Subtilisin variants and related methods

Abstract

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

Description

[Technical Field]

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

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 403,330, filed September 2, 2022, and U.S. Provisional Patent Application No. 63 / 492,614, filed March 28, 2023, which applications are incorporated by reference in their entireties.

[0003] Reference to an electronically submitted sequence listing The contents of the sequence list, an XML file (Name: NB42132PCT_SequenceListing.xml; Size: 38,509 bytes; Creation Date: August 25, 2023), constitutes part of this application and is incorporated herein by reference in its entirety. [Background technology]

[0004] Proteases (also known as proteinases) are enzyme proteins capable of degrading other proteins. Proteases are capable of proteolysis, which begins protein metabolism by hydrolyzing the peptide bonds that connect the amino acids in the peptide or polypeptide chain that makes up the protein. This activity of proteases as protein-digesting enzymes is called proteolytic activity. Many well-known methods exist for measuring proteolytic activity (Kalisz, "Microbial Proteinases," in: Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology, (1988)). For example, proteolytic activity can be confirmed by a comparative assay that analyzes the hydrolysis ability of each protease against a commercially available substrate. Representative substrates useful for analyzing protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and keratin azure (Sigma-Aldrich K8500). Ratio assays utilizing these substrates are well known in the art (see, for example, WO 99 / 34011 and US Pat. No. 6,376,450, 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. Based on their structure, they are further divided into chymotrypsin-like (trypsin-like) and subtilisin-like enzymes. The prototype subtilisin (EC number 3.4.21.62) was first isolated from Bacillus subtilis. Subtilisins and their homologs belong to the S8 peptidase family of the MEROPS classification scheme (Rawlings, ND et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates, and inhibitors. Nucleic Acids Res 44, D343-D350). Members of the S8 family share a catalytic triad consisting of Asp, His, and Ser in the following order: Although many useful mutant proteases have been developed for cleaning applications, there remains a need for improved protease variants. Summary of the Invention [Means for solving the problem]

[0006] One embodiment relates to a subtilisin variant comprising one, two or more substitutions selected from the group consisting of X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X117R, X127S, X127T, X128K, X128P, X128R, X129Q, X155E, X161Q, X181E, X181Q, X202V, X203E, X203N, X217S, X221Q, X260W and X264H at positions numbered according to SEQ ID NO: 1, which variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8. In some embodiments, the subtilisin variant has at least 25% improved stability in detergents compared to the parent subtilisin SEQ ID NO:1 and / or has a net charge of -4 to +2 at pH 8 compared to a subtilisin having the amino acid sequence of SEQ ID NO:1.

[0007] In another embodiment, the disclosure is directed to a variant subtilisin, wherein the variant comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 and is selected from the group consisting of X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X117R, X127S, X127T, X128K, X128P, X1 and X28R, X129Q, X155E, X161Q, X181E, X181Q, X202V, X203E, X203N, X217S, X221Q, X260W, and X264H, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO: 1, and wherein the variant has a net charge of -4 to +2 at pH 8 relative to a subtilisin having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the subtilisin variant has at least 25% improved stability in detergent compared to the parent subtilisin SEQ ID NO: 1.

[0008] Still other embodiments relate to methods of producing the variants described herein, comprising stably transforming a host cell with an expression vector comprising a polynucleotide encoding one or more of the subtilisin variants described herein.Still other embodiments relate to polynucleotides comprising a nucleic acid sequence encoding one or more of the subtilisin variants described herein. DETAILED DESCRIPTION OF THE INVENTION

[0009] explanation In one embodiment, the disclosure provides one or more subtilisin variants comprising one, two, three or more amino acid substitutions at positions selected from the group consisting of: positions 9, 17, 45, 68, 78, 86, 87, 96, 100, 103, 108, 115, 117, 127, 128, 129, 155, 161, 181, 202, 203, 217, 221, 260, and 264, numbered according to their correspondence with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variants provided herein exhibit one or more improved properties, such as improved stability or improved cleaning performance, or both improved stability and improved cleaning performance, when compared to a subtilisin having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the subtilisin variant comprises at least two, three or more substitutions selected from the group consisting of X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X117R, X127S, X127T, X128K, X128P, X128R, X129Q, X155E, X161Q, X181E, X181Q, X202V, X203E, X203N, X217S, X260W, X221Q, and X264H at positions numbered according to SEQ ID NO:1, and the variant has at least 75% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8. In some embodiments, the subtilisin variants further comprise one or more additional mutations selected from the group consisting of X24Q, X77N, X86D, X165Q, X184Q, X258D, and X258P at positions numbered according to SEQ ID NO: 1. The subtilisin variants provided herein are used in the preparation of cleaning compositions (e.g., dishwashing compositions or laundry detergent compositions). Furthermore, the subtilisin variants provided herein are also used in cleaning methods (e.g., dishwashing methods or laundry cleaning methods) that use such variants or compositions comprising such subtilisin variants.

[0010] Unless otherwise specified herein, one or more subtilisin variants described herein can be produced and used by a variety of techniques used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA technology, and industrial enzyme use and development. Terms and abbreviations not defined should adhere to their ordinary meanings used in the art. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Any definitions provided herein should be interpreted in the context of the present specification as a whole. As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Unless otherwise specified, nucleic acid sequences are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxy orientation. Every numerical range used herein includes every narrower numerical range that falls within such broader numerical range, as if all narrower numerical ranges were expressly written herein.

[0011] As used herein in connection with a numerical value, the term "about" refers to a range of ±0.5 of the numerical value, unless the term is otherwise specifically defined in the context. For example, the phrase "a pH value of about 6" refers to a pH value of 5.5 to 6.5, unless the pH value is otherwise specifically defined.

[0012] 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 the starting amino acid position and all possible substitutions that may be present at that position. Reference to a "position:amino acid substitution" (e.g., 1S / T / G, 3G, 17T, etc.) includes the starting amino acid at that position and one or more amino acids with which that starting amino acid may be substituted. Position references can be expressed in several formats. For example, position 003 can also be expressed as position 03 or 3. Reference to a starting amino acid or a substituting amino acid can further be expressed by separating multiple starting or substituting amino acids with a force slash (" / "). 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 amino acid proline (P) or serine (S) at position 197. When referring to an amino acid at a position, X refers to any amino acid at the specified position.

[0013] The amino acid residue positions of a given amino acid sequence are numbered relative 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 amino acid residue positions. For example, the amino acid sequences of one or more subtilisin variants described herein are aligned with the amino acid sequence of SEQ ID NO: 1 using an alignment algorithm as described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with the amino acid residue of SEQ ID NO: 1 is conveniently numbered with reference to the position number of the corresponding amino acid residue. For example, a sequence alignment algorithm as described herein identifies positions where insertions or deletions occur in a subject sequence when compared to a query sequence (also referred to as a "reference sequence"). Sequence alignment with other subtilisin amino acid sequences can be determined, for example, using an amino acid alignment as set forth in Figure 1 of PCT Publication No. WO 2018118917.

[0014] The terms "protease" and "proteinase" refer to enzymes capable of degrading proteins and peptides. Proteases perform "proteolysis" by hydrolyzing the peptide bonds that link amino acids together in the peptide or polypeptide chain that forms a protein. This activity of proteases as protein-digesting enzymes is referred to as "proteolytic activity." There are many well-known procedures for measuring proteolytic activity. For example, proteolytic activity can be determined by comparative assays that analyze the ability of each protease to hydrolyze an appropriate substrate. Representative substrates useful for analyzing protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and keratin azure (Sigma-Aldrich K8500). Colorimetric assays using these substrates are well known in the art (see, e.g., WO 99 / 34011 and U.S. Pat. No. 6,376,450). The pNA peptidyl assay (see, e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) can also be used to determine active enzyme concentration. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes a soluble synthetic substrate, e.g., succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA). The rate of yellow color production from the hydrolysis reaction is measured at 405 nm or 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a sample of purified protein. The enzyme activity on the substrate divided by the protein concentration is the enzyme-specific activity.

[0015] As used herein, the term "Bacillus" includes all species within the genus "Bacillus" known to those skilled in the art, such as B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. alkaloids, B. arginine ... lophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, B. pumilus, B. xiamenensis, B. sp. TY-145, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomic reorganization. Thus, this genus is intended to include species that have been reclassified, including, but not limited to, organisms such as B. stearothermophilus, now referred to as "Geobacillus stearothermophilus," or B. polymyxa, now Paenibacillus polymyxa.The production of resistant endospores under stressful environmental conditions is considered a defining feature of the genus Bacillus, but this characteristic also applies to the recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0016] 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 contain an origin of replication, a multicloning site, and a selection marker. The process of introducing a vector into a target cell is typically referred to as transformation. In some embodiments, the present invention includes vectors comprising a DNA sequence encoding a serine protease polypeptide (e.g., a precursor or mature serine protease polypeptide) operably linked to an appropriate prosequence (e.g., a secretory sequence, a 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.

[0017] As used herein, the term "introduced" in the context of introducing a nucleic acid sequence into a cell refers to any method suitable for transferring a nucleic acid sequence into a cell. Such methods of introduction include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic change of a cell resulting from the uptake, optional genomic integration, and expression of genetic material (e.g., DNA).

[0018] The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid molecule of the present disclosure. Expression can also refer to the translation of mRNA into a polypeptide. Thus, the term "expression" includes any step involved in "producing a polypeptide," such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0019] The phrase "expression cassette" or "expression vector" refers to a recombinantly or synthetically produced nucleic acid construct or vector for the expression of a nucleic acid of interest (e.g., a foreign nucleic acid or a transgene) in a target cell. The nucleic acid of interest usually expresses a protein of interest. An expression vector or expression cassette usually contains a promoter nucleotide sequence that drives or promotes the expression of the foreign nucleic acid. An expression vector or expression cassette also usually contains other specific nucleic acid elements that allow transcription of the specific nucleic acid in the target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Some expression vectors have the ability to integrate and express heterologous DNA fragments into a host cell or the genome of a host cell. Numerous prokaryotic and eukaryotic expression vectors are commercially available. It is within the knowledge of one of ordinary skill in the art to select an appropriate expression vector for expressing a protein from a nucleic acid sequence incorporated into the expression vector.

[0020] As used herein, a nucleic acid is "operably linked" to another nucleic acid sequence when it is placed into a functional relationship with the other nucleic acid sequence. For example, a promoter or enhancer is operably linked to a nucleotide coding sequence if the promoter affects the transcription of the coding sequence. A ribosome binding site may be operably linked to a coding sequence if it is positioned so as to promote transcription of the coding sequence. Typically, "operably linked" DNA sequences are contiguous. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional methods.

[0021] The term "gene" refers to a polynucleotide (e.g., a DNA fragment) that encodes a polypeptide and includes regions preceding and following the coding region. In some instances, a gene includes intervening sequences (introns) between individual coding segments (exons).

[0022] The term "recombinant," when used in reference to a cell, 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 not found in the same form within the native (non-recombinant) form of the cell, or a recombinant cell can contain a native gene (found in the native form of the cell) that has been modified and reintroduced into the cell. A recombinant cell can contain nucleic acid endogenous to the cell without removing the nucleic acid from the cell; such modifications include modifications achieved by gene replacement, site-specific mutagenesis, and related techniques known to those of ordinary skill in the art. Recombinant DNA technology includes techniques for producing recombinant DNA in vitro and introducing the recombinant DNA into cells capable of expressing or propagating the recombinant DNA, thereby producing a recombinant polypeptide. "Recombination" or "recombining" of polynucleotides or nucleic acids generally refers to the assembly or combination of two or more nucleic acids or polynucleotide strands or fragments to create a new polynucleotide or nucleic acid.

[0023] A nucleic acid or polynucleotide is said to "encode" a polypeptide if, in its natural state, or when manipulated by methods known to those of skill in the art, it can be transcribed and / or translated to produce the polypeptide or a fragment thereof. The antisense strand of such a nucleic acid is also said to encode the sequence.

[0024] The terms "host strain" and "host cell" refer to an appropriate host for an expression vector containing a DNA sequence of interest.

[0025] A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. Amino acids are referred to throughout this disclosure using the one-letter or three-letter codes defined in accordance with the IUPAC-IUB Joint Commission on Biological Nomenclature. 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.

[0026] 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; these are sometimes referred to as intramolecular chaperones. Cleavage of the prosequence or propeptide sequence results in the mature, active protease. Bacterial serine proteases are often expressed as proenzymes. Examples of modified propeptides are described, for example, in WO 2016 / 205710.

[0027] The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that may be involved in the secretion or direct export of the mature or precursor form of a protein. Signal sequences are generally located at the N-terminus of the precursor or mature protein sequence. Signal sequences may be endogenous or foreign. Signal sequences are usually absent from the mature protein. Signal sequences are generally cleaved from the protein by a signal peptidase after the protein has been exported.

[0028] The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide that does not include signal peptide and propeptide sequences.

[0029] 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 a prosequence. A precursor may also have additional polypeptides associated with post-translational activity (e.g., polypeptides that have been cleaved from it to leave the mature form of the protein or peptide).

[0030] With respect to polypeptides, the term "wild-type" refers to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. Similarly, with respect to polynucleotides, the term "wild-type" refers to a naturally occurring polynucleotide that does not contain an artificial substitution, insertion, or deletion at one or more nucleosides. However, a polynucleotide encoding a wild-type polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes a wild-type or parent polypeptide.

[0031] The term "parent" with respect to a polypeptide refers to a naturally occurring or wild-type polypeptide, or a naturally occurring polypeptide with artificial substitutions, insertions, or deletions at one or more amino acid positions that serve as a basis for introducing substitutions or additional substitutions to generate the mutant enzymes provided herein. The term "parent" with respect to a polypeptide also includes a polypeptide that has protease activity and serves as a starting polypeptide for modification, such as substitutions, additions, and / or deletions, resulting in a variant with 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 includes any wild-type, parent, or reference polypeptide. Similarly, the term "parent" with respect to a polynucleotide can refer to a naturally occurring polynucleotide or a polynucleotide that contains an artificial substitution, insertion, or deletion at one or more nucleotides. The term "parent" with respect to a polynucleotide also includes a polynucleotide that encodes a polypeptide with protease activity and serves as a starting polynucleotide for modification to result in a variant protease with modifications, such as substitutions, additions, and / or deletions, compared to the starting polynucleotide. That is, a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes a wild-type, parent, or reference polypeptide. In some embodiments, a parent polypeptide herein includes a polypeptide having the amino acid sequence set forth in SEQ ID NO:1.

[0032] The term "naturally occurring" refers, for example, to a sequence and the residues therein (e.g., a polypeptide sequence and the amino acids therein, or a nucleotide sequence and the nucleotides therein) that exist in nature. Conversely, the term "non-naturally occurring" refers, for example, to a sequence and the residues therein (e.g., a polypeptide sequence and the amino acids therein, or a nucleotide sequence and the nucleic acids therein) that do not exist in nature.

[0033] As used herein, "corresponding to" or "corresponding to" or "corresponding" in reference to an amino acid residue position 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 "region corresponding to" generally refers to an analogous position in a related or reference protein.

[0034] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by a strain of organism of interest, but also to proteins encoded by DNA sequences isolated from such a strain and produced in a host organism containing such DNA sequence. Furthermore, the term refers to proteins encoded by DNA sequences of synthetic and / or cDNA origin and having characteristics that identify the protein of interest. By way of example, "protease derived from Bacillus" refers to enzymes with proteolytic activity naturally produced by Bacillus, as well as serine proteases produced by Bacillus sources but produced by other host cells transformed by genetic engineering techniques with nucleic acids encoding the serine proteases.

[0035] The term "identical" in the context of two polynucleotide or polypeptide sequences refers to identical nucleotides or amino acids in the two sequences when aligned for maximum correspondence, as determined using sequence comparison or analysis algorithms described below and known in the art.

[0036] The phrases "% identity" or "percent identity" or "PID" refer to protein sequence identity. Percent identity can be determined using standard techniques known in the art. The percent amino acid identity shared by sequences of interest can be determined by aligning the sequences and directly comparing sequence information using programs such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described, for example, in Altschul et al., J Mol Biol, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993). The 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.

[0037] As used herein, "homologous protein" or "homologous protease" refers to proteins that share significant similarity 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, with a threshold (E-value cutoff) of 0.001. (Altschul et al., "Gapped BLAST and PSI BLAST: a new generation of protein database search programs," Nucleic Acids Res, Set 1;25(17):3389-402 (1997)). BLAST programs use several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences in terms of biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997; and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). Typical default BLAST parameters for nucleic acid sequence searches include a neighboring word threshold of 11; an E-value cutoff of 10; a scoring matrix of NUC.3.1 (match 1, mismatch 3); a gap opening of 5; and a gap extension of 2. Typical default BLAST parameters for amino acid sequence searches include a word size of 3; an E-value cutoff of 10; a scoring matrix of BLOSUM62; a gap opening of 11; and a gap extension of 1. This information can be used to group protein sequences and / or construct phylogenetic trees from them.Amino acid sequences can be entered into programs such as the Vector NTI Advance suite, and guide trees can be generated using the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol, 4:406-425, 1987). Phylogenetic tree construction can be calculated using the Kimura sequence distance correction, ignoring gaps. Programs such as AlignX can display calculated distance values ​​in parentheses following the molecule names shown on the phylogenetic tree.

[0038] Understanding homology between molecules can reveal information about a molecule's evolutionary history and function; homology of a newly sequenced protein to a previously characterized protein strongly indicates the biochemical function of the new protein. Two molecules are said to be homologous if they are derived from a common ancestral molecule. 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 groups (phylogenies) of proteins for which a common ancestry can be inferred. This common ancestry is usually based on sequence alignment and mechanistic similarity. Superfamilies usually include several protein families (exhibiting sequence similarity within the family). The term "protein clan" is commonly used to refer to protease superfamilies based on the MEROPS protease classification system. As used herein, the term "subtilisin" includes members 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).

[0039] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994). The default parameters for the CLUSTAL W algorithm include: Gap opening penalty = 10.0; Gap extension penalty = 0.05; Protein weight matrix = BLOSUM series; DNA weight matrix = IUB; Delay divergent sequences (%) = 40; Gap separation distance = 8; DNA transitions weight = 0.50; List of hydrophilic residues = GPSNDQEKR; Use negative matrix = OFF; Toggle Residue specific penalties = ON; Toggle hydrophilic penalties = ON; and Toggle end gap separation penalty. The CLUSTAL algorithm includes deletions occurring at either end. For example, a variant with a 5 amino acid deletion at one end of a 500 amino acid polypeptide (or within the polypeptide) has a percent sequence identity of 99% (495 / 500 identical residues x 100) to the "reference" polypeptide. Such a variant would be included in variants having "at least 99% sequence identity" to the polypeptide.

[0040] A nucleic acid or polynucleotide is "isolated" when it is at least partially or completely separated from other components, including, but not limited to, other proteins, nucleic acids, cells, etc. Similarly, polypeptides and polynucleotides are "isolated" when they are at least partially or completely separated from other components, including, but not limited to, other proteins, nucleic acids, cells, etc. On a molar basis, an isolated species is enriched in other species in a composition. For example, an isolated species may comprise at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (on a molar basis) of all macromolecular species present. Preferably, the species of interest is purified to substantial homogeneity (i.e., contaminating species in the composition cannot be detected by conventional detection methods). Purity and homogeneity can be determined using several techniques well known in the art, such as agarose electrophoresis or polyacrylamide gel electrophoresis of nucleic acid or protein samples, respectively, followed by visualization immediately after staining. If desired, high-resolution techniques, such as high-performance liquid chromatography (HPLC) or similar means, can be used to purify the material.

[0041] 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 will form a distinct band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to substantially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, 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 this context, a composition is enriched for a molecule if the concentration of that molecule is substantially increased after application of a purification or concentration procedure. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other particular material or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0042] The term "cleaning activity" refers to the cleaning performance achieved by a serine protease polypeptide, variant, or reference subtilisin under conditions used in the proteolysis, hydrolysis, cleaning, or other processes disclosed. In some embodiments, the cleaning performance of a serine protease or reference subtilisin can be determined by using various assays for cleaning one or more various enzyme-sensitive items or surface stains (e.g., stains resulting from food, grass, blood, ink, milk, oil, and / or egg proteins). The cleaning performance of one or more subtilisin variants or reference subtilisins described herein can be measured by subjecting the stains on the item or surface to standard cleaning conditions and assessing the extent to which the stain is removed using various chromatographic, spectrophotometric, or other quantitative methods. Exemplary cleaning assays and methods are known in the art and include, but are not limited to, those described in WO 99 / 34011 and U.S. Pat. No. 6,605,458, as well as those included in Examples 6 and 7 below.

[0043] As used herein, "surface properties" may be used in reference to properties such as charge and hydrophobicity and hydrophilicity exhibited by the surface of a protein.

[0044] The present disclosure includes subtilisin variants having one or more modifications to surface-exposed amino acids. The surface modifications in the enzyme variants may be useful in detergent compositions by having a minimum performance index for cleaning performance, enzyme stability in detergent compositions, and enzyme thermal stability, and by having at least one of these characteristics improved relative to the parent subtilisin enzyme. 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). The net charge of an amino acid at a desired pH can be calculated using the pKa value of a titratable chemical group in the amino acid, as described in Hass and Mulder (Hass, MAS and Mulder, FAA (2015) Annu. Rev. Biophys. 44:53-75). Subtilisin variants with a net charge of -4 to +2 at pH 8 relative to the subtilisin having the amino acid sequence of SEQ ID NO: 1 may find benefit in laundry cleaning applications.

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

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

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

[0048] A cleaning composition or cleaning formulation as used herein, unless otherwise indicated, refers to a granular or powdered all-purpose or heavy-duty cleaning agent, in particular a detergent; a liquid, granular, gel, solid, tablet, paste or unit dose all-purpose cleaning agent, in particular a so-called heavy-duty liquid (HDL) type detergent or heavy-duty dry (HDL) type detergent. dry (HDD) detergents; liquid detergents for tightly woven fabrics; hand or manual dishwashing detergents, e.g., high-foam detergents; household or commercial hand or manual dishwashing, automatic dishwashing, or dish or tableware detergents (including various tablet, powder, solid, granular, liquid, gel, and rinse aid types); liquid cleaners and disinfectants for humans and other animals, e.g., antibacterial hand washes, cleaning wands, mouthwashes, denture cleaners, car cleaners, carpet cleaners, bathroom cleaners, hair shampoos and / or hair rinses; shower gels, foam baths, and metal cleaners; and cleaning aids such as bleach additives and "stain sticks" or pre-treats. In some embodiments, the granular compositions are in "compact" form, and in some embodiments, the liquid compositions are in "concentrated" form.

[0049] The terms "detergent composition" or "detergent formulation" are used in reference to compositions used in wash media for cleaning soiled or stained objects, including certain fabric and / or non-fabric objects or items. In some embodiments, detergents of the present disclosure comprise one or more subtilisin variants described herein and further comprise one or more surfactants, transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, optical brighteners, caking inhibitors, masking agents, enzyme stabilizers, calcium, enzyme activators, antioxidants, and / or solubilizers. In some cases, the builder salts are mixtures of silicates and phosphates, preferably with more silicates (e.g., sodium metasilicate) than phosphates (e.g., sodium tripolyphosphate). Some embodiments relate to cleaning or detergent compositions that are phosphate-free (e.g., phosphate or phosphate builder). The cleaning composition may also contain a biological ingredient, such as one or more microorganisms or microbial extracts (as described in WO2018060475 and U.S. Pat. No. 10,968,556). Microorganisms can be used as the sole biologically active ingredient, or in combination with one or more enzymes described herein. For example, as described in WO2012 / 112718, the Bacillus strain with accession number PTA-7543 can be used to reduce malodors. Other purposes may include in situ production of desirable biological compounds or inoculating / populating a specific location with microorganisms to competitively prevent other undesirable microorganisms from populating the same location (competitive exclusion).

[0050] The phrases "substantially boron-free composition" or "substantially boron-free cleaning agent" refer to a composition or cleaning agent, respectively, that contains trace amounts of boron, 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, possibly of other composition or cleaning agent ingredients.

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

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

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

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

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

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

[0057] In one embodiment, a subtilisin variant is provided, wherein the variant comprises two, three, four or more amino acid substitutions at positions selected from the group consisting of positions 9, 17, 45, 68, 78, 86, 87, 96, 100, 103, 108, 115, 117, 127, 128, 129, 155, 161, 181, 202, 203, 217, 221, 260 and 264 numbered according to SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8.

[0058] In another embodiment, a subtilisin variant is provided, wherein the variant comprises any one of the following amino acids at positions X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X117R, X127S, X127T, X128K, X128P, X130L, X131L, X132L, X133L, X134L, X135L, X136L, X137L, X138L, X139L, X140L, X141L, X142L, X143L, X144L, X145L, X146L, X147L, X148L, X149L, X150L, ​​X151L, X152L, X153L, X154L, X155L, X156L, X157L, X158L, X159L, X160L, X161L, X162L, X163L, X164L, X165L, X166L, X167L, X168L, X169L, X170L, X171L, X172L, X173L, X174L, X175L, X176L, X177L, X178L, X179L, X180L, X181L, X182L, X183L, X184L, X185L, X186L, X187L, X188L, X189L, X190L, X191L, X192L, X1 The variant comprises one, two, three, or four or more amino acid substitutions selected from the group consisting of X128R, X129Q, X155E, X161Q, X181E, X181Q, X202V, X203E, X203N, X217S, X221Q, X260W, and X264H, and the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8.

[0059] In other implementations, the subtilisin variant further comprises an additional substitution selected from the group consisting of X24Q, X77N, X86D, X165Q, X184Q, X258D, and X258P at positions numbered according to the amino acid sequence of SEQ ID NO: 1.

[0060] In one embodiment, subtilisin variants are provided, wherein said variants are selected from the group consisting of X9T-X17H, X9T-X45R, X9T-X68S, X9T-X78I, X9T-X86E, X9T-X87A, X9T-X96D, X9T-X100E, X9T-X100N, X9T-X103F, X9T-X103I, X9T-X108Q, X9T-X115L, X9T-X117R, X9T-X127S, X9T-X127T, X9T-X128K, X9T-X128P, X9T-X128R, X9T-X129Q, X9T-X155E, X9T-X161Q, X9T-X181E, X9T-X181Q, X9T-X202V, X9T-X203E, X9T-X203N, X9T-X217S, X9 T-X260W, X9T-X221Q, X9T-X264H, X17H-X45R, X17H-X68S, X17H-X78I, X17H -X86E, X17H-X87A, X17H-X96D, X17H-X100E, X17H-X100N, X17H-X103F, X17 H-X103I, X17H-X108Q, X17H-X115L, X17H-X117R, X17H-X127S, X17H-X127T, X17H-X128K, X17H-X128P, X17H-X128R, X17H-X129Q, X17H-X155E, X17H-X1 61Q, X17H-X181E, X17H-X181Q, X17H-X202V, X17H-X203E, X17H-X203N, X17 H-X217S, X17H-X260W, X17H-X221Q, X17H-X264H, X45R-X68S, X45R-X78I, X 45R-X86E, X45R-X87A, X45R-X96D, X45R-X100E, X45R-X100N, X45R-X103F, X 45R-X103I, X45R-X108Q, X45R-X115L, X45R-X117R, X45R-X127S, X45R-X12 7T, X45R-X128K, X45R-X128P, X45R-X128R, X45R-X129Q, X45R-X155E, X45R- X161Q, X45R-X181E, X45R-X181Q, X45R-X202V, X45R-X203E, X45R-X203N, X 45R-X217S, X45R-X260W, X45R-X221Q, X45R-X264H, X68S-X78I, X68S-X86E,X68S-X87A、X68S-X96D、X68S-X100E、X68S-X100N、X68S-X103F、X68S-X103I、X68S-X108Q、X68S-X115L、X68S-X117R、X68S-X127S、X68S-X127T、X68S-X128K、X68S-X128P、X68S-X128R、X68S-X129Q、X68S-X155E、X68S-X161Q、X68S-X181E、X68S-X181Q、X68S-X202V、X68S-X203E、X68S-X203N、X68S-X217S、X68S-X260W、X68S-X221Q、X68S-X264H、X78I-X86E、X78I-X87A、X78I-X96D、X78I-X100E、X78I-X100N、X78I-X103F、X78I-X103I、X78I-X108Q、X78I-X115L、X78I-X117R、X78I-X127S、X78I-X127T、X78I-X128K、X78I-X128P、X78I-X128R、X78I-X129Q、X78I-X155E、X78I-X161Q、X78I-X181E、X78I-X181Q、X78I-X202V、X78I-X203E、X78I-X203N、X78I-X217S、X78I-X260W、X78I-X221Q、X78I-X264H、X86E-X87A、X86E-X96D、X86E-X100E、X86E-X100N、X86E-X103F、X86E-X103I、X86E-X108Q、X86E-X115L、X86E-X117R、X86E-X127S、X86E-X127T、X86E-X128K、X86E-X128P、X86E-X128R、X86E-X129Q、X86E-X155E、X86E-X161Q、X86E-X181E、X86E-X181Q、X86E-X202V、X86E-X203E、X86E-X203N、X86E-X217S、X86E-X260W、X86E-X221Q、X86E-X264H、X87A-X96D、X87A-X100E、X87A-X100N、X87A-X103F、X87A-X103I、X87A-X108Q、X87A-X115L、X87A-X117R、X87A-X127S、X87A-X127T、X87A-X128K、X87A-X128P、X87A-X128R、X87A-X129Q、X87A-X155E、X87A-X161Q、X87A-X181E、X87A-X181Q、X87A-X202V、X87A-X203E、X87A-X203N、X87A-X217S、X87A-X260W、X87A-X221Q、X87A-X264H、X96D-X100E、X96D-X100N、X96D-X103F、X96D-X103I、X96D-X108Q、X96D-X115L、X96D-X117R、X96D-X127S、X96D-X127T、X96D-X128K、X96D-X128P、X96D-X128R、X96D-X129Q、X96D-X155E、X96D-X161Q、X96D-X181E、X96D-X181Q、X96D-X202V、X96D-X203E、X96D-X203N、X96D-X217S、X96D-X260W、X96D-X221Q、X96D-X264H、X100E-X100N、X100E-X103F、X100E-X103I、X100E-X108Q、X100E-X115L、X100E-X117R、X100E-X127S、X100E-X127T、X100E-X128K、X100E-X128P、X100E-X128R、X100E-X129Q、X100E-X155E、X100E-X161Q、X100E-X181E、X100E-X181Q、X100E-X202V、X100E-X203E、X100E-X203N、X100E-X217S、X100E-X260W、X100E-X221Q、X100E-X264H、X100N-X103F、X100N-X103I、X100N-X108Q、X100N-X115L、X100N-X117R、X100N-X127S、X100N-X127T、X100N-X128K、X100N-X128P、X100N-X128R、X100N-X129Q、X100N-X155E、X100N-X161Q、X100N-X181E、X100N-X181Q、X100N-X202V、X100N-X203E、X100N-X203N、X100N-X217S、X100N-X260W、X100N-X221Q、X100N-X264H、X103F-X103I、X103F-X108Q、X103F-X115L、X103F-X117R、X103F-X127S、X103F-X127T、X103F-X128K、X103F-X128P、X103F-X128R、X103F-X129Q、X103F-X155E、X103F-X161Q、X103F-X181E、X103F-X181Q、X103F-X202V、X103F-X203E、X103F-X203N、X103F-X217S、X103F-X260W、X103F-X221Q、X103F-X264H、X103I-X108Q、X103I-X115L、X103I-X117R、X103I-X127S、X103I-X127T、X103I-X128K、X103I-X128P、X103I-X128R、X103I-X129Q、X103I-X155E、X103I-X161Q、X103I-X181E、X103I-X181Q、X103I-X202V、X103I-X203E、X103I-X203N、X103I-X217S、X103I-X260W、X103I-X221Q、X103I-X264H、X108Q-X115L、X108Q-X117R、X108Q-X127S、X108Q-X127T、X108Q-X128K、X108Q-X128P、X108Q-X128R、X108Q-X129Q、X108Q-X155E、X108Q-X161Q、X108Q-X181E、X108Q-X181Q、X108Q-X202V、X108Q-X203E、X108Q-X203N、X108Q-X217S、X108Q-X260W、X108Q-X221Q、X108Q-X264H、X115L-X117R、X115L-X127S、X115L-X127T、X115L-X128K、X115L-X128P、X115L-X128R、X115L-X129Q、X115L-X155E、X115L-X161Q、X115L-X181E、X115L-X181Q、X115L-X202V、X115L-X203E、X115L-X203N、X115L-X217S、X115L-X260W、X115L-X221Q、X115L-X264H、X117R-X127S、X117R-X127T、X117R-X128K、X117R-X128P、X117R-X128R、X117R-X129Q、X117R-X155E、X117R-X161Q、X117R-X181E、X117R-X181Q、X117R-X202V、X117R-X203E、X117R-X203N、X117R-X217S、X117R-X260W、X117R-X221Q、X117R-X264H、X127S-X127T、X127S-X128K、X127S-X128P、X127S-X128R、X127S-X129Q、X127S-X155E、X127S-X161Q、X127S-X181E、X127S-X181Q、X127S-X202V、X127S-X203E、X127S-X203N、X127S-X217S、X127S-X260W、X127S-X221Q、X127S-X264H、X127T-X128K、X127T-X128P、X127T-X128R、X127T-X129Q、X127T-X155E、X127T-X161Q、X127T-X181E、X127T-X181Q、X127T-X202V、X127T-X203E、X127T-X203N、X127T-X217S、X127T-X260W、X127T-X221Q、X127T-X264H、X128K-X128P、X128K-X128R、X128K-X129Q、X128K-X155E、X128K-X161Q、X128K-X181E、X128K-X181Q、X128K-X202V、X128K-X203E、X128K-X203N、X128K-X217S、X128K-X260W、X128K-X221Q、X128K-X264H、X128P-X128R、X128P-X129Q、X128P-X155E、X128P-X161Q、X128P-X181E、X128P-X181Q、X128P-X202V、X128P-X203E、X128P-X203N、X128P-X217S、X128P-X260W、X128P-X221Q、X128P-X264H、X128R-X129Q、X128R-X155E、X128R-X161Q、X128R-X181E、X128R-X181Q、X128R-X202V、X128R-X203E、X128R-X203N、X128R-X217S、X128R-X260W、X128R-X221Q、X128R-X264H、X129Q-X155E、X129Q-X161Q、X129Q-X181E、X129Q-X181Q、X129Q-X202V、X129Q-X203E, X129Q-X203N, X129Q-X217S, X129Q-X260W, X129Q-X221Q, X129Q-X264H, X155E-X161Q, X155E-X181E, X155E-X181Q, X155E-X202V, X155E-X203E, X155E-X203N, , X155E-X217S, X155E-X260W, X155E-X221Q, X155E-X264H, X161Q-X181E, X161Q-X181Q, , X161Q-X203E, X161Q-X203N, X161Q-X217S, X161Q-X260W, X161Q-X221Q, X161Q-X264H, X181E-X181Q , X181E-X202V, X181E-X203E, X181E-X203N, X181E-X217S, X181E-X260W, X181E-X221Q, , X181Q-X202V, X181Q-X203E, X181Q-X203N, X181Q-X217S, X181Q-X260W, X181Q-X221Q, X181Q-X264H , X202V-X203E, X202V-X203N, X202V-X217S, X202V-X260W, X202V-X221Q, X202V-X264H, X203E-X203N , X203E-X217S, X203E-X260W, X203E-X221Q, X203E-X264H, X203N-X217S, X203N-X260W, , X203N-X264H, X217S-X260W, X217S-X221Q, X217S-X264H, X260W-X221Q, X260W-X264H, and X221Q-X264H, where each position is numbered according to SEQ ID NO: 1, and where the variants have at least 75% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8.

[0061] In another embodiment, the subtilisin variants X017H-X096D-X127T, X017H-X096D-X103F-X202V, X127T-X128K-X129Q-X184Q, X009T-X103F-X202V-X203E, X096D-X100E-X127T-X202V, X087A-X155E-X165Q, X009T-X078I-X103F-X127T, X087A-X202V-X203E, X045R-X161Q-X181Q-X203E, X155E-X221Q, X078I-X103F, X096D- X100E-X127T-X217S, X127T-X128K-X129Q, X115L-X127T, X009T-X078I-X0 96D-X202V, X045R-X086E-X155E-X202V, X045R-X078I-X103F-X202V, X009T -X202V-X203E-X221Q, X202V-X203E-X221Q-X264H, X096D-X127T, X009T-X 017H-X103F-X202V, X045R-X127T-X128P-X129Q, X100E-X127T-X202V, X202 V-X221Q, X100N-X103I-X127T, X127T-X128K-X129Q-X217S, X127S-X128K- X129Q-X202V, X078I-X096D, X128K-X155E, X096D-X103F-X165Q, X086E-X07 7N-X078I, X045R-X078I-X086E-X202V, X096D-X100E-X127S, X078I-X127T -X128K-X129Q, X127S-X128K-X129Q, X017H-X086E-X202V-X203E, X161Q-X1 81Q-X202V-X203E, X045R-X096D-X100E, X045R-X103F-X127T-X202V, X096 D-X100E-X117R-X127T, X087A-X165Q-X202V, X127T-X128P-X129Q, X087A-X 221Q, X115L-X202V-X203E, X045R-X078I-X096D-X202V, X127T-X128R-X12 9Q-X165Q, X100E-X103I, X086E-X155E-X202V-X203E, X096D-X103I-X127T,X017H-X096D-X103F、X045R-X078I-X096D-X103F、X108Q-X127T、X078I-X086E-X202V-X203E、X078I-X103F-X127T-X202V、X181E-X202V-X203E-X258D、X096D-X202V-X203E、X096D-X103I-X127T-X221Q、X087A-X165Q-X202V-X203E、X009T-X127T-X128K-X129Q、X100E-X127T、X100N-X221Q、X127T-X128K-X129Q-X202V、X087A-X100N、X100E-X117R-X127T、X077N-X127T-X128K-X129Q、X017H-X096D-X127T-X202V、X096D-X202V-X221Q、X045R-X096D-X103F-X127T、X096D-X127T-X203E、X045R-X086E-X202V-X203E、X078I-X086E-X155E-X203E、X045R-X221Q、X017H-X096D-X103F-X127T、X017H-X103F-X127T-X202V、X078I-X202V-X221Q、X045R-X096D-X127T、X045R-X096D-X127T-X202V、X009T-X078I-X103F-X202V、X100N-X115L、X086E-X202V、X096D-X103F-X127T、X078I-X096D-X127T、X115L-X165Q-X202V、X078I-X086E、X045R-X078I-X096D-X127T、X009T-X096D-X127T、X127T-X128R-X129Q-X202V、X078I-X096D-X202V、X096D-X103I-X203E、X127S-X128P-X129Q-X202V、X096D-X127T-X217S、X045R-X078I、X024Q-X078I-X202V、X202V-X203N、X009T-X077N-X078I、X203E-X264H、X077N-X078I-X165Q-X202V、X045R-X077N-X165Q-X202V、X165Q-X202V-X203E-X258P、X009T-X078I-X202V、X045R-X127T, X077N-X078I-X165Q-X203E, X045R-X078I-X202V-X203E, X127T-X165Q-X202V-X203N, X202V-X264H, X009T -X202V-X203E, X077N-X202V-X203E, X202V-X203E-X217S, X103I-X202V, X078I-X203N, X009T-X017H-X078I-X165Q, X017H -X078I, X086D-X202V-X203E, X202V-X203E-X260W, X165Q-X202V-X203E-X217S, X127T-X165Q-X202V-X203E, X009T-X017 H-X078I-X127T, X045R-X155E, X009T-X017H-X078I-X202V, X009T-X078I, X086D-X155E-X202V-X203E, X009T-X017H-X202 V-X203E, X024Q-X165Q-X202V-X203E, X009T-X017H-X077N-X202V, X017H-X202V-X203E, X009T-X127T-X202V-X203E, X10 3I-X203E, X078I-X155E-X202V-X203E, X096D-X100E-X103I, X017H-X078I-X127T-X202V, X009T-X096D, X128R-X155E, X12 7T-X128R, X078I-X202V, X009T-X202V, X017H-X202V, X009T-X203E, X078I-X165Q-X202V, X078I-X202V-X203E-X217S, and X077N-X078I-X221Q are provided, where each position is numbered according to SEQ ID NO: 1, and where the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8.

[0062] In another implementation, a subtilisin variant is provided, wherein the variant comprises X221Q, X100N, X115L, and X087A at positions numbered according to SEQ ID NO:1, wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8.

[0063] In another embodiment, subtilisin variants are provided, wherein the variants are Q017H-N096D-G127T, Q017H-N096D-Y103F-A202V, G127T-A128K-S129Q-N184Q, P009T-Y103F-A202V-G203E, N096D-S100E-G127T-A202V, V087A-S155E-G165Q, P009T-T078I-Y103F-G127T, V087A-A202V-G203E, V045R-T161Q-S181Q-G203E, S155E-M221Q, T07 8I-Y103F, N096D-S100E-G127T-N217S, G127T-A128K-S129Q, T115L-G127T , P009T-T078I-N096D-A202V, V045R-S086E-S155E-A202V, V045R-T078I-Y 103F-A202V, P009T-A202V-G203E-M221Q, A202V-G203E-M221Q-K264H, N09 6D-G127T, P009T-Q017H-Y103F-A202V, V045R-G127T-A128P-S129Q, S100E- G127T-A202V, A202V-M221Q, S100N-Y103I-G127T, G127T-A128K-S129Q-N2 17S, G127S-A128K-S129Q-A202V, T078I-N096D, A128K-S155E, N096D-Y103 F-G165Q, S086E-T077N-T078I, V045R-T078I-S086E-A202V, N096D-S100E- G127S, T078I-G127T-A128K-S129Q, G127S-A128K-S129Q, Q017H-S086E-A20 2V-G203E, T161Q-S181Q-A202V-G203E, V045R-N096D-S100E, V045R-Y103F -G127T-A202V, N096D-S100E-G117R-G127T, V087A-G165Q-A202V, G127T-A 128P-S129Q, V087A-M221Q, T115L-A202V-G203E, V045R-T078I-N096D-A20 2V, G127T-A128R-S129Q-G165Q, S100E-Y103I, S086E-S155E-A202V-G203E,N096D-Y103I-G127T, Q017H-N096D-Y103F, V045R-T078I-N096D-Y103F , S108Q-G127T, T078I-S086E-A202V-G203E, T078I-Y103F-G127T-A202 V, S181E-A202V-G203E-S258D, N096D-A202V-G203E, N096D-Y103I-G12 7T-M221Q, V087A-G165Q-A202V-G203E, P009T-G127T-A128K-S129Q, S10 0E-G127T, S100N-M221Q, G127T-A128K-S129Q-A202V, V087A-S100N, S1 00E-G117R-G127T, T077N-G127T-A128K-S129Q, Q017H-N096D-G127T-A 202V, N096D-A202V-M221Q, V045R-N096D-Y103F-G127T, N096D-G127T- G203E, V045R-S086E-A202V-G203E, T078I-S086E-S155E-G203E, V045R- M221Q, Q017H-N096D-Y103F-G127T, Q017H-Y103F-G127T-A202V, T078I -A202V-M221Q, V045R-N096D-G127T, V045R-N096D-G127T-A202V, P009 T-T078I-Y103F-A202V, S100N-T115L, S086E-A202V, N096D-Y103F-G12 7T, T078I-N096D-G127T, T115L-G165Q-A202V, T078I-S086E, V045R-T07 8I-N096D-G127T, P009T-N096D-G127T, G127T-A128R-S129Q-A202V, T078I-N096D-A202V, N096D-Y103I-G203E, G127S-A128P-S129Q-A202V, N096D-G127T-N217S, and T077N-T078I-M221Q, where each position is numbered according to SEQ ID NO: 1, and where the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8.

[0064] In another implementation, a subtilisin variant is provided, wherein the variant comprises M221Q, S100N, T115L, or S087A at positions numbered according to SEQ ID NO:1, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8.

[0065] In other embodiments, the subtilisin variants provided herein comprise an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 and including at least one of the following amino acids numbered according to the amino acid sequence of SEQ ID NO: 1: X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X116L, X117L, X118L, X119L, X120L, X121L, X122L, X123L, X124L, X125L, X126L, X127L, X128L, X129L, X130L, X131L, X132L, X133L, X134L, X135L, X136L, X137L, X138L, X139L, X140L, X141L, X142L, X143L, X144L, X145L, X146L, X147L, X148L, X149L, X150L, ​​X151L, X152L, X153L, X154L, X155L, X156L, X157L, X158L, X159L, X160L, X161L, X162L, X163L, X164L, X165L, X166L, X167L, X168L, X169L, X170L, X171L, X172L, X173L, X174L, X175L, X176L, X17 and X264H, wherein the subtilisin variant has a net charge of -4 to +2 at pH 8 relative to a subtilisin having the amino acid sequence of SEQ ID NO: 1. In yet additional embodiments, such subtilisin variants may further comprise one or more substitutions selected from the group consisting of X24Q, X77N, X86D, X165Q, X184Q, X258D, and X258P, wherein positions are numbered according to the amino acid sequence of SEQ ID NO: 1.

[0066] In other embodiments, the subtilisin variants provided herein are variants that contain a set of substitutions selected from the group consisting of those variants listed in Tables 3, 4, 5, 6, 7, 8 and 15, and have at least 75% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8 at positions numbered according to SEQ ID NO: 1.

[0067] In another embodiment, one or more subtilisin variants described herein are directed, with the proviso that one or more substitutions are non-naturally occurring. Further embodiments relate to one or more subtilisin variants described herein, which are (i) variants derived from B. licheniformis subtilisin, (ii) isolated variants, (iii) variants with proteolytic activity, or (iv) variants comprising a combination of (i)-(iii). Yet another embodiment is directed to one or more subtilisin variants described herein, wherein the variants are derived from a parent or reference polypeptide that (i) has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, or (ii) has 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. In yet another embodiment, the parent comprises the amino acid sequence of SEQ ID NO: 1. Still further embodiments relate to one or more subtilisin variants described herein, wherein the variants (i) comprise an amino acid sequence that has less than 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1.

[0068] In still yet further embodiments, one or more subtilisin variants described herein combine one or more improved properties compared to a reference or parent subtilisin, wherein the improved properties are selected from improved cleaning performance in detergents, improved stability in detergents or buffers, and improved cleaning performance over time, and combinations thereof. Aged cleaning performance refers to the difference in soil removal of aged test samples (pre-incubated with enzyme) compared to "fresh" soil washes of the enzyme (pre-incubated with detergent at an elevated temperature, such as 37°C, for an extended period, such as 3-8 weeks). Thus, enzymes that exhibit improved cleaning performance over time will have a smaller difference between aged samples and freshly prepared samples compared to the same evaluation performed with the reference / parent enzyme.

[0069] In another embodiment, the parent subtilisin comprises the amino acid sequence of SEQ ID NO: 1. In another embodiment, the parent subtilisin is a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8. In yet another embodiment, the improved property is: (i) improved cleaning performance in detergents in which the variant, compared to the parent subtilisin, removes blood / milk / ink stains on woven cotton (CS-05), aged blood stains on cotton (CS-01), aged chocolate rice pudding stains on cotton (CS-100), aged whole egg stains with carbon black on cotton (CS-39), aged chocolate soy drink stains on cotton (CS-45), grass stains on cotton (CS-07), chocolate milk stains with carbon black on cotton (C-03), milk stains with carbon black on cotton (C-11), blood / milk / ink stains on polycotton (EMPA 116), aged blood stain on polyester / cotton (KCS-01), and any one combination thereof; and / or (ii) improved stability, wherein the variant has higher residual activity compared to the parent or reference subtilisin. In yet another embodiment, cleaning performance in a detergent is measured according to the Wash Performance Assay of Example 2, and / or stability is measured according to the Detergent Stability Assay of Example 2. In one embodiment, the improved property is improved cleaning performance at lower temperatures (such as 20°C) in a detergent, and the variant has improved cleaning performance compared to the parent subtilisin on stains selected from the group consisting of aged blood stain on cotton (CS-01), aged chocolate rice pudding stain on cotton (CS-100), aged whole egg stain with carbon black on cotton (CS-39), aged chocolate soy milk stain on cotton (CS-45), grass stain on cotton (CS-07), blood / milk / ink stain on polycotton (EMPA 116), and any one combination thereof.In one embodiment, the improved property is improved cleaning performance at elevated temperatures (such as 37°C) in a detergent, and the variant has improved cleaning performance compared to the parent subtilisin on a stain selected from the group consisting of chocolate milk stain with carbon black on cotton (C-03), blood / milk / ink stain on woven cotton (CS-05), milk stain with carbon black on cotton (C-11), aged blood stain on cotton (CS-01), chocolate rice pudding, grass stain on cotton (CS-07), aged stain on cotton (CS-100), aged whole egg stain with carbon black on cotton (CS-39), blood / milk / ink stain on polycotton (EMPA 116), aged blood / milk / ink stain on polyester / cotton (KCS-01), and any one combination thereof. The terms "enhanced stability" or "improved stability" in the context of oxidation-, chelating-, denaturing-, detergent-, heat-, and / or pH-stable proteases refer to a higher retained proteolytic activity of a subtilisin variant over time compared to a reference or parent subtilisin protease, e.g., a wild-type or parent protease such as SEQ ID NO: 1 or SEQ ID NO: 8. Autolysis has been identified as one manner in which subtilisin activity is lost 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.)

[0070] The terms "thermostable" and "thermostable" and "thermostable" with respect to protease variants refer to a protease that retains more residual activity compared to a parent or reference protease after exposure to altered temperatures for a period of time under conditions typically found during proteolysis, hydrolysis, cleaning, or other processing (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., % remaining activity. "Altered temperatures" encompass both increases and decreases in temperature. In some embodiments, the variant proteases provided herein retain a residual activity of at least about 5 minutes, at least about 20 minutes, at least about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, about 660 minutes, about 720 minutes, about 780 minutes, about 840 minutes, about 900 minutes, about 960 minutes, about 1000 minutes, about 120 ...400 minutes, about 1600 minutes, about 1800 minutes, about 2400 minutes, about 2800 minutes, about 2800 minutes, about 2800 minutes, about 3000 minutes, about 3200 minutes, about 3200 minutes, about 3400 minutes, about 3600 The variant subtilisins provided herein maintain 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 over a predetermined time period after exposure to temperatures between 40°C and 80°C for about 20 minutes, about 1080 minutes, about 1140 minutes, or about 1200 minutes. In some embodiments, the variant subtilisins provided herein have a residual activity that is greater than the activity of the parent protease or reference protease using the method described in Example 2. In some embodiments, the variant subtilisins provided herein have at least a 10% improved residual activity compared to the parent subtilisin when measured after 20 minutes in a liquid detergent at 43-63°C.

[0071] The subtilisin variants provided herein can be used to prepare a variety of compositions, such as enzyme compositions, cleaning or detergent compositions, etc. The enzyme compositions comprise the subtilisin variants provided herein. The enzyme compositions can be in any form, such as granules, liquid formulations, enzyme slurries, etc.

[0072] Enzyme granules can be made by, for example, rotary atomization, wet granulation, dry granulation, spray drying, disk granulation, extrusion, pan coating, spheronization, drum granulation, fluidized bed agglomeration, high shear granulation, fluidized bed spray coating, crystallization, precipitation, emulsion gelation, rotary disk atomization and other casting methods, and prilling granulation. The core of the granule may be the granule itself or the inner core of a layered granule.

[0073] The core may comprise one or more water-soluble or water-dispersible materials (such as, but not limited to, sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, sugars (such as sucrose, lactose, glucose, granular sucrose, maltodextrin, and fructose), plasticizers (such as polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (such as cellulose and cellulose derivatives, such as hydroxypropylmethylcellulose, carboxymethylcellulose, and hydroxyethylcellulose), phosphates, calcium, protease inhibitors, and combinations thereof. Suitable dispersible materials include, but are not limited to, clay, nonpareils (combinations of sugar and starch, such as starch-sucrose nonpareils-ASNP), talc, silicates, carboxymethylcellulose, starch, and combinations thereof.

[0074] 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.

[0075] In some embodiments, the core comprises a subtilisin variant provided herein. In other embodiments, the core comprises one or more enzymes in addition to a protease. In other embodiments, the core is inactive and does not contain any enzymes.

[0076] 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 water-dispersible materials listed herein. The enzyme may be or may include a protease to be stabilized, in which case the enzyme titer should further include a stabilizer.

[0077] 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 Application Publication No. 20100124586, WO 9932595, and U.S. Patent No. 5,324,649).

[0078] In some embodiments, the coating layer comprises one or more of the following materials: inorganic salts (e.g., sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid (e.g., sucrose, lactose, glucose, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives such as hydroxypropyl methylcellulose, carboxymethyl cellulose, and hydroxyethyl cellulose), clays, nonpareils (combinations of sugars and starches), silicates, carboxymethyl cellulose, phosphates, starches (e.g., corn starch), fats, oils (e.g., rapeseed oil and paraffin oil), lipids, vinyl polymers, vinyl copolymers, polyvinyl alcohol (PVA), plasticizers (e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate, and water), anti-agglomerating agents (e.g., talc, clay, amorphous silica, and titanium dioxide), anti-foaming agents (FOAMBLAST). 882® and EROL 6000K®, and talc. U.S. Patent Application Publication No. 20100124586, WO 9932595, and U.S. Patent No. 5,324,649 detail suitable ingredients for the coating layer.

[0079] In some embodiments, the coating layer comprises a sugar (e.g., sucrose, lactose, glucose, granular sucrose, maltodextrin, and fructose). In some embodiments, the coating layer comprises a polymer such as polyvinyl alcohol (PVA). PVA suitable for incorporation into the coating layer of the multi-layered granules includes partially hydrolyzed, fully hydrolyzed, and intermediately hydrolyzed PVAs with low to high viscosities. In some embodiments, the coating layer comprises an inorganic salt, such as, for example, sodium sulfate.

[0080] 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.

[0081] In some embodiments, the enzyme granules comprise an enzyme selected from the group consisting of acyltransferase, alpha amylase, beta amylase, alpha galactosidase, arabinosidase, arylesterase, beta galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta 1,4-glucanase, endo-beta mannase, esterase, exo-mannase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polygalacturonase, polyesterase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof. Generally, at least one enzyme coating layer comprises at least one subtilisin variant as provided herein.

[0082] The above list of enzymes is merely exemplary and is not meant to be exhaustive. Any enzyme can be used in the granules described herein, including wild-type, recombinant, and mutant enzymes of bacterial, fungal, and yeast origin, as well as acidic, neutral, or alkaline enzymes.

[0083] Another embodiment relates to a method of cleaning a surface, the method comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition comprising one or more subtilisin variants. In some embodiments, the surface or item in need of cleaning comprises proteinaceous soils on the surface. In some embodiments, the surface or item in need of cleaning comprises proteinaceous soils. The term "soil" refers to any type of soil attached to the surface of an item, such as a hard surface item (e.g., dishware), medical implement, textile, etc. In some embodiments, the soil is a proteinaceous soil. As used herein, "proteinaceous soil" refers to a soil or stain that contains protein.

[0084] Further embodiments relate to methods of cleaning proteinaceous soils comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition comprising one or more subtilisin variants provided herein.

[0085] In another embodiment, the present invention relates to a method for cleaning stains selected from the group consisting of blood / milk / ink stains on woven cotton fabric (CS-05), aged blood stains on cotton fabric (CS-01), aged chocolate rice pudding stains on cotton fabric (CS-100), aged whole egg stains with carbon black on cotton fabric (CS-39), aged chocolate soy drink stains on cotton fabric (CS-45), grass stains on cotton fabric (CS-07), chocolate milk stains with carbon black on cotton fabric (C-03), milk stains with carbon black on cotton fabric (C-11), blood / milk / ink stains on polycotton fabric (EMPA 116), aged blood stains on polyester / cotton (KCS-01), and any one combination thereof, said method comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition comprising one or more such subtilisin variants.

[0086] Another embodiment relates to a method for cleaning stains at low temperatures (e.g., 20°C or 25°C), comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition comprising one or more such subtilisin variants. In one embodiment, the method is a method for cleaning stains selected from the group consisting of aged blood stain on cotton (CS-01), aged chocolate rice pudding stain on cotton (CS-100), aged whole egg stain with carbon black on cotton (CS-39), aged chocolate soy milk stain on cotton (CS-45), grass stain on cotton (CS-07), blood / milk / ink stain on polycotton (EMPA 116), and any one combination thereof, at low temperatures (e.g., 20°C or 25°C), comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition comprising one or more such subtilisin variants. Another embodiment relates to a method of cleaning soils at elevated temperatures (such as 37°C) comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition comprising one or more such subtilisin variants.In one embodiment, the method is for cleaning a stain selected from the group consisting of chocolate milk stain with carbon black on cotton (C-03), blood / milk / ink stain on woven cotton (CS-05), milk stain with carbon black on cotton (C-11), aged blood stain on cotton (CS-01), chocolate rice pudding, grass stain on cotton (CS-07), aged stain on cotton (CS-100), aged whole egg stain with carbon black on cotton (CS-39), blood / milk / ink stain on polycotton (EMPA 116), aged blood / milk / ink stain on polyester / cotton (KCS-01), and any one combination thereof, at an elevated temperature (such as 37° C.), comprising contacting the surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein or a composition comprising one or more such subtilisin variants.

[0087] Various changes can be made to one or more subtilisin variants described herein, such as the insertion, deletion, and / or substitution of one or more conservative or non-conservative amino acids (including cases where such changes do not substantially alter the enzymatic activity of the variant). Similarly, the nucleic acids of the invention can also be made to one or more substitutions of one or more nucleotides in one or more codons, such that particular codons encode the same or different amino acids, resulting in silent mutations (e.g., where the nucleotide mutation does not change the encoded amino acid) or non-silent mutations, such as 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 will not substantially alter the enzymatic activity of the resulting encoded polypeptide enzyme compared to the polypeptide enzyme encoded by the original nucleic acid sequence. The nucleic acid sequences described herein can also be modified to include one or more codons that provide for optimal expression in an expression system (e.g., a bacterial expression system), while, if desired, the one or more codons still encode the same amino acid.

[0088] Described herein are one or more isolated, non-naturally occurring, or recombinant polynucleotides consisting of 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 the recombinant production (e.g., expression) of one or more subtilisin variants described herein, typically by expression of a plasmid expression vector containing a sequence encoding one or more subtilisin variants or fragments thereof described herein. One embodiment provides a nucleic acid encoding one or more subtilisin variants described herein, wherein the variants are in a mature form that has proteolytic activity. In some embodiments, one or more subtilisin variants described herein are recombinantly expressed with a homologous propeptide sequence. In other embodiments, one or more subtilisin variants described herein are recombinantly expressed with a heterologous or native propeptide sequence (e.g., a propeptide sequence from B. licheniformis (SEQ ID NO: 4)).

[0089] One or more nucleic acid sequences described herein can be produced by using any suitable synthesis, manipulation, and / or isolation method, or a combination thereof. For example, one or more polynucleotides described herein can be made using standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques well known to those of skill in the art. Such techniques typically involve synthesizing fragments of up to 50 or more nucleobases, which are then ligated to form the substantially desired contiguous nucleic acid sequence. Synthesis of one or more polynucleotides described herein can also be facilitated by any suitable method known in the art, including, but not limited to, 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 performed 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 for synthesizing nucleic acids and related principles are described, for example, in Itakura et al., Annu. Rev. Biochem. 53:323 (1984); and Itakura et al., Science 198:1056 (1984).

[0090] 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, polymerase chain reaction (e.g., PCR), etc. One or more polynucleotides described herein can also be obtained by screening a cDNA library with one or more oligonucleotide probes capable of hybridizing to or PCR amplifying a polynucleotide encoding one or more subtilisin variants described herein, or a recombinant polypeptide or active fragment thereof. Methods for screening and isolating cDNA clones and PCR amplification methods 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.

[0091] Further embodiments relate to one or more vectors comprising one or more subtilisin variants described herein (e.g., polynucleotides encoding one or more subtilisin variants described herein); expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequences described herein; isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequences described herein; isolated or recombinant cells comprising one or more polynucleotide sequences described herein; and compositions comprising one or more such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof.

[0092] Some embodiments relate to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein, which contain one or more nucleic 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 comprise bacterial cells, including, but not limited to, Bacillus cells, such as B. subtilis or B. licheniformis cells. Other embodiments relate to recombinant cells (e.g., recombinant host cells) comprising one or more subtilisins described herein.

[0093] In some embodiments, one or more vectors described herein are expression vectors or expression cassettes that include one or more polynucleotide sequences described herein operably linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., a promoter operably linked to one or more polynucleotide sequences described herein). The vectors may include a transcription terminator and / or a selection gene (e.g., an antibiotic resistance gene) that allows for continuous cultural maintenance of plasmid-infected host cells by growth in antimicrobial-containing medium.

[0094] Expression vectors may be derived from plasmids or viral DNA, or in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (see Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990); suitable replicating plasmids for B. subtilis include those described on p. 92). (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).

[0095] 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 optionally 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 as an autonomous extrachromosomal element within the cell. Some embodiments provide both extrachromosomal nucleic acid elements and incoming nucleotide sequences that are integrated into the genome of the host cell. The vectors described herein are useful for production of one or more subtilisin variants described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variants described herein is present on an integrating vector that allows for integration and, optionally, amplification of the polynucleotide encoding the variant into the host chromosome. Examples of integration sites are well known to those of skill in the art. In some embodiments, transcription of a polynucleotide encoding one or more subtilisin variants described herein is driven 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.Examples of promoters suitable for use in bacterial host cells include, but are not limited to, the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII, and rrnIp2 promoters; the promoters of the B. stearothermophilus maltogenic amylase gene, and the B. amyloliquefaciens (BAN) amylase gene. Examples of promoters include the Bacillus subtilis alkaline protease gene, the Bacillus subtilis clausii alkaline protease gene, the Bacillus pumilus xylosidase gene, the Bacillus thuringiensis cryIIIA gene, and the Bacillus licheniformis alpha-amylase gene. Additional promoters include, but are not limited to, the A4 promoter, the PR or PL promoters of phage lambda, and the lac, trp, or tac promoters of E. coli.

[0096] One or more subtilisin variants described herein can be produced in suitable microbial host cells, such as bacteria and fungi. In some embodiments, one or more subtilisin variants described herein can be produced by Gram-positive bacteria. In some embodiments, the host cell is Bacillus spp., Streptomyces spp., Escherichia spp., Aspergillus spp., Trichoderma spp., Pseudomonas spp., Corynebacterium spp., Saccharomyces spp., or Pichia spp. In some embodiments, one or more of the subtilisin variants described herein are produced by a Bacillus sp. host cell. Examples of Bacillus host cells that find use in the production of one or more of the subtilisin variants described herein include, but are not limited to, B. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B. pumilis, B. thuringiensis, B. clausii, and B. megaterium, as well as other organisms within the genus Bacillus. In some embodiments, B. subtilis host cells are used to produce the variants described herein.U.S. Pat. No. 5,264,366 and U.S. Pat. No. 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 also be used.

[0097] Some bacterial strains that can be used to produce one or more subtilisin variants described herein include non-recombinant (i.e., wild-type) Bacillus sp. strains, as well as various naturally occurring and / or recombinant strains. In some embodiments, the host strain is a recombinant strain, and a polynucleotide encoding one or more subtilisin variants described herein has been introduced into the host. In some embodiments, the host strain is a B. subtilis host strain, particularly a recombinant B. subtilis host strain. Many B. subtilis strains are known, including, but not limited to, strains 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753-PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211 (see, e.g., Hoch et al., Genetics 73:215-228

[1973] ; see also U.S. Pat. Nos. 4,450,235, 4,302,544, and EP 0134048. The use of B. subtilis as an expression host cell is well known in the art (see, e.g., Palva et al., Gene 19:81-87 (1982); Fahnestock and Fischer, J. Bacteriol., 165:796-804 (1986); and Wang et al., Gene 69:39-47 (1988)).

[0098] In some embodiments, the Bacillus host cell is a bacterium of the genus Bacillus that contains a mutation or deletion in at least one of the following genes: degU, degS, degR, and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU(Hy)32 (see, e.g., Msadek et al., J. Bacteriol. 172:824-834 (1990) and Olmos et al., Mol. Gen. Genet. 253:562-567 (1997)). In some embodiments, the Bacillus host 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 results in the same phenotype as a mutation in the oppA gene is contemplated for use in some embodiments of the mutant Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, altered Bacillus host cells that can be used to produce one or more subtilisin variants described herein are Bacillus host strains that already contain mutations in one or more of the above genes. Additionally, host cells of Bacillus species that contain mutations and / or deletions of endogenous protease genes are also used. In some embodiments, the Bacillus host cell contains deletions of the aprE and nprE genes.In other embodiments, the Bacillus sp. host cell comprises a deletion of five protease genes, while in other embodiments, the Bacillus sp. host cell comprises a deletion of nine protease genes (e.g., U.S. Patent Application Publication No. 2005 / 0202535).

[0099] Host cells are 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 transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmid is subsequently isolated from the E. coli cells and transformed into Bacillus cells. However, an intervening microorganism, e.g., E. coli, need not necessarily be used; in some embodiments, the DNA construct or vector is directly introduced into the Bacillus host.

[0100] Exemplary methods for introducing one or more of the nucleic acid sequences described herein into Bacillus cells are described, for example, in Ferrari et al., "Genetics," in 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. 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 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 (e.g., Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol. 154:1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169:1205-1211 (1987)). In this method, an incoming donor plasmid recombines with homologous regions of a resident "helper" plasmid in a process that mimics chromosomal transformation.

[0101] In addition to commonly used methods, in some embodiments, host cells are directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more subtilisin variants described herein (i.e., no intermediate cells are used to amplify or otherwise manipulate the DNA construct or vector prior to introduction into the 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 inserting it into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In additional embodiments, the DNA construct or vector is not introduced into a plasmid, but is co-transformed with a plasmid. In a further embodiment, the selectable marker is deleted from the mutant Bacillus strain using 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)).

[0102] In some embodiments, the transformed cells are cultured in a conventional nutrient medium. Suitable specific culture conditions, such as temperature and pH, are known to those of skill in the art and are well described in the scientific literature. Some embodiments provide a culture (e.g., a cell culture) comprising one or more subtilisin variants or nucleic acid sequences described herein.

[0103] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more subtilisin variants described herein are 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. In some embodiments, the variants produced by the cells are recovered from the culture medium by conventional procedures, including, but not limited to, separating the host cells from the culture medium by centrifugation or filtration, precipitating the protein components of the supernatant or filtrate with a salt (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).

[0104] In some embodiments, one or more subtilisin variants produced by recombinant host cells are 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)). Such purification-facilitating domains include, but are not limited to, metal-chelating proteins such as histidine-tryptophan modules, which enable purification by immobilized metals (see, e.g., Porath, Protein Expr. Purif. 3:263-281 (1992)), protein A domains, which enable purification by immobilized immunoglobulins, and domains used 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, CA) between the purification domain and the heterologous protein can also be used to facilitate purification.

[0105] The variant proteins of the present invention can be produced in host cells by secretion or intracellular expression, for example, using methods known in the art. Fermentation, separation, and concentration techniques are well known in the art, and conventional methods can be used to prepare concentrated enzyme-containing solutions. The host cells can be further treated, for example, by heating, altering the pH or salt content, or treating with enzymes, including hen egg white lysozyme, T4 lysozyme, and enzymes described in WO2022047149, to release the enzyme or improve cell separation. To restore production scale, the variant polypeptides can be concentrated or partially purified, generally as described above, by removing cells via polymer flocculation. Alternatively, the enzymes can be concentrated or purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment. However, for some applications, the enzymes do not need to be concentrated or purified; the whole broth culture can be dissolved and used without further processing. The enzymes can then be processed, for example, into granules.

[0106] A variety of methods can be used to determine the production levels of one or more mature subtilisin variants described herein in a host cell. Such methods include, but are not limited to, methods utilizing protease-specific polyclonal or monoclonal antibodies. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), and fluorescence-activated cell sorter (FACS). These and other assays are well known in the art (e.g., Maddox et al., J. Exp. Med. 158:1211 (1983)).

[0107] Some other embodiments provide methods for generating or producing one or more mature subtilisin variants described herein. Mature subtilisin variants do not include signal peptide or propeptide sequences. Some methods include generating or producing one or more subtilisin variants described herein in a recombinant bacterial host cell, such as a Bacillus sp. cell (e.g., a B. subtilis cell). Other embodiments provide methods for 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 the production of the variants. Some such methods further include recovering the variants from the culture.

[0108] Further embodiments provide methods for producing one or more subtilisin variants described herein, 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 Bacillus cells); and (b) culturing the cells in a culture medium under conditions suitable for producing the variant encoded by the expression vector. Some such methods further comprise (c) isolating the variant from the cells or culture medium.

[0109] Further embodiments relate to methods of improving the cleaning performance or stability of a subtilisin, comprising modifying the subtilisin to include one or more substitutions, or a combination of substitutions, as provided herein.

[0110] Unless otherwise specified, all ingredient or composition levels presented herein are adjusted based on the activity level of that ingredient or composition and do not include impurities, such as residual solvents or by-products, that may be present in commercially available sources. The weight of the enzyme ingredient is based on total active protein. All percentages and ratios are calculated by weight unless otherwise specified. All percentages and ratios are calculated based on the total composition unless otherwise specified. The compositions described herein include cleaning compositions, such as detergent compositions. In exemplary detergent compositions, the enzyme level is expressed as pure enzyme by weight of the total composition, and detergent contents are expressed by weight of the total composition unless otherwise specified.

[0111] In one embodiment, one or more subtilisin variants described herein are useful in cleaning applications such as, but not limited to, cleaning dishes or dishware, fabrics, medical implements, and hard surface items (e.g., tables, tabletops, walls, furniture, floors, and ceiling hard surfaces). In other embodiments, one or more subtilisin variants described herein are useful in disinfecting applications such as, but not limited to, disinfecting automatic dishwashers or washing machines. In one embodiment, the cleaning composition is a cleaning composition comprising one or more subtilisin variants described herein, wherein the cleaning composition is a composition selected from the group consisting of a laundry detergent, a fabric softener, a dishwashing detergent (e.g., an automatic dishwashing detergent or a hand wash detergent), a hard surface cleaning detergent, and a medical implement cleaning composition.

[0112] Another embodiment is directed to a composition comprising one or more subtilisin variants described herein. In some embodiments, the composition is a cleaning composition. In other embodiments, the composition is a detergent composition. In still other embodiments, the composition is selected from a laundry detergent composition, an automatic dishwashing (ADW) composition, a manual dishwashing detergent composition, a hard surface cleaning composition, an eyeglass cleaning composition, a medical instrument cleaning composition, a disinfecting (e.g., malodor or microbial) composition, and a personal care cleaning composition. In still other embodiments, the composition is a laundry detergent composition, an ADW composition, or a manual dishwashing detergent composition. Still further embodiments are directed to fabric cleaning compositions, while other embodiments are directed to non-fabric cleaning compositions. In some embodiments, the cleaning composition is boron-free. In other embodiments, the cleaning composition is phosphate-free. In still other embodiments, the composition comprises one or more subtilisin variants described herein and one or more excipients, adjuvants, and / or additional enzymes.

[0113] In another embodiment, the present disclosure provides a cleaning composition (e.g., an ADW composition) comprising a surfactant and at least one subtilisin variant as defined herein. Such compositions may further comprise one or more excipients, adjunct materials, and / or additional enzymes.

[0114] In still further embodiments, the compositions described herein contain phosphate, are phosphate-free, contain boron, are boron-free, or a combination thereof. In other embodiments, the compositions are boron-free. In some embodiments, the boron-free compositions are compositions to which no borate stabilizers have been added. In another embodiment, the boron-free compositions are compositions containing less than 5.5% boron. In yet a further embodiment, the boron-free compositions are compositions containing less than 4.5% boron. In yet another embodiment, the boron-free compositions are compositions containing less than 3.5% boron. In yet a further embodiment, the boron-free compositions are compositions containing less than 2.5% boron. In yet a further embodiment, the boron-free compositions are compositions containing less than 1.5% boron. In another embodiment, the boron-free compositions are compositions containing less than 1.0% boron. In yet a further embodiment, the boron-free compositions are compositions containing less than 0.5% boron. In other embodiments, the compositions are free or substantially free of enzyme stabilizers or peptide inhibitors.

[0115] In another embodiment, one or more compositions described herein are in the form of a gel, tablet, powder, granule, solid, liquid, unit dosage form, and combinations thereof. In yet another embodiment, one or more compositions described herein are in a form selected from a low-water compact formulation, a low-water HDL or unit dose (UD), or a high-water formulation or HDL. In some embodiments, the cleaning compositions described herein are in a unit dosage form. In other embodiments, the unit dosage form is selected from a pill, tablet, capsule, gel capsule, sachet, bag, multi-compartment bag, and pre-measured powder or liquid. In some embodiments, the unit dosage form is designed to control the release of the contents within the multi-compartment pouch (or other unit dosage form). Suitable unit dosage forms and controlled-release dosage forms are described, for example, in EP 2100949; WO 02 / 102955; U.S. Pat. No. 4,765,916; U.S. Pat. No. 4,972,017; and WO 04 / 111178. In some embodiments, the unit dosage form is a tablet or powder contained in a water-soluble film or pouch.

[0116] Examples of laundry detergent compositions include, but are not limited to, liquid and powder laundry detergent compositions. Examples of hard surface cleaning compositions include, but are not limited to, compositions used to clean hard surfaces such as non-dishware, non-food utensils, tables, tabletops, furniture, walls, floors, and ceilings. Examples of hard surface cleaning compositions are described, for example, in U.S. Patent Nos. 6,610,642, 6,376,450, and 6,376,450. Examples of personal care compositions include, but are not limited to, compositions used to clean dentures, teeth, hair, contact lenses, and skin. Examples of ingredients for such oral care compositions include, for example, those described in U.S. Patent No. 6,376,450.

[0117] In some embodiments, one or more subtilisin variants described herein clean at low temperatures. In other embodiments, one or more compositions described herein clean at low temperatures. In other embodiments, one or more compositions described herein comprise an effective amount of one or more subtilisin variants described herein as useful or effective for cleaning surfaces in need of proteinaceous soil removal.

[0118] In some embodiments, these adjunct materials are included, for example, to aid or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the cleaning composition, as in the case of fragrances, colorants, or dyes. Some embodiments are directed to compositions comprising one or more adjuncts and one or more subtilisin variants described herein. Other embodiments relate to compositions comprising one or more adjunct materials and one or more subtilisin variants described herein, where the adjunct materials are selected from the group consisting of bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilization systems), chelating agents, optical brighteners, soil release polymers, biodegradable polymers, dye transfer agents, dispersants, slime control agents, dyes, fragrances, colorants, fillers, photoactive agents, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, shrinkage inhibitors, anti-wrinkle agents ... conditioners, hydrolyzable surfactants, preservatives, antioxidants, shrink inhibitors, wrinkle inhibitors, disinfectants, mildew inhibitors, color spot inhibitors, silver care agents, tarnish inhibitors, rust inhibitors, alkalinity sources, solubilizers, carriers, processing aids, pigments, pH adjusters, surfactants, builders, chelating agents, dye transfer inhibitors, precipitation aids, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, clay soil removal / redeposition inhibitors, structural elasticizers, fabric softeners, carriers, hydrotropes, processing aids, pigments, and combinations thereof. Examples of adjunct materials and concentrations for use are described in U.S. Patent Nos. 5,576,282, 6,306,812, 6,326,348, 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014, and 5,646,101. In embodiments where one or more cleaning adjunct materials are incompatible with one or more of the subtilisin variants described herein, a method is employed in which the adjunct and the variant are kept separate (i.e., not in contact with each other) until the combination of the two components is appropriate.Such separation methods may include any suitable method known in the art (eg, gelcaps, encapsulation, tablets, physical separation, etc.).

[0119] Some embodiments relate to cleaning additive products comprising one or more subtilisin variants described herein. In some embodiments, the additive is packaged in a dosage form for addition to a cleaning process. In some embodiments, the additive is packaged in a dosage form for addition to a cleaning process where a source of peroxide is utilized and enhanced bleaching is desired.

[0120] Examples of fillers or carriers for granular compositions include, but are not limited to, various sulfates, carbonates, and silicates; talc; and clay. Examples of fillers or carriers for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols, such as polyols and diols (e.g., methanol, ethanol, propanol, and isopropanol). In some embodiments, the composition contains from about 5% to about 90% of such fillers or carriers. Such compositions can contain acidic fillers to lower the pH of solutions resulting from cleaning processes or applications.

[0121] In one embodiment, one or more cleaning compositions described herein comprise an effective amount of one or more subtilisin variants described herein, alone or in combination with one or more additional enzymes. Typically, cleaning compositions comprise at least about 0.0001 to about 20%, about 0.0001 to about 10%, about 0.0001 to about 1%, about 0.001 to about 1%, or about 0.01 to about 0.2% by weight of one or more subtilisin variants described herein. In another embodiment, one or more cleaning compositions described herein contain about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 2 mg, about 0.01 to about 1 mg, about 0.05 to about 1 mg, about 0.5 to about 10 mg, about 0.5 to about 5 mg, about 0.5 to about 4 mg, about 0.5 to about 3 mg, about 0.5 to about 2 mg, about 0.5 to about 1 mg, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 4 mg, about 0.1 to about 3 mg, about 0.1 to about 2 mg, about 0.1 to about 2 mg, about 0.1 to about 1 mg, or about 0.1 to about 0.5 mg of one or more subtilisin variants described herein per gram of composition.

[0122] The cleaning compositions described herein are typically formulated to provide a wash water pH of about 4.0 to about 11.5, even about 5.0 to about 11.5, 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 have a pH of about 3.0 to about 9.0, even about 3 to about 5. Granular laundry products are typically formulated to have a pH of about 8 to about 11. In some embodiments, the cleaning compositions of the present invention can be formulated to have an alkaline pH under wash conditions, for example, a pH of about 8.0 to about 12.0, or about 8.5 to about 11.0, or about 9.0 to about 11.0. In some embodiments, cleaning compositions of the present invention can be formulated to have 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 using a conventional pH meter when the cleaning composition is dissolved 1:100 (weight:weight) in deionized water at 20°C. Techniques for adjusting pH to recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0123] 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. Examples of encapsulating materials include, but are not limited to, carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin wax, and combinations thereof. When the encapsulating material is a carbohydrate, the encapsulating material is typically selected from monosaccharides, oligosaccharides, and combinations thereof. In some embodiments, the encapsulant is starch (see, e.g., EP 0922499, U.S. Pat. No. 4,977,252, U.S. Pat. No. 5,354,559, and U.S. Pat. No. 5,935,826). In some embodiments, the encapsulant is a microsphere made from a plastic, such as a thermoplastic, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof. Exemplary commercially available microspheres include, but are not limited to, EXPANCEL® (Stockviksverken, Sweden); and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (PQ Corp., Valley Forge, PA).

[0124] The wash conditions to which one or more subtilisin variants described herein may be exposed include various detergent formulations, wash water volumes, wash water temperatures, and wash durations. Low detergent concentration systems relate to washes with wash water containing less than about 800 ppm of detergent components. Medium detergent concentration systems relate to washes containing from about 800 ppm to about 2000 ppm of detergent components. High detergent concentration systems relate to washes containing more than about 2000 ppm of detergent components. In some embodiments, the "cold water wash" of the present invention utilizes "cold water washes" suitable for washing at temperatures of from about 10°C to about 40°C, or from about 20°C to about 30°C, or from about 15°C to about 25°C, as well as all other combinations within the range of from about 15°C to about 35°C, or from 10°C to 40°C.

[0125] Water hardness varies depending on the geography. Hardness is determined by the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) is a measure of the amount of Ca 2+ / Mg 2+ It is stated in grains per gallon ("gpg") of the mixture. Most water in the United States is hard, but hardness levels vary. Medium-hard water (60-120 ppm) to hard water (121-181 ppm) has hardness minerals between 60 and 181 ppm (ppm can be converted to grains per U.S. gallon by dividing ppm by 17.1). [Table A]

[0126] Other embodiments are directed to one or more cleaning compositions comprising from about 0.00001% to about 10% by weight of the composition of one or more subtilisin variants described herein and from about 99.999% to about 90.0% by weight of the composition of one or more adjuncts. In other embodiments, the cleaning composition comprises from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% by weight of the composition of one or more subtilisin variants, and from about 99.9999% to about 90.0%, from about 99.999% to about 98%, or from about 99.995% to about 99.5% by weight of the composition of one or more adjunct materials.

[0127] In other embodiments, the compositions described herein comprise one or more subtilisin variants described herein and one or more additional enzymes, including acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannase, esterase, exo-mannase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannase, metalloprotease, nuclease, or the like. The enzymes may be selected from cleavage enzymes (e.g., DNase and / or RNase), oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phosphodiesterases, phospholipases, phytases, polygalacturonases, polyesterases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xanthan lyases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof. Some embodiments relate to the use of enzymes (i.e., "cocktails") that combine traditional enzymes, such as amylases, lipases, cutinases, mannanases, and / or cellulases, with one or more subtilisin variants and / or one or more additional proteases described herein.

[0128] In other embodiments, one or more compositions described herein comprise one or more subtilisin variants described herein and one or more additional proteases. In one embodiment, the additional protease is a serine protease. In another embodiment, the additional protease is not immunologically related (e.g., the proteases do not cross-react in antibody-based tests known in the art for assessing shared immune epitopes). In another embodiment, the additional protease is a protease with a different net charge at pH 8. In another embodiment, the additional protease is a metalloprotease, a fungal subtilisin, an alkaline microbial protease, or a trypsin-like protease. Suitable additional proteases include those of animal, vegetable, or microbial origin. In some embodiments, the additional protease is a microbial protease. In another embodiment, the additional protease is a chemically modified or genetically engineered mutant. In another embodiment, the additional protease is an alkaline microbial protease or a trypsin-like protease. In other embodiments, the additional protease does not contain a cross-reactive epitope with the subtilisin variant as determined by antibody binding or other assays available in the art. Representative alkaline proteases include, for example, those from the Bacillus genus (e.g., BPN', Carlsberg, subtilisin 309, subtilisin 147, subtilisin PB92, subtilisin DY, subtilisin 168, B. gibsonii DSM 14391 subtilisin, B. gibsonii AGS 78407 subtilisin, B. pumilus DSM 18097 subtilisin, B. lentus DSM 5483, B. amyloliquefaciens, B. siamenensis, and Bacillus sp. TY-145 subtilisin), orExamples of additional proteases include, but are not limited to, alkaline proteases of fungal origin, such as those described in U.S. Patent No. 8,362,222. Examples of additional proteases include, but are not limited to, those described in WO 92 / 21760, WO 95 / 23221, WO 2008 / 010925, WO 09 / 149200, WO 09 / 149144, WO 09 / 149145, WO 10 / 056640, WO 10 / 056653, WO 2010 / 0566356, WO 11 / 072099, WO 2011 / 13022 pamphlet, WO 11 / 140364 pamphlet, WO 12 / 151534 pamphlet, WO 2015 / 038792 pamphlet, WO 2015 / 089447 pamphlet, WO 2015 / 089441 pamphlet, WO 2017 / 215925 pamphlet, U.S. Patent Application Publication No. U.S. Publication No. 2008 / 0090747, U.S. Patent No. 5,801,039, U.S. Patent No. 5,340,735, U.S. Patent No. 5,500,364, U.S. Patent No. 5,855,625, RE 34,606, U.S. Pat. No. 5,955,340, U.S. Pat. No. 5,700,676, U.S. Pat. No. 6,312,936, U.S. Pat. No. 6,482,628, U.S. Pat. No. 8,530,219, U.S. Provisional Patent Application No. 62 / 180673 and U.S. Provisional Patent Application No. 62 / 161077, and PCT Application Nos. PCT / US2015 / 021813, PCT / US2015 / 055900, PCT / US No. 2015 / 057497, PCT / US2015 / 057492, PCT / US2015 / 057512, PCT / US2015 / 057526, PCT / US2015 / 057520, PCT / US2015 / 057502, PCT / US2016 / 022282, International Publication No. 2016074925, International Publication No. 2020178102, International Publication No. 2022106400,International Publication No. 2016203064, European Patent No. 3380599, International Publication No. 2017215925, International Publication No. 201948495, International Publication No. 2020221578, International Publication No. 2016203064, U.S. Patent No. 7294499, International Publication No. 2016 / 097354, International Publication No. 2020112599, International Publication No. 2021175696, International Publication No. 2021175697, and PCT / US16 / 32514, and International Publication No. 1999014341 and WO 2014 / 194117. Exemplary additional proteases include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and the Fusarium proteases described in WO 89 / 06270. Representative commercially available proteases include MAXATASE®, MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX™, EXCELLASE™, PREFERENZ™ (e.g., P100, P110, P280, P300), EFFECTENZ™ (e.g., 1000, P1050, P2000), EXCELLENZ™ (e.g., P1000), ULTIMASE®,and PURAFAST™ Protease (Danisco / Genencor / IFF); ALCALASE®, ALCALASE® ULTRA, BLAZE®, BLAZE® variants, BLAZE® EVITY®, BLAZE® EVITY® 16L, CORONASE®, SAVINASE®, SAVINASE® ULTRA, SAVINASE® EVITY®, SAVINASE® EVERIS®, PRIMASE®, DURAZYM™, POLA Examples of suitable antibacterial agents include, but are not limited to, RZYME®, OVOZYME®, KANNASE®, LIQUANASE®, LIQUANASEEVERIS®, NEUTRASE®, PROGRESSUNO®, RELASE®, and ESPERASE® (Novozymes); BLAP™ and BLAP™ variants (Henkel); LAVERGY™ PRO104L (BASF), KAP (B. alkaliphilus subtilisin (Kao)), and BIOTOUCH® (AB Enzymes).

[0129] Another embodiment relates to a composition comprising one or more subtilisin variants described herein and one or more lipases. In some embodiments, the composition comprises from about 0.00001% to about 10%, from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% lipase by weight of the composition. Exemplary lipases may be chemically modified or genetically engineered mutants.Representative lipases include, but are not limited to, those derived from bacteria or fungi, such as H. lanuginosa lipase (see, for example, European Patent Nos. 258068 and 305216), T. lanuginosa lipase (see, for example, International Publication Nos. WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase (see, for example, European Patent Nos. 2014 / 059360 and 2015 / 010009), and Rhizomucor miehei lipase (see, for example, European Patent Nos. 2014 / 059360 and 2015 / 010009). Rhizomucormiehei lipase (see, for example, EP 238023), Candida lipase, such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, for example, EP 214761), P. alcaligenes and P. pseudoalcaligenes Pseudomonas lipases such as Pseudomonas alcaligenes lipase (see, for example, EP 218272), P. cepacia lipase (see, for example, EP 331376), P. stutzeri lipase (see, for example, DE 1,372,034), P. fluorescens lipase, Bacillus s) Lipases (e.g., B. subtilis lipase (Dartoise et al., Biochem. Biophys. Acta 1131:253-260 (1993)), B. stearothermophilus lipase (see, for example, JP-A-64-744992), and B. pumilus lipase (see, for example, WO 91 / 16422).Examples of cloned lipases include, but are not limited to, Penicillium camembertii lipase (see, e.g., Yamaguchi et al., Gene 103:61-67 (1991)), Geotrichum candidum lipase (see, e.g., Schimada et al., J. Biochem., 106:383-388 (1989)), and various Rhizopus lipases, such as R. delemar lipase (see, e.g., Hass et al., Gene 109:117-113 (1991)), R. niveus lipase (Kugimiya et al., J. Biochem., 109:118-119 (1991)), and R. hydroxybenzoate lipase (see, e.g., Hass et al., Gene 109:119-120 (1991)). al., Biosci. Biotech. Biochem. 56:716-719 (1992)), and Rhizopus oryzae lipase. Other lipolytic enzymes, such as cutinases, may also be used in one or more of the compositions described herein, including, but not limited to, cutinases derived from Pseudomonas mendocina (see, e.g., WO 88 / 09367) and / or cutinases derived from Fusarium solanipisi (see, e.g., WO 90 / 09446). Representative commercially available lipases include, but are not limited to, M1 LIPASE™, LUMA FAST™, LIPOMAX™, and PREFERENZ™ L100 (IFF); LIPEX™, LIPOCLEAN™, LIPOLASE™, and LIPOLASE™ ULTRA (Novozymes); and LIPASE P™ (Amano Pharmaceutical Co. Ltd.).

[0130] Still other embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more amylases. In one embodiment, the composition comprises about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% amylase by weight of the composition. Any amylase (e.g., alpha and / or beta) suitable for use in alkaline solutions can be usefully included in such compositions. Exemplary amylases may be chemically modified or genetically engineered mutants. Exemplary amylases include those described, for example, in GB 1,296,839, WO 9100353, WO 9402597, WO 94183314, WO 9510603, WO 9526397, WO 9535382, WO 9605295, WO 9623873, WO 9623874, WO 9630481, WO 9710342, WO 9741213, WO 9743424, WO 9813481, WO 9826078, WO 990270, 2, International Publication No. 9909183, International Publication No. 9919467, International Publication No. 9923211, International Publication No. 9929876, International Publication No. 9942567, International Publication No. 9943793, International Publication No. 9943794, International Publication No. 9946399, International Publication No. 0029560, International Publication No. 0060058, International Publication No. 0060059, International Publication No. 0060060, International Publication No. 0114532, International Publication No. 0134784, International Publication No. 0164852, International Publication No. 0166712, International Publication No. 0188107,International Publication No. 0196537, International Publication No. 02092797, International Publication No. 0210355, International Publication No. 0231124, International Publication No. 2004055178, International Publication No. 2004113551, International Publication No. 2005001064, International Publication No. 2005003311, International Publication No. 2005018336, International Publication No. 2005019443, International Publication No. 2005066338, International Publication No. 200 6002643, International Publication No. 2006012899, International Publication No. 2006012902, International Publication No. 2006031554, International Publication No. 2006063594, International Publication No. 2006066594, International Publication No. 2006066596, International Publication No. 2006136161, International Publication No. 2008000825, International Publication No. 2008088493, International Publication No. 2008092919, International Publication No. International Publication No. 2008101894, International Publication No. 2008 / 112459, International Publication No. 2009061380, International Publication No. 2009061381, International Publication No. 2009100102, International Publication No. 2009140504, International Publication No. 2009149419, International Publication No. 2010 / 059413, International Publication No. 2010088447, International Publication No. 2010091221, International Publication No. 2010104675, International Publication No. 2010115021, International Publication No. 10115028, International Publication No. 2010117511, International Publication No. 2011076123, International Publication No. 2011076897, International Publication No. 2011080352, International Publication No. 2011080353, International Publication No. 2011080354, International Publication No. 2011082425, International Publication No. 2011082429, International Publication No. 2011087836,These include, but are not limited to, those of bacterial or fungal origin, such as the amylases described in WO2011098531, WO2013063460, WO2013184577, WO2014099523, WO2014164777, WO2015077126, WO2022 / 175435, and WO2018184004. Exemplary commercially available amylases include, but are not limited to, AMPLIFY®, DURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, AMPLIFY PRIME®, STAINZYME ULTRA® EVITY®, and BAN™ (Novozymes); EFFECTENZ™ S 1000, POWERASE™, PREFERENZ™ S 100, PREFERENZ™ S 110, PREFERENZ™ S 210, EXCELLENZ™ S 2000, RAPIDASE®, MAXAMYL® P (IFF), and the like. In some embodiments, the subtilisin variants provided herein may be combined with one or more amylases selected from the group consisting of AA707, AA560, AAI10, BspAmy24, SP722, and CspAmy1, and variants thereof, and combinations thereof.

[0131] Yet another embodiment relates to a composition comprising one or more subtilisin variants described herein and one or more cellulases. In one embodiment, the composition comprises about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulase by weight of the composition. Any suitable cellulase can be used in the compositions described herein. Exemplary cellulases may be chemically or genetically modified mutants. Exemplary cellulases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO2005054475, WO2005056787, U.S. Pat. No. 7,449,318, U.S. Pat. No. 7,833,773, U.S. Pat. No. 4,435,307, EP0495257, and U.S. Provisional Patent Application No. 62 / 296,678. Exemplary commercially available cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®, CAREZYME®, ENDOLASE®, RENOZYME®, CAREZYME® PREMIUM (Novozymes); REVITALENZ™ 100, REVITALENZ™ 200 / 220, REVITALENZ® 2000 (IFF); and KAC-500(B)™ (Kao Corporation). In some embodiments, the cellulase is included as a portion or fragment of a mature wild-type or mutant cellulase, where a portion of the N-terminus has been removed (see, e.g., U.S. Pat. No. 5,874,276).

[0132] Yet another embodiment relates to a composition 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. An example of a mannanase may be a variant that has been modified chemically or by genetic engineering. Non-limiting examples of mannanases include those of bacterial or fungal origin, such as those described in WO 2016 / 007929; U.S. Patent Nos. 6,566,114; 6,602,842; and 6,440,991; and U.S. Provisional Patent Applications Nos. 62 / 251516, 62 / 278383, and 62 / 278387. Exemplary commercially available mannanases include, but are not limited to, MANNAWAY® (Novozymes), EFFECTENZ™ M 1000, EFFECTENZ™ M 2000, PREFERENZ® M 100, MANNASTAR®, PURABRITE™ (IFF), and BIOTOUCH® (AB Enzymes).

[0133] Further embodiments relate 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. Examples of nucleases include those described in International Publication No. 2015181287, International Publication No. 2015155350, International Publication No. 2016162556, International Publication No. 2017162836, International Publication No. 2017060475 (e.g., SEQ ID NO: 21), International Publication No. 2018184816, International Publication No. 2018177936, International Publication No. 2018177938, International Publication No. 2018 / 185269, International Publication No. 2018185285, Examples of such compounds include, but are not limited to, those described in International Publication No. 2018177203, International Publication No. 2018184817, International Publication No. 2019084349, International Publication No. 2019084350, International Publication No. 2019081721, International Publication No. 2018076800, International Publication No. 2018185267, International Publication No. 2018185280, International Publication No. 2018206553, and International Publication No. 2020099490.Other nucleases that can be used in combination with the subtilisin variants provided herein in the compositions and methods provided herein include those described in Nijland R, Hall MJ, Burgess JG (2010) Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase. PLoS ONE 5(12) and Whitchurch, CB, Tolker-Nielsen, T., Ragas, PC, Mattick, JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295:1487.

[0134] Still further, still further embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more peroxidases and / or peroxidases. 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% peroxidase or oxidase by weight of the composition. Peroxidases may be used in combination with hydrogen peroxide or a source thereof (e.g., percarbonate, perborate, or persulfate), and oxidases may be used in combination with oxygen. Peroxidases and oxidases are used alone or in combination with enhancers for "bleaching" (i.e., to prevent textile dye transfer from dyed fabrics to other fabrics when the fabrics are washed together in the wash liquor) (see, e.g., WO 94 / 12621 and WO 95 / 01426). Exemplary peroxidases and / or oxidases may be chemically or genetically modified mutants. Exemplary peroxidases / oxidases include, but are not limited to, those of plant, bacterial, or fungal origin.

[0135] Another embodiment relates 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.

[0136] Another embodiment relates to a composition comprising one or more subtilisin variants described herein and a modified polysaccharide biopolymer having the benefit of improved cleaning / whitening or fabric surface modification.

[0137] In yet another embodiment, one or more compositions described herein contain one or more subtilisin variants and one or more additional enzymes, each independently, in an amount up to about 10% by weight of the composition, with the remainder of the cleaning composition being one or more adjuncts.

[0138] In some embodiments, one or more compositions described herein are used as cleaning additives, and the additives are in solid or liquid form. Such additive products are intended to supplement and / or enhance the performance of conventional cleaning compositions and can be added at any stage of the cleaning process. In some embodiments, the laundry detergent compositions have a density in the range of about 400 to about 1200 g / liter, while in other embodiments, the density is in the range of about 500 to about 950 g / liter, measured at 20°C.

[0139] Some embodiments relate to laundry detergent compositions comprising one or more subtilisin variants described herein and one or more adjunct materials selected from surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaching agents, additional enzymes, sizing agents, dispersants, lime soap dispersants, stain suspending agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry compositions also comprise a fabric softener.

[0140] Further embodiments relate to manual dishwashing compositions comprising one or more subtilisin variants described herein and one or more adjunct materials selected from surfactants, organic polymeric compounds, slime enhancers, Group II metal ions, solvents, hydrotropes, and additional enzymes.

[0141] Other embodiments relate to one or more compositions described herein, wherein the composition is a compact granular fabric cleaning composition or a heavy-duty liquid (HDL) fabric cleaning composition for use in laundering colored fabrics or providing softness through cleaning performance. Examples of fabric cleaning compositions and / or methods for making same are described in U.S. Patent Nos. 6,610,642 and 6,376,450. Other examples of cleaning compositions are described in, for example, U.S. Patent Nos. 6,605,458; 6,294,514; 5,929,022; 5,879,584; 5,691,297; 5,565,145; 5,574,005; 5,569,645; 5,565,422; and 5,565,422. Nos. 16,448; 5,489,392; and 5,486,303; 4,968,451; 4,597,898; 4,561,998; 4,550,862; 4,537,706; 4,515,707; and 4,515,705.

[0142] In some embodiments, the cleaning composition comprises acidifying particles or aminocarboxylic acid builders. Examples of aminocarboxylic acid builders include aminocarboxylic acids, aminocarboxylic acid salts, and derivatives thereof. In some embodiments, the aminocarboxylic acid builders are aminopolycarboxylic acid builders, such as those represented by the general formula MOOC-CHR-N(CHCOOM) (wherein R is C 1~12and M is an alkali metal). In some embodiments, the aminocarboxylic acid builder is methylglycine diacetate (MGDA), GLDA (glutamic acid-N,N-diacetate), iminodisuccinic acid (IDS), carboxymethyl inulin and salts and derivatives thereof, aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodiacetic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-( The acidifying agent may be 2-sulfoethyl)glutamic acid (SEGL), IDA (iminodiacetic acid) and its salts and derivatives, such as N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), and alkali metal salts and derivatives thereof. In some embodiments, the acidifying particles have a weighted geometric mean particle size of about 400 μm to about 1200 μm and a bulk density of at least 550 g / L. In some embodiments, the acidifying particles comprise at least about 5% builder.

[0143] In some embodiments, the acidified particles can contain any acid, including organic and mineral acids. The organic acid can have one or two carboxyls, and in some cases, up to 15 carbon atoms, particularly up to 10 carbon atoms, such as formic acid, acetic acid, propionic acid, capric acid, oxalic acid, succinic acid, adipic acid, maleic acid, fumaric acid, sebacic acid, malic acid, lactic acid, glycolic acid, tartaric acid, and glyoxylic acid. In some embodiments, the acid is citric acid. Mineral acids include hydrochloric acid and sulfuric acid. In some cases, the acidified particles are high-activity particles containing a high concentration of aminocarboxylic acid builder. Sulfuric acid has also been found to further contribute to the stability of the microparticles.

[0144] In further embodiments, the present invention relates to cleaning compositions comprising one or more subtilisin variants and one or more surfactants and / or surfactant systems, wherein the surfactants are selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some embodiments, the surfactant is present in a concentration of about 0.1 to about 60% by weight of the cleaning composition; in alternative embodiments, the concentration is about 1 to about 50%, and in yet other embodiments, the concentration is about 5 to about 40%.

[0145] In some embodiments, one or more compositions described herein comprise one or more detergent builders or builder systems. In one embodiment, the composition comprises at least about 0.1% or more, or from about 0.1% to about 90%, from about 0.1% to about 80%, from about 3% to about 60%, from about 5% to about 40%, or from about 10% to about 50% by weight of builder. Builders include, but are not limited to, alkali metal, ammonium, and alkanolammonium salts of polyphosphates, alkali metal silicates, alkali metal and alkaline earth metal carbonates, aluminosilicates, polycarboxylate compounds, etherhydroxypolycarboxylates, copolymers of maleic anhydride and ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethyloxysuccinic acid, various ammonium and substituted ammonium salts of polyacetic acid (e.g., ethylenediaminetetraacetic acid and nitrilotriacetic acid), polycarboxylates (e.g., mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, polycarboxylic acids such as carboxymethyloxysuccinic acid, and the like, and soluble salts thereof. In some such compositions, the builder is a water-soluble hardness ion complex ( For example, sequestering builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate, sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixtures of sodium tripolyphosphate and potassium tripolyphosphate). Examples of builders are described, for example, in EP 2100949. In some embodiments, the builder includes a phosphate builder and a non-phosphate builder. In some embodiments, the builder is a phosphate builder. In some embodiments, the builder is a non-phosphate builder. In some embodiments, the builder includes a mixture of a phosphate builder and a non-phosphate builder. Examples of phosphate builders include, but are not limited to, monophosphates, diphosphates, tripolyphosphates, or oligomeric polyphosphates, including alkali metal salts of these compounds, including sodium salts.In some embodiments, the builder may be sodium tripolyphosphate (STPP). Additionally, the composition may contain carbonate and / or citrate. Other suitable non-phosphate builders include homopolymers and copolymers of polycarboxylic acids and their partially or fully neutralized salts, monomeric polycarboxylic acids, and hydroxycarboxylic acids, and their salts. In some embodiments, salts of the above compounds include ammonium and / or alkali metal salts, i.e., lithium, sodium, and potassium salts, including sodium salts. Suitable polycarboxylic acids include acyclic, alicyclic, heterocyclic, and aromatic carboxylic acids, which in some embodiments may contain at least two carboxyl groups, each separated from the other by no more than two carbon atoms, in some cases.

[0146] In some embodiments, one or more compositions described herein comprise one or more chelating agents. In one embodiment, the composition comprises from about 0.1% to about 15%, or from about 3% to about 10%, by weight of the composition, of a chelating agent. Examples of chelating agents include, but are not limited to, copper, iron, manganese, and mixtures thereof.

[0147] In some embodiments, one or more compositions described herein include one or more deposition aids. Examples of deposition aids include, but are not limited to, polyethylene glycol; polypropylene glycol; polycarboxylates; soil-repellent polymers such as polyethylene terephthalate; clays such as kaolinite, montmorillonite, attapulgite, illite, bentonite, halloysite, and mixtures thereof.

[0148] In other embodiments, one or more compositions described herein include one or more anti-redeposition agents or nonionic surfactants (which can prevent soil redeposition) (see, e.g., EP 2100949). For example, in ADW compositions, nonionic surfactants can be used for surface modification purposes, particularly for upholstery fabrics, to prevent filming and staining, and to improve gloss. These nonionic surfactants can also be used to prevent soil redeposition. In some embodiments, the nonionic surfactant can be an ethoxylated nonionic surfactant, an epoxy-capped poly(oxyalkylated) alcohol, and an amine oxide surfactant.

[0149] In some embodiments, one or more compositions described herein comprise one or more dye transfer inhibitors. Examples of polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone, polyvinylimidazole, and mixtures thereof. In one embodiment, the composition comprises from about 0.0001% to about 10%, from about 0.01% to about 5%, or from about 0.1% to about 3% of the dye transfer inhibitor by weight of the composition.

[0150] In some embodiments, one or more compositions described herein comprise one or more silicates. Examples of silicates include, but are not limited to, sodium silicates, such as sodium disilicate, sodium metasilicate, and crystalline phyllosilicates. In some embodiments, the silicate is present in an amount of about 1% to about 20%, or about 5% to about 15% by weight of the composition.

[0151] In some further embodiments, one or more compositions described herein comprise one or more dispersants. The water-soluble organic material may include, but is not limited to, a homopolymeric or copolymeric acid or salt thereof, in which the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms.

[0152] In some further embodiments, one or more compositions described herein comprise one or more enzyme stabilizers. In some embodiments, the enzyme stabilizer is a water-soluble source of calcium and / or magnesium ions. In some embodiments, the enzyme stabilizer comprises an oligosaccharide, a polysaccharide, and an inorganic divalent metal salt, e.g., an alkaline earth metal salt such as a calcium salt. In some embodiments, the enzymes used herein are stabilized by the presence in the final composition of a water-soluble source of zinc(II), calcium(II), and / or magnesium(II) ions to provide such ions to the enzyme, as well as other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and oxovanadium(IV)). Chlorides and sulfates are also used in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described, for example, in WO 07 / 145964. In some embodiments, reversible protease inhibitors, such as boron-containing compounds (e.g., borates, 4-formylphenylboronic acid and phenylboronic acid derivatives such as those described in WO 96 / 41859) and / or peptide aldehydes, such as those further described in WO 2009 / 118375 and WO 2013004636, are also used.

[0153] Peptide aldehydes can be used as protease stabilizers in detergent formulations, as described above (WO 199813458, WO 2011036153, U.S. Patent Application Publication No. 20140228274). Examples of peptide aldehyde stabilizers are peptide aldehydes, ketones, or halomethyl ketones, which may be "N-capped" with, for example, a ureido, carbamate, or urea moiety, or "doubly N-capped" with, for example, a carbonyl, ureido, oxamide, thioureido, dithiooxamide, or thiooxamide moiety (EP 2358857B1). The molar ratio of these inhibitors to protease is 0.1:1 to 100:1, for example, 0.5:1 to 50:1, 1:1 to 25:1, or 2:1 to 10:1. Other examples of protease stabilizers are benzophenone or benzoic acid anilide derivatives, which may contain a carboxyl group (U.S. Pat. No. 7,968,508 B2). The molar ratio of these stabilizers to protease is preferably in the range of 1:1 to 1000:1, particularly preferably 1:1 to 500:1, particularly preferably 1:1 to 100:1, and most preferably 1:1 to 20:1.

[0154] In some embodiments, one or more compositions described herein comprise one or more bleaching agents, bleach activators, and / or bleach catalysts. In some embodiments, one or more compositions described herein comprise one or more inorganic and / or organic bleaching compounds. Examples of inorganic bleaching agents include, but are not limited to, perhydrate salts, such as perborates, percarbonates, perphosphates, persulfates, and persilicates. In some embodiments, the inorganic perhydrate salts are alkali metal salts. In some embodiments, the inorganic perhydrate salts are present as crystalline solids without additional protection, while in other embodiments, the salts are coated. Bleach activators are typically organic peracid precursors that enhance bleaching activity during washes at temperatures of 60°C or less. Examples of bleach activators include compounds that, under perhydrolysis conditions, yield aliphatic peroxycarboxylic acids having from about 1 to about 10 carbon atoms or from about 2 to about 4 carbon atoms and / or optionally substituted perbenzoic acids. Examples of bleach activators are described in, for example, EP 2100949. Examples of bleach catalysts include, but are not limited to, manganese triazacyclononane complexes and related complexes, as well as cobalt complexes, copper complexes, manganese complexes, and iron complexes. Examples of additional bleach catalysts are described in, for example, U.S. Patent No. 4,246,612; U.S. Patent No. 5,227,084; U.S. Patent No. 4,810,410; International Publication No. WO99 / 06521; and EP 2100949.

[0155] In some embodiments, one or more compositions described herein contain one or more catalytic metal complexes. In some embodiments, a metal-containing bleach catalyst is used. In some embodiments, the metal bleach catalyst comprises a catalyst system containing a transition metal cation of predetermined bleach catalytic activity (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation), an auxiliary metal cation having little or no bleach catalytic activity (e.g., zinc or aluminum cation), and a sequestering agent having a predetermined stability constant for the catalyst and auxiliary metal cation, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and water-soluble salts thereof (see, e.g., U.S. Pat. No. 4,430,243). In some embodiments, one or more compositions described herein are catalyzed by a manganese compound. Such compounds and amounts used are described, for example, in U.S. Pat. No. 5,576,282. In additional embodiments, a cobalt bleach catalyst is used and included in one or more compositions described herein. Various cobalt bleach catalysts are described, for example, in US Pat. Nos. 5,597,936 and 5,595,967.

[0156] In some additional embodiments, one or more compositions described herein comprise a transition metal complex of a macropolycyclic rigid ligand (MRL). As a practical matter, and not by way of limitation, in some embodiments, the compositions and cleaning processes described herein are adjusted to provide an active MRL in the cleaning solution of at least parts per billion, from about 0.005 ppm to about 25 ppm, from about 0.05 ppm to about 10 ppm, or from about 0.1 ppm to about 5 ppm. Exemplary MRLs include, but are not limited to, specialized bridged ultrarigid ligands such as 5,12-diethyl-1,5,8,12-tetraazabicyclo(6.6.2)hexadecane. Examples of metal MRLs are described, for example, in WO 2000 / 32601 and U.S. Pat. No. 6,225,464.

[0157] In another embodiment, one or more compositions described herein include one or more metal care agents. In some embodiments, the composition includes about 0.1% to about 5% by weight of the composition of the metal care agent. Exemplary metal care agents include, for example, aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). Additional examples of metal care agents are described, for example, in EP 2100949, WO 94 / 26860, and WO 94 / 26859. In some compositions, the metal care agent is a zinc salt.

[0158] In some embodiments, the cleaning composition is a heavy-duty liquid (HDL) composition comprising one or more subtilisin variants described herein. HDL liquid laundry detergents can include an inhibitor surfactant (10-40%) consisting of an anionic inhibitor surfactant selected from the group of linear, branched, or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof; and optionally a nonionic surfactant selected from the group of linear, branched, or random chain, substituted or unsubstituted alkyl alkoxylated alcohols, e.g., C8-C9. 18 Alkyl alkoxylated alcohol and / or C6-C 12 a nonionic surfactant selected from the group consisting of alkylphenol alkoxylates, optionally in a weight ratio of anionic suds suppressor surfactant (hydrophilicity index (HIc) of 6.0 to 9) to nonionic surfactant of greater than 1:1. Suitable suppressor surfactants also include cationic suppressor surfactants (selected from alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and / or mixtures thereof); zwitterionic and / or amphoteric suppressor surfactants (selected from alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants; and mixtures thereof.

[0159] In another embodiment, the cleaning composition is a non-unit-dose liquid or gel cleaner, which may be aqueous, typically containing at least 20% and up to 95% water by weight, e.g., up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, or up to about 35% water by weight. Other types of liquids, including but not limited to, alkanols, amines, diols, ethers, and polyols, can be included in the aqueous liquid or gel. The aqueous liquid or gel cleaner may contain 0-30% organic solvents. The liquid or gel cleaner may be non-aqueous.

[0160] The composition may optionally comprise a surfactant-enhancing polymer consisting of an amphiphilic alkoxylated grease cleaning polymer selected from the group of branched hydrophilic and hydrophobic alkoxylated polymers, such as alkoxylated polyalkyleneimines, in the range of 0.05% to 10% by weight, and / or a random graft polymer typically comprising a hydrophilic backbone consisting of monomers selected from the group consisting of unsaturated C1 to C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyhydric alcohols such as glycerol, and mixtures thereof; and 25 It comprises a hydrophobic side chain selected from the group consisting of alkyl groups, polypropylene, polybutylene, vinyl esters of saturated C2-C6 monocarboxylic acids, C1-C6 alkyl esters of acrylic or methacrylic acid, and mixtures thereof.

[0161] The compositions may comprise additional polymers, such as soil release polymers comprising anionic end-capped polyesters, such as SRP1; polymers comprising at least one monomer unit selected from saccharides, dicarboxylic acids, polyols, and combinations thereof, in a random or block configuration; random or block ethylene terephthalate-based polymers and copolymers thereof, such as Repel-o-tex SF, SF-2, and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325, Marloquest The SL may contain: an anti-redeposition polymer (0.1% to 10% by weight, for example, a carboxylic acid polymer, for example, a polymer containing at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof; a vinylpyrrolidone homopolymer; and / or polyethylene glycol having a molecular weight in the range of 500 to 100,000 Da); a cellulosic polymer (for example, an alkyl cellulose; an alkyl alkoxyalkyl cellulose; a carboxyalkyl cellulose; an alkyl carboxyalkyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, and methylcarboxymethyl cellulose; and mixtures thereof); and a polymeric carboxylate (for example, a maleate / acrylate random copolymer or a polyacrylate homopolymer).

[0162] The composition may further comprise a saturated or unsaturated fatty acid, preferably a saturated or unsaturated C 12 ~C 24Fatty acids (0-10% by weight); precipitation aids (examples of which include polysaccharides, cellulose polymers, polydiallyldimethylammonium halides (DADMAC), random or block copolymers of DADMAC with vinylpyrrolidone, acrylamide, imidazole, imidazolinium halides, and mixtures thereof, cationic celluloses such as cationic guar gum, cationic hydroxyethyl cellulose, cationic starch, cationic polyacrylamide, and mixtures thereof).

[0163] The composition can further comprise a dye transfer inhibitor, examples of which include manganese phthalocyanine, peroxidase, polyvinylpyrrolidone polymer, polyamine N-oxide polymer, copolymer of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone, and polyvinylimidazole, and / or mixtures thereof; chelating agents, including ethylenediaminetetraacetic acid (EDTA); diethylenetriamine penta methylene phosphonic acid (DTPMP); hydroxyethane diphosphonic acid (HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methylglycine diacetic acid (MGDA); diethylenetriamine penta acetic acid (DTPA); propylene diamine tetracetic acid (PDT). A; 2-hydroxypyridine-N-oxide (HPNO); or methylglycine diacetate (MGDA); glutamic acid N,N-diacetate (N,N-dicarboxymethylglutamic acid tetrasodium salt (GLDA); nitrilotriacetic acid (NTA); 4,5-dihydroxy-m-benzenedisulfonic acid; citric acid and any salt thereof;Examples include N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), and derivatives thereof;

[0164] The composition may further comprise silicone or fatty acid based suds suppressors; enzyme stabilizers; hue dyes, calcium and magnesium cations, visual signal ingredients, antifoaming agents (0.001 to about 4.0% by weight), and / or structurants / thickeners (0.01 to 5% by weight) selected from the group consisting of di- and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulosic materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.

[0165] In some embodiments, the cleaning composition is a heavy-duty powder (HDD) composition comprising one or more subtilisin variants described herein. HDD powder laundry detergents can include nonionic deterrent surfactants (straight-chain, branched-chain, or random-chain, substituted or unsubstituted C8-C9 alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof) that can comprise deterrent surfactants including anionic deterrent surfactants (straight-chain, branched-chain, or random-chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof). 18 Alkyl ethoxylates and / or C6-C 12alkylphenol alkoxylates), cationic deterrent surfactants (selected from alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and mixtures thereof); zwitterionic and / or amphoteric deterrent surfactants (selected from alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof; builders (phosphate-free builders, e.g., zeolite builders, are exemplified by 0 to 10% by weight of phosphate builders, such as sodium tripolyphosphate in the range of 0 to less than 10% by weight; citric acid, citrates, and nitrilotriacetic acid or salts thereof in the range of less than 15% by weight; silicates (sodium or potassium silicate, or sodium metasilicate, or layered silicate (SKS-6) in the range of 0 to less than 10% by weight); carbonates (sodium carbonate and / or sodium bicarbonate in the range of 0 to less than 10% by weight); and bleaching agents (photobleaches, such as sulfonated zinc phthalocyanine, phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes, and mixtures thereof; hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED, and nonanoyloxybenzenesulfonate-NOBS, nitrile quats, and mixtures thereof); hydrogen peroxide; hydrogen peroxide sources (inorganic perhydrate salts, e.g., perborates, percarbonates, mono- or tetrahydrate sodium salts of persulfates, perphosphates, or persilicates; preformed hydrophilic and / or hydrophobic peracids (selected from percarboxylic acids and salts, percarboxylic acids and salts, perimidic acids and salts, peroxymonosulfates and salts, and mixtures thereof); and / or bleach catalysts (e.g., imine-based bleach boosters, e.g., iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulfonylimines; N-phosphonylimines; N-acylimines; thiadiazole dioxides; perfluoroimines;cyclic sugar ketones and mixtures thereof), metal-containing bleach catalysts (e.g., cations of copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese, auxiliary metal cations such as zinc or aluminum, and sequestering agents such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts);

[0166] The composition may further comprise additional detergent ingredients, such as perfume microcapsules, starch-encapsulated perfume accords, enzyme stabilizers, hueing agents, additional polymers including fabric integrity and cationic polymers, dye lock components, fabric softeners, whitening agents (e.g., CI fluorescent brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids, and / or cyclodextrins.

[0167] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a cleaning composition selected from the group consisting of laundry detergents, bar soaps, fabric softeners, dish detergents, medical implement detergents, and hard surface cleaning detergents.

[0168] In some embodiments, the present invention is directed to cleaning compositions comprising at least two proteases in combination with one or more additional cleaning composition components, such as, but not limited to, liquid laundry compositions described in WO2022106404.

[0169] In some embodiments, one or more subtilisin variants described herein are selected from the group consisting of liquid laundry detergent compositions, such as those described in U.S. Pat. No. 1,046,919 B2, WO 2021 / 223552, WO 2022 / 167251, WO 2022 / 074037, WO 2021 / 123184, WO 2021 / 037895, WO 2022 / 10372, WO 2020 / 264077, WO 2022 / 106404, and / or WO 2017 / 54983; compressed liquid laundry compositions (U.S. Pat. No. 10,683,474 B2); fatty alkyl esters; The compositions may be part of or added to liquid laundry detergent compositions, including, but not limited to, water soluble unit dose articles comprising teralkoxylate nonionic surfactants and alkoxylated alcohol nonionic surfactants (U.S. Patent Application Publication No. 20220162523A1); liquid laundry detergent compositions comprising improved alkyl benzene sulfonate surfactants (WO 2021 / 108307); liquid laundry detergent compositions comprising benzyl benzoate (WO 2020 / 223959); and / or detergent compositions comprising branched surfactants (WO 2021 / 247801), water soluble unit dose articles comprising amphiphilic graft polymers and polyester terephthalates (WO 2019 / 032257).

[0170] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition comprising an alkyl ether carboxylic acid, a betaine, an anionic surfactant, and a nonionic surfactant to provide softening benefits (WO 2013 / 087286).

[0171] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition comprising a sulfite radical scavenger, a protease stabilizer / inhibitor, or a combination thereof (WO 2022 / 157311).

[0172] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition described in US20210317387A1, WO2021 / 219296, WO2021 / 127662, WO2021 / 041685, US11208619, or US20220186144.

[0173] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition comprising a dispersin variant, such as, but not limited to, the liquid laundry detergent compositions described in U.S. Patent Application Publication No. 20210317387A1.

[0174] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition, such as (but not limited to) a highly alkaline fabric detergent, such as the liquid laundry detergent compositions described in WO 2021 / 219296.

[0175] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition, such as, but not limited to, a low-density unit dose detergent with encapsulated perfume, such as, but not limited to, the detergent compositions described in WO 2021 / 127662.

[0176] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a liquid laundry detergent composition comprising polyethylene glycol and an organic acid, such as, but not limited to, the detergent compositions described in WO 2021 / 041685.

[0177] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a cleaning composition comprising polyethylene glycol and an organic acid, such as, but not limited to, the cleaning compositions described in WO 2021 / 041685.

[0178] In some embodiments, cleaning compositions comprising one or more subtilisin variants described herein are cleaning compositions effective against proteinaceous soils, such as, but not limited to, the cleaning compositions described in U.S. Pat. No. 1,120,8619.

[0179] In some embodiments, a cleaning composition comprising one or more subtilisin variants described herein is a detergent composition comprising an antifouling polymer, such as, but not limited to, the detergent compositions described in U.S. Patent Application Publication No. 20220186144.

[0180] Examples of laundry detergent compositions include those shown in the following examples or in the following tables: [Table B]

[0181] In some embodiments, the cleaning composition is an ADW cleaning composition comprising one or more subtilisin variants described herein. The ADW cleaning composition may contain two or more nonionic surfactants selected from ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohols, and amine oxide surfactants in an amount of 0-10 wt. %; and a builder in the range of 5-60 wt. %. Phosphates (mono-phosphates, di-phosphates, tri-polyphosphates or oligomeric polyphosphates), sodium tripolyphosphate-STPP or phosphate-free builders (amino acid-based compounds, such as MGDA (methyl-glycine-diacetic acid) and its salts and derivatives, GLDA (glutamic acid-N,N-diacetic acid) and its salts and derivatives, IDS (iminodisuccinic acid) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives and mixtures thereof, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), B-alaninediacetic acid (B-ADA) and their salts), 0.5 to 50% by weight; about 0.1 to about 50% by weight of sulfonated / carboxylated polymers (which provide dimensional stability to the product); about 0.1 to about 10% by weight of drying aids (polyesters) Polyols, especially anionic polyesters, optionally with additional monomers having 3 to 6 functionality, especially acid, alcohol or ester functionality, that contribute to polycondensation, reactive cyclic carbonate and urea type polycarbonates, polyurethane and / or polyurea-polyorganosiloxane compounds or precursor compounds thereof; 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 inorganic salts (e.g., perhydrates such as perborates, percarbonates, perphosphates, persulfates and persilicates) and organic salts (e.g., organic peroxyacids including diacyl and tetraacyl peroxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid and diperoxyhexadecanedioic acid); about 0.1 to about 10% by weight of a bleaching catalyst (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn, and Fe bispyridylamine and related complexes, pentamine cobalt(III) acetate and related complexes); about 0.1 to 5% by weight of a benzatriazole, metal salts and complexes, silicates; about 0.01 to 5.0 mg of active enzymes (acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase) per gram of ADW detergent composition. The composition may comprise two or more non-ionic surfactants selected from enzymes ranging from enzymes such as hydroxylase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, peroxidase, phenoloxidase, phosphatase, phospholipase, phytase, polyesterase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and mixtures thereof; and enzyme stabilizer components (selected from oligosaccharides, polysaccharides, and inorganic divalent metal salts).

[0182] An example of an ADW composition is shown in the table below. [Table C]

[0183] Further embodiments relate to compositions and methods for treating fabrics (e.g., bleaching fabrics) using one or more subtilisin variants described herein. Methods for treating textiles are well known in the art (see, e.g., U.S. Pat. No. 6,077,316). For example, the feel and appearance of fabrics can be improved by a method comprising contacting the fabric in a solution with a variant described herein. The fabric can be treated with the solution under pressure.

[0184] One or more subtilisin variants described herein can be applied during or after weaving of a textile, or in the desizing stage, or in one or more additional textile processing steps. During weaving of textiles, yarns are subjected to significant mechanical strain. Before weaving on a loom, warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. One or more subtilisin variants described herein can be applied during weaving or after weaving to remove these sizing starches or starch derivatives. After weaving, the variants can be used to remove the sizing coating before further processing of the textile to ensure a uniform and wash-resistant result. One or more subtilisin variants described herein can be used alone or in combination with other desizing agents and / or desizing enzymes to desize textiles, such as cotton-containing textiles, e.g., as detergent additives in aqueous compositions. Amylases can also be used in combination with subtilisin variants in compositions and methods for creating a stonewashed appearance on indigo-dyed denim fabrics and garments. To produce garments, textiles can be cut and sewn into garments or garments, which are then finished. Various enzymatic finishing methods have been developed, particularly for producing denim jeans. Finishing denim garments typically begins with an enzymatic desizing step, during which the garment is subjected to the action of proteolytic enzymes to soften the fabric and make the cotton more accessible to subsequent enzymatic finishing steps. One or more subtilisin variants described herein can be used in methods for finishing denim garments (e.g., "biostoning"), enzymatic desizing and fabric softening methods, and / or finishing processes.

[0185] The present disclosure also provides methods for cleaning the surface of an article, the methods comprising contacting the article with at least one subtilisin variant provided herein (or a composition comprising such a subtilisin variant). In some embodiments, the article may have, for example, a proteinaceous soil on its surface. In some embodiments, the proteinaceous soil may consist of egg or egg-based soil, such as creme brûlée, baked cheese, BMI, or other protein-containing substances.

[0186] Non-limiting examples of the compositions and methods disclosed herein are as follows.

[0187] 1. Subtilisin mutants, including X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X117R, X127S, X127T, X128K, X128P, X128R, X129Q, X155E, X161Q, X181E, X181Q, X202V, A subtilisin variant comprising at least two, three or more substitutions selected from the group consisting of X203E, X203N, X217S, X221Q, X260W, and X264H, wherein these positions are numbered according to SEQ ID NO:1, and wherein the variant has at least 75% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8.

[0188] 2. A subtilisin variant according to embodiment 1, wherein the variant a) has at least 25% improved stability in detergent compared to the parent subtilisin SEQ ID NO: 1; and / or b) has a net charge of -4 to +2 at pH 8 compared to a subtilisin having the amino acid sequence of SEQ ID NO: 1.

[0189] 3. A subtilisin variant according to embodiment 1 or 2, wherein the variant further comprises one or more additional mutations selected from the group consisting of X24Q, X77N, X86D, X165Q, X184Q, X258D and X258P, wherein the positions are numbered according to SEQ ID NO: 1.

[0190] 4. A subtilisin variant according to any one of embodiments 1 to 3, wherein the variant is selected from the group consisting of X9T-X17H, X9T-X45R, X9T-X68S, X9T-X78I, X9T-X86E, X9T-X87A, X9T-X96D, X9T-X100E, X9T-X100N, X9T-X103F, X9T-X103I, X9T-X108Q, X9T-X115L, X9T-X117R, X9T-X127S, X9T-X127T, X9T-X128K, X9T-X128P, X9T-X128R, X9T-X129Q, X9T-X155E, X9T-X161Q , X9T-X181E, X9T-X181Q, X9T-X202V, X9T-X203E, X9T-X203N, X9T-X217S, X 9T-X260W, X9T-X221Q, X9T-X264H, X17H-X45R, X17H-X68S, X17H-X78I, X17H -X86E, X17H-X87A, X17H-X96D, X17H-X100E, X17H-X100N, X17H-X103F, X17 H-X103I, X17H-X108Q, X17H-X115L, X17H-X117R, X17H-X127S, X17H-X127T, X17H-X128K, X17H-X128P, X17H-X128R, X17H-X129Q, X17H-X155E, X17H-X1 61Q, X17H-X181E, X17H-X181Q, X17H-X202V, X17H-X203E, X17H-X203N, X17 H-X217S, X17H-X260W, X17H-X221Q, X17H-X264H, X45R-X68S, X45R-X78I, X 45R-X86E, X45R-X87A, X45R-X96D, X45R-X100E, X45R-X100N, X45R-X103F, X 45R-X103I, X45R-X108Q, X45R-X115L, X45R-X117R, X45R-X127S, X45R-X12 7T, X45R-X128K, X45R-X128P, X45R-X128R, X45R-X129Q, X45R-X155E, X45R- X161Q, X45R-X181E, X45R-X181Q, X45R-X202V, X45R-X203E, X45R-X203N, X 45R-X217S, X45R-X260W, X45R-X221Q, X45R-X264H, X68S-X78I, X68S-X86E,X68S-X87A、X68S-X96D、X68S-X100E、X68S-X100N、X68S-X103F、X68S-X103I、X68S-X108Q、X68S-X115L、X68S-X117R、X68S-X127S、X68S-X127T、X68S-X128K、X68S-X128P、X68S-X128R、X68S-X129Q、X68S-X155E、X68S-X161Q、X68S-X181E、X68S-X181Q、X68S-X202V、X68S-X203E、X68S-X203N、X68S-X217S、X68S-X260W、X68S-X221Q、X68S-X264H、X78I-X86E、X78I-X87A、X78I-X96D、X78I-X100E、X78I-X100N、X78I-X103F、X78I-X103I、X78I-X108Q、X78I-X115L、X78I-X117R、X78I-X127S、X78I-X127T、X78I-X128K、X78I-X128P、X78I-X128R、X78I-X129Q、X78I-X155E、X78I-X161Q、X78I-X181E、X78I-X181Q、X78I-X202V、X78I-X203E、X78I-X203N、X78I-X217S、X78I-X260W、X78I-X221Q、X78I-X264H、X86E-X87A、X86E-X96D、X86E-X100E、X86E-X100N、X86E-X103F、X86E-X103I、X86E-X108Q、X86E-X115L、X86E-X117R、X86E-X127S、X86E-X127T、X86E-X128K、X86E-X128P、X86E-X128R、X86E-X129Q、X86E-X155E、X86E-X161Q、X86E-X181E、X86E-X181Q、X86E-X202V、X86E-X203E、X86E-X203N、X86E-X217S、X86E-X260W、X86E-X221Q、X86E-X264H、X87A-X96D、X87A-X100E、X87A-X100N、X87A-X103F、X87A-X103I、X87A-X108Q、X87A-X115L、X87A-X117R、X87A-X127S、X87A-X127T、X87A-X128K、X87A-X128P、X87A-X128R、X87A-X129Q、X87A-X155E、X87A-X161Q、X87A-X181E、X87A-X181Q、X87A-X202V、X87A-X203E、X87A-X203N、X87A-X217S、X87A-X260W、X87A-X221Q、X87A-X264H、X96D-X100E、X96D-X100N、X96D-X103F、X96D-X103I、X96D-X108Q、X96D-X115L、X96D-X117R、X96D-X127S、X96D-X127T、X96D-X128K、X96D-X128P、X96D-X128R、X96D-X129Q、X96D-X155E、X96D-X161Q、X96D-X181E、X96D-X181Q、X96D-X202V、X96D-X203E、X96D-X203N、X96D-X217S、X96D-X260W、X96D-X221Q、X96D-X264H、X100E-X100N、X100E-X103F、X100E-X103I、X100E-X108Q、X100E-X115L、X100E-X117R、X100E-X127S、X100E-X127T、X100E-X128K、X100E-X128P、X100E-X128R、X100E-X129Q、X100E-X155E、X100E-X161Q、X100E-X181E、X100E-X181Q、X100E-X202V、X100E-X203E、X100E-X203N、X100E-X217S、X100E-X260W、X100E-X221Q、X100E-X264H、X100N-X103F、X100N-X103I、X100N-X108Q、X100N-X115L、X100N-X117R、X100N-X127S、X100N-X127T、X100N-X128K、X100N-X128P、X100N-X128R、X100N-X129Q、X100N-X155E、X100N-X161Q、X100N-X181E、X100N-X181Q、X100N-X202V、X100N-X203E、X100N-X203N、X100N-X217S、X100N-X260W、X100N-X221Q、X100N-X264H、X103F-X103I、X103F-X108Q、X103F-X115L、X103F-X117R、X103F-X127S、X103F-X127T、X103F-X128K、X103F-X128P、X103F-X128R、X103F-X129Q、X103F-X155E、X103F-X161Q、X103F-X181E、X103F-X181Q、X103F-X202V、X103F-X203E、X103F-X203N、X103F-X217S、X103F-X260W、X103F-X221Q、X103F-X264H、X103I-X108Q、X103I-X115L、X103I-X117R、X103I-X127S、X103I-X127T、X103I-X128K、X103I-X128P、X103I-X128R、X103I-X129Q、X103I-X155E、X103I-X161Q、X103I-X181E、X103I-X181Q、X103I-X202V、X103I-X203E、X103I-X203N、X103I-X217S、X103I-X260W、X103I-X221Q、X103I-X264H、X108Q-X115L、X108Q-X117R、X108Q-X127S、X108Q-X127T、X108Q-X128K、X108Q-X128P、X108Q-X128R、X108Q-X129Q、X108Q-X155E、X108Q-X161Q、X108Q-X181E、X108Q-X181Q、X108Q-X202V、X108Q-X203E、X108Q-X203N、X108Q-X217S、X108Q-X260W、X108Q-X221Q、X108Q-X264H、X115L-X117R、X115L-X127S、X115L-X127T、X115L-X128K、X115L-X128P、X115L-X128R、X115L-X129Q、X115L-X155E、X115L-X161Q、X115L-X181E、X115L-X181Q、X115L-X202V、X115L-X203E、X115L-X203N、X115L-X217S、X115L-X260W、X115L-X221Q、X115L-X264H、X117R-X127S、X117R-X127T、X117R-X128K、X117R-X128P、X117R-X128R、X117R-X129Q、X117R-X155E、X117R-X161Q、X117R-X181E、X117R-X181Q、X117R-X202V、X117R-X203E、X117R-X203N、X117R-X217S、X117R-X260W、X117R-X221Q、X117R-X264H、X127S-X127T、X127S-X128K、X127S-X128P、X127S-X128R、X127S-X129Q、X127S-X155E、X127S-X161Q、X127S-X181E、X127S-X181Q、X127S-X202V、X127S-X203E、X127S-X203N、X127S-X217S、X127S-X260W、X127S-X221Q、X127S-X264H、X127T-X128K、X127T-X128P、X127T-X128R、X127T-X129Q、X127T-X155E、X127T-X161Q、X127T-X181E、X127T-X181Q、X127T-X202V、X127T-X203E、X127T-X203N、X127T-X217S、X127T-X260W、X127T-X221Q、X127T-X264H、X128K-X128P、X128K-X128R、X128K-X129Q、X128K-X155E、X128K-X161Q、X128K-X181E、X128K-X181Q、X128K-X202V、X128K-X203E、X128K-X203N、X128K-X217S、X128K-X260W、X128K-X221Q、X128K-X264H、X128P-X128R、X128P-X129Q、X128P-X155E、X128P-X161Q、X128P-X181E、X128P-X181Q、X128P-X202V、X128P-X203E、X128P-X203N、X128P-X217S、X128P-X260W、X128P-X221Q、X128P-X264H、X128R-X129Q、X128R-X155E、X128R-X161Q、X128R-X181E、X128R-X181Q、X128R-X202V、X128R-X203E、X128R-X203N、X128R-X217S、X128R-X260W、X128R-X221Q、X128R-X264H、X129Q-X155E、X129Q-X161Q、X129Q-X181E、X129Q-X181Q、X129Q-X202V、X129Q-X203E, X129Q-X203N, X129Q-X217S, X129Q-X260W, X129Q-X221Q, X129Q-X264H, X155E-X161Q, X155E-X181E, X155E-X181Q, X155E-X202V, X155E-X203E, X155E-X203N, , X155E-X217S, X155E-X260W, X155E-X221Q, X155E-X264H, X161Q-X181E, X161Q-X181Q, 02V, X161Q-X203E, X161Q-X203N, X161Q-X217S, X161Q-X260W, X161Q-X221Q, X161Q-X264H, -X181Q, X181E-X202V, X181E-X203E, X181E-X203N, X181E-X217S, X181E-X260W, X181E-X221Q, 81E-X264H, X181Q-X202V, X181Q-X203E, X181Q-X203N, X181Q-X217S, X181Q-X260W, X181Q-X221Q , X181Q-X264H, X202V-X203E, X202V-X203N, X202V-X217S, X202V-X260W, X202V-X221Q, X202V-X2 64H, X203E-X203N, X203E-X217S, X203E-X260W, X203E-X221Q, X203E-X264H, X203N-X217S, -X260W, X203N-X221Q, X203N-X264H, X217S-X260W, X217S-X221Q, X217S-X264H, X260W-X221Q, X260W-X264H, and X221Q-X264H, wherein each position is numbered according to SEQ ID NO: 1.

[0191] 5. A subtilisin variant according to any one of embodiments 1 to 4, wherein the variants are Q017H-N096D-G127T, Q017H-N096D-Y103F-A202V, G127T-A128K-S129Q-N184Q, P009T-Y103F-A202V-G203E, N096D-S100E-G127T-A202V, V087A-S155E-G165Q, P009T-T078I-Y103F-G127T, V087A-A202V-G203E, V045R-T161Q-S181Q-G203E, S155E-M221Q, T0 78I-Y103F, N096D-S100E-G127T-N217S, G127T-A128K-S129Q, T115L-G127 T, P009T-T078I-N096D-A202V, V045R-S086E-S155E-A202V, V045R-T078I- Y103F-A202V, P009T-A202V-G203E-M221Q, A202V-G203E-M221Q-K264H, N0 96D-G127T, P009T-Q017H-Y103F-A202V, V045R-G127T-A128P-S129Q, S100E -G127T-A202V, A202V-M221Q, S100N-Y103I-G127T, G127T-A128K-S129Q-N 217S, G127S-A128K-S129Q-A202V, T078I-N096D, A128K-S155E, N096D-Y10 3F-G165Q, S086E-T077N-T078I, V045R-T078I-S086E-A202V, N096D-S100E -G127S, T078I-G127T-A128K-S129Q, G127S-A128K-S129Q, Q017H-S086E-A2 02V-G203E, T161Q-S181Q-A202V-G203E, V045R-N096D-S100E, V045R-Y103 F-G127T-A202V, N096D-S100E-G117R-G127T, V087A-G165Q-A202V, G127T-A 128P-S129Q, V087A-M221Q, T115L-A202V-G203E, V045R-T078I-N096D-A20 2V, G127T-A128R-S129Q-G165Q, S100E-Y103I, S086E-S155E-A202V-G203E,N096D-Y103I-G127T、Q017H-N096D-Y103F、V045R-T078I-N096D-Y103F、S108Q-G127T、T078I-S086E-A202V-G203E、T078I-Y103F-G127T-A202V、S181E-A202V-G203E-S258D、N096D-A202V-G203E、N096D-Y103I-G127T-M221Q、V087A-G165Q-A202V-G203E、P009T-G127T-A128K-S129Q、S100E-G127T、S100N-M221Q、G127T-A128K-S129Q-A202V、V087A-S100N、S100E-G117R-G127T、T077N-G127T-A128K-S129Q、Q017H-N096D-G127T-A202V、N096D-A202V-M221Q、V045R-N096D-Y103F-G127T、N096D-G127T-G203E、V045R-S086E-A202V-G203E、T078I-S086E-S155E-G203E、V045R-M221Q、Q017H-N096D-Y103F-G127T、Q017H-Y103F-G127T-A202V、T078I-A202V-M221Q、V045R-N096D-G127T、V045R-N096D-G127T-A202V、P009T-T078I-Y103F-A202V、S100N-T115L、S086E-A202V、N096D-Y103F-G127T、T078I-N096D-G127T、T115L-G165Q-A202V、T078I-S086E、V045R-T078I-N096D-G127T、P009T-N096D-G127T、G127T-A128R-S129Q-A202V、T078I-N096D-A202V、N096D-Y103I-G203E、G127S-A128P-S129Q-A202V、N096D-G127T-N217S、V045R-T078I、A024Q-T078I-A202V、A202V-G203N、P009T-T077N-T078I、G203E-K264H、T077N-T078I-G165Q-A202V、V045R-T077N-G165Q-A202V、G165Q-A202V-G203E-S258P、P009T-T078I-A202V, V045R-G127T, T077N-T078I-G165Q-G203E, V045R-T078I-A202V-G203E, G127T-G165Q-A202V-G203 N, A202V-K264H, P009T-A202V-G203E, T077N-A202V-G203E, A202V-G203E-N217S, Y103I-A202V, T078I-G203N, P009T-Q01 7H-T078I-G165Q, Q017H-T078I, S086D-A202V-G203E, A202V-G203E-F260W, G165Q-A202V-G203E-N217S, G127T-G165Q-A 202V-G203E, P009T-Q017H-T078I-G127T, V045R-S155E, P009T-Q017H-T078I-A202V, P009T-T078I, S086D-S155E-A202V- G203E, P009T-Q017H-A202V-G203E, A024Q-G165Q-A202V-G203E, P009T-Q017H-T077N-A202V, Q017H-A202V-G203E, P009 T-G127T-A202V-G203E, Y103I-G203E, T078I-S155E-A202V-G203E, N096D-S100E-Y103I, Q017H-T078I-G127T-A202V, P00 1. A subtilisin variant comprising a series of substitutions selected from the group consisting of 9T-N096D, A128R-S155E, G127T-A128R, T078I-A202V, P009T-A202V, Q017H-A202V, P009T-G203E, T078I-G165Q-A202V, T078I-A202V-G203E-N217S, and T077N-T078I-M221Q, wherein each position is numbered according to SEQ ID NO: 1.

[0192] 6. A subtilisin variant according to any one of embodiments 1 to 5, wherein the variant comprises an amino acid sequence having less than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1.

[0193] 7. A subtilisin variant according to any of embodiments 1 to 6, wherein the variant has one or more improved properties compared to a parent or reference subtilisin; wherein the improved properties are selected from improved cleaning performance in a detergent, improved stability in a detergent; improved cleaning performance over time, and combinations thereof.

[0194] 8. A subtilisin variant according to embodiment 7, wherein the improved properties are: (i) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning aged blood stains on cotton (CS-01) compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (ii) improved cleaning performance in a detergent, wherein the variant has a PI of ≥ 1.1 in cleaning an aged chocolate rice pudding stain (CS-100) on cotton fabric compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (iii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 for cleaning aged whole egg stain with carbon black (CS-39) on cotton fabric compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (iv) improved cleaning performance in a detergent, wherein the variant has a PI of ≥ 1.1 for cleaning an aged chocolate soy milk stain (CS-45) on cotton fabric compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (v) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 for cleaning grass stains (CS-07) on cotton fabric compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (vi) improved cleaning performance in a detergent, wherein the variant has a PI of ≥ 1.1 in cleaning chocolate milk stain with carbon black (C-03) on cotton fabric compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (vii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning blood / milk / ink stains on cotton (CS-05) compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (viii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 for cleaning milk with carbon black stains (C-11) on cotton fabric compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (ix) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning blood / milk / ink stains (EMPA116) on polycotton fabric compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (x) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning aged blood stains (KCS-01) on polyester / cotton compared to a subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (xi) improved stability, in which the variant has a higher residual activity compared to a subtilisin having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8, when measured according to the stability assay of Example 2; and / or (xii) A subtilisin variant that has improved cleaning performance over time, as measured as the difference in soil removal measured on time-course test samples (wherein the enzyme is pre-incubated in the detergent at an elevated temperature, such as 37°C, for an extended period, such as 3 to 8 weeks), compared to the cleaning performance over time of SEQ ID NO: 8.

[0195] 9. A subtilisin variant according to embodiment 7, wherein the improved properties are: (i) proteolytic activity in the DMC assay; and / or (ii) has improved stability in detergents, wherein the variant has a higher residual activity compared to the subtilisin having the amino acid sequence of SEQ ID NO: 1; and / or (iii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning blood / milk / ink stains (EMPA116) on polycotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 1; (iv) a net charge of −4 to +2 at pH 8; wherein said subtilisin variant has at least 75% sequence identity with SEQ ID NO:1.

[0196] 10. An enzyme composition comprising one or more subtilisin variants according to any one of embodiments 1 to 9.

[0197] 11. The enzyme composition of embodiment 10, comprising an acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-beta 1,4-glucanase, endo-beta-mannase, esterase, exo-mannase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannase, metalloprotease, nuclease (e.g., The enzyme composition further comprises one or more other enzymes selected from: an enzyme selected from: a DNase and / or an RNase), an oxidase, an oxidoreductase, a pectate lyase, a pectin acetylesterase, a pectinase, a pentosanase, a perhydrolase, a peroxidase, a phenoloxidase, a phosphatase, a phospholipase, a phosphodiesterase, a phytase, a polygalacturonase, a polyesterase, an additional protease, a pullulanase, a reductase, a rhamnogalacturonase, a beta-glucanase, a tannase, a transglutaminase, a xanthan lyase, a xylan acetylesterase, a xylanase, a xyloglucanase, a xylosidase, and any combination or mixture thereof.

[0198] 12. A polynucleotide comprising a nucleic acid sequence encoding a variant according to any one of embodiments 1 to 9, said polynucleotide optionally being isolated.

[0199] 13. The polynucleotide of embodiment 12, wherein the nucleic acid sequence is operably linked to a promoter.

[0200] 14. An expression vector or cassette comprising a polynucleotide according to embodiment 12.

[0201] 15. A recombinant host cell comprising the polynucleotide of embodiment 12.

[0202] 16. A cleaning composition comprising a subtilisin variant according to any one of embodiments 1 to 9 and at least one auxiliary agent.

[0203] 17. The cleaning composition of embodiment 16, wherein the cleaning composition is a composition selected from the group consisting of laundry detergents, fabric softener detergents, dishwashing detergents (e.g., automatic or hand dishwashing detergents), hard surface cleaning detergents, and medical equipment cleaning compositions.

[0204] 18. The cleaning composition according to any one of embodiments 16-17, wherein the cleaning composition does not contain a synthetic or peptidic protease stabilizer.

[0205] 19. A cleaning method comprising the step of contacting a surface or item in need of cleaning with an effective amount of a subtilisin variant according to any one of embodiments 1 to 9 or the enzyme composition according to any one of embodiments 10-11 or 16-17; and optionally further rinsing the surface or item after contacting the surface or item with the variant or the enzyme composition.

[0206] 20. The method of embodiment 19, wherein the article is tableware, fabric, or a medical device.

[0207] The following examples are provided to demonstrate and illustrate certain preferred embodiments and aspects of the present disclosure and should not be construed as limiting. [Example]

[0208] Example 1 Expression of AprL subtilisin mutants The Bacillus licheniformis subtilisin (AprL) is set forth in SEQ ID NO: 1. This AprL subtilisin wild-type sequence was used as the starting point for protein engineering. All AprL subtilisin variants were expressed using the following DNA fragments: The DNA fragment contains, in sequential 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 engineered variants are described in more detail in WO 2020112609), a nucleotide sequence encoding the aprE signal peptide sequence (SEQ ID NO: 3), the B. licheniformis AprL propeptide (SEQ ID NO: 4), a sequence corresponding to the gene encoding mature AprL subtilisin (SEQ ID NO: 5), a Bacillus amyloliquefaciens (BPN') terminator and its flanking sequence (SEQ ID NO: 6), and a sequence corresponding to the 3' AprE flanking sequence containing the kanamycin gene expression cassette (SEQ ID NO: 7). This DNA fragment was constructed using standard molecular biology techniques. Linear DNA of the expression cassette was used to transform competent B. subtilis cells of an appropriate strain.

[0209] The transformation mixture was plated on 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. For protein assays, transformed cells were grown in 96-well microtiter plates (MTPs) in culture medium (a semi-synthetic medium based on MOPS buffer, with urea as the main nitrogen source, glucose as the main carbon source, 1% soytone added to promote cell growth, and antibiotic selection) at 37°C, 300 rpm, and 80% humidity in a shaking incubator for 3 days. After centrifugation and filtration, the clarified culture supernatant containing the target protease was used for the assay.

[0210] Alternatively, mutant AprL proteases were also expressed in B. licheniformis strains suitable for protein expression by integrating an expression cassette of the protease gene into the serA locus of B. licheniformis. The AprL subtilisin variant comprises a 5' serA flanking sequence (SEQ ID NO: 9) containing a variant of the B. subtilis rrnIp2 promoter sequence (SEQ ID NO: 16) (see WO2020112609), operably linked to DNA encoding the B. subtilis aprE 5' UTR (SEQ ID NO: 10), operably linked to DNA encoding the AprL signal peptide (SEQ ID NO: 11), operably linked to DNA encoding the AprL propeptide (SEQ ID NO: 4), DNA encoding the mature AprL subtilisin or variant of interest, the B. licheniformis amyL terminator (SEQ ID NO: 12), and operably linked to a 3' serA flanking sequence (SEQ ID NO: 13). A second DNA expression cassette consisting of a 5' lysA flanking sequence (SEQ ID NO: 14) containing a variant of the B. subtilis rrnIp2 promoter sequence (SEQ ID NO: 17) operably linked to DNA encoding the B. subtilis aprE 5' UTR (SEQ ID NO: 10), operably linked to DNA encoding the UTR (SEQ ID NO: 11), the aprL signal peptide (SEQ ID NO: 11), operably linked to DNA encoding the AprL propeptide (SEQ ID NO: 4), operably linked to DNA encoding the mature AprL subtilisin or variant of interest, operably linked to an amyL terminator (SEQ ID NO: 12), and operably linked to a lysA 3' flanking region (SEQ ID NO: 15).

[0211] Recombinant B. licheniformis cells can be constructed in various ways, for example, by using pBl.comK in combination with a linear fragment (see WO 2019 / 40412). Briefly, B. licheniformis cells containing pBl.comK were made competent by methods well known in the art (see, for example, WO 2021 / 146411). One hundred microliters of competent cells were mixed with 10 μl of the linear DNA fragment. The mixture was incubated at 37°C and 1400 rpm for 1.5 hours. The mixture was plated on a selective medium (e.g., minimal medium without lysine or serine) to isolate strains transformed with the fragment of interest.

[0212] In some cases, mutant proteases were isolated from B. licheniformis cultures grown in flat-bottom shake flasks at 37°C in a humidified incubator for approximately 86 hours. For some strains, an MES-based medium containing soytone and essential salts and minerals was used, while for other strains, an MOPS-based medium containing soytone and essential salts and minerals was used. To harvest the protein for stability and performance testing, the culture broth was centrifuged at 4000 rpm for 30 minutes at 4°C. After filtration through 0.45 μM and 0.2 μM filters, the clear supernatant was obtained and frozen, then stored at -80°C for sample storage.

[0213] Example 2 Assay Protein measurement The concentrations of AprL subtilisin variants in the culture supernatants were measured by UHPLC using a Zorbax 300 SB-C3 column and a linear gradient of 0.1% trifluoroacetic acid (solution A) and 0.07% trifluoroacetic acid acetonitrile (solution B), with detection at 220 nm. The culture supernatants were diluted with 10 mM NaCl, 0.1 mM CaCl, 0.005% Tween®-80 for loading onto the column. The protein concentrations of the samples were calculated using a standard curve of the purified reference enzyme (SEQ ID NO: 8).

[0214] Protease activity The protease activity of the AprL subtilisin mutants was assessed by measuring the hydrolysis of the AAPF-pNA synthetic peptide substrate or dimethylcasein substrate.

[0215] For the AAPF assay, the reagent solution used was: 100 mM Tris pH 8.6, 0.005% Tween®-80 (Tris buffer), and 160 mM suc-AAPF-pNA (suc-AAPF-pNA stock solution) (Sigma: S-7388) in DMSO. To prepare a working dilution solution, 1 mL of suc-AAPF-pNA stock solution was added to 100 mL of Tris buffer and mixed. Enzyme samples were added to a microtiter plate (MTP) containing a 1 mg / mL suc-AAPF-pNA working solution, and activity was measured at 405 nm over 3–5 min using the kinetic mode of a SpectraMax plate reader at room temperature (RT). Protease activity was expressed in mOD / min.

[0216] Stability assays for liquid laundry detergents The liquid laundry detergents used in the stability assay were Persil Small & Mighty Non-Bio Liquid Detergent “Persil Non-Bio” (PNB, Unilever), China National Standard HDL (CNS, Table 1 ), and Test Detergent A (Table 2 ). [Table 1] [Table 2]

[0217] The stability of the subtilisin variants described herein was measured by diluting the variants in 10% or 20% (v / v) detergent solution and measuring the proteolytic activity of the variants before and after the thermal incubation step using the AAPF assay described above. Test samples were incubated at 45°C to 75°C for 20 minutes in a 384-well thermal cycler, and stability results were calculated as the percentage of remaining activity for each enzyme sample by comparing the ratio of mOD / min under stressed and unstressed conditions.

[0218] Example 3 Stability of AprL mutants in detergents The stability of the AprL mutants generated in this study was evaluated in several liquid laundry detergents using the method described in Example 2. Tables 3, 4, and 5 show the results for enzyme samples expressed in a B. subtilis host and obtained as clear supernatants from 96-well cultures using the method described in Example 1. For comparison, AprL-wild-type subtilisin (SEQ ID NO: 1) and mutant subtilisin CMT24 (SEQ ID NO: 8) were included. [Table 3] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0219] Tables 6, 7, and 8 show the results for enzyme samples produced by expression in a B. licheniformis host using the method described in Example 1, obtained from shake flask cultures in clarified supernatant, and evaluated for stability in detergents using the method described in Example 2. [Table 6-1] [Table 6-2] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 8]

[0220] This testing of AprL subtilisin mutants identified a number of amino acid substitutions that significantly improved stability in high temperature detergents, as can be seen from the data in Tables 3, 4, 5, 6, 7, and 8. The parental strain AprL-WT (wild type) and a previously studied AprL mutant, CMT24 (SEQ ID NO: 8), were used for comparison. The detergent and enzyme compositions evaluated lacked protease stabilizers.

[0221] Example 4 Evaluation of the temporal stability of AprL mutants in liquid detergents A time-course study of enzyme stability in HDL detergents was performed using Test Detergent A to compare the stability of PREFERENZ® P 100, the previously disclosed subtilisin variant CMT24 (SEQ ID NO: 8) (the variant disclosed as SQCBV419 in Patent Application WO 2017 / 210295), and the AprL variants described herein. The proteases were added at 0.07 wt.% CMT24-05693, 0.07 wt.% CMT24, and 0.04 wt.% PREFERENZ® P 100, based on the active protein in the detergent, and mixed at room temperature. Samples were then stored in a rotating incubator at 37°C. Aliquots were taken at the start of incubation (time zero) and at additional time points up to 8 weeks. Residual enzyme activity was measured using the AAPF substrate as described in Example 2, and results are reported as percent (%) remaining activity. Table 9 shows the results at 1, 4 and 8 weeks comparing the variant CMT24-05693 with the reference molecules CMT24 and PREFERENZ® P 100. The novel variant CMT24-05693 shows a significant improvement in stability compared to both reference enzymes.

Table 9

[0222] Additional AprL variants were evaluated against the reference enzyme CMT24, and the percent remaining activity after incubation for a period of time was determined using the method described above. Results for samples tested in Persil Non Bio (PNB) detergent are shown in Tables 10 and 11. Culture supernatants of cells expressing various variants were used in this study. The variants shown in Table 10 were expressed in a B. subtilis host, and the variants shown in Table 11 were expressed in a B. licheniformis host. For the data in Table 10, the protease dosage in the detergent ranged from 0.02% to 0.08%. Mutant CMT24-07674 consists of the following substitutions: P009T-Q017H-T077N-T078I-N096D-G127T-A128K-S129Q-G165Q-N184Q-A202V-G203E-S258P. Mutant CMT24-07675 consists of the following substitutions: P009T-Q017H-T077N-T078I-N096D-G127T-A128K-S129Q-G165Q-N184Q-A202V-G203E-N217S. Mutant CMT24-02667 is composed of the following substitutions: P009T-Q017H-V045R-T077N-T078I-N096D-S108Q-G127T-A128P-G165Q-N184Q-A202V-G203E-N217S-S258P. Mutant CMT24-02843 is composed of the following substitutions: Q017H-T077N-T078I-N096D-S108Q-G117R-G127T-A128P-G165Q-N184Q-A202V-G203E-N217S-S258P. Mutant CMT24-02609 is composed of the following substitutions: P009T-Q017H -V045R-T077N-T078I-N096D-G127T-A128P-G165Q-N184Q-A202V-G203E-N217S-S258P, and mutant CMT-05698 consists of the substitutions P009T-Q017H-V045R-A068S-T077N-T078I-N096D-S108Q-T115L-G127T-A128K-S129Q-G165Q-N184Q-A202V-G203E-S258P-K264H, in both cases relative to the AprL wild type (SEQ ID NO: 1).As shown in Tables 10 and 11, all new AprL variants show significantly higher stability in HDL detergents compared to the reference enzyme CMT24. [Table 10]

[0223] Table 11 below shows cases where the incubation periods (hereafter indicated in days) for some tests were similar but not identical, and an asterisk (*) is used to indicate the number of days for the test in each column. [Table 11]

[0224] Example 5 Laundry cleaning performance of various AprL mutants over time The cleaning performance of subtilisin variants was tested using a process of aging the detergent before measuring cleaning activity. The protocol for measuring laundry cleaning after aging is as follows:

[0225] Samples were prepared by adding the AprL subtilisin variant to the detergent solution at 1.9 ppm active protein. Additionally, 100% Persil Non Bio detergent was supplemented with 0.11 ppm amylase PREFERENZ S210 and 0.11 ppm PspMan138 mutant mannanase TL1219 (U.S. Provisional Application No. 63 / 403332, filed September 2, 2022). The enzyme-enriched detergent was added at a rate of 2.7 g per liter of detergent solution and agitated end-over-end for at least 1 hour. The samples were then stored in a 37°C incubator and aged for 4 weeks. After aging, the samples were evaluated for cleaning performance in a Miele commercial washing machine (Model PW6065 Plus). The wash cycle lasted 55 minutes, including 25 minutes of the main wash at 30°C. The wash included water containing 250 ppm calcium:magnesium with a hardness of 3:1, and approximately 3 kg of total clean ballast, technical soil monitor, and synthetic stain ballast. After the wash process, the technical stains were dried using a gentle cycle. Stain cleaning efficacy was evaluated photometrically on the dried stains using a MACH-5. L*a*b* measurements of each stain taken before and after washing were used to determine the stain removal index (%SRI) for each aged sample. %SRI was calculated using the following formula, where w is the white standard, i is the stain before washing, and f is the stain after washing:

number

[0226] The stains used in this evaluation were: C-03 Chocolate Milk / Soot, C-05 Blood / Milk / Ink, CS-07 Grass, CS-39 Whole Egg / Pigment / Heat Aging. Technical stains were purchased from the Center for Testmaterials BV, Vlaardingen, The Netherlands.

[0227] Table 12 shows the %SRI (stain removal rate) of aged samples of various AprL variants compared to the reference molecule CMT24 in the test stain set. [Table 12]

[0228] As shown in Table 12, all AprL variants showed improved cleaning efficacy over time on multiple protease-sensitive stains compared to the reference protein CMT24 (SEQ ID NO: 8).

[0229] Example 6 Full-scale cleaning performance of AprL mutants Wash performance was measured on enzyme-reactive stains commercially available from the Center for Test Materials (CFT BV, The Netherlands). Tests were performed using a Miele W1935 WPS Ecoline washing machine on the "Cotton, Short" wash cycle at 20°C and 30°C.

[0230] The wash process lasted 109 minutes, of which 50 minutes were the main wash. The load consisted of 3 kg of cotton ballast, synthetic earthen ballast (4xSBL2004), earthen ballast, and technical soil. The water was maintained at 14°GH (using 3Ca:1Mg). The Stain Removal Index (SRI)% was calculated as shown in Example 5. The technical soils used in this study were: CS-01 (blood aged on cotton), CS-100 (chocolate rice pudding aged on cotton), CS-39 (whole egg and carbon black aged on cotton), CS-45 (soy chocolate drink aged on cotton), and CS-07 (grass on cotton). The technical soils were purchased from the Center for Test Materials BV, Vlaardingen, The Netherlands.

[0231] In the study shown in Table 13, samples were prepared by adding protease to 100% Persil Non Bio (PNB) detergent at an active protein level equivalent to 0.95 ppm in a wash solution that also contained 0.07 ppm amylase PREFERENZ S210 and 0.01 ppm mannanase PREFERENZ M 100. The detergent with the enzyme was dosed at a rate of 2.8 g per liter of wash solution and stirred for at least 1 hour.

[0232] Table 13 shows the results of testing the AprL variant CMT24-05693 and the CMT24 reference protease, as described above. The standard deviation (StdDev) of the results, averaged over four replicates (two soils x two machines), is shown at the bottom of the table. The variant CMT24-05693 demonstrates significant advantages across soils compared to the related commercial enzyme, CMT24. To directly compare the new variant CMT24-05693 with the reference molecule CMT24, a performance index (PI) value was calculated by subtracting the zero protease %SRI response from the enzyme addition results and calculating the ratio of the delta %SRI for CMT24-05693 and the reference molecule CMT24. Results are shown in the column labeled PI. A PI value of 1.0 indicates equivalent performance, while a PI value of 1.1 or greater indicates significant cleaning benefit. [Table 13]

[0233] Additional wash performance tests were conducted at 30°C under the same wash conditions as described above and reported in Table 14. The technical stains used in this test were: C-03 (chocolate milk with carbon black on cotton), C-05 (blood / milk / ink on cotton), C-11 (milk and carbon black on cotton), CS-01 (blood aged on cotton), CS-07 (grass on cotton), CS-100 (chocolate rice pudding aged on cotton), CS-39 (whole egg and carbon black aged on cotton), EMPA 116 (blood / milk / ink on polycotton), and KCS-01 (blood aged on polyester / cotton). Samples were prepared by adding protease to 100% Persil Non Bio detergent at 0.07 ppm amylase PREFERENZ S210 and 0.01 ppm mannanase PREFERENZ M 100, equivalent to 2.85 ppm active protein. The enzyme-enriched detergent was added at a rate of 2.8 g per liter of detergent solution and stirred for at least 1 hour.

[0234] Table 14 shows the results of testing several AprL variants and CMT24 protease on a variety of technical stains at 30°C in the presence of amylase and mannanase. The standard deviation for each data set is shown at the bottom of the table. The cumulative effect across stains was calculated and displayed as a total. PI values ​​were also displayed for each evaluation. The results show an improved benefit for all AprL variants compared to CMT24 protease. [Table 14]

[0235] Example 7 Cleaning performance and stability in detergents of additional AprL variants Additional AprL variants shown in Table 15 were generated by expression in a B. subtilis host as described in Example 1, and clarified culture supernatants were used for microtiter-scale testing. Detergent stability was tested at 10% Test Detergent A (45°C) and 20% CNS (48°C) using the methods described in Example 2. Cleaning performance was tested as described below. Protease variants were tested for cleaning performance against the reference AprL wild-type parent strain (SEQ ID NO: 1).

[0236] Washing Performance Assay For the cleaning performance assay, test detergent A was diluted to 6.0 g / L in 5 mM HEPES (pH 8.2) with a hardness of 6 gpg. Technical EMPA-116 (blood / milk / ink on woven cotton fabric) was purchased from the Center for Test Materials BV, Vlaardingen, The Netherlands. This swatch was punched into small circular swatches and distributed into microtiter plates (MTPs). The MTPs containing the microswatches were first filled with the detergent solution. The parent enzyme and variants were then 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, 50 microliters of the supernatant was transferred to a new MTP, and the absorbance was read at 600 nm using a SpectraMax plate reader. The absorbance results were obtained by subtracting the blank control (no enzyme) value from each sample value. For each condition and subtilisin variant, 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 concentrations of the variant were determined using a standard curve of the parent protease. This standard curve was included in the study and constructed using a Langmuir fit or a Hill sigmoidal fit, as appropriate.

[0237] The proteolytic activity of AprL subtilisin mutants was also tested by measuring the hydrolysis of dimethylcasein (DMC) substrate. For the DMC assay, the reagent solution used was: 2.5% dimethylcasein (DMC, Sigma C-9801) dissolved in carbonate buffer (100 mM sodium carbonate pH 9.2 + 100 mM sodium chloride) and 0.075% TNBSA (2,4,6-trinitrobenzenesulfonic acid, Thermo Scientific) diluted in carbonate buffer. 27 μL of DMC substrate was placed in a Greiner PS-microwell 384 MTP, followed by 27 μL of TNBSA. The reaction was initiated by adding 6 μL of the appropriately diluted protease culture supernatant and mixing. After a 3-minute incubation at room temperature, proteolytic activity was measured at 405 nm over a 3-minute period using a SpectraMax plate reader in kinetic mode at room temperature. The relative proteolytic activity of the proteases was captured as mOD*min-1.

[0238] The net charge of each mutant was calculated using the reported amino acid pKa values ​​set to 0 for parent AprL at pH 8 by Hass and Mulder (Hass, MAS and Mulder, FAA (2015) Annu. Rev. Biophys. 44:53-75).

[0239] Table 15 shows the proteolytic activity, cleaning performance, detergent stability, and calculated net charge at pH 8 of the AprL variants compared to the AprL reference parent. [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6] [Table 15-7]

[0240] 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.

[0241] All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present disclosure. To the extent headings are used, they should not be construed as necessarily limiting.

Claims

1. Subtilisin mutants, including X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X117R, X127S, X127T, X128K, X128P, X128R, X129Q, X155E, X161Q, X181E, X181Q, X202V, X 1. A subtilisin variant comprising at least two, three or more amino acid substitutions selected from the group consisting of X203E, X203N, X217S, X221Q, X260W, and X264H, wherein said positions are numbered according to SEQ ID NO: 1, and wherein said variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

8.

2. 2. The subtilisin variant of claim 1, wherein the variant a) has at least 25% improved stability in detergent compared to the parent subtilisin SEQ ID NO: 1; and / or b) has a net charge of -4 to +2 at pH 8 compared to the subtilisin having the amino acid sequence of SEQ ID NO:

1.

3. 2. The subtilisin variant of claim 1, wherein the variant further comprises one or more additional mutations selected from the group consisting of X24Q, X77N, X86D, X165Q, X184Q, X258D, and X258P, wherein the positions are numbered according to SEQ ID NO:

1.

4. 2. The subtilisin variants according to claim 1, wherein the variants are selected from the group consisting of X9T-X17H, X9T-X45R, X9T-X68S, X9T-X78I, X9T-X86E, X9T-X87A, X9T-X96D, X9T-X100E, X9T-X100N, X9T-X103F, X9T-X103I, X9T-X108Q, X9T-X115L, X9T-X117R, X9T-X127S, X9T-X127T, X9T-X128K, X9T-X128P, X9T-X128R, X9T-X129Q, X9T-X155E, X9T-X161Q, X9T-X181 E, X9T-X181Q, X9T-X202V, X9T-X203E, X9T-X203N, X9T-X217S, X9T-X260W, X9T-X221Q, X9T-X264H, X17H-X45R, X17H-X68S, X17H-X78I, X17H-X86E, X17 H-X87A, X17H-X96D, X17H-X100E, X17H-X100N, X17H-X103F, X17H-X103I, X 17H-X108Q, X17H-X115L, X17H-X117R, X17H-X127S, X17H-X127T, X17H-X128 K, X17H-X128P, X17H-X128R, X17H-X129Q, X17H-X155E, X17H-X161Q, X17H- X181E, X17H-X181Q, X17H-X202V, X17H-X203E, X17H-X203N, X17H-X217S, X1 7H-X260W, X17H-X221Q, X17H-X264H, X45R-X68S, X45R-X78I, X45R-X86E, X 45R-X87A, X45R-X96D, X45R-X100E, X45R-X100N, X45R-X103F, X45R-X103I, X45R-X108Q, X45R-X115L, X45R-X117R, X45R-X127S, X45R-X127T, X45R-X1 28K, X45R-X128P, X45R-X128R, X45R-X129Q, X45R-X155E, X45R-X161Q, X45R -X181E, X45R-X181Q, X45R-X202V, X45R-X203E, X45R-X203N, X45R-X217S, X45R-X260W, X45R-X221Q, X45R-X264H, X68S-X78I, X68S-X86E, X68S-X87A,X68S-X96D、X68S-X100E、X68S-X100N、X68S-X103F、X68S-X103I、X68S-X108Q、X68S-X115L、X68S-X117R、X68S-X127S、X68S-X127T、X68S-X128K、X68S-X128P、X68S-X128R、X68S-X129Q、X68S-X155E、X68S-X161Q、X68S-X181E、X68S-X181Q、X68S-X202V、X68S-X203E、X68S-X203N、X68S-X217S、X68S-X260W、X68S-X221Q、X68S-X264H、X78I-X86E、X78I-X87A、X78I-X96D、X78I-X100E、X78I-X100N、X78I-X103F、X78I-X103I、X78I-X108Q、X78I-X115L、X78I-X117R、X78I-X127S、X78I-X127T、X78I-X128K、X78I-X128P、X78I-X128R、X78I-X129Q、X78I-X155E、X78I-X161Q、X78I-X181E、X78I-X181Q、X78I-X202V、X78I-X203E、X78I-X203N、X78I-X217S、X78I-X260W、X78I-X221Q、X78I-X264H、X86E-X87A、X86E-X96D、X86E-X100E、X86E-X100N、X86E-X103F、X86E-X103I、X86E-X108Q、X86E-X115L、X86E-X117R、X86E-X127S、X86E-X127T、X86E-X128K、X86E-X128P、X86E-X128R、X86E-X129Q、X86E-X155E、X86E-X161Q、X86E-X181E、X86E-X181Q、X86E-X202V、X86E-X203E、X86E-X203N、X86E-X217S、X86E-X260W、X86E-X221Q、X86E-X264H、X87A-X96D、X87A-X100E、X87A-X100N、X87A-X103F、X87A-X103I、X87A-X108Q、X87A-X115L、X87A-X117R、X87A-X127S、X87A-X127T、X87A-X128K、X87A-X128P、X87A-X128R、X87A-X129Q、X87A-X155E、X87A-X161Q、X87A-X181E、X87A-X181Q、X87A-X202V、X87A-X203E、X87A-X203N、X87A-X217S、X87A-X260W、X87A-X221Q、X87A-X264H、X96D-X100E、X96D-X100N、X96D-X103F、X96D-X103I、X96D-X108Q、X96D-X115L、X96D-X117R、X96D-X127S、X96D-X127T、X96D-X128K、X96D-X128P、X96D-X128R、X96D-X129Q、X96D-X155E、X96D-X161Q、X96D-X181E、X96D-X181Q、X96D-X202V、X96D-X203E、X96D-X203N、X96D-X217S、X96D-X260W、X96D-X221Q、X96D-X264H、X100E-X100N、X100E-X103F、X100E-X103I、X100E-X108Q、X100E-X115L、X100E-X117R、X100E-X127S、X100E-X127T、X100E-X128K、X100E-X128P、X100E-X128R、X100E-X129Q、X100E-X155E、X100E-X161Q、X100E-X181E、X100E-X181Q、X100E-X202V、X100E-X203E、X100E-X203N、X100E-X217S、X100E-X260W、X100E-X221Q、X100E-X264H、X100N-X103F、X100N-X103I、X100N-X108Q、X100N-X115L、X100N-X117R、X100N-X127S、X100N-X127T、X100N-X128K、X100N-X128P、X100N-X128R、X100N-X129Q、X100N-X155E、X100N-X161Q、X100N-X181E、X100N-X181Q、X100N-X202V、X100N-X203E、X100N-X203N、X100N-X217S、X100N-X260W、X100N-X221Q、X100N-X264H、X103F-X103I、X103F-X108Q、X103F-X115L、X103F-X117R、X103F-X127S、X103F-X127T、X103F-X128K、X103F-X128P、X103F-X128R、X103F-X129Q、X103F-X155E、X103F-X161Q、X103F-X181E、X103F-X181Q、X103F-X202V、X103F-X203E、X103F-X203N、X103F-X217S、X103F-X260W、X103F-X221Q、X103F-X264H、X103I-X108Q、X103I-X115L、X103I-X117R、X103I-X127S、X103I-X127T、X103I-X128K、X103I-X128P、X103I-X128R、X103I-X129Q、X103I-X155E、X103I-X161Q、X103I-X181E、X103I-X181Q、X103I-X202V、X103I-X203E、X103I-X203N、X103I-X217S、X103I-X260W、X103I-X221Q、X103I-X264H、X108Q-X115L、X108Q-X117R、X108Q-X127S、X108Q-X127T、X108Q-X128K、X108Q-X128P、X108Q-X128R、X108Q-X129Q、X108Q-X155E、X108Q-X161Q、X108Q-X181E、X108Q-X181Q、X108Q-X202V、X108Q-X203E、X108Q-X203N、X108Q-X217S、X108Q-X260W、X108Q-X221Q、X108Q-X264H、X115L-X117R、X115L-X127S、X115L-X127T、X115L-X128K、X115L-X128P、X115L-X128R、X115L-X129Q、X115L-X155E、X115L-X161Q、X115L-X181E、X115L-X181Q、X115L-X202V、X115L-X203E、X115L-X203N、X115L-X217S、X115L-X260W、X115L-X221Q、X115L-X264H、X117R-X127S、X117R-X127T、X117R-X128K、X117R-X128P、X117R-X128R、X117R-X129Q、X117R-X155E、X117R-X161Q、X117R-X181E、X117R-X181Q、X117R-X202V、X117R-X203E、X117R-X203N、X117R-X217S、X117R-X260W、X117R-X221Q、X117R-X264H、X127S-X127T、X127S-X128K、X127S-X128P、X127S-X128R、X127S-X129Q、X127S-X155E、X127S-X161Q、X127S-X181E、X127S-X181Q、X127S-X202V、X127S-X203E、X127S-X203N、X127S-X217S、X127S-X260W、X127S-X221Q、X127S-X264H、X127T-X128K、X127T-X128P、X127T-X128R、X127T-X129Q、X127T-X155E、X127T-X161Q、X127T-X181E、X127T-X181Q、X127T-X202V、X127T-X203E、X127T-X203N、X127T-X217S、X127T-X260W、X127T-X221Q、X127T-X264H、X128K-X128P、X128K-X128R、X128K-X129Q、X128K-X155E、X128K-X161Q、X128K-X181E、X128K-X181Q、X128K-X202V、X128K-X203E、X128K-X203N、X128K-X217S、X128K-X260W、X128K-X221Q、X128K-X264H、X128P-X128R、X128P-X129Q、X128P-X155E、X128P-X161Q、X128P-X181E、X128P-X181Q、X128P-X202V、X128P-X203E、X128P-X203N、X128P-X217S、X128P-X260W、X128P-X221Q、X128P-X264H、X128R-X129Q、X128R-X155E、X128R-X161Q、X128R-X181E、X128R-X181Q、X128R-X202V、X128R-X203E、X128R-X203N、X128R-X217S、X128R-X260W、X128R-X221Q、X128R-X264H、X129Q-X155E、X129Q-X161Q、X129Q-X181E、X129Q-X181Q、X129Q-X202V、X129Q-X203E、X129Q-X203N、X129Q-X217S、X129Q-X260W、X129Q-X221Q、X129Q-X264H、X155E-X161Q、X155E-X181E、X155E-X181Q、X155E-X202V、X155E-X203E、X155E-X203N、X155E-、 X217S, X155E-X260W, X155E-X221Q, X155E-X264H, X161Q-X181E, X161Q-X181Q, X161Q-X202V, 61Q-X203E, X161Q-X203N, X161Q-X217S, X161Q-X260W, X161Q-X221Q, X161Q-X264H, X181E-X181 Q, X181E-X202V, X181E-X203E, X181E-X203N, X181E-X217S, X181E-X260W, X181E-X221Q, X264H, X181Q-X202V, X181Q-X203E, X181Q-X203N, X181Q-X217S, X181Q-X260W, X181Q-X221Q, 1Q-X264H, X202V-X203E, X202V-X203N, X202V-X217S, X202V-X260W, X202V-X221Q, X202V-X264H , X203E-X203N, X203E-X217S, X203E-X260W, X203E-X221Q, X203E-X264H, X203N-X217S, 60W, X203N-X221Q, X203N-X264H, X217S-X260W, X217S-X221Q, X217S-X264H, X260W-X221Q, X260W-X264H, and X221Q-X264H, wherein said positions are numbered according to SEQ ID NO:

1.

5. 2. The subtilisin variants according to claim 1, wherein the variants are selected from the group consisting of Q017H-N096D-G127T, Q017H-N096D-Y103F-A202V, G127T-A128K-S129Q-N184Q, P009T-Y103F-A202V-G203E, N096D-S100E-G127T-A202V, V087A-S155E-G165Q, P009T-T078I-Y103F-G127T, V087A-A202V-G203E, V045R-T161Q-S181Q-G203E, S155E-M221Q, T078I-Y1 03F, N096D-S100E-G127T-N217S, G127T-A128K-S129Q, T115L-G127T, P00 9T-T078I-N096D-A202V, V045R-S086E-S155E-A202V, V045R-T078I-Y103F -A202V, P009T-A202V-G203E-M221Q, A202V-G203E-M221Q-K264H, N096D-G 127T, P009T-Q017H-Y103F-A202V, V045R-G127T-A128P-S129Q, S100E-G12 7T-A202V, A202V-M221Q, S100N-Y103I-G127T, G127T-A128K-S129Q-N217 S, G127S-A128K-S129Q-A202V, T078I-N096D, A128K-S155E, N096D-Y103F- G165Q, S086E-T077N-T078I, V045R-T078I-S086E-A202V, N096D-S100E-G1 27S, T078I-G127T-A128K-S129Q, G127S-A128K-S129Q, Q017H-S086E-A202 V-G203E, T161Q-S181Q-A202V-G203E, V045R-N096D-S100E, V045R-Y103F- G127T-A202V, N096D-S100E-G117R-G127T, V087A-G165Q-A202V, G127T-A1 28P-S129Q, V087A-M221Q, T115L-A202V-G203E, V045R-T078I-N096D-A202 V, G127T-A128R-S129Q-G165Q, S100E-Y103I, S086E-S155E-A202V-G203E,N096D-Y103I-G127T、Q017H-N096D-Y103F、V045R-T078I-N096D-Y103F、S108Q-G127T、T078I-S086E-A202V-G203E、T078I-Y103F-G127T-A202V、S181E-A202V-G203E-S258D、N096D-A202V-G203E、N096D-Y103I-G127T-M221Q、V087A-G165Q-A202V-G203E、P009T-G127T-A128K-S129Q、S100E-G127T、S100N-M221Q、G127T-A128K-S129Q-A202V、V087A-S100N、S100E-G117R-G127T、T077N-G127T-A128K-S129Q、Q017H-N096D-G127T-A202V、N096D-A202V-M221Q、V045R-N096D-Y103F-G127T、N096D-G127T-G203E、V045R-S086E-A202V-G203E、T078I-S086E-S155E-G203E、V045R-M221Q、Q017H-N096D-Y103F-G127T、Q017H-Y103F-G127T-A202V、T078I-A202V-M221Q、V045R-N096D-G127T、V045R-N096D-G127T-A202V、P009T-T078I-Y103F-A202V、S100N-T115L、S086E-A202V、N096D-Y103F-G127T、T078I-N096D-G127T、T115L-G165Q-A202V、T078I-S086E、V045R-T078I-N096D-G127T、P009T-N096D-G127T、G127T-A128R-S129Q-A202V、T078I-N096D-A202V、N096D-Y103I-G203E、G127S-A128P-S129Q-A202V、N096D-G127T-N217S、V045R-T078I、A024Q-T078I-A202V、A202V-G203N、P009T-T077N-T078I、G203E-K264H、T077N-T078I-G165Q-A202V、V045R-T077N-G165Q-A202V、G165Q-A202V-G203E-S258P、P009T-T078I-A202V, V045R-G127T, T077N-T078I-G165Q-G203E, V045R-T078I-A202V-G203E, G127T-G165Q-A202V-G203 N, A202V-K264H, P009T-A202V-G203E, T077N-A202V-G203E, A202V-G203E-N217S, Y103I-A202V, T078I-G203N, P009T-Q01 7H-T078I-G165Q, Q017H-T078I, S086D-A202V-G203E, A202V-G203E-F260W, G165Q-A202V-G203E-N217S, G127T-G165Q-A 202V-G203E, P009T-Q017H-T078I-G127T, V045R-S155E, P009T-Q017H-T078I-A202V, P009T-T078I, S086D-S155E-A202V- G203E, P009T-Q017H-A202V-G203E, A024Q-G165Q-A202V-G203E, P009T-Q017H-T077N-A202V, Q017H-A202V-G203E, P009 T-G127T-A202V-G203E, Y103I-G203E, T078I-S155E-A202V-G203E, N096D-S100E-Y103I, Q017H-T078I-G127T-A202V, P00 9T-N096D, A128R-S155E, G127T-A128R, T078I-A202V, P009T-A202V, Q017H-A202V, P009T-G203E, T078I-G165Q-A202V, T078I-A202V-G203E-N217S, and T077N-T078I-M221Q, wherein said positions are numbered according to SEQ ID NO:

1.

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

1.

7. 2. The subtilisin variant of claim 1, wherein the variant has one or more improved properties compared to a parent or reference subtilisin; wherein the improved properties are selected from improved cleaning performance in a detergent, improved stability in a detergent; improved cleaning performance over time, and combinations thereof.

8. 8. A subtilisin variant according to claim 7, wherein the improved properties are (i) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning aged blood stains on cotton (CS-01) compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (ii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning aged chocolate rice pudding stain (CS-100) on cotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (iii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning aged whole egg stain with carbon black (C-S-39) on cotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (iv) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning aged chocolate soy milk stain (C-S-45) on cotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (v) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning grass stains (CS-07) on cotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (vi) improved cleaning performance in a detergent, wherein the variant has a PI of ≥ 1.1 in cleaning chocolate milk with carbon black stain (C-03) on cotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (vii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning blood / milk / ink stains on woven cotton fabric (CS-05) compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (viii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning milk with carbon black stains (C-11) on cotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (ix) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning blood / milk / ink stains (EMPA116) on polycotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (x) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning aged blood stains (KCS-01) on polyester / cotton compared to the subtilisin having the amino acid sequence of SEQ ID NO: 8; and / or (xi) improved stability, in which the variant has a higher residual activity compared to the subtilisin having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8, when measured according to the stability assay of Example 2; and / or (xii) A subtilisin variant that has improved cleaning performance over time, as measured as the difference in soil removal measured on aged test samples (wherein the enzyme is pre-incubated in the detergent for an extended period of time, such as 3-8 weeks, at an elevated temperature, such as 37°C), compared to the cleaning performance of SEQ ID NO:

8.

9. 8. A subtilisin variant according to claim 7, wherein the improved properties are (i) proteolytic activity in the DMC assay; and / or (ii) improved stability in detergents, wherein the variant has a higher residual activity compared to the subtilisin having the amino acid sequence of SEQ ID NO: 1; and / or (iii) improved cleaning performance in detergents, wherein the variant has a PI of ≥ 1.1 in cleaning blood / milk / ink stains (EMPA116) on polycotton fabric compared to the subtilisin having the amino acid sequence of SEQ ID NO: 1; (iv) a net charge of −4 to +2 at pH 8; wherein said subtilisin variant has at least 75% sequence identity with SEQ ID NO:

1.

10. 10. An enzyme composition comprising one or more subtilisin variants according to claim 1.

11. 11. The enzyme composition of claim 10, comprising an enzyme selected from the group consisting of acyltransferase, alpha amylase, beta amylase, alpha galactosidase, arabinosidase, arylesterase, beta galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo beta 1,4-glucanase, endo beta mannase, esterase, exomannase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannase, metalloprotease, nuclease (e.g., The enzyme composition further comprises one or more other enzymes selected from: an enzyme selected from the group consisting of DNase and / or RNase, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polygalacturonase, polyesterase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof.

12. 10. A polynucleotide comprising a nucleic acid sequence encoding the variant of claim 1, said polynucleotide optionally being isolated.

13. 13. The polynucleotide of claim 12, wherein the nucleic acid sequence is operably linked to a promoter.

14. An expression vector or cassette comprising the polynucleotide of claim 12.

15. A recombinant host cell comprising the polynucleotide of claim 12.

16. A cleaning composition comprising the subtilisin variant of claim 1 and at least one adjuvant.

17. 17. The cleaning composition of claim 16, wherein the cleaning composition is a composition selected from the group consisting of a laundry detergent, a fabric softener detergent, a dishwashing detergent (e.g., an automatic or hand dishwashing detergent), a hard surface cleaning detergent, and a medical implement cleaning composition.

18. 20. The cleaning composition of claim 17, wherein the cleaning composition does not include a synthetic or peptidic protease stabilizer.

19. 11. A cleaning method comprising the further step of contacting a surface or item in need of cleaning with an effective amount of a subtilisin variant according to claim 1 or an enzyme composition according to claim 10; and optionally rinsing the surface or item after contacting the surface or item with the variant or enzyme composition.

20. 20. The method of claim 19, wherein the article is tableware, fabric, or medical equipment.

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