additional surfactants, builders, bleach components, colorants, chelating agents, dye transfer agents, deposition aids, dispersants, additional enzymes (optionally encapsulated), enzyme stabilizers, catalytic materials, optical brighteners, photoactivators, fluorescent agents, fabric hueing agents (tint dyes), fabric conditioners, preformed peracids, cleaning polymers (such as polymeric dispersants), stain release polymers and / or clay stain removal / anti-redeposition agents, fabric conditioning polymers including polyquaternium 10 (CathEC), fillers, The present invention also includes salts, hydrotropes, brighteners, suds suppressors, structural elasticity agents, softeners, preservatives, antioxidants, shrinkage inhibitors, disinfectants, mildew inhibitors, discoloration inhibitors, corrosion inhibitors, alkalinity sources, solubilizers, carriers, processing aids, pigments, dyes, fragrances, and pH adjusters, encapsulating agents, polymers, antimicrobial agents including Tinosan HP100, probiotics, or one or more microorganisms or microbes or microbial extracts or microbial spores, or mixtures thereof. 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.

[0113] For example, these include bleach components such as bleach activators, bleach boosters such as imine bleach activators, bleach catalysts, hydrogen peroxide, hydrogen peroxide sources such as percarbonates and / or perborates, especially percarbonates coated with materials such as carbonates and / or sulfates, silicates, borosilicates, and any mixtures thereof, preformed peracids, including preformed peracids in encapsulated form, transition metal catalysts; suds suppressors or suds suppressor systems, for example silicone-based suds suppressors and / or fatty acid-based suds suppressors; softening agents such as clays, silicones, and / or quaternary ammonium compounds; flocculating agents such as polyethylene oxide; dye transfer inhibitors such as polyvinylpyrrolidone, poly 4-vinylpyridine N-oxide, and / or copolymers of vinylpyrrolidone and vinylimidazole; imidazoles and epichlorohydrin; soil dispersants and soil anti-redeposition aids such as alkoxylated polyamines and ethoxylated ethyleneimine polymers; anti-redeposition ingredients such as polyesters; carboxylate polymers, for example, maleic acid polymers or copolymers of maleic acid and acrylic acid; perfumes which may be free and / or encapsulated, for example, in perfume microcapsules and / or in the form of starch-encapsulated accords and / or perfume spray-ons; soap rings; aesthetic particles; aesthetic dyes; fillers such as sodium sulfate and / or citrus fibers (although it may be preferred that the composition be substantially free of fillers); 1.6R and 2.silicates such as sodium silicates, including 0R sodium silicate, or sodium metasilicate; copolyesters of dicarboxylic acids and diols; cellulosic polymers such as methylcellulose, carboxymethylcellulose, hydroxyethoxycellulose, or other alkyl or alkylalkoxycelluloses; solvents such as 1,2 propanediol, monoethanolamine, and the like; polyethylene glycol, diethylene glycol, ethanol, dipropylene glycol, tripropylene glycol, glycerol, sorbitol, and any mixtures thereof; hydrotropes, such as sodium cumene sulfonate, sodium xylene sulfonate, toluenesulfonate, and the like. sodium sulfonate, and any mixtures thereof; organic acids and salts thereof, such as citric acid / citrate salts; microorganisms such as Bacillus strains having deposit accession number PTA-7543, which may be used to reduce malodors, for example, as described in WO 2012 / 112718, and exemplary commercially available microbial products include, but are not limited to, Microvia™ (Novozymes), or inoculation / inoculation of a locus with a microorganism, for example, that provides in situ production of a desired biological compound or to competitively prevent other undesirable microorganisms from colonizing the same locus (competitive exclusion); and any combination thereof.

[0114] Preferably, the composition comprises one or more selected from the group consisting of: (i) a fragrance (preferably comprising at least some fragrance in microcapsules); (ii) an amylase; (iii) an amphiphilic cleaning polymer; or (iv) a mixture thereof.

[0115] Preferably, the pH of the cleaning composition is 6-10, 6.5-8.9, or 7-8, and the pH of the cleaning composition is measured as a 10% concentration in demineralized water at 20°C. When liquid, the cleaning composition can be Newtonian or non-Newtonian. In some examples, the liquid laundry detergent composition is non-Newtonian. Without wishing to be bound by theory, non-Newtonian liquids have different properties than Newtonian liquids; more specifically, the viscosity of non-Newtonian liquids depends on shear rate, while Newtonian liquids have a constant viscosity regardless of the applied shear rate. It is believed that the decrease in viscosity of non-Newtonian liquids upon the application of shear further facilitates dissolution of the liquid detergent. The liquid laundry detergent compositions described herein can have any suitable viscosity, depending on factors such as the formulated ingredients and the purpose of the composition.

[0116] Anionic surfactants The detergent composition preferably comprises 1 to 60% by weight of anionic surfactant. Preferred anionic surfactants are sulfonate and sulfate surfactants, preferably alkylbenzene sulfonates and / or (optionally alkoxylated) alkyl sulfates. Particularly preferred anionic surfactants include linear alkylbenzene sulfonates (LAS). Preferred alkyl sulfates include alkyl ether sulfates, particularly C-9-15 alcohol ether sulfates, especially those with an average degree of ethoxylation of 0.5 to 7, preferably 1 to 5, or 1 to 3, or 2 to 3. Exemplary alkyl fractions of alkyl sulfate anionic surfactants are derived from aliphatic alcohols, oxo-synthetic alcohols, Guerbet alcohols, or mixtures thereof. In some examples, the laundry detergent composition comprises 10% to 50%, 15% to 45%, 20% to 40%, or 30% to 40% by weight of the laundry detergent composition of non-soap anionic surfactant. The alkyl sulfates, alkyl alkoxylated sulfates, and alkyl benzene sulfonates may be linear or branched, including dialkyl-substituted or mid-chain branched, substituted or unsubstituted, and derived from petrochemicals or biological sources. Preferably, the branching group is alkyl. Typically, the alkyl is selected from methyl, ethyl, propyl, butyl, pentyl, cyclic alkyl groups, and mixtures thereof. Single or multiple alkyl branches may be present on the hydrocarbyl backbone of the starting alcohol used to produce the sulfated anionic surfactant used in the detergent of the present invention. Most preferably, the branched sulfated anionic surfactant is selected from alkyl sulfates, alkyl ethoxy sulfates, and mixtures thereof.

[0117] In preferred compositions, the anionic surfactant comprises an alkylbenzene sulfonate and, optionally, further ethoxylated alkyl sulfate, preferably having an ethoxylation degree of 0 to 7, more preferably 0.5 to 3. Examples of suitable surfactants include isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, alkenesulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES), alcohol ethoxy sulfates or fatty Also suitable anionic surfactants are fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid, or salts of fatty acids (soaps), and combinations thereof.

[0118] Other suitable anionic surfactants include alkyl ether carboxylates, typically containing a C10-C26 linear or branched chain alkyl alcohol, preferably a C10-C20 linear chain, and most preferably a C16-C18 linear chain alkyl alcohol, and 2 to 20, preferably 7 to 13, more preferably 8 to 12, and most preferably 9.5 to 10.5 ethoxylates. The acid form or a salt form, such as a sodium or ammonium salt, may be used, and the alkyl chain may contain one cis or trans double bond.

[0119] Other suitable anionic surfactants are rhamnolipids, which may have one rhamnose sugar ring or two rhamnose sugar rings.

[0120] Anionic surfactants are preferably added to detergent compositions in the form of salts. Preferred cations are alkali metal ions such as sodium and potassium. However, salt forms of anionic surfactants can also be formed in situ by neutralizing the acid form of the surfactant with an alkali, such as sodium hydroxide, or an amine, such as mono-, di-, or triethanolamine. More preferred agents for neutralizing the acid form of anionic surfactants include ammonia, amines, oligumin, or alkanolamines. Alkanolamines are preferred. Suitable non-limiting examples include monoethanolamine, diethanolamine, triethanolamine, and other linear or branched alkanolamines known in the art. For example, highly preferred alkanolamines include 2-amino-1-propanol, 1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amine neutralization can be complete or partial; for example, a portion of the anionic surfactant can be neutralized with sodium or potassium, and a portion of the anionic surfactant can be neutralized with an amine or alkanolamine.

[0121] The surfactant preferably comprises a surfactant system comprising an anionic surfactant and one or more additional surfactants, which may be semi-polar and / or cationic and / or zwitterionic and / or ampholytic and / or amphoteric and / or semi-polar nonionic and / or nonionic, including mixtures thereof. Preferably, the surfactant comprises an anionic and a nonionic surfactant, preferably in an anionic to nonionic weight ratio of 30:1 to 1:2, preferably 20:1 to 2:3 or 1:1. Preferably, the surfactant comprises an anionic and an amphoteric surfactant, most preferably an amine oxide, preferably in an anionic to amphoteric weight ratio of 100:1 to 5:1, more preferably 50:1 to 10:1.

[0122] Suitable nonionic surfactants include alcohol ethoxylates (AEs), alcohol propoxylates, propoxylated fatty alcohols (PFAs), alkoxylated fatty acid alkyl esters such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APEs), nonylphenol ethoxylates (NPEs), alkyl polyglycosides (APGs), alkoxylated amines, fatty acid monoethanolamides (FAMs), fatty acid diethanolamides (FADAs), ethoxylated fatty acid monoethanolamides (EFAMs), propoxylated fatty acid monoethanolamides (PFAMs), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides (GAs), or fatty acid glucamides (FAGAs)), and products available under the trade names SPAN and TWEEN, and combinations thereof. Alcohol ethoxylates are particularly preferred, preferably having a C9-18 or preferably C12-15 alkyl chain, and preferably having an average degree of ethoxylation of 3 to 9, more preferably 3 to 7. Commercially available nonionic detergent surfactants include Plurafac™, Lutensol™, and Pluronic™ from BASF, the Dehypon™ series from Cognis, and the Genapol™ series from Clariant. In some examples, the nonionic surfactant is selected from alcohol alkoxylates, oxo-synthetic alcohol alkoxylates, Guerbet alcohol alkoxylates, alkylphenol alcohol alkoxylates, or mixtures thereof. In some examples, the laundry detergent composition comprises 0.01% to 10%, 0.01% to 8%, 0.1% to 6%, or 0.15% to 5% of the nonionic surfactant by weight of the liquid laundry detergent composition.

[0123] Suitable amphoteric or zwitterionic surfactants include amine oxides and / or betaines. Preferred amine oxides are alkyl dimethyl amine oxides or alkylamidopropyl dimethyl amine oxides, more preferably alkyl dimethyl amine oxides, especially coco dimethyl amine oxide. The amine oxides can have a straight-chain or mid-chain branched alkyl moiety. Typical straight-chain amine oxides include water-soluble amine oxides containing one R1 C8-18 alkyl moiety and two R2 and R3 moieties selected from the group consisting of C1-3 alkyl groups and C1-3 hydroxyalkyl groups. Preferably, the amine oxide is characterized by the formula R1-N(R2)(R3)O, where R1 is a C8-18 alkyl and R2 and R3 are selected from the group consisting of methyl, ethyl, propyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl, and 3-hydroxypropyl. Linear amine oxide surfactants include, in particular, linear C10-C18 alkyl dimethyl amine oxides and linear C8-C12 alkoxy ethyl dihydroxy ethyl amine oxides. Other suitable surfactants include betaines, such as alkyl betaines, alkyl amido betaines, amidazolinium betaines, sulfobetaines (INCI sultaines), and phosphobetaines. Preferred compositions according to the invention contain 0.1 to 15% by weight, preferably 0.5 to 5 or 10% by weight, of the amine oxide surfactant.

[0124] The cleaning composition preferably comprises 0.5% to about 40% by weight of a nonionic surfactant, preferably 1 to 30% by weight of the composition. Other examples of weight ratios of anionic surfactant to nonionic surfactant are 1:2 to 20:1, 1:1 to 17.5:1, 2:1 to 15:1, or 2.5:1 to 13:1. Examples of weight ratios of linear alkylbenzene sulfonate to alkyl sulfate anionic surfactant are 1:2 to 9:1, 1:1 to 7:1, 1:1 to 5:1, or 1:1 to 4:1.

[0125] In some examples, the cleaning composition comprises 1.5% to 20%, 2% to 15%, 3% to 10%, or 4% to 8% by weight of the cleaning composition of a soap, in some examples a fatty acid salt, in some examples an amine-neutralized fatty acid salt, and in some examples the amine is an alkanolamine selected from, for example, monoethanolamine, diethanolamine, triethanolamine or mixtures thereof, in some examples monoethanolamine.

[0126] The cleaning composition preferably comprises one or more additional enzymes. Suitable additional enzymes include acyltransferases, alginate lyases, aminopeptidases, amylases, carbohydrases, carboxypeptidases, catalases, cellulases, chitinases, carrageenases, cellobiohydrolases, chondroitinases, cutinases, cyclodextrin endoglucanases, dispersins, endo-β-1,4-glucanases, endo-β-mannanases, esterases, exo-mannanases, fructo- enzymes, glycosyltransferases, esterases, α-galactosidases, β-galactosidases, α-glucosidases, β-glucosidases, galactanases, glucoamylases, hemicellulases, hexosaminidases, hyaluronidases, keratinases, haloperoxidases, hexosaminidases, invertases, laccases, ligninases, lactases, lipases, mannanases, mannosidases, nucleases, oxidases , pectin degrading enzymes, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, additional proteolytic enzymes (proteases), transglutaminase, lipoxygenase, lysozyme, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectinase, pentosanase, perhydrolase, peroxidase, phenol oxidase, phosphatase, phosphodiesterase, phospholipase, phytase, polygalacturonase, polyesterase, additional protease, pullulanase, reductase, rhamnogalacturonase, tannase, transglutaminase, xylosidase, xylanase, xyloglucanase, xanthan lyase, xanthanase, xylan acetyl esterase. Preferably, the cleaning composition comprises an additional enzyme selected from a nuclease such as a DNase or an RNase, a mannanase, a xyloglucanase, a xanthan lyase, a xanthanase, an amylase, and mixtures thereof.

[0127] Preferably, the composition comprises an additional enzyme selected from an additional protease, amylase, xanthan lyase, xyloglucanase, xanthanase, mannanase, and mixtures thereof.

[0128] Preferably, the composition comprises an additional protease. In one embodiment, the additional protease is a serine protease. In another embodiment, the additional protease is a metalloprotease, a fungal subtilisin, or an alkaline microbial protease or a trypsin-like protease. Suitable additional proteases include those of animal, plant, or microbial origin. In some embodiments, the additional protease is a microbial protease. In other embodiments, the additional protease is a chemically or genetically modified variant. In another embodiment, the additional protease is an alkaline microbial protease or a trypsin-like protease. In other embodiments, the additional protease does not contain a cross-reactive epitope with the subtilisin variant, as determined by antibody binding or other assays available in the art. Exemplary alkaline proteases include, for example, subtilisins derived from Bacillus (e.g., B. lentus, B. gibsonii, B. pumilus, TY-145, Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168) or fungal sources, such as those described in U.S. Patent No. 8,362,222. Additional suitable proteases are discussed in more detail below.

[0129] 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.

[0130] Still further embodiments are directed to compositions comprising one or more subtilisin variants described herein and one or more amylases. In one embodiment, the composition comprises from about 0.00001% to about 10%, 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% amylase by weight of the composition.

[0131] 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 from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulase by weight of the composition. Any suitable cellulase may be used in the compositions described herein.

[0132] 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% mannanase by weight of the composition. Exemplary mannanases can be chemically or genetically modified variants.

[0133] In some embodiments, the cleaning compositions described herein further comprise a suitable pectin-degrading enzyme. As used herein, "pectin degrading enzymes" include arabinanases (EC 3.2.1.99), galactanases (EC 3.2.1.89), polygalacturonases (EC 3.2.1.15), exo-polygalacturonases (EC 3.2.1.67), exo-poly-α-galacturonosidases (EC 3.2.1.82), pectin lyases (EC 4.2.2.10), pectinesterases (EC 3.1.1.11), pectate lyases (EC 4.2.2.2), exo-polygalacturonate lyases (EC 4.2.2.9), and hemicellulases, such as endo-1,3-β-xylosidases (EC 3.2.1.32), xylan-1,4-β-xylosidases (EC These enzymes include pectin methylesterases (EC 3.2.1.37), xanthan lyases, and α-L-arabinofuranosidases (EC 3.2.1.55). Pectin degrading enzymes are naturally occurring mixtures of the above enzymatic activities. Thus, pectic enzymes include pectin methylesterases, which hydrolyze pectin methyl ester bonds; polygalacturonases, which cleave glycosidic bonds between galacturonic acid molecules; and pectin transeliminases or lyases, which act on pectic acid to effect non-hydrolytic cleavage of the α-1,4 glycosidic bond to form unsaturated derivatives of galacturonic acid.

[0134] Suitable pectin-degrading enzymes include those of plant, fungal, or microbial origin. In some embodiments, the cleaning compositions described herein further comprise from about 0.00001% to about 10%, from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% of a pectin-degrading enzyme, by weight of the composition.

[0135] In some other embodiments, the cleaning compositions described herein further comprise a suitable β-glucanase. Suitable β-glucanases include, but are not limited to, those derived from plants, fungi, or bacteria. Some embodiments include chemically or genetically modified variants. As used herein, β-glucanase refers to an endo-β-1,4-glucanase activity (e.g., an endo-1,4 β-D-glucanase) that catalyzes the hydrolysis of the β-1,4-bond connecting two glucosyl residues in a β-glucan. Non-limiting examples of β-glucanases as defined herein include cellulases (e.g., EC 3.2.1.4) that have endo-cellulase activity on the β-1,4 bond between a D-glucose unit and a licheninase (or lichenase) (e.g., EC 3.2.1.73) that hydrolyzes the (1,4)-β-D-glucosidic bond in β-D-glucans containing both (1,3)- and (1,4)-bonds. β-Glucanases (e.g., EC 3.2.1.4) can endohydrolyze (1,4)-β-D-glucosidic bonds in, for example, cellulose, lichenin, and cereal β-D-glucans, and also hydrolyze 1,4-linkages in β-D-glucans containing 1,3-linkages. Examples of useful β-glucanases are described in the genus Bacillus (e.g., B. agaradhaerens, B. akibai, B. mojavensis; WO 2021148364).

[0136] In some other embodiments, the cleaning compositions described herein further comprise a suitable xyloglucanase. Suitable xyloglucanases include, but are not limited to, those derived from plants, fungi, or bacteria. In some embodiments, these include chemically or genetically modified variants. As used herein, "xyloglucanase" encompasses the family of enzymes described by Vincken and Voragen of Wageningen University (Vincken et al. (1994) Plant Physiol., 104, 99-107) and capable of degrading xyloglucan as described in Hayashi et al. (1989) Annu. Rev. Plant. Physiol. Plant Mol. Biol., 40, 139-168. Vincken et al. demonstrated the removal of the xyloglucan coating from the cellulose of isolated apple cell walls by a xyloglucanase (endo-IV-glucanase) purified from Trichoderma viride. This enzyme enhances the enzymatic degradation of cellulose embedded in cell walls and acts synergistically with pectic enzymes. Rapidase LIQ+ from DSM contains xyloglucanase activity. In some embodiments, the cleaning compositions described herein further comprise between about 0.00001% and about 10%, between about 0.0001% and about 10%, between about 0.001% and about 5%, between about 0.001% and about 2%, or between about 0.005% and about 0.5% xyloglucanase, by weight of the composition. In certain other embodiments, the xyloglucanase for certain applications is an alkaline xyloglucanase, i.e., an enzyme having at least 10%, at least 25%, or at least 40% of its maximum enzymatic activity at a pH in the range of 7 to 12. In certain other embodiments, the xyloglucanase is an enzyme having maximum activity at a pH of between about 7.0 and about 12.

[0137] 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). 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 nuclease by weight of the composition.

[0138] In some other embodiments, the cleaning compositions described herein further comprise a suitable peroxidase / oxidase. Suitable peroxidases / oxidases include those derived from plants, bacteria, or fungi, including chemically modified or genetically engineered variants of the protein. Examples of useful peroxidases include peroxidases from the genus Coprinus, such as those from Coprinus cinereus and variants thereof, as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0139] 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.

[0140] In yet another embodiment, one or more of the subtilisin variants and one or more additional enzymes described herein contained in one or more of the compositions described herein may each independently range from 0.001 to about 10% by weight of the composition, with the remainder of the cleaning composition being one or more adjunct materials.

[0141] The additional enzyme may be derived from, for example, an Aspergillus species, such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae; a Fusarium species, such as Fusarium bactridioides, Fusarium cerealis, or Fusarium cloacae; crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum toruloseum, Fusarium trichothecioides, or Fusarium venenatum;It may be produced by microorganisms belonging to the genus Humicola, such as Humicola insolens or Humicola lanuginosa; or Trichoderma, such as Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0142] Preferably, the composition comprises a lipase or a mixture of two or more lipases, a peroxidase or a mixture of two or more peroxidases, one or more starch-degrading enzymes, such as alpha-amylase, glucoamylase, maltogenic amylase, and / or cellulase, or a mixture thereof.

[0143] Generally, the properties of the selected enzyme should be compatible with the selected detergent (i.e., pH optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme should be present in an effective amount. If present, additional enzymes are preferably present in the cleaning composition in an amount of at least 0.0001 mg, preferably from about 0.005 to about 10 mg, more preferably from about 0.001 to about 10 mg, and especially from about 0.002 to about 5 mg active additional enzyme per gram of composition.

[0144] Protease: The compositions of the present invention may include an additional protease. A mixture of two or more proteases can contribute to improved cleaning over a wider temperature, cycle duration, and / or substrate range. Suitable additional proteases include metalloproteases and serine proteases, including neutral or alkaline microbial serine proteases such as subtilisin (EC 3.4.21.62). Suitable proteases include those of animal, plant, or microbial origin. In one aspect, such suitable proteases may be of microbial origin. Suitable proteases include chemically or genetically modified variants of the aforementioned suitable proteases. In one aspect, suitable proteases may be serine proteases, such as alkaline microbial proteases and / or trypsin-type proteases. Examples of suitable neutral or alkaline proteases include: Additional subtilisins (EC 3.4.21.62), in particular those described in WO 2004067737, WO 2015091989, WO 2015091990, WO 2015024739, WO 2015143360, U.S. Pat. Nos. 6,312,936 (B1), 5,679,630, 4,760,025, German Patent Application Publication No. 102006022216 (A1), Bacillus species, such as Bacillus lentus (B. those from the genus Bacillus, such as B. lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, B. pumilus, B. gibsonii, and B. akibaii; Specifically, these include the mutations S9R, A15T, V66A, A188P, V199I, Q239R, N255D, X9E, X200L, X256E, X9R, X19L, X60D (Savinase numbering system); subtilisins from B. pumilus, such as those described in German Patent Application Publication No. 102006022224(A1), International Publication Nos. 2020 / 221578, 2020 / 221579, and 2020 / 221580, and variants comprising an amino acid substitution at at least one of positions 9, 130, 133, 144, 224, 252, and 271 (BPN' numbering system).

[0145] Trypsin- or chymotrypsin-type proteases such as trypsin (e.g., porcine or bovine), including the Fusarium proteases described in WO 89 / 06270 and the chymotrypsin proteases from Cellumonas described in WO 05 / 052161 and WO 05 / 052146.

[0146] Metalloproteases, in particular those from Bacillus amyloliquefaciens as described in WO 07 / 044993(A2), those from Bacillus, Brevibacillus, Thermoactinomyces, Geobacillus, Paenibacillus, Lysinibacillus or Streptomyces spp. as described in WO 2014194032, WO 2014194054 and WO 2014194117, those from Kribella aluminosa as described in WO 2015193488, alluminosa, and those derived from the genera Streptomyces and Lysobacter as described in WO 2016075078.

[0147] TY145, NCIMB40339, as described in WO 92 / 17577 (Novozymes A / S), including variants of the Bacillus sp. TY145 subtilase described in WO 2015024739 and WO 2016066757.

[0148] Particularly preferred proteases for the cleaning compositions of the present invention have at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, especially 100% identity to the wild-type enzyme from Bacillus lentus and are designated S9R, A15T, V68A, N76D, N87S, S99D, S99SD, S99S, S99T, S99R, S99T, S99S, S99T ... and / or M222S, Q245R, and / or M222S.

[0149] Most preferably, the protease is selected from the group of proteases comprising the following mutations (BPN' numbering system) relative to either PB92 wild type (SEQ ID NO: 2 of WO 08 / 010925) or subtilisin 309 wild type (sequence according to the PB92 backbone but containing the natural mutation N87S): (i) G118V + S128L + P129Q + S130A (ii)S101M+G118V+S128L+P129Q+S130A (iii)N76D+N87R+G118R+S128L+P129Q+S130A+S188D+N248R (iv)N76D+N87R+G118R+S128L+P129Q+S130A+S188D+V244R (v)N76D+N87R+G118R+S128L+P129Q+S130A (vi) V68A+N87S+S101G+V104N (vii)S99AD (viii)S9R+A15T+V68A+N218D+Q245R

[0150] Suitable commercially available protease enzymes include those sold by Novozymes A / S (Denmark) under the trade names Alcalase®, Savinase®, Primase®, Durazym®, Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®, Ovozyme®, Neutrase®, Everlase®, Coronase®, Blaze®, Blaze Ultra® and Esperase®; and those sold under the trade names Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®, Excellase®, Ultimase® and Purafect those sold by DuPont under the trade names Opticlean and Optimase; and those available from Henkel / Kemira, namely, BLAP (the sequence of which is shown in Figure 29 of U.S. Pat. No. 5,352,604 and which has the mutations S99D+S101R+S103A+V104I+G159S, hereafter referred to as BLAP), BLAP R (BLAP with S3T+V4I+V199M+V205I+L217D), BLAP X (BLAP with S3T+V4I+V205I) and BLAP F49 (BLAP with S3T+V4I+A194P+V199M+V205I+L217D) with additional mutations 101E / D, S156D, L262E, 206A / L / S / T / , 209K / V / W, 215W, 216N / S / T; KAP (Bacillus alkalophilus subtilisin with mutations A230V+S256G+S259N) from Kao and Lavergy®, Lavergy® Pro, Lavergy® C Bright from BASF.

[0151] Particularly preferred for use herein are commercially available proteases selected from the group consisting of Properase®, Blaze®, Ultimase®, Everlase®, Savinase®, Excellase®, Blaze Ultra®, BLAP and BLAP variants.

[0152] Preferred concentrations of additional protease in the products of the present invention include from about 0.001 to about 10, more preferably from about 0.002 to about 7, especially from about 0.005 to about 6 mg of active protease per gram of composition.

[0153] Lipase: The composition preferably includes a lipase. The presence of oils and / or fats can further increase the recovery of stains containing mannans and other polysaccharides. Therefore, the presence of lipase in the enzyme package can further improve the removal of such stains. Suitable lipases include those of bacterial, fungal, or synthetic origin, including chemically modified or genetically engineered variants of the protein. Examples of useful lipases include lipases from the genus Humicola (also known as Thermomyces), such as Humicola lanuginosa (T. lanuginosus) or Humicola insolens; lipases from Pseudomonas alcaligenes (P. alcaligenes), Pseudomonas pseudoalcaligenes, Pseudomonas cepacia, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas sp. strain SD705, Pseudomonas wiscosinensis (P. Pseudomonas lipases derived from Bacillus wisconsinensis; Bacillus lipases derived from Bacillus subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), Bacillus stearothermophilus, or B. pumilus.

[0154] The lipase may be a "first cycle lipase" as described in U.S. Patent No. 6,939,702 (B1) and U.S. Patent Application Publication No. 2009 / 0217464. In one embodiment, the lipase is a first wash lipase, preferably a variant of the wild-type lipase from Thermomyces lanuginosus, containing the T231R and N233R mutations. The wild-type sequence is 269 amino acids (amino acids 23-291) of Swissprot accession number Swiss-Prot O59952 (from Thermomyces lanuginosus (Humicola lanuginosa)). Preferred lipases include those sold under the trade names Lipex®, Lipolex®, and Lipoclean®.

[0155] Other suitable lipases include, for example, Liprl 139 as described in WO 2013 / 171241, TfuLip2 as described in, for example, WO 2011 / 084412 and WO 2013 / 033318, Pseudomonas stutzeri lipase as described in, for example, WO 2018228880, Microbulbifer thermotolerans lipase as described in, for example, WO 2018228881, Sulfobacillus acidocaldarius lipase as described in, for example, EP 3299457, acidocaldarius lipases, such as LIP062 lipase as described in WO2018209026, PinLip lipase as described in WO2017036901, and Absidia species lipases as described in WO2017005798.

[0156] Suitable lipases include (a) Substitution T231R and (b) Substitution N233R or N233C and (c) a variant of SEQ ID NO: 5 comprising at least three additional substitutions selected from E1C, D27R, N33Q, G38A, F51V, G91Q, D96E, K98L, K98I, D111A, G163K, H198S, E210Q, Y220F, D254S, I255A, and P256T; wherein the positions correspond to those of SEQ ID NO:5, the lipase variant has at least 90% but less than 100% sequence identity with a polypeptide having the amino acid sequence of SEQ ID NO:5, and the variant has lipase activity.

[0157] One preferred lipase is a variant of SEQ ID NO:5 that includes the following substitutions: T231R, N233R, D27R, G38A, D96E, D111A, G163K, D254S, and P256T.

[0158] One preferred lipase is a variant of SEQ ID NO: 5 containing the following substitutions: T231R, N233R, N33Q, G91Q, E210Q, I255A.

[0159] Suitable lipases are commercially available from Novozymes, for example as Lipex Evity 100L, Lipex Evity 200L (both liquid ingredients) and Lipex Evity 105T (granules), which have a different structure to the products Lipex 100L, Lipex 100T and Lipex Evity 100T, which are outside the scope of the present invention.

[0160] Cellulases: Suitable cellulases include those of bacterial or fungal origin, including chemically modified or genetically engineered variants of the protein. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as fungal cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and 5,691,178.

[0161] In one aspect, preferred enzymes include endoglucanases derived from microorganisms that exhibit endo-β-1,4-glucanase activity (EC 3.2.1.4), preferably selected from the group comprising: (a) a bacterial polypeptide endogenous to a member of the genus Bacillus having a sequence that is at least 90%, 94%, 97%, or even 99% identical to the amino acid sequence of SEQ ID NO:2 in U.S. Patent No. 7,141,403(B2), with preferred substitutions including one or more positions corresponding to positions 292, 274, 266, 265, 255, 246, 237, 224, and 221 of the mature polypeptide of SEQ ID NO:2, wherein the variant has cellulase activity; (b) a glycosyl hydrolase having enzymatic activity on both xyloglucan and amorphous cellulose substrates, the glycosyl hydrolase being selected from GH family 5, 7, 12, 16, 44, or 74; (c) a glycosyl hydrolase having a sequence that is at least 90%, 94%, 97%, or even 99% identical to the amino acid sequence of SEQ ID NO: 3 of WO 09 / 148983; (d) a variant exhibiting at least 70% identity to SEQ ID NO: 5 in WO2017106676. Preferred substitutions include one or more positions corresponding to positions 4, 20, 23, 29, 32, 36, 44, 51, 77, 80, 87, 90, 97, 98, 99, 102, 112, 116, 135, 136, 142, 153, 154, 157, 161, 163, 192, 194, 204, 208, 210, 212, 216, 217, 221, 222, 225, 227, and 232; (e) and mixtures thereof.

[0162] Suitable endoglucanases are sold under the trade names Celluclean® and Whitezyme® (Novozymes A / S, Bagsvaerd, Denmark). Examples include Celluclean® 5000L, Celluclean® Classic 400L, Celluclean® Classic 700T, Celluclean® 4500T, Whitezyme® 1.5T, and Whitezyme® 2.0L.

[0163] Other commercially available cellulases include Celluzyme®, Carezyme®, Carezyme® Premium (Novozymes A / S), Clazinase®, Puradax HA®, Revitalenz® 1000, Revitalenz® 2000 (Genencor International Inc.), KAC-500® (Kao Corporation), Biotouch® FCL, Biotouch® DCL, Biotouch® DCC, Biotouch® NCD, Biotouch® FCC, Biotouch® FLX1 (AB Enzymes).

[0164] Suitable glucanases include endo-β-1,3-glucanases, preferably from EC class 3.2.1.39, preferably obtained from microorganisms of the genus Paenibacillus, Zobellia galactanivorans, Thermotoga petrophila, or Trichoderma, preferably Paenibacillus or Zobellia galactanivorans, most preferably Paenibacillus.

[0165] Amylase: Preferably, the composition of the present invention comprises an amylase. Suitable α-amylases include those of bacterial or fungal origin, including chemically or genetically modified mutants (variants). Preferred alkaline α-amylases are those derived from strains of the genus Bacillus, such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis, or other Bacillus species, such as Bacillus species NCBI12289, NCBI12512, NCBI12513, DSM9375 (U.S. Pat. No. 7,153,818), DSM12368, DSMZ No. 12649, KSM AP1378 (WO 97 / 00324), KSM K36, or KSM K38 (EP 1,022,334). Preferred amylases include: (a) the variants described in U.S. Pat. No. 5,856,164 and WO 99 / 23211, WO 96 / 23873, WO 00 / 60060, WO 06 / 002643, and WO 2017 / 192657, in particular the AA560 enzyme described as SEQ ID NO: 12 in WO 06 / 002643; and one or more substitutions at positions 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 202, 214, 231, 246, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484, preferably D183. * and G184 * Mutants that also contain deletions of (b) variants exhibiting at least 85%, preferably 90%, identity to SEQ ID NO: 4 in WO 06 / 002643, the wild-type enzyme from Bacillus sp. SP722, in particular variants in which positions 183 and 184 are deleted, and variants described in WO 00 / 60060, WO 2011 / 100410 and WO 2013 / 003659, in particular SEQ ID NO: 4 in WO 06 / 002643, which is incorporated herein by reference; those with one or more substitutions at positions 51, 52, 54, 109, 304, 140, 189, 134, 195, 206, 243, 260, 262, 284, 347, 439, 469, 476, and 477. (c) Variants exhibiting at least 90% identity to the wild-type enzyme from Bacillus sp. 707 (SEQ ID NO: 7 in U.S. Pat. No. 6,093,562), particularly those containing one or more of the following mutations: M202, M208, S255, R172, and / or M261. Preferably, the amylase contains one or more of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N, and / or R172Q. Particularly preferred are those containing the M202L or M202T mutations. Further relevant mutations / deletions based on the SP707 backbone are W48, A51, V103, V104, A113, R118, N125, V131, T132, E134, T136, E138, R142, S154, V165, R182, G182, H183, E190, D192, T193, I206, M208, D209, E212, V213, V214, N215, L217, R218, N219, and N220. Includes 9, V222, T225, T227, G229, I235, K242, Y243, S244, F245, T246, I250, S255, A256, H286, V291, T316, V317, V318, N417, T418, A419, H420, P421, I428, M429, F440, R443, N444, K445, Q448, S451, A465, N470, S472. (d) a variant described in WO 09 / 149130, preferably SEQ ID NO: 1 or SEQ ID NO: 2 in WO 09 / 149130, which exhibits at least 90% identity to the wild-type enzyme from Geobacillus Stearophermophilus, or a truncated version thereof. (e) a variant described in WO 10 / 115021, in particular SEQ ID NO: 2 in WO 10 / 115021, which exhibits at least 75%, or at least 85%, or at least 90%, or at least 95% identity with the α-amylase from Bacillus sp. TS-23. (f) Variants exhibiting at least 89% identity to SEQ ID NO: 1 in WO2016091688, in particular those containing a deletion at positions H183+G184 and further containing one or more mutations at positions 405, 421, 422, and / or 428. (g) Variants described in WO 2014099523, in particular those exhibiting at least 60% amino acid sequence identity with "PcuAmyl α-amylase" from Paenibacillus curdlanolyticus YK9 (SEQ ID NO: 3 in WO 2014099523). (h) Variants described in WO 2014099523, in particular "CspAmy2 amylase" from Cytophaga species (showing at least 60% amino acid sequence identity with SEQ ID NOs: 1 and 6 in WO 2014164777, in particular those containing one or more of the following deletions and / or mutations based on SEQ ID NO: 1 in WO 2014164777: R178 * , G179 * , T38N, N88H, N126Y, T129I, N134M, F153W, L171R, T180D, E187P, I203Y, G476K, G477E, Y303D. (i) A variant exhibiting at least 85% identity with AmyE from Bacillus subtilis (SEQ ID NO: 1 in WO 2009149271). (j) A variant exhibiting at least 90% identity with the wild-type amylase derived from Bacillus sp. KSM-K38 having accession number AB051102. (k) variants described in WO2016180748, in particular those exhibiting at least 80% identity with the mature amino acid sequence of AAI10 from Bacillus sp. as set forth in SEQ ID NO: 7 in WO2016180748; those exhibiting at least 80% identity with the mature amino acid sequence of the amylase from Alicyclobacillus sp. as set forth in SEQ ID NO: 8 in WO2016180748, and those exhibiting at least 80% identity with the mature amino acid sequence of SEQ ID NO: 13 in WO2016180748, in particular those having the following mutations: *, N54S, V56T, K72R, G109A, F113Q, R116Q, W167F, Q172G, A174S, G184T, N195F, V206L, K391A, P473R, G476K. (l) Variants described in WO 2018060216, in particular those exhibiting at least 70% identity to the mature amino acid sequence of SEQ ID NO: 4 in WO 2018060216, and fusion molecules of Bacillus amyloliquefaciens and Bacillus licheniformis. In particular, those containing one or more substitutions at positions H1, N54, V56, K72, G109, F113, R116, T134, W140, W159, W167, Q169, Q172, L173, A174, R181, G182, D183, G184, W189, E194, N195, V206, G255, N260, F262, A265, W284, F289, S304, G305, W347, K391, Q395, W439, W469, R444, F473, G476, and G477.

[0166] A preferred amylase is a genetically engineered enzyme in which one or more amino acids susceptible to bleaching oxidation are replaced with amino acids less susceptible to oxidation. Specifically, methionine residues are preferably replaced with any other amino acid. Specifically, the methionine most susceptible to oxidation is preferably replaced. Preferably, the methionine at position 202 in SEQ ID NO: 11 is replaced. Preferably, the methionine at this position is replaced with threonine or leucine, preferably leucine.

[0167] Suitable commercially available α-amylases include DURAMYL®, LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®, STAINZYME®, STAINZYME PLUS®, FUNGAMYL®, ATLANTIC®, ACHIEVE ALPHA®, AMPLIFY® PRIME, INTENSA®, and BAN® (Novozymes A / S, Bagsvaerd, Denmark), KEMZYM® AT 9000 (Biozym Biotech Trading GmbH, Wehlistrasse 27b A-1200 Wien, Austria), RAPIDASE®, PURASTAR®, ENZYSIZE®, OPTISIZE HT PLUS®, POWERASE®, and PREFERENZ. Examples include the S® series (including PREFERENZ S1000® and PREFERENZ S2000®), PURASTAR OXAM® (DuPont, Palo Alto, California), and KAM® (Kao, 14-10 Nihonbashi Kayabacho, 1-chome, Chuo-ku, Tokyo 103-8210, Japan).

[0168] Preferably, the composition contains at least 0.01 mg, preferably about 0.05 to about 10, more preferably about 0.1 to about 6, especially about 0.2 to about 5 mg of active amylase per gram of composition.

[0169] Peroxidases / Oxidases: Suitable peroxidases / oxidases include those derived from plants, bacteria, or fungi, including chemically modified or genetically engineered variants of the protein. Examples of useful peroxidases include peroxidases from the genus Coprinus, such as those from Coprinus cinereus and variants thereof, as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0170] Commercially available peroxidases include GUARDZYME® (Novozymes A / S).

[0171] Pectate lyases: Suitable pectate lyases include those sold under the trade names Pectawash®, Pectaway®, X-Pect® (all Novozymes A / S, Bagsvaerd, Denmark), Preferenz® F1000 (DuPont Industrial Biosciences).

[0172] Mannanase. The composition preferably contains one or more mannanase enzymes. As used herein, the term "mannanase" or "galactomannanase" refers to a mannanase enzyme that catalyzes the hydrolysis of 1,4-β-D-mannosidic bonds in mannans, galactomannans, glucomannans, and galactoglucomannans, defined in accordance with what is known in the art as mannan endo-1,4-β-mannosidase, also known as β-mannanase and endo-1,4-mannanase. Mannanases are classified as EC 3.2.1.78 according to enzyme nomenclature and belong to glycosyl hydrolase families 5, 26, and 113. Many suitable mannanases belong to glycosyl hydrolase family 5. Commercially available mannanases include those sold under the trade name Mannaway® (Novozymes A / S), such as Mannaway® 200L and Mannaway Evity 4.0T. Other commercially available mannanases include Effectenz® M1000, Mannastar® 375, Preferenz M100, and Purabrite® (all from DuPont Industrial Biosciences) and Biotouch M7 (AB Enzymes). Other suitable mannanases belong to glycosyl hydrolase family 26, including those described in WO 2018191135, WO 2015040159, WO 2017021515, WO 2017021516, WO 2017021517, and WO 2019081515. Suitable mixtures of mannanases include the combination of glycosyl hydrolase family 5 mannanase and glycosyl hydrolase family 26 mannanase described in WO 2019081515.

[0173] Nuclease: Preferably, the composition comprises a nuclease enzyme, such as RNase or DNase, or a mixture thereof. A nuclease enzyme is an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. The nuclease enzyme herein is preferably a deoxyribonuclease or ribonuclease enzyme or a functional fragment thereof. A functional fragment or portion refers to a portion of a nuclease enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone, and thus is a region of the nuclease protein that retains catalytic activity. It therefore includes truncated but functional versions of the enzyme and / or mutants and / or derivatives and / or homologs in which functionality is maintained.

[0174] Preferably, the nuclease enzyme is a deoxyribonuclease preferably selected from any of the following classes: EC 3.1.21.x (where x=1, 2, 3, 4, 5, 6, 7, 8 or 9), EC 3.1.22.y (where y=1, 2, 4 or 5), EC 3.1.30.z (where z=1 or 2), EC 3.1.31.1, and mixtures thereof.

[0175] DNases: Suitable DNases include those set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9 of WO 2017162836 (Novozymes) and SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 of WO 2018108865, DNase wild-type and mutant forms defined by 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54, as well as mutant forms of Bacillus cibi DNase, including those described in International Publication No. 2018011277 (Novozymes), which are incorporated herein by reference. Preferred DNases are as claimed in European Patent No. 3476935(A).

[0176] RNases: Suitable RNases include wild-type and variants of DNases as defined by SEQ ID NOs: 3, 6, 9, 12, 15, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 72 and 73 of WO2018178061 (Novozymes) and SEQ ID NOs: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 and 104 of WO2020074499 (Novozymes), which are incorporated herein by reference.

[0177] Hexosaminidase: The composition preferably additionally comprises one or more hexosaminidase enzymes. The term hexosaminidase includes "dispersin" and the abbreviation "Dsp" and refers to a polypeptide having hexosaminidase activity. Suitable enzymes are found in EC 3.2.1., which catalyze the hydrolysis of β-1,6-glycosidic bonds in N-acetyl-glucosamine polymers found in microbial soils. The term "hexosaminidase" includes polypeptides having N-acetylglucosaminidase activity and β-N-acetylglucosaminidase activity. Hexosaminidase activity can be determined according to Assay II described in WO2018184873. Suitable hexosaminidases include those described in International Publication Nos. 2017186936, 2017186937, 2017186943, 2017207770, 2018184873, 2019086520, 2019086528, 2019086530, 2019086532, 2019086521, and 2019 Examples of suitable hexosaminidases include those disclosed in WO 2020207944, WO 2020002604, WO 2020002608, WO 2020007863, WO 2020007875, WO 2020008024, WO 2020070063, WO 2020070249, WO 2020088957, WO 2020088958, and WO 2020207944. Variants of the Terribacillus saccharophilus hexosaminidase defined by SEQ ID NO: 1 in WO 2020207944 are preferred, particularly the variants with improved thermostability disclosed therein.

[0178] Xanthan gum-degrading enzymes: The compositions may contain one or more xanthan gum-degrading enzymes. Enzymes suitable for degrading xanthan gum-based stains include xanthan endoglucanases, optionally in combination with xanthan lyases. As used herein, the term "xanthan endoglucanase" refers to an enzyme exhibiting endo-β-1,4-glucanase activity that can catalyze the hydrolysis of the 1,4-linked β-D-glucose polymer backbone of xanthan gum, optionally in combination with a suitable xanthan lyase enzyme. Suitable xanthan endoglucanases are described in WO 2013167581, WO 2015181299, WO 2015181292, WO 2017046232, WO 2017046260, WO 201837062, WO 201837065, WO 2019038059, and WO 2019162000. As used herein, the term "xanthan lyase" refers to an enzyme that cleaves the β-D-mannosyl-β-D-1,4-glucuronosyl bond in xanthan gum. Such enzymes belong to the EC 4.2.2.12 enzyme class. Suitable xanthan gelases are described in WO 2015001017, WO 2018037061, WO 201837064, WO 2019038060, WO 2019162000, and WO 2019038057.

[0179] Galactanase: Preferably, the composition comprises a galactanase, i.e., an extracellular polymer-degrading enzyme, including an endo-β-1,6-galactanase enzyme. The term "endo-β-1,6-galactanase" or "polypeptide with endo-β-1,6-galactanase activity" refers to endo-β-1,6-galactanase activity (EC 3.2.1.164) from glycoside hydrolase family 30 that catalyzes the hydrolytic cleavage of 1,6-3-D-galactooligosaccharides with a degree of polymerization (DP) greater than 3 and their acidic derivatives bearing a 4-O-methyl glucosyluronate or glucosyluronate group at the non-reducing end. For purposes of this disclosure, endo-β-1,6-galactanase activity is determined in Assay I according to the procedure described in WO2015185689. Suitable examples from classification EC3.2.1.164 are described in WO2015185689, e.g., mature polypeptide SEQ ID NO:2.

[0180] The additional enzymes may be included in the detergent composition by adding a separate enzyme additive containing the additional enzymes or a mixed enzyme additive containing two, some, or all of the additional enzymes. Such enzyme additives may be in the form of granules, liquids, or slurries, and preferably further include an enzyme stabilizer.

[0181] Preferably, the or each additional enzyme is present in the composition in an amount of at least 0.0001% to about 0.1% by weight of pure active enzyme protein, such as from about 0.0001% to about 0.01% by weight, from about 0.001% to about 0.01% by weight, or from about 0.001% to about 0.01% by weight, or from about 0.001% to about 0.01% by weight, based on the weight of the composition.

[0182] Fabric Hueing Agents. The composition may include a fabric hueing agent (sometimes referred to as a tinting agent, bluing agent, or whitening agent / dye). Typically, the hueing agent imparts a blue or purple hue to the fabric. Hueing agents can be used either alone or in combination to create a particular hue and / or tint different types of fabric. This can be achieved, for example, by mixing a red and a green-blue dye to produce a blue or purple hue. The hueing agent may be selected from any known chemical class of dyes, including, but not limited to, acridines, anthraquinones (including polycyclic quinones), azines, azos including premetallized azos (e.g., monoazos, diazos, trisazos, tetrakisazos, polyazos), benzodifurans and benzodifuranones, carotenoids, coumarins, cyanines, diazahemicyanines, diphenylmethanes, formasans, hemicyanines, indigoids, methanes, naphthalimides, naphthoquinones, nitro and nitroso, oxazines, phthalocyanines, pyrazoles, stilbenes, styryls, triarylmethanes, triphenylmethanes, xanthenes, and mixtures thereof. Azo dyes, particularly monoazo or bis-azo dyes, triarylmethane dyes, and anthraquinone dyes are preferred.

[0183] Suitable fabric hueing agents include dyes, dye-clay conjugates, and organic and inorganic pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes classified in the Colour Index (CI) classification of direct dyes, basic dyes, reactive or hydrolysis reactive dyes, solvent dyes, or disperse dyes. Examples of suitable small molecule dyes include, for example, dyes classified in the Colour Index (CI) classification of Society of Dyes and Colourists, Bradford, UK) numbers: Direct Violet dyes 9, 35, 48, 51, 66, and 99; Direct Blue dyes 1, 71, 80, and 279; Acid Red dyes 17, 73, 52, 88, and 150; Acid Violet dyes 15, 17, 24, 43, 49, 50, and 51; Acid Blue dyes 15, 17, 25, 29, 40, 45, 75, 80, 83, 90, and 11 3, Acid Black Dye 1, Basic Violet Dyes 1, 3, 4, 10, and 35, Basic Blue Dyes 3, 16, 22, 47, 66, 75, and 159, Disperse or Solvent Dyes such as those described in EP 1794275 or EP 1794276, or the dyes disclosed in U.S. Pat. No. 7,208,459 B2, and mixtures thereof.

[0184] Preferred are polymeric dyes, including polymers containing covalently attached (sometimes referred to as conjugated) chromogens (dye-polymer conjugates), such as polymers having chromogens copolymerized into the backbone of the polymer, and mixtures thereof, including those described in WO 2011 / 98355, WO 2011 / 47987, U.S. Patent No. 2012 / 090102, WO 2010 / 145887, WO 2006 / 055787, and WO 2010 / 142503.

[0185] Preferred polymeric dyes include alkoxylated, preferably ethoxylated, azo, anthraquinone, or triarylmethane dyes. Particularly preferred are polymeric dyes selected from the group consisting of ethoxylated thiophene azo dyes, such as the fabric substantive colorant sold under the name Liquitint® (Milliken, Spartanburg, South Carolina, USA), and dye-polymer conjugates formed from at least one reactive dye and a polymer selected from the group consisting of polymers containing a moiety selected from the group consisting of hydroxyl moieties, primary amine moieties, secondary amine moieties, thiol moieties, and mixtures thereof. Suitable polymeric dyes include polymeric dyes selected from the group consisting of Liquitint® Violet CT, carboxymethylcellulose (CMC) (covalently bonded to a reactive blue, reactive violet, or reactive red dye, such as CMC conjugated with CI Reactive Blue 19 sold under the trade name AZO-CM-CELLULOSE, product code S-ACMC by Megazyme, Wicklow, Ireland), alkoxylated triphenyl-methane polymeric colorants, alkoxylated thiophene polymeric colorants, and mixtures thereof. The composition may also include an aesthetic dye / colorant.Aesthetic colorants include Liquitint® Blue AH, Liquitint® Blue BB, Liquitint® Blue 275, Liquitint® Blue 297, Liquitint® Blue BB, Cyan 15, Liquitint® Green 101, Liquitint® Orange 272, Liquitint® Orange 255, Liquitint® Pink AM, Liquitint® Pink AMC, Liquitint® Pink ST, Li Liquitint® Violet 129, Liquitint® Violet LS, Liquitint® Violet 291, Liquitint® Yellow FT, Liquitint® Blue Buf, Liquitint® Pink AM, Liquitint® Pink PV, Acid Blue 80, Acid Blue 182, Acid Red 33, Acid Red 52, Acid Violet 48, Acid Violet 126, Acid Blue 9, Acid Blue 1, and mixtures thereof.

[0186] Preferred hueing dyes include the alkoxylated thiophene azo whiteners found in U.S. Patent No. 2008 / 0177090, and can optionally be anionic, such as those selected from Examples 1-42 in Table 5 of WO 2011 / 011799. Other preferred dyes are disclosed in U.S. Patent No. 8,138,222.

[0187] Preferred hueing dyes can be provided by incorporating into the composition leuco compounds (leuco dyes) containing a leuco moiety. These uncolored dyes convert (i) over time in the composition, (ii) in the wash liquor, and / or (iii) upon deposition on washed fabrics after washing to produce colored forms of the dyes that result in increased color and whiter appearance of the fabrics. In one aspect, the leuco moiety is selected from the group consisting of diarylmethane leuco moieties, triarylmethane leuco moieties, oxazine moieties, thiazine moieties, hydroquinone moieties, and arylaminophenol moieties. The leuco compound can include a leuco moiety and an alkyleneoxy moiety covalently bonded to the leuco moiety, wherein the alkyleneoxy moiety comprises at least one ethylene oxide group, and preferably the alkyleneoxide moiety also comprises at least one propylene oxide group. In one aspect, preferred leuco compounds include those conforming to the structure of formula (CVIII):

[0188] [ka] In the formula, R 8 is H or CH3, and each subscript b independently averages from about 1 to 2. Other suitable leuco dyes are disclosed in U.S. Patent Nos. 10,377,976, 10,377,977, 10,351,709, 10,385,294, 10,472,595, 10,479,961, 10,501,633, 10,577,570, 10,590,275, 10,633,618, 10,647,854, and 10,676,699, each of which is incorporated by reference in its entirety.

[0189] Suitable pigments include those selected from the group consisting of Ultramarine Blue (CI Pigment Blue 29), Ultramarine Violet (CI Pigment Violet 15), and mixtures thereof. Pigments and / or dyes may also be added to add color for aesthetic reasons. Preferred are organic blue, purple, and / or green pigments.

[0190] Builders: The detergent compositions may further contain builders such as builders based on carbonates, bicarbonates or silicates, which may be zeolites such as Zeolite A, Zeolite MAP (Maximum Aluminum type P). Laundry-safe zeolites are preferably of the formula Na 12 (AlO2) 12 (SiO2) 12 ·27H2O, with particle sizes typically ranging from 1 to 10 μm for zeolite A and 0.7 to 2 μm for zeolite MAP. Another builder is the strongly alkaline sodium metasilicate (Na2SiO3·nH2O or Na2SiO5·nH2O), preferably used in dishwashing. In preferred embodiments, the amount of detergent builder may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, or greater than 50%, or less than 80% or less than 65%. In dishwashing detergents, the builder concentration is typically 40 to 65%, particularly 50 to 65%, or even 75 to 90%.

[0191] Encapsulating Agent: The composition may comprise an encapsulated benefit agent comprising a core and a shell having an inner surface and an outer surface, the shell encapsulating the core. The core may comprise a material selected from the group consisting of fragrances, brighteners, dyes, insect repellents, silicones, waxes, fragrances, vitamins, softeners, skin care agents (in one embodiment, paraffin), enzymes, antimicrobial agents, bleaching agents, sensates, and mixtures thereof. The shell may comprise a material selected from the group consisting of polyethylene, polyamide, polystyrene, polyisoprene, polycarbonate, polyester, polyacrylate, aminoplast (in one aspect, the aminoplast may comprise polyurea, polyurethane, and / or polyureaurethane; in one aspect, the polyurea may comprise polyoxymethylene urea and / or melamine formaldehyde), polyolefin, polysaccharide (in one aspect, the polysaccharide may comprise alginate and / or chitosan), gelatin, shellac, epoxy resin, vinyl polymer, water-insoluble inorganic material, silicone, and mixtures thereof. A preferred encapsulant comprises a core containing a perfume. Such an encapsulant is a perfume microcapsule.

[0192] Enzyme stabilizer: The composition may contain an enzyme stabilizer. However, the composition of the present invention is particularly suitable for enabling the reduction of enzyme stabilizers. In particular, the cleaning composition of the present invention may be a boron-free cleaning composition. Suitable enzyme stabilizers may be selected from the group consisting of: (a) inorganic salts selected from the group consisting of calcium salts, magnesium salts, and mixtures thereof; (b) carbohydrates selected from the group consisting of oligosaccharides, polysaccharides, and mixtures thereof, and sugars or sugar alcohols; (c) phenylboronic acid and derivatives thereof, such as aromatic borate esters, or phenylboronic acid derivatives, such as 4-formylphenylboronic acid, or peptide aldehydes, such as di-, tri-, or tetrapeptide aldehydes or aldehyde analogs (any of the forms B1-B0-R, where R is H, CH3, CX3, CHX2, or CH2X (X=halogen), B0 is a single amino acid residue (preferably an optionally substituted aliphatic or aromatic side chain), and B1 has an N-terminal protecting group. (d) a mass-efficient reversible protease inhibitor selected from the group consisting of: (a) a reversible protease inhibitor consisting of one or more amino acid residues (preferably 1, 2 or 3) or as described in WO 09118375, WO 98 / 13459, optionally comprising: (b) a reversible protease inhibitor, such as a boron-containing compound; (c) a polyol, such as propylene glycol or glycerol 1-2 propanediol; (d) a calcium formate and / or sodium formate; (e) a protein-type protease inhibitor, such as RASI, BASI, WASI (rice, barley and wheat bifunctional α-amylase / subtilisin inhibitor) or CI2 or SSI, and (e) any combination thereof. It has been found that the cleaning compositions of the present invention allow for the reduction or omission of boron-containing protease stabilizers, such as borates and phenylboronic acid and their derivatives. Thus, in preferred embodiments, the cleaning composition contains less than 1.0% boron, or less than 0.5% by weight boron, or more preferably less than 0.2% by weight boron. Most preferably, the composition is free of boron-containing compounds, i.e., no boron-containing compounds are added. In other embodiments, the composition is free or substantially free of enzyme stabilizers or peptide inhibitors.

[0193] Structuring Agents: In one aspect, the composition may comprise a structuring agent selected from the group consisting of di- and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulosic materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.

[0194] Polymers: The composition preferably comprises one or more polymers. Preferred examples are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers, and amphiphilic polymers, and mixtures thereof.

[0195] Amphiphilic cleaning polymer: Preferably, the composition comprises an amphiphilic cleaning polymer. Preferred suitable polymers have the following general structure: bis((C2H5O)(C2H4O)n)(CH3)-N + -C x H 2x -N + Compounds having the formula -(CH3)-bis((C2H5O)(C2H4O)n) where n is 20-30 and x is 3-8, or sulfated or sulfonated variants thereof.

[0196] As used herein, the term "amphiphilic alkoxylated grease-cleaning polymer" refers to an alkoxylated polymer that has balanced hydrophilic and hydrophobic properties to remove grease particles from fabrics and surfaces. A specific embodiment of the amphiphilic alkoxylated grease-cleaning polymer of the present invention comprises a core structure and a plurality of alkoxylate groups attached to the core structure. These may comprise an alkoxylated polyalkyleneimine, preferably having an inner polyethylene oxide block and an outer polypropylene oxide block. The polymer is preferably present in an amount of 0.1 to 8 wt. %, more preferably 0.5 to 5 wt. %.

[0197] The core structure may comprise a polyalkyleneimine structure comprising repeat units of formulae (I), (II), (III), and (IV) in condensed form:

[0198] [ka] where # in each case indicates a link between a nitrogen atom and a group A of two adjacent repeating units of formula (I), (II), (III) or (IV). 1 means half of the bond between the free bond position of * in each case means half of the bond to one of the alkoxylate groups, A 1 are independently selected from straight-chain or branched C2-C6-alkylene; the polyalkyleneimine structure consists of 1 repeat units of formula (I), x repeat units of formula (II), y repeat units of formula (III), and y+1 repeat units of formula (IV), where x and y in each instance have values ​​ranging from 0 to about 150; and the average weight average molecular weight Mw of the polyalkyleneimine core structure has a value ranging from about 60 to about 10,000 g / mol.

[0199] Alternatively, the core structure may comprise a polyalkanolamine structure that is a condensation product of at least one compound selected from N-(hydroxyalkyl)amines of formula (Ia) and / or (Ib):

[0200] [ka] wherein A is independently selected from C1-C6-alkylene; R 1 , R 1* , R 2 , R 2* , R 3 , R 3* , R 4 , R 4* , R 5 and R 5*are independently selected from hydrogen, alkyl, cycloalkyl, or aryl, the last three radicals of which may be optionally substituted; R 6 is selected from hydrogen, alkyl, cycloalkyl, or aryl, the last three radicals of which may be optionally substituted.

[0201] The multiple alkyleneoxy groups attached to the core structure are independently selected from alkyleneoxy units of formula (V):

[0202] [ka] During the ceremony, * in each case means half of the bond to the nitrogen atom of a repeating unit of formula (I), (II), or (IV), and A 2 is, in each occurrence, independently selected from 1,2-propylene, 1,2-butylene, and 1,2-isobutylene; A 3 is 1,2-propylene; R, at each occurrence, is independently selected from hydrogen and C1-C4-alkyl; m has an average value ranging from 0 to about 2; n has an average value ranging from about 20 to about 50; and p has an average value ranging from about 10 to about 50.

[0203] Carboxylate polymer: The composition preferably also includes one or more carboxylate polymers, such as a maleate / acrylate random copolymer or a polyacrylate homopolymer. In one embodiment, the carboxylate polymer is a polyacrylate homopolymer having a molecular weight of 4,000 Da to 9,000 Da or 6,000 Da to 9,000 Da.

[0204] Soil Release Polymers: The composition preferably also includes one or more soil release polymers having a structure defined by one of the following structures (I), (II) or (III): (I) -[(OCHR 1 -CHR 2 ) a -O-OC-Ar-CO-] d (II) -[(OCHR 3 -CHR 4 ) b -O-OC-sAr-CO-] e (III) -[(OCHR 5 -CHR 6 ) c -OR 7 ] f During the ceremony, a, b, and c are 1 to 200; d, e, and f are 1 to 50; Ar is 1,4-substituted phenylene; sAr is 1,3-substituted phenylene substituted at the 5-position with SO3Me; Me is Na, Li, K, Mg / 2, Ca / 2, Al / 3, ammonium, mono-, di-, tri-, or tetra-alkylammonium (the alkyl groups are C1-C 18 Alkyl or C2-C 10 hydroxyalkyl), or mixtures thereof; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently H or C1 to C 18 n- or iso-alkyl; R 7 is a straight or branched chain C1 to C 18 Alkyl, or straight or branched C2-C 30 Alkenyl or cycloalkyl groups having 5 to 9 carbon atoms, or C8 to C 30 Aryl group or C6-C 30 It is an arylalkyl group.

[0205] Suitable soil release polymers are polyester soil release polymers such as Repel-o-tex polymers, including Repel-o-tex SF, SF-2, and SRP6, supplied by Rhodia. Other suitable soil release polymers include Texcare polymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325, supplied by Clariant. Other suitable soil release polymers are Marloquest polymers, such as Marloquest SL, supplied by Sasol.

[0206] Cellulosic polymer: The composition also preferably includes one or more cellulosic polymers, including those selected from alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, and alkyl carboxyalkyl celluloses. In one embodiment, the cellulosic polymer is selected from the group consisting of carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methylcarboxymethyl cellulose, and mixtures thereof. In one embodiment, the carboxymethyl cellulose has a degree of carboxymethyl substitution of 0.5 to 0.9 and a molecular weight of 100,000 Da to 300,000 Da.

[0207] The composition preferably comprises one or more organic acids selected from the group consisting of acetic acid, adipic acid, aspartic acid, carboxymethyloxymalonic acid, carboxymethyloxysuccinic acid, citric acid, formic acid, glutaric acid, hydroxyethyliminodiacetic acid, iminodiacetic acid, lactic acid, maleic acid, malic acid, malonic acid, oxydiacetic acid, oxydisuccinic acid, succinic acid, sulfamic acid, tartaric acid, tartaric-disuccinic acid, tartaric-monosuccinic acid, or mixtures thereof. Preferably, the detergent composition comprises an organic acid selected from the group consisting of acetic acid, lactic acid, and citric acid.

[0208] The composition preferably comprises an amine. Non-limiting examples of amines include, but are not limited to, etheramines, cyclic amines, polyamines, oligoamines (e.g., triamines, diamines, pentamines, tetraamines), or combinations thereof. The compositions described herein may comprise an amine selected from the group consisting of oligoamines, etheramines, cyclic amines, and combinations thereof. In some embodiments, the amine is not an alkanolamine. In some embodiments, the amine is not a polyalkyleneimine.

[0209] Examples of suitable oligoamines include, but are not limited to, diethylenetriamine (DETA), 4-methyldiethylenetriamine (4-MeDETA), dipropylenetriamine (DPTA), 5-methyldipropylenetriamine (5-MeDPTA), triethylenetetraamine (TETA), 4-methyltriethylenetetraamine (4-MeTETA), 4,7-dimethyltriethylenetetraamine (4,7-Me2TETA), 1,1,4,7,7-pentamethyldiethylenetriamine (M5-DETA), tripropylenetetraamine (TPTA), tetraethylenetetraamine (TETA), tetraethylenetri ... Examples of suitable amines include tetraethylenepentamine (TEPA), tetrapropylenepentamine (TPPA), pentaethylenehexamine (PEHA), pentapropylenehexamine (PPHA), hexaethyleneheptamine (HEHA), hexapropyleneheptamine (HPHA), N,N'-bis(3-aminopropyl)ethylenediamine, 1,1,4,7,7-pentamethyldiethylenetriamine (M5-DETA), dipropylenetriamine (DPTA), or mixtures thereof, most preferably diethylenetriamine (DETA). DETA may be preferred due to its low molecular weight and / or relatively low manufacturing costs.

[0210] The oligoamines of the present disclosure can have a molecular weight of about 100 to about 1200 Da, or about 100 to about 900 Da, or about 100 to about 600 Da, or about 100 to about 400 Da, preferably about 100 Da to about 250 Da, and most preferably about 100 Da to about 175 Da, or even about 100 Da to about 150 Da. For purposes of this disclosure, molecular weight is determined using the free base form of the oligoamine.

[0211] Etheramine: The cleaning compositions described herein may contain an etheramine. The cleaning compositions may contain from about 0.1% to 10%, or from about 0.2% to about 5%, or from about 0.5% to about 4% of the etheramine by weight of the composition.

[0212] The etheramines of the present disclosure may have a weight average molecular weight of less than about 1000 grams / mole, or from about 100 to about 800 grams / mole, or from about 200 to about 450 grams / mole, or from about 290 to about 1000 grams / mole, or from about 290 to about 900 grams / mole, or from about 300 to about 700 grams / mole, or from about 300 to about 450 grams / mole. The etheramines of the present disclosure may have a weight average molecular weight of from about 150, or about 200, or about 350, or about 500 grams / mole to about 1000, or about 900, or about 800 grams / mole.

[0213] Alkoxylated Phenolic Compound: The cleaning composition of the present disclosure may include an alkoxylated phenolic compound. The alkoxylated phenolic compound may be selected from the group consisting of an alkoxylated polyarylphenol compound, an alkoxylated polyalkylphenol compound, and mixtures thereof. The alkoxylated phenolic compound may be an alkoxylated polyarylphenol compound. The alkoxylated phenolic compound may be an alkoxylated polyalkylphenol compound.

[0214] The alkoxylated phenolic compound may be present in the cleaning composition at a concentration of from about 0.2% to about 10% or from about 0.5% to about 5% by weight of the cleaning composition.

[0215] The alkoxylated phenolic compound may have a weight average molecular weight of 280-2880.

[0216] The composition may optionally contain from about 0.001 to about 2% by weight of an antioxidant present in the composition. Preferably, the antioxidant is present at a concentration in the range of 0.01 to 0.08% by weight. Mixtures of antioxidants may also be used.

[0217] Antioxidants are substances such as those described in Kirk-Othmer (Vol. 3, p. 424) and Ullmann's Encyclopedia (Vol. 3, p. 91).

[0218] One class of antioxidants that may be used in the present invention are alkylated phenols having the general formula:

[0219] [ka] In the formula, R is C1 to C 22 R1 is a straight or branched chain alkyl, preferably methyl or branched chain C3-C6 alkyl, C1-C6 alkoxy, preferably methoxy, R1 is a C3-C6 branched chain alkyl, preferably tert-butyl, and x is 1 or 2. Hindered phenol compounds are a preferred class of alkylated phenols having this formula.

[0220] Examples of such hindered phenol antioxidants may include, but are not limited to, 2,6-bis(1-methylpropyl)phenol; 2,6-bis(1,1-dimethylethyl)-4-methyl-phenol (hydroxybutylated toluene, also known as "BHT"); 2-(1,1-dimethylethyl)-1,4-benzenediol; 2,4-bis(1,1-dimethylethyl)-phenol; 2,6-bis(1,1-dimethylethyl)-phenol; 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, methyl ester; 2-(1,1-dimethylethyl)-1,4-benzenediol; 2-(1,1-dimethylethyl)-4,6-dimethylphenol;3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, 1,1'-[2,2-bis[[3-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1-oxopropoxy]methyl]-1,3-propanediyl] ester;3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octadecyl ester;2, 2'-Methylenebis[6-(1,1-dimethylethyl)-4-methylphenol;2-(1,1-dimethylethyl)-phenol;2,4,6-Tris(1,1-dimethylethyl)-phenol;4,4'-Methylenebis[2,6-bis(1,1-dimethylethyl)-phenol;4,4',4''-[(2,4,6-trimethyl-1,3,5-benzenetriyl)tris(methylene)]tris[2,6-bis(1,1-dimethylethyl)-phenol];N,N'-1,6-Hexanediyl Rubis[3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanamide; 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzoic acid, hexadecyl ester; P-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylphosphonic acid, diethyl ester; 1,3,5-tris[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione;3,5-Bis(1,1-5 dimethylethyl)-4-hydroxybenzenepropanoic acid, 2-[3-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1-oxopropyl]hydrazide;3-(1,1-dimethylethyl)-4-hydroxy-5-methylbenzenepropanoic acid, 1,1'-[1,2-ethanediylbis(oxy-2,1-ethanediyl)]ester;4-[(dimethylamino)methyl]-2,6-bis(1,1-dimethylethyl)phenol;4-[[4,6-bis(octylthio)-1,3,5-triazin-2-yl ]Amino]-2,6-bis(1,1-dimethylethyl)phenol;3,5-Bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, 1,1'-(thiodi-2,1-ethanediyl) ester;3,5-Bis(1,1-dimethylethyl)-4-hydroxybenzoic acid, 2,4-bis(1,1-dimethylethyl)phenyl ester;3,5-Bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, 1,1'-(1,6-hexanediyl) ester;3-(1,1-dimethylethyl)-4-hydroxy-5-methylbenzenepropanoic acid Panic acid, 1,1'-[2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diylbis(2,2-dimethyl-2,1-ethanediyl)] ester; 3-(1,1-dimethylethyl)-b-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-b-methylbenzenepropanoic acid, 1,1'-(1,2-ethanediyl) ester; 2-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]-2-butylpropanedioic acid, 1,3-bis(1,2,2,6,6-pentamethyl-4-piperazinyl) lysinyl) ester;3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, 1-[2-[3-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1-oxopropoxy]ethyl]-2,2,6,6-tetramethyl-4-piperidinyl ester;3,4-dihydro-2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-(2R)-2H-1-benzopyran-6-ol;2,6-dimethylphenol;2,3,5-trimethyl-1,4-benzenediol;2,4,6-Trimethylphenol; 2,3,6-Trimethylphenol; 4,4'-(1-methylethylidene)-bis[2,6-dimethylphenol]; 1,3,5-tris[[4-(1,1-dimethylethyl)-3-hydroxy-2,6-dimethylphenyl]methyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione; 4,4'-methylenebis[2,6-dimethylphenol]; and mixtures thereof.

[0221] Preferably, the hindered phenol antioxidant contains at least one phenolic -OH group with at least one C3 to C6 branched alkyl in the ortho position relative to the at least one phenolic -OH group. More preferably, the hindered phenol antioxidant is an ester of 3,5-bis(1,1-dimethylethyl)-4-hydroxy-benzenepropanoic acid, and most preferably a C1 to C22 linear alkyl ester of 3,5-bis(1,1-dimethylethyl)-4-hydroxy-benzenepropanoic acid. Commercially available C1-C22 linear alkyl esters of 3,5-bis(1,1-dimethylethyl)-4-hydroxy-benzenepropanoic acid include RALOX®, a methyl ester of 3,5-bis(1,1-dimethylethyl)-4-hydroxy-benzenepropanoic acid manufactured by Raschig USA (Texas, USA), and TINOGARD® TS, an octadecyl ester of 3,5-bis(1,1-dimethylethyl)-4-hydroxy-benzenepropanoic acid manufactured by BASF (Ludwigshafen, Germany).

[0222] Furthermore, the antioxidant used in the composition may be selected from the group consisting of α-, β-, γ-, δ-tocopherol, ethoxyquin, 2,2,4-trimethyl-1,2-dihydroquinoline, 2,6-di-tert-butylhydroquinone, tert-butylhydroxyanisole, lignosulfonic acid and its salts, and mixtures thereof. It should be noted that ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) is sold under the name Raluquin™ by the company Raschig™.

[0223] Other types of antioxidants that can be used in the compositions are 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox™) and 1,2-benzisothiazolin-3-one (Proxel GXL™).

[0224] A further class of antioxidants that may be suitable for use in the compositions are benzofuran or benzopyran derivatives having the formula:

[0225] [ka] wherein R1 and R2 are each independently alkyl, or R1 and R2 can join together to form a C5-C6 cyclic hydrocarbyl moiety; B is absent or CH2; R4 is a C1-C6 alkyl; and R5 is hydrogen or -C(O)R3, where R3 is hydrogen or a C1-C6 alkyl. 19 R6 is a C1-C6 alkyl, R7 is hydrogen or a C1-C6 alkyl, and X is -CH2OH or -CH2A, where A is a nitrogen-containing unit, phenyl, or substituted phenyl. Preferred nitrogen-containing A units include amino, pyrrolidino, piperidino, morpholino, piperazino, and mixtures thereof. The cleaning compositions of the present disclosure may include a tannin selected from the group consisting of gallotannins, ellagitannins, complex tannins, condensed tannins, and combinations thereof.

[0226] Bleaching System: The compositions may contain a bleaching system comprising a source of HO, such as a perborate or percarbonate, which may be combined with a peracid-forming bleach activator, such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise, for example, an amide, imide, or sulfone type peroxyacid. Generally, when a bleaching agent is used, the compositions of the present invention may comprise from about 0.1% to about 30%, or even from about 0.1% to about 25%, by weight of the subject cleaning composition, of bleaching agent.

[0227] Chelating Agents: The compositions preferably include a chelating agent in an amount of from 0.005% to about 15%, or even from about 3.0% to about 10% chelating agent by weight of the composition. Suitable chelating agents include copper, iron, and / or manganese chelating agents, and mixtures thereof. Preferred chelating (complexing) agents include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid)), 1,2-dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, ethylenediamine, diethylenetriamine, ethylenediaminedisuccinic acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), iminodisuccinic acid (IDS), carboxymethyl inulin; oxidized alpha-1,3-glucan; and salts, derivatives, and mixtures thereof. Preferred chelating agents are selected from the group consisting of methyl-glycine-diacetic acid (MGDA), its salts and derivatives, glutamic acid-N,N-diacetic acid (GLDA), its salts and derivatives, iminodisuccinic acid (IDS), its salts and derivatives, carboxymethyl inulin, its salts and derivatives, and mixtures thereof. MGDA and its salts are particularly preferred, including in particular the trisodium salt of MGDA.

[0228] Solvent: When the composition is liquid, it typically contains less than 15% or less than 12% water by weight of the liquid laundry detergent composition. In some examples, the laundry detergent composition is a liquid laundry detergent composition containing a non-aqueous solvent selected from 1,2-propanediol, dipropylene glycol, tripropylene glycol, glycerol, sorbitol, polyethylene glycol, or a mixture thereof. In some examples, the liquid laundry detergent composition contains 10% to 40% or 15% to 30% non-aqueous solvent by weight of the liquid laundry detergent composition.

[0229] In some embodiments, cleaning compositions comprising one or more protease variants described herein are liquid laundry detergent compositions containing sulfite radical scavengers, protease stabilizers / inhibitors, or combinations thereof (WO 2022 / 157311).

[0230] In some embodiments, a cleaning composition comprising one or more protease variants described herein is a liquid laundry detergent composition such as those described in U.S. Patent No. 20210317387(A1), WO 2021 / 219296, WO 2021 / 127662, WO 2021 / 041685, U.S. Patent No. 11208619, U.S. Patent No. 20220186144, WO 2022 / 043045, WO 2022 / 043138, WO 2023 / 117494, WO 2023 / 088776, WO 2023 / 227332, and WO 2024 / 002922.

[0231] In some embodiments, cleaning compositions comprising one or more protease variants described herein are liquid laundry detergent compositions comprising dispersion variants, such as, but not limited to, the liquid laundry detergent compositions described in U.S. Patent No. 20210317387(A1).

[0232] In some embodiments, cleaning compositions comprising one or more protease variants described herein are liquid laundry detergent compositions that are highly alkaline fabric cleaners, such as, but not limited to, the liquid laundry detergent compositions described in WO 2021 / 219296.

[0233] In some embodiments, cleaning compositions comprising one or more protease variants described herein are liquid laundry detergent compositions that are low-density unit dose detergents with encapsulated fragrance, such as, but not limited to, the detergent compositions described in WO 2021 / 127662.

[0234] In some embodiments, cleaning compositions comprising one or more protease variants described herein are liquid laundry detergent compositions containing polyethylene glycol and an organic acid, such as, but not limited to, the detergent compositions described in WO 2021 / 041685.

[0235] In some embodiments, cleaning compositions comprising one or more protease variants described herein are detergent compositions containing polyethylene glycol and an organic acid, such as, but not limited to, the detergent compositions described in WO 2021 / 041685.

[0236] In some embodiments, cleaning compositions comprising one or more protease variants described herein are detergent compositions that are effective against proteinaceous stains, such as, but not limited to, the detergent compositions described in U.S. Pat. No. 1,120,8619.

[0237] In some embodiments, cleaning compositions comprising one or more protease variants described herein are detergent compositions containing soil release polymers, such as, but not limited to, the detergent compositions described in U.S. Patent No. 20220186144.

[0238] Preferably, the composition is substantially free of boron, i.e., contains trace amounts of boron, possibly from other composition or detergent ingredients, e.g., 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.

[0239] The cleaning aid may include a biopolymer. In some examples, the biopolymer is a glucan. In further examples, the glucan is an α-1,6-glucan or an α-glucan derivative. As used herein, the terms "α-1,6-glucan," "poly α-1,6-glucan," "α-1,6-glucan polymer," "dextran," and the like refer to a water-soluble α-glucan comprising glucose monomer units linked together by glycosidic bonds, wherein at least about 40% of the glycosidic bonds are α-1,6. In some embodiments, the α-1,6-glucan comprises about or at least about 90%, 95%, or 100% α-1,6 glycosidic bonds. Other linkages that may be present in α-1,6-glucans include α-1,2, α-1,3, and / or α-1,4 linkages.

[0240] The dextran herein can be, for example, that disclosed (e.g., molecular weight, linkage / branching profile, method of manufacture) in U.S. Patent Application Publication Nos. 2016 / 0122445, 2017 / 0218093, 2018 / 0282385, 2020 / 0165360, or 2019 / 0185893, each of which is incorporated herein by reference. In some embodiments, dextran for ester derivatization can be produced in a suitable reaction comprising glucosyltransferase (GTF) 0768 (SEQ ID NO: 1 or 2 in U.S. Patent Application Publication No. 2016 / 0122445), GTF 8117, GTF 6831, or GTF 5604 (these latter three GTF enzymes are SEQ ID NOs: 30, 32, and 33, respectively, in U.S. Patent Application Publication No. 2018 / 0282385), or a GTF comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of GTF 0768, GTF 8117, GTF 6831, or GTF 5604.

[0241] Dextrans herein can have, for example, α-1,2, α-1,3, and / or α-1,4 branches. In some embodiments, about, at least about, or less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, or 50% of the total glycosidic linkages of the branched dextran are α-1,2, α-1,3, and / or α-1,4 glycosidic branching linkages. Such branches are typically predominantly (>90% or >95%) or entirely (100%) a single glucose monomer in length. In some embodiments, dextrans with α-1,2-branching can be produced enzymatically according to the procedures of U.S. Patent Application Publication Nos. 2017 / 0218093 or 2018 / 0282385 (both incorporated herein by reference), where an α-1,2-branching enzyme, such as GTFJ18T1 or GTF9905, can be added during or after dextran production. In some embodiments, any other enzyme known to produce α-1,2-branching can be used. Dextrans with α-1,3-branching can be prepared, for example, as disclosed in Vuillemin et al. (2016, J. Biol. Chem. 291:7687-7702) or International Patent Application Publication No. WO 2021 / 007264, both of which are incorporated herein by reference.

[0242] The terminology used herein with respect to "esters" (e.g., α-glucan ester derivatives) may be as disclosed, for example, in U.S. Patent Application Publication Nos. 2014 / 0187767, 2018 / 0155455, or 2020 / 0308371, or International Patent Application Publication No. 2021 / 252575, each of which is incorporated herein by reference. Terms such as "α-glucan ester derivatives," "α-glucan ester compounds," and "α-glucan esters" are used interchangeably herein. An α-glucan ester derivative herein is an α-glucan ester that is esterified with one or more organic groups (e.g., hydrophobic organic groups), such that the derivative has a degree of substitution (DoS) with one or more organic groups of up to about 3.0. An α-glucan ester derivative is a compound having the moiety -C G The term "ester" is used herein by including -O-CO-C-, where "-C G "-" represents a carbon atom of a monomer unit (e.g., glucose) of an α-glucan ester derivative (where such carbon atom is bonded to a hydroxyl group [-OH] in the α-glucan precursor of the ester), and "-CO-C-" is included in the acyl group. An example of an α-glucan ester derivative herein is benzoyl α-glucan.

[0243] The hydrophobic acyl group of the α-glucan ester derivatives herein may be as disclosed, for example, in U.S. Patent Application Publication Nos. 2014 / 0187767, 2018 / 0155455, or 2020 / 0308371, or International Patent Application Publication No. 2021 / 252575, each of which is incorporated herein by reference.

[0244] The term "ether" (e.g., α-glucan ether derivative) used herein may be as disclosed, for example, in U.S. Patent Application Publication Nos. 2016 / 0311935, 2018 / 0237816, or 2020 / 0002646, or International Patent Application Publication Nos. 2021 / 257786 or 2021 / 252569, each of which is incorporated herein by reference. Terms such as "α-glucan ether derivative," "α-glucan ether compound," and "α-glucan ether" are used interchangeably herein. The α-glucan ether derivative herein is an α-glucan ether that has been etherified with one or more organic groups (e.g., charged organic groups such as cationic groups), such that the derivative has a DoS with the one or more organic groups of up to about 3.0. The α-glucan ether derivative has the moiety -C G The term "ether" is used herein by including -O-C-, where "-C G "-" represents a carbon atom of a monomer unit (typically glucose) of an α-glucan ether derivative (such carbon atom is bonded to a hydroxyl group [-OH] in the α-glucan precursor of the ether), and "-C-" is a carbon atom of an organic group.

[0245] The α-glucan ether derivatives of the present disclosure can be substituted with at least one positively charged organic group herein ether-linked to the α-glucan. The positively charged organic group can be, for example, any of the groups disclosed in U.S. Patent Application Publication Nos. 2016 / 0311935, 2018 / 0237816, or 2020 / 0002646, or International Patent Application Publication No. 2021 / 257786, which are incorporated herein by reference. The positively charged organic group can include, for example, a substituted ammonium group. Examples of substituted ammonium groups include primary, secondary, tertiary, and quaternary ammonium groups. In some embodiments, one or more positively charged organic groups can include a trimethylammonium hydroxypropyl group.

[0246] The α-glucan derivatives (e.g., esters or ethers) and / or products comprising such derivatives are biodegradable in some embodiments. Such biodegradability can be, for example, about, at least about, or at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90 days after testing, as determined by the carbon dioxide evolution test method (OECD Guideline 301B, incorporated herein by reference). It can be 90%, 5-60%, 5-80%, 5-90%, 40-70%, 50-70%, 60-70%, 40-75%, 50-75%, 60-75%, 70-75%, 40-80%, 50-80%, 60-80%, 70-80%, 40-85%, 50-85%, 60-85%, 70-85%, 40-90%, 50-90%, 60-90%, or 70-90%, or any value between 5% and 90%.

[0247] In another embodiment, the cleaning composition is a non-unit-dose liquid or gel detergent, which may be aqueous, typically containing at least 20% and up to 95% by weight of water, e.g., up to about 70% by weight of water, up to about 65% by weight of water, up to about 55% by weight of water, up to about 45% by weight of water, or up to about 35% by weight of water. Other types of liquids may be included in the aqueous liquid or gel, including, but not limited to, alkanols, amines, diols, ethers, and polyols. Aqueous liquid or gel detergents may contain 0-30% organic solvents. Liquid or gel detergents may also be non-aqueous.

[0248] The compositions may also contain other conventional detergent ingredients such as, for example, fabric conditioners including clays, suds boosters, suds suppressors, corrosion inhibitors, soil settling agents, soil redeposition inhibitors, dyes, disinfectants, optical brighteners, hydrotropes, anti-tarnish agents, organic solvents such as ethanol, or fragrances.

[0249] When the composition is in the form of a unit dose article, the water-soluble unit dose article typically comprises at least one water-soluble film oriented to create at least one unit dose internal compartment, the at least one unit dose internal compartment containing the detergent composition. In some examples, the consumer product comprises a container and at least one water-soluble unit dose article, optionally at least two water-soluble unit dose articles, optionally at least 20 water-soluble unit dose articles, or optionally at least 30 water-soluble unit dose articles. In some examples, the water-soluble unit dose article is in the form of a pouch. In some examples, the water-soluble unit dose article contains a unit dose of the composition in a volume sufficient to provide a benefit in the end use. In some examples, the water-soluble unit dose article comprises a water-soluble film shaped so that the unit dose article contains at least one internal compartment surrounded by a water-soluble film. The at least one compartment contains a cleaning composition. The water-soluble film is sealed to prevent leakage of the cleaning composition from the compartment during storage. However, when the water-soluble unit dose article is added to water, the water-soluble film dissolves, releasing the contents of the internal compartment into the cleaning solution. A unit dose article may include two or more compartments, at least two compartments, or at least three compartments, or at least four compartments, or even at least five compartments. The compartments may be arranged in a nested orientation, i.e., one on top of the other. Alternatively, the compartments may be positioned in a side-by-side orientation, i.e., one adjacent to the other. The compartments may be oriented in a "tire and rim" configuration, i.e., a first compartment is positioned adjacent to a second compartment, but the first compartment at least partially surrounds but does not completely enclose the second compartment. Alternatively, one compartment may be completely enclosed within another compartment. In some examples, a unit dose article includes at least two compartments, one of which is smaller than the other. In some examples, when a unit dose article includes at least three compartments, two of the compartments may be smaller than the third compartment, and in some examples, the smaller compartment is nested on top of the larger compartment. The nested compartments are, in some examples, oriented side-by-side.In some examples, each individual unit dose article may have a weight of 10 g to 40 g, or even 15 g to 35 g. The water-soluble film may be soluble or dispersible in water. Before being formed into a unit dose article, the water-soluble film has a thickness of 20 to 150 micrometers in some examples, 35 to 125 micrometers in other examples, 50 to 110 micrometers in further examples, and about 76 micrometers in still further examples. Examples of water-soluble film materials include polymeric materials. The film material may be obtained, for example, by casting, blow molding, extrusion, or blow-extrusion of a polymeric material. In some examples, the water-soluble film comprises a polyvinyl alcohol homopolymer or polyvinyl alcohol copolymer, e.g., a blend of polyvinyl alcohol homopolymers and / or polyvinyl alcohol copolymers, preferably selected from sulfonated and carboxylated anionic polyvinyl alcohol copolymers, particularly carboxylated anionic polyvinyl alcohol copolymers, e.g., a blend of polyvinyl alcohol homopolymer and carboxylated anionic polyvinyl alcohol copolymer, or a blend of two or more, preferably two, polyvinyl alcohol homopolymers. In some examples, the water-soluble film is one supplied by Monosol under product reference numbers M8630, M8900, M8779, or M8310. In some examples, the film may be opaque, transparent, or translucent. The film may include printed areas. The printed areas can be obtained using techniques such as flexographic printing or inkjet printing. The film may also include an aversive agent, e.g., a bittering agent. Suitable bittering agents include, but are not limited to, naringin, sucrose octaacetate, quinine hydrochloride, denatonium benzoate, or mixtures thereof. Exemplary concentrations of aversive agents include, but are not limited to, 1-5000 ppm, 100-2500 ppm, or 250-2000 ppm. The water-soluble film or the water-soluble unit dose article, or both, may be coated with a lubricant.In some examples, the lubricant is selected from talc, zinc oxide, silica, siloxane, zeolite, silicic acid, alumina, sodium sulfate, potassium sulfate, calcium carbonate, magnesium carbonate, sodium citrate, sodium tripolyphosphate, potassium citrate, potassium tripolyphosphate, calcium stearate, zinc stearate, magnesium stearate, starch, modified starch, clay, kaolin, gypsum, cyclodextrin, or mixtures thereof. 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.

[0250] The cleaning compositions described herein are typically formulated to provide wash water with a pH of about 4.0 to about 11.5, or even about 5.0 to about 11.5, or even about 5.0 to about 8.0, or even about 7.5 to about 10.5 during use in aqueous cleaning operations. Liquid product formulations are typically formulated to provide a pH of about 3.0 to about 9.0, or even about 3 to about 5. Granular laundry products are usually formulated to provide a pH of about 8 to about 11. In some embodiments, the cleaning compositions of the present invention can be formulated to provide an alkaline pH under wash conditions, e.g., 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 provide a neutral pH under wash conditions, e.g., a pH of about 5.0 to about 8.0, or about 5.5 to about 8.0, or about 6.0 to about 8.0, or about 6.0 to about 7.5. In some embodiments, neutral pH conditions can be measured when the cleaning composition is dissolved 1:100 (weight:weight) in deionized water at 20° C. and measured using a conventional pH meter. Techniques for controlling pH at recommended use concentrations include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0251] Suitable low pH cleaning compositions typically have a net pH of about 3.0 to about 5.0, or even about 3.5 to about 4.5. Low pH cleaning compositions typically do not contain surfactants that hydrolyze in such pH environments. Such surfactants include sodium alkyl sulfate surfactants containing at least one ethylene oxide moiety, or even about 1 to about 16 moles of ethylene oxide. Such cleaning compositions typically contain a pH adjuster, such as sodium hydroxide, monoethanolamine, or hydrochloric acid, in an amount sufficient to provide such cleaning compositions with a net pH of about 3.0 to about 5.0. Such compositions typically contain at least one acid-stable enzyme. In some embodiments, the composition is a liquid; in other embodiments, the composition is a solid. The pH of such liquid compositions is typically measured as net pH. The pH of such solid compositions is measured as a 10% solids solution of the composition in distilled water. In these embodiments, unless otherwise specified, all pH measurements are taken at 20°C.

[0252] Suitable high pH cleaning compositions typically have a net pH of about 9.0 to about 11.0, or even a net pH of 9.5 to 10.5. Such cleaning compositions typically contain a pH adjuster, e.g., sodium hydroxide, monoethanolamine, or hydrochloric acid, in an amount sufficient to provide such cleaning compositions with a net pH of about 9.0 to about 11.0. Such compositions typically contain at least one base-stable enzyme. In some embodiments, the composition is a liquid; in other embodiments, the composition is a solid. The pH of such liquid compositions is typically measured as net pH. The pH of such solid compositions is measured as a 10% solids solution of the composition in distilled water. In these embodiments, all pH measurements are taken at 20°C unless otherwise specified.

[0253] 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 variants during washing. In some embodiments, encapsulation enhances the performance of the variants and / or additional enzymes. In some embodiments, the encapsulating material typically encapsulates at least a portion of the subtilisin variants described herein. Typically, the encapsulating material is water-soluble and / or water-dispersible. In some embodiments, the encapsulating material has a glass transition temperature (Tg) of 0°C or higher. Exemplary encapsulating materials include, but are not limited to, carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin wax, and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, and combinations thereof. In some embodiments, the encapsulating material is starch (see, 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 encapsulating material is a microsphere made from a plastic, such as a thermoplastic, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof. Exemplary commercially available microspheres include, but are not limited to, EXPANCEL® (Stockviksverken, Sweden); PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (PQ Corp., Valley Forge, PA).

[0254] In some embodiments, one or more compositions described herein find use as detergent additives, wherein the additive is in solid or liquid form. Such additive products are intended to supplement and / or enhance the performance of conventional detergent compositions and can be added at any stage of the cleaning process. In some embodiments, the density of the laundry detergent composition, measured at 20° C., ranges from about 400 to about 1200 g / liter, and in other embodiments, ranges from about 500 to about 950 g / liter of composition.

[0255] When the cleaning composition is an ADW detergent composition comprising one or more subtilisin variants described herein, the composition preferably 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 wt. %; phosphate (monophosphate, diphosphate, tripolyphosphate, or oligomeric polyphosphate), sodium tripolyphosphate-STPP, or a phosphate-free builder ( Amino acid compounds, such as MGDA (methylglycine diacetic acid) and its salts and derivatives, GLDA (glutamic acid-N,N-diacetic acid) and its salts and derivatives, IDS (iminodisuccinic acid) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives, and mixtures thereof, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and β-alanine diacetic acid (B-ADA) and their salts, homopolymers and copolymers of polycarboxylic acids and their partially or fully neutralized salts, and monomeric polycarboxylic acids. and builders in the range of 5 to 60% by weight including hydroxycarboxylic acids and their salts in the range of 0.5 to 50% by weight; sulfonated / carboxylated polymers in the range of about 0.1 to about 50% by weight (to provide dimensional stability to the product); drying aids in the range of about 0.1 to about 10% by weight (polyesters, especially anionic polyesters, polycarbonates, polyurethanes and / or polyureas, optionally with further monomers having 3 to 6 functional groups, specifically acid, alcohol or ester functional groups, to promote polycondensation, or polyorganosiloxane compounds or reactive cyclic crystalline carbonates and their precursor compounds of the urea type; 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; bleaching agents of inorganic (e.g., perhydrated salts such as perborates, percarbonates, perphosphates, persulfates, and persilicates) and organic (e.g., diacyl peroxides and tetraacyl peroxides, especially organic peroxyacids including diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); about 0.bleach activator-organic peracid precursor in the range of 1 to about 10% by weight; bleach catalysts (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn, and Fe bispyridylamine and related complexes, and pentamine acetate cobalt(III) and related complexes); metal care agents (selected from benzatriazoles, metal salts and complexes, and silicates) in the range of about 0.1 to 5% by weight; enzymes (acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, β-glucanase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactosidase ... lactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, peroxidase, phenoloxidase, phosphatase, phosphodiesterase, phospholipase, phytase, polyesterase, polygalacturonase, additional protease, pullulanase, reductase, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and mixtures thereof); enzyme stabilizer component (selected from oligosaccharides, polysaccharides, and inorganic divalent metal salts).

[0256] Exemplary Automatic Dish Washing (ADW) compositions are provided in the table below.

[0257] [Table 1]

[0258] How to use The present invention also provides a method of treating / cleaning a surface, particularly a fabric surface, which method comprises, in a contacting step, contacting the surface with an aqueous wash solution comprising a subtilisin variant as described herein and a cleaning adjunct. The surface may have egg-containing stains or proteinaceous stains, such as egg-based stains such as creme brulee, baked cheese, BMI, or other protein-containing materials.

[0259] Preferably, the amount of the subtilisin variant in the cleaning solution is 0.01 ppm to 10 ppm, preferably 0.1 ppm to 1 ppm. Preferably, the cleaning solution also contains an anionic surfactant, preferably in an amount of 0.05 to 50 g / L, more preferably 0.2 g / L to 5 g / L, or 0.5 g / L to 3 g / L.

[0260] An aqueous wash solution may be formed by adding the above-described composition to water, for example in a washing machine or hand washing process. Alternatively, the aqueous wash solution may be formed by adding the subtilisin variant and cleaning adjuvants as separate components to water to form the wash solution. Surfaces, particularly fabrics, may then optionally be washed and / or rinsed and / or dried.

[0261] The subtilisin variant enzyme and any additional enzymes may be present in the wash liquor in an amount corresponding to 0.001 to 100 mg of active enzyme protein per liter of wash liquor, preferably 0.005 to 5 mg of active enzyme protein per liter of wash liquor, more preferably 0.01 to 1 mg of enzyme protein per liter of wash liquor, and especially 0.1 to 1 mg of enzyme protein per liter of wash.

[0262] During the contacting step or a subsequent step, it may be preferable to use mechanical agitation to facilitate cleaning and removal of dissolved soil by-products from the surface, especially when the surface is a fabric surface. The pH of the wash liquor is preferably about 7 or about 8 to about 10.5. The composition can typically be used at a concentration of about 500 ppm to about 15,000 ppm in solution to form the wash liquor. The wash liquor preferably has a temperature of about 5°C to about 40°C, or preferably 10 to 35°C, or 30°C, or 25°C. When the surface is a fabric surface, the water to fabric ratio is typically about 1:1 to about 30:1. The compositions and methods herein are particularly useful for treating any synthetic or natural fabric surface, including blended fabrics such as cotton, wool, silk, polyester, nylon, elastane, or polycotton.

[0263] The following examples are provided to demonstrate and illustrate certain preferred embodiments and aspects of the present disclosure and are not to be construed as limiting.

[0264] Example 1 Generation of enzyme mutants Bacillus amyloliquefaciens (BPN') wild-type subtilisin and its mutants were produced as described below. The amino acid sequence of the mature BPN' parent enzyme is set forth as SEQ ID NO: 1. All BPN' subtilisin variants were expressed using a DNA fragment containing, in consecutive order, the 5' AprE flanking region containing a variant of the B. subtilis rrnIp2 promoter sequence (SEQ ID NO:2) (the B. subtilis rrnIp2 promoter and the engineered variants are described more fully in patent application WO 2020112609), a nucleotide sequence encoding the aprE signal peptide sequence (SEQ ID NO:3), a nucleotide sequence encoding the B. amyloliquefaciens propeptide (SEQ ID NO:4), a sequence corresponding to the gene encoding mature BPN' subtilisin, the BPN' terminator (SEQ ID NO:5), and a 3' AprE flanking sequence containing a kanamycin resistance gene expression cassette (SEQ ID NO:6). This DNA fragment was assembled using standard molecular biology techniques. The linear DNA of the expression cassette was used to transform competent B. subtilis cells of a suitable strain. A library of BPN' subtilisin variants was generated by the method described above.

[0265] The transformation mixture was plated onto LA plates containing 1.6% skim milk and 5 ppm kanamycin and incubated overnight at 37° C. Single colonies were picked and grown in Luria broth at 37° C. under antibiotic selection.

[0266] For protein expression experiments, transformed cells were grown in 96-well microtiter plates (MTPs) in culture medium (a concentrated, semi-defined medium based on MOPS buffer containing urea as the main nitrogen source, glucose as the main carbon source, supplemented with 1% soytone for robust cell growth, and containing antibiotic selection) at 32°C, 250 rpm, and 70% humidity in a shaking incubator for 3 days. After centrifugation and filtration, the clarified culture supernatant containing the protease of interest was used for the assay.

[0267] Example 2 Enzyme assay Protein concentration determination: An Agilent Infinity II 1290 UHPLC equipped with an Agilent 300 SB-C3 RRHD (1.8 μm 2.1 × 50 mm) column was used to quantify protein concentration. The column temperature was 65 °C, and the sample was eluted from the column using a gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.07% TFA in acetonitrile. Absorbance was measured at 220 nm, and peaks were integrated using OpenLab software (Agilent Technologies, USA). Protein concentrations of the samples were calculated based on a standard curve of the parent protease.

[0268] Protease activity: The protease activity of parent subtilisin and its variants was tested by measuring the hydrolysis of the N-suc-AAPF-pNA substrate. The AAPF assay used a reagent solution of 160 mM suc-AAPF-pNA (suc-AAPF-pNA stock solution) (Sigma: S-7388) in 100 mM Tris (pH 8.6), 0.005% Tween®-80, and DMSO. To prepare the working solution, 1 mL of suc-AAPF-pNA stock solution was added to 100 mL of Tris buffer and mixed. The enzyme sample was added to a microtiter plate (MTP) containing a 1.6 mM suc-AAPF-pNA working solution, and activity was assayed by measuring absorbance at 405 nm over 3 to 5 minutes using a SpectraMax plate reader in kinetic mode at room temperature. Protease activity was expressed as mOD / min.

[0269] Wash Performance Assay: The liquid laundry detergent used in the wash performance and stability assays was Persil Small & Mighty Non-Bio Liquid Detergent "Persil Non-Bio" (PNB, Unilever), which was purchased from a UK supermarket on September 26, 2014. For the wash performance assay, PNB detergent was diluted to 2.7 g / L in deionized water, 5 mM HEPES (pH 8.2) with a water hardness of 12 gpg (3Ca:1Mg). This detergent, when tested for elemental boron content, contains ≦5 mg / Kg of boron and is therefore considered boron-free.

[0270] The protease variants were tested for cleaning performance compared to the parent (BPN') on industrial stains C-05 (blood / milk / ink on cotton fabric) and CS-39 (complete egg carbon with carbon black, aged, on cotton fabric) (both purchased from the Center for Test Materials BV, Vlaardingen, Netherlands). The stains were punched into small circular swatches and dispensed into Costar 9017 or Greiner 655101 microtiter plates (MTPs). The MTPs containing the microswatches were first filled with detergent. Then, a volume of the parent enzyme and variants were added to a final volume of 200 microliters. The assay was performed for 25 minutes at 25°C with gentle shaking. After the incubation period, 100–150 microliters of the supernatant was transferred to a fresh MTP, and the absorbance was read at 600 nm for BMI swatches and 405 nm for whole egg swatches using a SpectraMax plate reader. Absorbance results were obtained by subtracting the blank control (no enzyme) value from each sample value. For each condition and subtilisin variant in Example 2, the cleaning performance index (PI) was calculated by dividing the blank-subtracted absorbance of the variant by the absorbance of the parent protease at the same concentration. The blank-subtracted absorbance values ​​of the parent protease at the corresponding concentration of the variant were included in the test and determined using a standard curve of the parent protease generated using a Langmuir fit or a Hillsigmoid fit, as appropriate.

[0271] General sample setup for stability assays: Subtilisin enzymes were tested for stability in 10% solutions (v / v) of detergent, PNB, detergent A (Det A), detergent B (Det B), and / or detergent C (Det C). The composition of detergent A is listed in Table 1. The composition of detergent B is listed in Table 2. The composition of detergent C is listed in Table 5. BPN' and BPN' variants were tested at 40°C, 51°C, 53°C, 54°C, 56°C, 60°C, and / or 66°C. Elevated temperatures were set to allow for the determination of residual activity of stressed samples compared to unstressed samples during a 20-minute incubation period, in a range appropriate for discerning differences between the variant enzymes and their parent or reference variants. The exact temperature required to achieve the desired stability range may depend on the instrument manufacturer and the setup used. Incubation of samples across a temperature gradient may help determine this temperature. By fitting the residual activity across the temperature gradient to an appropriate curve fit, it is possible to compare the relative stability of samples at any temperature within the test range. Enzyme samples were mixed with diluted detergent, and protease activity against the AAPF substrate was immediately measured and used as the unstressed value. Samples were then placed in a PCR plate, sealed, and incubated at elevated temperature for 20 minutes using a thermocycler, then assayed for AAPF activity to obtain the stressed value. Residual activity was calculated as the ratio of stressed to unstressed activity. All enzyme samples were assayed in triplicate for the assay. A lower cutoff of 200 ppm protein expression was applied, and data with a CV (coefficient of variation) of 20% or less were analyzed.

[0272] [Table 2]

[0273] [Table 3]

[0274] Example 3 Mutant subtilisins with increased stability in the presence of detergents Mutants of subtilisin BPN' were tested against the parent enzyme or a reference mutant to determine the relative improvement in stability when measured in detergent at 54°C, 56°C, 60°C, or 66°C for 20 minutes, as described in Example 2, and reported as a percentage of stressed to unstressed enzyme activity. Tables 3 and 4 show the test results obtained for a series of BPN' mutants that have significantly enhanced stability compared to wild-type BPN' (SEQ ID NO: 1).

[0275] The subtilisin variants in this example generally exhibited strong cleaning performance.

[0276] [Table 4]

[0277] [Table 5-1]

[0278] [Table 5-2]

[0279] [Table 5-3]

[0280] [Table 5-4]

[0281] Example 4 Further mutant subtilisins with increased stability in the presence of detergents Additional variants of subtilisin BPN' were tested against the parent enzyme (wild-type BPN', SEQ ID NO: 1) to determine the relative improvement in stability in various detergent conditions using the method described in Example 2. Stability was measured for 20 minutes at 51°C for detergent C (the composition of detergent C (Det. C) is shown in Table 5), 53°C for detergent B, and 40°C for PBN detergent using the method described in Example 2.

[0282] [Table 6]

[0283] The stability improvement results of the BPN' mutants are reported as percent (%) residual activity after stress and are shown in Table 6.

[0284] [Table 7-1]

[0285] [Table 7-2]

[0286] [Table 7-3]

[0287] The cumulative contribution of substitutions was visualized by plotting the number of mutations against percent (%) residual activity for the mutants listed in Table 6 for each detergent and temperature stress. Data for mutants tested in detergent C at 51°C are shown in Figure 1. Data for mutants tested in detergent B at 53°C are shown in Figure 2. Data for mutants tested in PNB detergent at 40°C are shown in Figure 3.

[0288] Detergent Example Examples 1-6. Granular laundry detergent compositions designed for hand washing or top loading washing machines.

[0289] [Table 8]

[0290] Examples 7-13. Granular laundry detergent compositions designed for front-loading automatic washing machines.

[0291] [Table 9] * DNase is given as mg of active enzyme per 100 g of detergent.

[0292] Examples 14 to 29. Heavy-duty liquid laundry detergent compositions

[0293] [Table 10]

[0294] [Table 11-1]

[0295] [Table 11-2]

[0296] Examples 30-36. Unit dose laundry detergent compositions. Such unit dose formulations may contain one or more compartments.

[0297] [Table 12]

[0298] Example 37. Multi-compartment unit dose composition The following are multi-compartment water-soluble unit dose laundry articles comprising a larger bottom compartment with two smaller compartments in a side-by-side configuration stacked on top of the bottom compartment, following the Ariel 3-in-1 pod design, as commercially available in the UK in January 2020. The following compositions are encapsulated in a polyvinyl alcohol-based water-soluble outer film, more specifically a water-soluble film comprising a blend of polyvinyl alcohol homopolymer and a carboxylated anionic polyvinyl alcohol copolymer, and a water-soluble intermediate film comprising a blend of polyvinyl alcohol homopolymer or a blend of polyvinyl alcohol homopolymer and a carboxylated anionic polyvinyl alcohol copolymer.

[0299] [Table 13]

[0300] Ingredients and Notes for the Compositions of Examples 1 to 29 Linear alkylbenzene sulfonates with an average aliphatic carbon chain length of C11 to C18. C12-18 dimethylhydroxyethylammonium chloride. AE3S is a C12-15 alkyl ethoxy (3) sulfate. AE7 is a C12-15 alcohol ethoxylate with an average degree of ethoxylation of 7. AE9 is a C12-16 alcohol ethoxylate with an average degree of ethoxylation of 9. HSAS is a mid-chain branched primary alkyl sulfate having a carbon chain length of about 16-17, as disclosed in US Pat. Nos. 6,020,303 and 6,060,443. Polyacrylate MW4500 is supplied by BASF. The carboxymethylcellulose is Finnfix® V supplied by CP Kelco (Arnhem, Netherlands). CHEC is a cationically modified hydroxyethyl cellulose polymer. An example of a phosphonate chelating agent is diethylenetetraaminepentaacetic acid (DTPA) hydroxyethane diphosphonate (HEDP). Savinase®, Natalase®, Stainzyme®, Lipex®, Celluclean™, Mannaway®, and Whitezyme® are all products of Novozymes (Bagsvaerd, Denmark). Purafect® and Purafect Prime® are products of Genencor International (Palo Alto, California, USA). Optical Brightener 1 is Tinopal® AMS, Optical Brightener 2 is Tinopal® CBS-X, Direct Violet 9 is Pergasol® Violet BN-Z, and NOBS is sodium nonanoyloxybenzenesulfonate. TAED is tetraacetylethylenediamine. S-ACMC is carboxymethyl cellulose conjugated with CI Reactive Blue 19, trade name AZO-CM-CELLULOSE. The soil release agent is Repel-o-tex® PF. The acrylic acid / maleic acid copolymer has a molecular weight of 70,000 and an acrylate:maleate ratio of 70:30. EDDS is the sodium salt of ethylenediamine-N,N'-disuccinic acid, (S,S) isomer, a suds suppressor aggregate supplied by Dow Corning (Midland, Michigan, USA). HSAS is a medium-chain branched alkyl sulfate. Liquitint® Violet CT is a polymeric color pigment supplied by Milliken (Spartanburg, South Carolina, USA). The polyethoxylated azothiophene dye is Violet DD™ polymeric hue dye supplied by Milliken (Spartanburg, South Carolina, USA). 1 Alkoxylated ethylenediamine is bis((C2H5O)(C2H4O)n)(CH3)-N+-CxH2x-N+-(CH3)-bis((C2H5O)(C2H4O)n); n is 20-30 and x is 3-8, and is optionally sulfated or sulfonated. 2 The random graft copolymer is a polyvinyl acetate-grafted polyethylene oxide copolymer having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is about 6000, the weight ratio of polyethylene oxide to polyvinyl acetate is about 40-60, and there is no more than one grafting point per 50 ethylene oxide units. 3 Polyethyleneimine (MW=600) with 20 ethoxylate groups per —NH, e.g. Lutensol FP620 from BASF. 4 An example of an amphiphilic alkoxylated polymer is polyethyleneimine (MW600) prepared from a polymer derivatized to contain 24 ethoxylate groups per —NH and 16 propoxylate groups per —NH. 5 A polyethylene glycol graft polymer comprising a polyethylene glycol backbone (Pluriol E6000) and hydrophobic vinyl acetate side chains, comprising 40% by weight of the polyethylene glycol backbone polymer system and 60% by weight of the grafted vinyl acetate side chain polymer system. 6 Lutensit Z96 (a zwitterionic polyamine from BASF - a zwitterionic hexamethylenediamine according to the formula: 100% quaternized, approximately 40% of the polyethoxy (EO24) groups are sulfonated).

[0301] [ka] 4 Amylase is given as mg of active enzyme per 100g of detergent unless otherwise specified. 5 DNase as described herein (mg of active enzyme per 100g of detergent unless otherwise indicated). DNase may contain trace amounts of superoxide dismutase impurity. 6 Alginate lyase (given as mg of active enzyme per 100 g of detergent unless otherwise indicated). 7 Hexosaminidase as described herein (given as mg of active enzyme per 100 g of detergent unless otherwise indicated). 8 Pel-ase as described herein (given as mg of active enzyme per 100 g of detergent unless otherwise indicated). 9 Psl-ase as described herein (given as mg of active enzyme per 100 g of detergent unless otherwise indicated). Proxel GXL, 1,2-benzisothiazolin-3-one in 20% water in dipropylene glycol, supplied by Lonza. b N,N-bis(hydroxyethyl)-N,N-dimethylammonium chloride fatty acid ester. The iodine value of the parent fatty acid of this material is 18-22. The material obtained from Evonik contains impurities in the form of free fatty acids, the monoester form of N,N-bis(hydroxyethyl)-N,N-dimethylammonium chloride fatty acid ester, and fatty acid esters of N,N-bis(hydroxyethyl)-N-methylamine. c MP10®, supplied by Dow Corning, 8% active. d Expressed as 100% encapsulated perfume oil as described in US Pat. No. 8,765,659. eRheovis® CDE, a cationic polymeric thickener, is supplied by BASF. f N,N-dimethyloctanamide and N,N-dimethyldecanamide in a weight ratio of approximately 55:45, trade name Steposol® M-8-10, manufactured by Stepan.

[0302] Dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 mm" is intended to mean "approximately 40 mm."

[0303] While this disclosure has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0304] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure. To the extent section headings are used, they should not be construed as necessarily limiting.

Claims

1. 1. A cleaning composition comprising: (a) a subtilisin variant comprising two or more substitutions selected from the group consisting of X006W, X024K, X055P, X109Q, X162Q, X182Q, X183N, X204Q, X206Y, X222Q, X248A, or X254A, wherein the subtilisin variant has at least 80% identity to the amino acid sequence of SEQ ID NO:1, and wherein positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1; and (b) a cleaning adjunct.

2. 2. The cleaning composition of claim 1, wherein the substitution is selected from the group consisting of Y006W, S024K, T055P, N109Q, S162Q, S182Q, S183N, S204Q, Q206Y, M222Q, S248A, or T254A.

3. 3. The detergent composition of claim 1 or 2, wherein the variant comprises one, two or more further substitutions from the group consisting of X003Q, X022Y, X024Q, X033T, X045V, X053G, X076D, X078N, X087D, X101N, X118R, X128A, X128S, X145R, X166Q, X169A, X217Q or X218S.

4. 4. The cleaning composition of any one of claims 1 to 3, wherein the variant comprises one, two or more further substitutions from the group consisting of S003Q, T022Y, S024Q, S033T, A045V, S053G, N076D, S078N, S087D, S101N, N118R, G128A, G128S, S145R, G166Q, G169A, Y217Q, or N218S.

5. The subtilisin mutants include X006W-X024K, X006W-X055P, X006W-X109Q, X006W-X162Q, X006W-X183N, X006W-X182Q, X006W-X204Q, X006W-X206Y, X006W-X222Q, X006W-X248A, X006W-X254A, X024K-X055P, X024K-X109Q, X024K-X162Q, X024K-X183N, X024K-X182Q, X024K-X204Q, and X024 K-X206Y, X024K-X222Q, X024K-X248A, X024K-X254A, X055P-X109Q,X055P-X162Q, 206Y, X055P-X222Q, X055P-X248A, X055P-X254A, X109Q-X162Q,X109Q-X183N, Q, X109Q-X248A, X109Q-X254A, X162Q-X183N, X162Q-X182Q, X162Q-X204Q, 183N-X182Q, X183N-X204Q, X183N-X206Y, X183N-X222Q, X183N-X248A, X183N-X254A, 5. The cleaning composition of claim 1, comprising two mutations selected from the group consisting of Q-X248A, X182Q-X254A, X204Q-X206Y, X204Q-X222Q, X204Q-X248A, X204Q-X254A, X206Y-X222Q, X206Y-X248A, X206Y-X254A, X222Q-X248A, X222Q-X254A, and X248A-X254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1 (BPN').

6. The subtilisin mutants include X006W-X024K-X055P, X006W-X024K-X109Q, X006W-X024K-X162Q, X006W-X024K-X183N, X006W-X024K-X182Q, X006W-X024K-X204Q, X006W-X024K-X206Y, X006W-X024K-X222Q, X006W-X024K-X248A, X006W-X024K-X254A, X006W-X055P-X109Q, X006W-X055P-X162Q, and X006W-X055P-X183N. , X006W-X055P-X182Q, X006W-X055P-X204Q, X006W-X055P-X206Y, X006W- X055P-X222Q, X006W-X055P-X248A, X006W-X055P-X254A, X006W-X109Q-X1 62Q, X006W-X109Q-X183N, X006W-X109Q-X182Q, X006W-X109Q-X204Q, X00 6W-X109Q-X206Y, X006W-X109Q-X222Q, X006W-X109Q-X248A, X006W-X109Q -X254A, X006W-X162Q-X183N, X006W-X162Q-X182Q, X006W-X162Q-X204Q, X006W-X162Q-X206Y, X006W-X162Q-X222Q, X006W-X162Q-X248A, X006W-X1 62Q-X254A, X006W-X183N-X182Q, X006W-X183N-X204Q, X006W-X183N-X20 6Y, X006W-X183N-X222Q, X006W-X183N-X248A, X006W-X183N-X254A, X006W -X182Q-X204Q, X006W-X182Q-X206Y, X006W-X182Q-X222Q, X006W-X182Q- X248A, X006W-X182Q-X254A, X006W-X204Q-X206Y, X006W-X204Q-X222Q, X0 06W-X204Q-X248A, X006W-X204Q-X254A, X006W-X206Y-X222Q, X006W-X206 Y-X248A, X006W-X206Y-X254A, X006W-X222Q-X248A, X006W-X222Q-X254A,X006W-X248A-X254A、X024K-X055P-X109Q、X024K-X055P-X162Q、X024K-X055P-X183N、X024K-X055P-X182Q、X024K-X055P-X204Q、X024K-X055P-X206Y、X024K-X055P-X222Q、X024K-X055P-X248A、X024K-X055P-X254A、X024K-X109Q-X162Q、X024K-X109Q-X183N、X024K-X109Q-X182Q、X024K-X109Q-X204Q、X024K-X109Q-X206Y、X024K-X109Q-X222Q、X024K-X109Q-X248A、X024K-X109Q-X254A、X024K-X162Q-X183N、X024K-X162Q-X182Q、X024K-X162Q-X204Q、X024K-X162Q-X206Y、X024K-X162Q-X222Q、X024K-X162Q-X248A、X024K-X162Q-X254A、X024K-X183N-X182Q、X024K-X183N-X204Q、X024K-X183N-X206Y、X024K-X183N-X222Q、X024K-X183N-X248A、X024K-X183N-X254A、X024K-X182Q-X204Q、X024K-X182Q-X206Y、X024K-X182Q-X222Q、X024K-X182Q-X248A、X024K-X182Q-X254A、X024K-X204Q-X206Y、X024K-X204Q-X222Q、X024K-X204Q-X248A、X024K-X204Q-X254A、X024K-X206Y-X222Q、X024K-X206Y-X248A、X024K-X206Y-X254A、X024K-X222Q-X248A、X024K-X222Q-X254A、X024K-X248A-X254A、X055P-X109Q-X162Q、X055P-X109Q-X183N、X055P-X109Q-X182Q、X055P-X109Q-X204Q、X055P-X109Q-X206Y、X055P-X109Q-X222Q、X055P-X109Q-X248A、X055P-X109Q-X254A、X055P-X162Q-X183N、X055P-X162Q-X182Q、X055P-X162Q-X204Q、X055P-X162Q-X206Y、X055P-X162Q-X222Q、X055P-X162Q-X248A、X055P-X162Q-X254A、X055P-X183N-X182Q、X055P-X183N-X204Q、X055P-X183N-X206Y、X055P-X183N-X222Q、X055P-X183N-X248A、X055P-X183N-X254A、X055P-X182Q-X204Q、X055P-X182Q-X206Y、X055P-X182Q-X222Q、X055P-X182Q-X248A、X055P-X182Q-X254A、X055P-X204Q-X206Y、X055P-X204Q-X222Q、X055P-X204Q-X248A、X055P-X204Q-X254A、X055P-X206Y-X222Q、X055P-X206Y-X248A、X055P-X206Y-X254A、X055P-X222Q-X248A、X055P-X222Q-X254A、X055P-X248A-X254A、X109Q-X162Q-X183N、X109Q-X162Q-X182Q、X109Q-X162Q-X204Q、X109Q-X162Q-X206Y、X109Q-X162Q-X222Q、X109Q-X162Q-X248A、X109Q-X162Q-X254A、X109Q-X183N-X182Q、X109Q-X183N-X204Q、X109Q-X183N-X206Y、X109Q-X183N-X222Q、X109Q-X183N-X248A、X109Q-X183N-X254A、X109Q-X182Q-X204Q、X109Q-X182Q-X206Y、X109Q-X182Q-X222Q、X109Q-X182Q-X248A、X109Q-X182Q-X254A、X109Q-X204Q-X206Y、X109Q-X204Q-X222Q、X109Q-X204Q-X248A、X109Q-X204Q-X254A、X109Q-X206Y-X222Q、X109Q-X206Y-X248A、X109Q-X206Y-X254A、X109Q-X222Q-X248A、X109Q-X222Q-X254A、X109Q-X248A-X254A、X162Q-X183N-X182Q、X162Q-X183N-X204Q、X162Q-X183N-X206Y、X162Q-X183N-X222Q、X162Q-X183N-X248A、X162Q-X183N-X254A、X162Q-X182Q-X204Q、X162Q-X182Q-X206Y、X162Q-X182Q-X222Q、X162Q-X182Q-X248A、X162Q-X182Q-X254A、X162Q-X204Q-X206Y、X162Q-X204Q-X222Q、X162Q-X204Q-X248A、X162Q-X204Q-X254A、X162Q-X206Y-X222Q、X162Q-X206Y-X248A、X162Q-X206Y-X254A、X162Q-X222Q-X248A、X162Q-X222Q-X254A、X162Q-X248A-X254A、X183N-X182Q-X204Q、X183N-X182Q-X206Y、X183N-X182Q-X222Q、X183N-X182Q-X248A、X183N-X182Q-X254A、X183N-X204Q-X206Y、X183N-X204Q-X222Q、X183N-X204Q-X248A、X183N-X204Q-X254A、X183N-X206Y-X222Q、X183N-X206Y-X248A、X183N-X206Y-X254A、X183N-X222Q-X248A、X183N-X222Q-X254A、X183N-X248A-X254A、X182Q-X204Q-X206Y、X182Q-X204Q-X222Q、X182Q-X204Q-X248A、X182Q-X204Q-X254A、X182Q-X206Y-X222Q、X182Q-X206Y-X248A、X182Q-X206Y-X254A、X182Q-X222Q-X248A、X182Q-X222Q-X254A、X182Q-X248A-X254A、X204Q-X206Y-X222Q、X204Q-X206Y-X248A、X204Q-X206Y-X254A、X204Q-X222Q-X248A、X204Q-X222Q-X254A、X204Q-X248A-X254A、X206Y-X222Q-X248A、X206Y-X222Q-X254A、X206Y-X248A-X254A、and X222Q-X248A-X254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1 (BPN').

7. The subtilisin mutants include Y006W-S024K, Y006W-T055P, Y006W-N109Q, Y006W-S162Q, Y006W-S183N, Y006W-S182Q, Y006W-S204Q, Y006W-Q206Y, Y006W-M222Q, Y006W-S248A, Y006W-T254A, S024K-T055P, S024K-N109Q, S024K-S162Q, S024K-S183N, S024K-S182Q, S024K-S204Q, and S024 K-Q206Y, S024K-M222Q, S024K-S248A, S024K-T254A, T055P-N109Q, T055P-S162Q, T055P-S183N, T055P-S182Q, T055P-S204Q, T055P-Q 206Y, T055P-M222Q, T055P-S248A, T055P-T254A, N109Q-S162Q, N109Q-S183N, N109Q-S182Q, N109Q-S204Q, N109Q-Q206Y, N109Q-M222 Q, N109Q-S248A, N109Q-T254A, S162Q-S183N, S162Q-S182Q, S162Q-S204Q, S162Q-Q206Y, S162Q-M222Q, S162Q-S248A, S162Q-T254A, S 183N-S182Q, S183N-S204Q, S183N-Q206Y, S183N-M222Q, S183N-S248A, S183N-T254A, S182Q-S204Q, S182Q-Q206Y, S182Q-M222Q, S182 7. The cleaning composition of any one of claims 1 to 6, comprising two mutations selected from the group consisting of Q-S248A, S182Q-T254A, S204Q-Q206Y, S204Q-M222Q, S204Q-S248A, S204Q-T254A, Q206Y-M222Q, Q206Y-S248A, Q206Y-T254A, M222Q-S248A, M222Q-T254A, and S248A-T254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1 (BPN').

8. The subtilisin mutants are Y006W-S024K-T055P, Y006W-S024K-N109Q, Y006W-S024K-S162Q, Y006W-S024K-S183N, Y006W-S024K-S182Q, Y006W-S024K-S204Q, Y006W-S024K-Q206Y, Y006W-S024K-M222Q, Y006W-S024K-S248A, Y006W-S024K-T254A, Y006W-T055P-N109Q, Y006W-T055P-S162Q, Y006W-T055P-S183N , Y006W-T055P-S182Q, Y006W-T055P-S204Q, Y006W-T055P-Q206Y, Y006W- T055P-M222Q, Y006W-T055P-S248A, Y006W-T055P-T254A, Y006W-N109Q-S1 62Q, Y006W-N109Q-S183N, Y006W-N109Q-S182Q, Y006W-N109Q-S204Q, Y00 6W-N109Q-Q206Y, Y006W-N109Q-M222Q, Y006W-N109Q-S248A, Y006W-N109Q -T254A, Y006W-S162Q-S183N, Y006W-S162Q-S182Q, Y006W-S162Q-S204Q, Y006W-S162Q-Q206Y, Y006W-S162Q-M222Q, Y006W-S162Q-S248A, Y006W-S1 62Q-T254A, Y006W-S183N-S182Q, Y006W-S183N-S204Q, Y006W-S183N-Q20 6Y, Y006W-S183N-M222Q, Y006W-S183N-S248A, Y006W-S183N-T254A, Y006W -S182Q-S204Q, Y006W-S182Q-Q206Y, Y006W-S182Q-M222Q, Y006W-S182Q- S248A, Y006W-S182Q-T254A, Y006W-S204Q-Q206Y, Y006W-S204Q-M222Q, Y0 06W-S204Q-S248A, Y006W-S204Q-T254A, Y006W-Q206Y-M222Q, Y006W-Q206 Y-S248A, Y006W-Q206Y-T254A, Y006W-M222Q-S248A, Y006W-M222Q-T254A,Y006W-S248A-T254A、S024K-T055P-N109Q、S024K-T055P-S162Q、S024K-T055P-S183N、S024K-T055P-S182Q、S024K-T055P-S204Q、S024K-T055P-Q206Y、S024K-T055P-M222Q、S024K-T055P-S248A、S024K-T055P-T254A、S024K-N109Q-S162Q、S024K-N109Q-S183N、S024K-N109Q-S182Q、S024K-N109Q-S204Q、S024K-N109Q-Q206Y、S024K-N109Q-M222Q、S024K-N109Q-S248A、S024K-N109Q-T254A、S024K-S162Q-S183N、S024K-S162Q-S182Q、S024K-S162Q-S204Q、S024K-S162Q-Q206Y、S024K-S162Q-M222Q、S024K-S162Q-S248A、S024K-S162Q-T254A、S024K-S183N-S182Q、S024K-S183N-S204Q、S024K-S183N-Q206Y、S024K-S183N-M222Q、S024K-S183N-S248A、S024K-S183N-T254A、S024K-S182Q-S204Q、S024K-S182Q-Q206Y、S024K-S182Q-M222Q、S024K-S182Q-S248A、S024K-S182Q-T254A、S024K-S204Q-Q206Y、S024K-S204Q-M222Q、S024K-S204Q-S248A、S024K-S204Q-T254A、S024K-Q206Y-M222Q、S024K-Q206Y-S248A、S024K-Q206Y-T254A、S024K-M222Q-S248A、S024K-M222Q-T254A、S024K-S248A-T254A、T055P-N109Q-S162Q、T055P-N109Q-S183N、T055P-N109Q-S182Q、T055P-N109Q-S204Q、T055P-N109Q-Q206Y、T055P-N109Q-M222Q、T055P-N109Q-S248A、T055P-N109Q-T254A、T055P-S162Q-S183N、T055P-S162Q-S182Q、T055P-S162Q-S204Q、T055P-S162Q-Q206Y、T055P-S162Q-M222Q、T055P-S162Q-S248A、T055P-S162Q-T254A、T055P-S183N-S182Q、T055P-S183N-S204Q、T055P-S183N-Q206Y、T055P-S183N-M222Q、T055P-S183N-S248A、T055P-S183N-T254A、T055P-S182Q-S204Q、T055P-S182Q-Q206Y、T055P-S182Q-M222Q、T055P-S182Q-S248A、T055P-S182Q-T254A、T055P-S204Q-Q206Y、T055P-S204Q-M222Q、T055P-S204Q-S248A、T055P-S204Q-T254A、T055P-Q206Y-M222Q、T055P-Q206Y-S248A、T055P-Q206Y-T254A、T055P-M222Q-S248A、T055P-M222Q-T254A、T055P-S248A-T254A、N109Q-S162Q-S183N、N109Q-S162Q-S182Q、N109Q-S162Q-S204Q、N109Q-S162Q-Q206Y、N109Q-S162Q-M222Q、N109Q-S162Q-S248A、N109Q-S162Q-T254A、N109Q-S183N-S182Q、N109Q-S183N-S204Q、N109Q-S183N-Q206Y、N109Q-S183N-M222Q、N109Q-S183N-S248A、N109Q-S183N-T254A、N109Q-S182Q-S204Q、N109Q-S182Q-Q206Y、N109Q-S182Q-M222Q、N109Q-S182Q-S248A、N109Q-S182Q-T254A、N109Q-S204Q-Q206Y、N109Q-S204Q-M222Q、N109Q-S204Q-S248A、N109Q-S204Q-T254A、N109Q-Q206Y-M222Q、N109Q-Q206Y-S248A、N109Q-Q206Y-T254A、N109Q-M222Q-S248A、N109Q-M222Q-T254A、N109Q-S248A-T254A、S162Q-S183N-S182Q、S162Q-S183N-S204Q、S162Q-S183N-Q206Y、S162Q-S183N-M222Q、S162Q-S183N-S248A、S162Q-S183N-T254A、S162Q-S182Q-S204Q、S162Q-S182Q-Q206Y、S162Q-S182Q-M222Q、S162Q-S182Q-S248A、S162Q-S182Q-T254A、S162Q-S204Q-Q206Y、S162Q-S204Q-M222Q、S162Q-S204Q-S248A、S162Q-S204Q-T254A、S162Q-Q206Y-M222Q、S162Q-Q206Y-S248A、S162Q-Q206Y-T254A、S162Q-M222Q-S248A、S162Q-M222Q-T254A、S162Q-S248A-T254A、S183N-S182Q-S204Q、S183N-S182Q-Q206Y、S183N-S182Q-M222Q、S183N-S182Q-S248A、S183N-S182Q-T254A、S183N-S204Q-Q206Y、S183N-S204Q-M222Q、S183N-S204Q-S248A、S183N-S204Q-T254A、S183N-Q206Y-M222Q、S183N-Q206Y-S248A、S183N-Q206Y-T254A、S183N-M222Q-S248A、S183N-M222Q-T254A、S183N-S248A-T254A、S182Q-S204Q-Q206Y、S182Q-S204Q-M222Q、S182Q-S204Q-S248A、S182Q-S204Q-T254A、S182Q-Q206Y-M222Q、S182Q-Q206Y-S248A、S182Q-Q206Y-T254A、S182Q-M222Q-S248A、S182Q-M222Q-T254A、S182Q-S248A-T254A、S204Q-Q206Y-M222Q、S204Q-Q206Y-S248A、S204Q-Q206Y-T254A、S204Q-M222Q-S248A、S204Q-M222Q-T254A、S204Q-S248A-T254A、Q206Y-M222Q-S248A、Q206Y-M222Q-T254A、Q206Y-S248A-T254A、and M222Q-S248A-T254A, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1 (BPN').

9. 9. The cleaning composition of any one of claims 1 to 8, wherein the subtilisin variant is derived from a parent or reference polypeptide having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:

1.

10. 10. The cleaning composition of any one of claims 1 to 9, wherein the subtilisin variant comprises an amino acid sequence having 81%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:

1.

11. 11. A cleaning composition according to any one of claims 1 to 10, wherein the subtilisin variant has improved stability when compared to a reference subtilisin lacking two, three, four or more substitutions.

12. 12. The cleaning composition of claim 11, wherein the improved stability is measured as a residual activity (fraction of 1) of greater than 0.2 after 20 minutes at 54, 56, 60, or 66°C in a 10% detergent solution compared to the respective parent according to the assay described in Example 2; or the improved stability is measured as a percent residual activity of about 20% or greater after 20 minutes at 40, 51, or 53°C in a 10% detergent solution according to the assay described in Example 2.

13. The cleaning composition according to any one of claims 1 to 12, wherein the subtilisin variant has protease activity.

14. A cleaning composition according to any one of claims 1 to 13, wherein the subtilisin variant does not have an amino acid sequence identical to the naturally occurring molecule.

15. 15. A method of cleaning a kitchen, bathroom, dish or fabric surface, comprising contacting said surface with an aqueous cleaning liquor comprising the composition of any one of claims 1 to 14.

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