Subtilisin variants and methods of use

Abstract

Disclosed herein are one or more subtilisin variants, nucleic acids encoding the same, and compositions and methods relating to the production and use thereof, including one or more subtilisin variants having improved stability and / or soil removal compared to one or more reference subtilisin.

Description

[0001] This application claims priority to U.S. Provisional Application 62 / 852,337, filed May 24, 2019, and U.S. Provisional Application 62 / 925,265, filed October 24, 2019, the entire contents of which are hereby incorporated by reference.

[0002] References to sequence lists submitted electronically

[0003] An official copy of the sequence list was submitted electronically via EFS-Web as an ASCII format sequence list, with the filename 20200520_NB41644PCT_SeqLst, created on [date missing]. May 20, 2020 and has 11 The file is in kilobytes in size and is submitted with this specification. The sequence list contained in the file in the ASCII format is part of this specification and is incorporated herein by reference in its entirety.

[0004] This document discloses one or more subtilisin protease variants, nucleic acids encoding them, and compositions and methods relating to their production and use, said one or more subtilisin protease variants including one or more subtilisin protease variants having improved stability and / or dirt removal properties compared to one or more reference subtilisin proteases. Background Technology

[0005] Proteases (also known as prions) are enzymes that have the ability to break down other proteins. Proteases have the ability to initiate protein catabolism 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 called proteolytic activity. Many well-known procedures exist for measuring proteolytic activity (Kalisz, "Microbial Proteinases," in: Fiechter (edited)). Advances in Biochemical Engineering / Biotechnology [Advances in Biochemical Engineering / Biotechnology], (1988). For example, proteolytic activity can be determined by a comparative assay of the ability of various proteases to hydrolyze commercial substrates. Exemplary substrates that can be used to analyze protease or proteolytic activity include, but are not limited to, dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111). Colorimetric assays using these substrates are well known in the art (see, for example, WO 99 / 34011 and U.S. Patent No. 6,376,450, both of which are incorporated herein by reference).

[0006] Serine proteases are enzymes containing serine residues as active sites to initiate the hydrolysis of protein peptide bonds (EC 3.4.21). Serine proteases comprise a wide variety of enzymes with broad specificity and biological functions, and based on their structure, they are further classified into chymotrypsin-like (trypsin-like) and subtilisin-like enzymes. The prototypical subtilisin (EC 3.4.21.62) was originally obtained from Bacillus subtilis. Subtilisin and its homologs are members of the S8 peptidase family according to the MEROPS classification scheme (Rawlings, ND et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic AcidsRes. 44, D343-D350). Members of the S8 family possess a catalytic triplet in their amino acid sequence of Asp, His, and Ser. Although many variant proteases have been developed for use in cleaning applications, there is still a need for improved protease variants. Summary of the Invention

[0007] One embodiment relates to a Bacillus gibberellinii subtilis protease variant comprising one, two, three, four or more amino acid substitutions selected from the group consisting of: S039E, S099R, S126A, D127E and F128G, and further comprising one or more additional substitutions selected from the group consisting of: N074D, N085R, N116R, G160Q, R179Q, N198A / G / L / Q / R / S / T / V, Q200L, R207Q, M211E / L / N / Q, N212Q / S, N242D, N253P and Q256E, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1. In one embodiment, the Bacillus subtilis protease variant comprises the substituted S039E-S099R-S126A-D127E-F128G. In some embodiments, the subtilis protease variant has at least 80% identity with the amino acid sequence of SEQ ID NO:2.In another embodiment, a *Bacillus gibelio* subtilis protease variant is provided, the variant comprising one, two, three, four or more amino acid substitutions selected from the group consisting of: S039E, S099R, S126A, D127E and F128G, wherein the substitution comprises i) at least one substitution selected from the group consisting of S039E, S099R, S126A, D127E and F128G; ii) a substitution selected from S039E-S099R, S039E-S126A, S039E-D127E, S039E-F iii) Combinations of substitutions selected from S039E-S099R-S126A, S099R-D127E, S099R-F128G, S126A-D127E, S126A-F128G, and D127E-F128G; iv) Combinations of substitutions selected from S099R-S126A-D127E, S099R-S126A-F128G, S099R-D127E-F128G and S126A-D127E-F128G; Combinations of F128G substitutions; and combinations of S039E-S099R-S126A-D127E-F128G, wherein said variants further comprise one or more additional substitutions selected from the group consisting of: N074D, N085R, N116R, G160Q, R179Q, N198A / G / L / Q / R / S / T / V, Q200L, R207Q, M211E / L / N / Q, N212Q / S, N242D, N253P, and Q256E, wherein said amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0008] Other embodiments include Bacillus subtilis protease variants containing one, two, three, four or more amino acid substitutions selected from the group consisting of: S039E, S099R, S126A, D127E and F128G, and further containing one or more additional substitutions, or a group of substitutions, selected from the group consisting of: N074D-M211L-N253P, R179Q-M211L-N253P, N074D-N253P, N085R-G160Q-R179Q-M211L-N212S-N253P, R179Q-N253P, G160Q-R179Q-M211L-N212 S-N253P, R179Q-M211L, G160Q-R179Q-M211L-N253P, G160Q-R179Q-N212S -N253P, N074D-M211L, M211L-N242D, G160Q-R179Q-M211L-N212S, N074D- R179Q-M211L-N253P, G160Q-R179Q-M211L, G160Q-R179Q-N253P, N074D-Q 200L-M211L, N074D-G160Q-N212S-N253P, N074D-G160Q-M211L-N253P, G16 0Q-R179Q, G160Q-R179Q-N212S, N074D-G160Q-N253P, N074D-G160Q-R179 Q-M211L-N212S-N253P, N074D-N085R-G160Q-R179Q-M211L, N074D-G160Q -M211L-N212S-N253P, N074D-N085R-N116R-Q200L-Q256E, N074D-G160Q- R179Q-N212S-N253P, N074D-G160Q-M211L-N212S, N074D-G160Q, N074D-G 160Q-R179Q-M211L-N253P, N074D-R179Q-M211L, N074D-G160Q-N212S, N0 74D-G160Q-M211L, N074D-G160Q-R179Q-N253P, N074D, N074D-G160Q-R17 9Q-M211L-N212S, N074D-N085R-M211L-N212S, N074D-G160Q-R179Q-N212 S. N074D-G160Q-R179Q-M211L, N074D-M211L-Q256E, N074D-G160Q-R179Q,R179Q-M211L-N212S-N253P、R179Q-M211L-N212S、N074D-N085R-R179Q-M211L-N212S、N074D-M211L-N212S、N074D-R179Q-M211L-N212S、N074D-M211L-N242D、N074D-Q200L-M211L-Q256E、N074D-Q200L-M211L-N242D-Q256E、N074D-Q200L、N074D-M211N、N074D-M211N-N212Q、N074D-M211N-N212Q-Q256E、N074D-M211N-Q256E、N074D-M211Q、N074D-M211Q-N212Q、N074D-M211Q-N212Q-Q256E、N074D-M211Q-Q256E、N074D-N198A-M211Q、N074D-N198A-M211Q-N212Q、N074D-N198A-M211Q-Q256E、N074D-N198G-M211Q、N074D-N198G-M211Q-N212Q、N074D-N198G-M211Q-Q256E、N074D-N198K-M211Q-N212Q、N074D-N198L-M211Q-N212Q、N074D-N198Q-M211Q-N212Q、N074D-N198R-M211Q-N212Q、N074D-N198T-M211Q-N212Q、N074D-N198V-M211Q-N212Q、N074D-N212Q-Q256E、N074D-Q256E、N074D-R207Q、N074D-R207Q-M211N、N074D-R207Q-M211N-N212Q、N074D-R207Q-M211N-N212Q-Q256E、N074D-R207Q-M211N-Q256E、N074D-R207Q-M211Q、N074D-R207Q-M211Q-N212Q、N074D-R207Q-M211Q-N212Q-Q256E、N074D-R207Q-N212Q、N074D-R207Q-N212Q-Q256E、N074D-R207Q-Q256E、N074D-N198S-M211Q、N074D-N198L-M211Q、M211E、M211Q、N212Q-N242D、M211Q-N212Q、M211E-N212Q-N242D、N198A-M211Q-N212Q、N074D-N198A-M211Q-N212Q and N074D-N198A-M211Q-N212Q, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1. In some embodiments, the subtilis protease variant has at least 80% identity with the amino acid sequence of SEQ ID NO:1 or 2.

[0009] Other embodiments relate to methods for producing the variants described herein, the methods comprising stably transforming host cells with an expression vector containing a polynucleotide encoding one or more subtilisin protease variants described herein. Other embodiments relate to polynucleotides containing a nucleic acid sequence encoding one or more subtilisin protease variants described herein.

[0010] manual

[0011] In one embodiment, this disclosure provides one or more Bacillus giganteus subtilisin protease variants comprising one or more amino acid substitutions as described in more detail below. In some embodiments, the variants provided herein exhibit one or more improved properties, such as improved cleaning performance, or improved stability, or both, when compared to subtilisin having the amino acid sequence of SEQ ID NO:2. The subtilisin protease variants provided herein can be used to prepare cleaning compositions (e.g., automatic dishwashing compositions). Furthermore, the subtilisin protease variants provided herein can also be used in cleaning methods (e.g., dishwashing methods) that use such variants or compositions comprising such subtilisin protease variants.

[0012] Unless otherwise indicated herein, one or more Substantiium subtilis protease variants described herein may be prepared and used by a variety of techniques used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA fields, and industrial enzyme use and development. Undefined terms and abbreviations shall conform to their conventional meaning as used in the art. Unless otherwise defined herein, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any definitions provided herein shall be interpreted as a whole within the context of the specification. Unless the context clearly indicates otherwise, the singular “a / an” and “the” as used herein include the plural. Unless otherwise indicated, nucleic acid sequences are written from left to right in a 5' to 3' orientation; and amino acid sequences are written from left to right in an amino to carboxyl orientation. Each numerical range used herein includes each narrower numerical range falling within such a wider numerical range, as if all such narrower numerical ranges were explicitly stated herein.

[0013] As used herein in conjunction with numerical values, the term “about” refers to a range of + / - 0.5, unless the term is specifically defined otherwise in the context. For example, the phrase “pH of about 6” refers to a pH value of 5.5 to 6.5, unless the pH value is specifically defined otherwise.

[0014] The nomenclature for amino acid substitutions in one or more Substance protease variants described herein uses one or more of the following: position; position: one or more amino acid substitutions; or one or more starting amino acids: position: one or more amino acid substitutions. References to "position" (e.g., 5, 8, 17, 22, etc.) cover any starting amino acid that may be present at such position, and any substitution that may be present at such position. References to "position: one or more amino acid substitutions" (e.g., 1S / T / G, 3G, 17T, etc.) cover any starting amino acid that may be present at such position and one or more amino acids that may substitute for such starting amino acid. References to positions can be listed in several forms; for example, position 003 can also be referred to as position 03 or 3. References to starting or substituted amino acids can be further indicated as several starting or substituted amino acids separated by foreslashes (" / "). For example, D275S / K indicates that position 275 is replaced by serine (S) or lysine (K), and P / S197K indicates that the starting amino acid at position 197, proline (P) or serine (S), is replaced by lysine (K). References to X as a positional amino acid refer to any amino acid at the listed positions.

[0015] The positions of amino acid residues in a given amino acid sequence are numbered by corresponding to the amino acid sequence of SEQ ID NO:1. That is, the amino acid sequence of SEQ ID NO:1 is used as a reference sequence. For example, the amino acid sequences of one or more Bacillus subtilis protease 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 a given amino acid sequence aligned (preferably, best aligned) with the amino acid residues in SEQ ID NO:1 is conveniently numbered by referring to the numerical position of the corresponding amino acid residue. When compared with a query sequence (sometimes also called a “reference sequence”), a sequence alignment algorithm such as that described herein will identify one or more positions in the subject sequence where an insertion or deletion has occurred. Sequence alignment with other subtilisin proteases can be determined using amino acid alignment, for example, as shown in Figure 1 of PCT application No. PCT / US2018 / 062768, filed November 28, 2018, which claims priority to U.S. Provisional Application No. 62 / 591,976, filed November 29, 2017, entitled "Highly Stable Subtilisin Enzymes".

[0016] The terms “protease” and “proteinase” refer to enzymes that have the ability to break down proteins and peptides. Proteases have the ability to “proteolyze” proteins 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 called “proteolytic activity.” Many well-known procedures exist for measuring proteolytic activity. For example, proteolytic activity can be determined by a comparative assay analyzing the ability of each protease to hydrolyze a suitable substrate. Exemplary substrates that can be used to analyze protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma-Aldrich C-9801), bovine collagen (Sigma-Aldrich C-9879), bovine elastin (Sigma-Aldrich E-1625), and keratin (Keratin Azure) (Sigma-Aldrich K8500). Colorimetric assays using these substrates are well known in the art (see, for example, WO 99 / 34011 and US6,376,450). The pNA peptide assay (see, for example, Del Mar et al., Anal Biochem, 99:316-320, 1979) can also be used to determine the concentration of active enzymes. This assay measures the rate at which p-nitroaniline is released when the enzyme hydrolyzes a soluble synthetic substrate such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (suc-AAPF-pNA). The rate of formation of the yellow color from the hydrolysis reaction is measured at 405 or 410 nm on a spectrophotometer, and this rate is proportional to the concentration of active enzyme. Additionally, absorbance measurements at 280 nm can be used to determine the total protein concentration in a purified protein sample. The activity of the substrate divided by the protein concentration gives the specific enzyme activity.

[0017] As used herein, “Bacillus” includes all species within the genus “Bacillus” as known to those skilled in the art, including but not limited to: Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus giganteus, and Bacillus thuringiensis. It should be recognized that the genus Bacillus is undergoing continuous taxonomic recombination. Therefore, the genus is intended to include species that have been reclassified, including but not limited to organisms such as *Geobacillus stearothermophilus* (now named *Geobacillus stearothermophilus*) or *B. polymyxa* (now named *Paenibacillus polymyxa*). The production of resistant endospores under stress conditions is considered a defining characteristic of the genus *Bacillus*, although this characteristic also applies to recently named genera such as *Alicyclobacillus*, *Amphibacillus*, *Aneurinibacillus*, *Anoxybacillus*, *Brevibacillus*, *Filobacillus*, *Gracilibacillus*, *Halobacillus*, *Paenibacillus*, *Salibacillus*, *Thermobacillus*, *Ureibacillus*, and *Virgibacillus*.

[0018] "Bacillus girdrensis subtilisin" includes any subtilisin derived from or derived from Bacillus girdrensis. In one embodiment, the Bacillus girdrensis subtilisin variants provided herein may be derived from Bacillus girdrensis clade subtilisin proteases (e.g., those described in WO 2015 / 089447 and WO2016 / 205755). Other Bacillus girdrensis subtilisin proteases include those described in U.S. Patent Application Publication No. 20090275493 and variants thereof, those described in International Patent Application Publication No. WO2016 / 087403 and variants thereof, and those described in U.S. Patent No. 7,449,187 and variants thereof. In other embodiments, Bacillus girdrensis subtilisin includes polypeptides having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1 or 2.

[0019] The term "vector" refers to a nucleic acid construct used to introduce or transfer one or more nucleic acids into target cells or tissues. Typically, vectors are used to introduce exogenous DNA into cells or tissues. Vectors include plasmids, cloning vectors, bacteriophages, viruses (e.g., viral vectors), granules, expression vectors, shuttle vectors, etc. Typically, vectors include an origin of replication, a multiple cloning site, and a selectivity marker. Typically, the process of inserting a vector into a target cell is called transformation. In some embodiments, the present invention includes: a vector comprising a DNA sequence encoding a serine protease polypeptide (e.g., a precursor or mature serine protease polypeptide) that is efficiently linked to a suitable pre-sequence (e.g., a secretory, signal peptide sequence, etc.), said vector being capable of expressing the DNA sequence in a suitable host, as well as folding and translocation of the recombinant polypeptide chain.

[0020] As used herein, in the context of introducing nucleic acid sequences into cells, the term "introduction" refers to any method suitable for transferring nucleic acid sequences into cells. Such introduction methods include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to genetic changes in cells caused by uptake, optional genome incorporation, and expression of genetic material (e.g., DNA).

[0021] The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecules of this disclosure. Expression can also refer to the translation of mRNA into a polypeptide. Therefore, the term "expression" includes any step involved in "polypeptide production," including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0022] The phrase "expression cassette" or "expression vector" refers to a nucleic acid construct or vector, recombinantly or synthetically produced, for expressing a target nucleic acid (e.g., a foreign nucleic acid or a transgene) in target cells. Typically, the target nucleic acid expresses a target protein. Typically, an expression vector or expression cassette contains a promoter nucleotide sequence that drives or promotes the expression of a foreign nucleic acid. Typically, an expression vector or expression cassette also includes other specified nucleic acid elements that allow the transcription of a 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 incorporate and express heterologous DNA fragments into host cells or the host cell genome. Many prokaryotic and eukaryotic expression vectors are commercially available. Selecting a suitable expression vector for protein expression from the nucleic acid sequence incorporated into the expression vector is within the knowledge of those skilled in the art.

[0023] As used herein, when a nucleic acid is placed in a functional relationship with another nucleic acid sequence, that nucleic acid is “effectively linked” to the other nucleic acid sequence. For example, if a promoter influences the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the nucleotide coding sequence. If a ribosome binding site is located to facilitate the translation of a coding sequence, then that ribosome binding site can be effectively linked to the coding sequence. Typically, “effectively linked” DNA sequences are contiguous. However, enhancers do not have to be contiguous. Linkage is achieved by joining at a convenient restriction site. If such a site is not present, synthetic oligonucleotide adaptors or linkers can be used according to standard practice.

[0024] The term "gene" refers to a polynucleotide (e.g., a segment of DNA) that encodes a polypeptide and includes regions before and after the coding region. In some cases, a gene includes spacer sequences (introns) between individual coding regions (exons).

[0025] When used in relation to cells, the term "recombinant" typically indicates that the cell has been modified by introducing a foreign nucleic acid sequence, or that the cell is derived from a cell that has been so modified. For example, a recombinant cell may contain genes that are not present in the same form in the natural (non-recombinant) form of the cell, or a recombinant cell may contain natural genes (found in the natural form of the cell) that have been modified and reintroduced into the cell. Recombinant cells may contain nucleic acids that are endogenous to cells that have been modified but have not had the nucleic acids removed from the cells; such modifications include those obtained through gene substitution, site-specific mutations, and related techniques known to those skilled in the art. Recombinant DNA technology includes techniques for producing recombinant DNA in vitro and transferring said recombinant DNA into cells in which it can be expressed or propagated to produce recombinant polypeptides. "Recombination" of polynucleotides or nucleic acids generally refers to the assembly or combination of two or more nucleic acid or polynucleotide chains or fragments to produce new polynucleotides or nucleic acids.

[0026] If a nucleic acid or polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof in its natural state or when manipulated by methods known to those skilled in the art, then the nucleic acid or polynucleotide may be said to "encode" the polypeptide. The antisense strand of such a nucleic acid may also be referred to as encoding a sequence.

[0027] The terms “host strain” and “host cell” refer to the appropriate host for an expression vector containing the target DNA sequence.

[0028] A "protein" or "peptide" is a polymeric sequence of amino acid residues. The terms "protein" and "peptide" are used interchangeably herein. Throughout this disclosure, single-letter and three-letter codes for amino acids are used in accordance with the definitions provided by the Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC-IUB. The single-letter X refers to any one of the twenty amino acids. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0029] The terms "presequence" or "prepeptide sequence" refer to the amino acid sequence between the signal peptide sequence and the mature protease sequence, essential for the proper folding and secretion of the protease; they are sometimes referred to as intramolecular chaperones. Cleavage of the presequence or prepeptide sequence yields the mature, active protease. Bacterial serine proteases are often represented as preenzymes. For example, examples of modified prepeptides are provided in WO 2016 / 205710.

[0030] The terms "signal sequence" and "signal peptide" refer to amino acid residue sequences that can participate in the secretion or directed transport of proteins in their mature or precursor forms. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. Signal sequences are generally absent in mature proteins. Typically, after protein transport, the signal sequence is cleaved from the protein by a signal peptidase.

[0031] The term "mature" form of a protein, polypeptide, or peptide refers to a functional form of a protein, polypeptide, or peptide that lacks a signal peptide sequence and a propeptide sequence.

[0032] The term "precursor" refers to a protein or peptide in its mature form that has a pre-sequence effectively linked to the amino or carboxyl terminus of the protein. A precursor may also have a "signal" sequence effectively linked to the amino terminus of the pre-sequence. A precursor may also have additional polypeptides involved in post-translational activity (e.g., polypeptides cleaved from which the mature protein or peptide is left).

[0033] Regarding polypeptides, the term "wild-type" refers to a naturally occurring polypeptide that does not contain artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, regarding polynucleotides, the term "wild-type" refers to a naturally occurring polynucleotide that does not contain artificial substitutions, insertions, or deletions at one or more nucleotide positions. However, polynucleotides encoding wild-type polypeptides are not limited to naturally occurring polynucleotides and encompass any polynucleotide encoding either wild-type or parental polypeptides.

[0034] Regarding polypeptides, the term "parent" includes reference to naturally occurring or wild-type polypeptides, or naturally occurring polypeptides in which one or more amino acid positions have been artificially substituted, inserted, or deleted. Regarding polypeptides, the term "parent" also includes any polypeptide with protease activity that acts as a starting polypeptide for modification (such as substitution, addition, and / or deletion) to produce a variant with one or more modifications compared to the starting polypeptide. That is, parent or reference polypeptides are not limited to naturally occurring wild-type polypeptides and encompass any wild-type, parent, or reference polypeptide. Similarly, regarding polynucleotides, the term "parent" can refer to naturally occurring polynucleotides or polynucleotides that do indeed include artificially substituted, inserted, or deleted polynucleotides at one or more nucleotide positions. Regarding polynucleotides, the term "parent" also includes any polynucleotide encoding a polypeptide with protease activity that acts as a starting polynucleotide for modification to produce a variant protease with modifications such as substitution, addition, and / or deletion compared to the starting polynucleotide. That is, polynucleotides encoding wild-type, parent, or reference polypeptides are not limited to naturally occurring polynucleotides and encompass any polynucleotide encoding wild-type, parent, or reference polypeptides. In some embodiments, the parental polypeptide comprises Bacillus subtilis protease. In some embodiments, the parental polypeptide herein comprises a polypeptide having the amino acid sequence shown in SEQ ID NO:1.

[0035] The term "naturally occurring" refers, for example, sequences and residues found in nature (e.g., polypeptide sequences and amino acid sequences or nucleotide sequences and nucleotide sequences). Conversely, the term "non-naturally occurring" refers, for example, sequences and residues not found in nature (e.g., polypeptide sequences and amino acid sequences or nucleotide sequences and nucleic acid sequences).

[0036] As used herein, "corresponding to" or "corresponds to" refers to an amino acid residue at the listed position in a protein or peptide, or an amino acid residue that is similar to, homologous to, or equivalent to the listed residue in a protein or peptide. As used herein, "corresponding region" generally refers to a similar position in a related or reference protein.

[0037] The terms "derived from" and "obtained from" refer not only to proteins produced or potentially produced by strains of the organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in host organisms containing such DNA sequences. Additionally, the terms refer to proteins encoded by DNA sequences derived from synthetic and / or cDNA and possessing the identifying characteristics of the protein in question. For example, "proteases derived from the genus Bacillus" refers to those enzymes with proteolytic activity naturally produced by the genus Bacillus, as well as serine proteases, such as those produced from Bacillus but produced by other host cells transformed with nucleic acids encoding serine proteases using genetic engineering techniques.

[0038] In the context of two polynucleotide or polypeptide sequences, the term “identity” refers to the fact that the nucleotides or amino acids in the two sequences are identical when compared to the maximum correspondence, as measured using sequence comparison or analysis algorithms described below and known in the art.

[0039] The phrase “% identity” or “percentage identity” or “PID” refers to protein sequence identity. Percentage identity can be determined using standard techniques known in the art. The percentage of amino acid identity shared by a target sequence can be determined by aligning sequences to directly compare sequence information (e.g., using programs such as BLAST, MUSCLE, or CLUSTAL). The BLAST algorithm is described, for example, in Altschul et al., J Mol Biol [Journal of Molecular Biology], 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 90:5873-5787 (1993). The percentage (%) 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 best / maximum alignment). The BLAST algorithm refers to the “reference” sequence as the “query” sequence.

[0040] As used herein, “homological protein” or “homological protease” refers to proteins that have different similarities in primary, secondary, and / or tertiary structures. When comparing proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology can be determined by 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, Group 1; 25(17):3389-402(1997)). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most relevant sequences based on biological similarity, but it is not recommended for query sequences with fewer than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997 and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searching include: adjacent word length threshold = 11; E-value cutoff = 10; scoring matrix = NUC.3.1 (match = 1, mismatch = -3); gap open = 5; and gap extension = 2. Exemplary default BLAST parameters for amino acid sequence searching include: word length = 3; E-value cutoff = 10; scoring matrix = BLOSUM62; gap open = 11; and gap extension = 1. Using this information, protein sequences can be grouped and / or phylogenetic trees can be constructed from them. Amino acid sequences can be input into programs such as the Vector NTI Advance suite, and guide trees can be created using the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol [Molecular Biology and Evolution], 4:406-425, 1987). The tree structure can be calculated using Kimura correction for sequence distances and ignoring positions with vacancies. Programs such as AlignX can display the calculated distance values ​​in parentheses after the molecule name displayed on the phylogenetic tree.

[0041] Understanding homology between molecules can reveal their evolutionary history and functional information; if a newly sequenced protein is homologous to a previously characterized protein, there is a strong indication of the new protein's biochemical function. Two molecules are said to be homologous if they derive from a common ancestor. Homologous molecules or homologs can be divided into two categories: paralogs and orthologs. Paralogs are homologs that exist within a single species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs that exist in different species and have very similar or identical functions. A protein superfamily is the largest group (clade) of proteins whose common ancestor can be inferred. This common ancestor is usually based on sequence alignment and mechanical similarity. Typically, a superfamily contains several protein families that show sequence similarity within the family. Based on the MEROPS protease classification system, the term "protein family" is often used for protease superfamilies. As used herein, the term “subtilisin” includes any member of the S8 serine protease family as described in the MEROPS-peptidase database (Rawlings, ND et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors [Nucleic Acids Res] 44, D343-D350).

[0042] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res [Nucleic Acids Research] 22:4673-4680, 1994). The default parameters for the CLUSTAL W algorithm include: Vacancy open penalty = 10.0; Vacancy extension penalty = 0.05; Protein weight matrix = BLOSUM series; DNA weight matrix = IUB; Delayed divergence sequence % = 40; Vacancy separation distance = 8; DNA conversion weight = 0.50; List hydrophilic residues = GPSNDQEKR; Use negative matrix = off; Switch special residue penalty = on; Switch hydrophilic penalty = on; and Switch end vacancy separation penalty = off. In the CLUSTAL algorithm, deletions occurring at any end are included. For example, a variant with a five-amino acid deletion at any end (or within) of a 500-amino acid polypeptide will have 99% (495 / 500 identical residues × 100) percentage sequence identity relative to the "reference" polypeptide. Such variants will be covered by variants that have “at least 99% sequence identity” with the polypeptide.

[0043] A nucleic acid or polynucleotide is "separated" when it is at least partially or completely separated from other components, including but not limited to other proteins, nucleic acids, cells, etc. Similarly, a polypeptide, protein, or peptide is "separated" when it is at least partially or completely separated from other components, including but not limited to other proteins, nucleic acids, cells, etc. The separated species is more abundant than other species in the composition, in molar terms. For example, the separated species may constitute at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% (in molar terms) of all present macromolecular species. Preferably, the target species is purified to substantially homogeneity (i.e., contaminant species are not detectable in the composition by conventional detection methods). Purity and homogeneity can be determined using many techniques well-known in the art, such as agarose or polyacrylamide gel electrophoresis of nucleic acid or protein samples, followed by visualization after staining. If necessary, high-resolution techniques such as high-performance liquid chromatography (HPLC) or similar methods can be used to purify the substances.

[0044] The term "purified," when applied to nucleic acids or peptides, generally means nucleic acids or peptides that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified peptides or polynucleotides form discrete bands in electrophoresis gels, chromatographic eluates, and / or media subjected to density gradient centrifugation). For example, a nucleic acid or peptide that produces substantially one band in an electrophoresis gel is "purified." Purified nucleic acids or peptides are at least about 50% pure, and typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., percentage by weight on a molar basis). In a relevant sense, a composition is enriched for the molecules when there is a significant increase in the concentration of the molecules after the application of purification or enrichment techniques. The term "enrichment" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid, or other specific substance or component in the composition at a relative or absolute concentration higher than that in the starting composition.

[0045] The term "tableware" refers to all forms of tableware, including all forms of tableware (such as plates, cups, glasses, and bowls); all forms of cutlery (such as spoons, knives, forks, and serving utensils); all forms of ceramics; all forms of plastics (such as melamine); and all metal, porcelain, glass, and acrylic products.

[0046] The term "cleaning activity" refers to the cleaning performance achieved by a serine protease polypeptide, variant, or reference subtilisin under the primary conditions during the proteolysis, hydrolysis, cleaning, or other processes disclosed herein. In some embodiments, the cleaning performance of the serine protease or reference subtilisin can be determined by various assays for cleaning one or more enzyme-sensitive stains (e.g., stains caused by food, grass, blood, ink, milk, oil, and / or egg protein) on an article or surface. The cleaning performance of one or more subtilisin variants or reference subtilisins described herein can be determined by subjecting stains on an article or surface to one or more standard washing conditions and assessing the extent of stain removal 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 US 6,605,458, as well as those included in the examples provided below.

[0047] The term "effective amount" used in the description of one or more subtilisin variants or reference subtilisin proteases herein refers to the amount of protease that achieves the desired level of enzymatic activity in a specific cleaning composition. Such effective amounts can be readily determined by those skilled in the art and are based on many factors, such as the specific protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, tablet, or stick) composition is required.

[0048] The term "auxiliary material" refers to any liquid, solid, or gaseous substance, or recombinant polypeptide or its active fragment, contained in a cleaning composition other than one or more Bacillus subtilis protease variants described herein. In some embodiments, the cleaning compositions of this disclosure include one or more cleaning auxiliary materials. Typically, each cleaning auxiliary material is selected depending on the specific type and form of the cleaning composition (e.g., liquid, granules, powder, stick, paste, spray, tablet, gel, foam, or other composition). Preferably, each cleaning auxiliary material is compatible with the protease used in the composition.

[0049] Cleaning compositions and cleaning formulations include any composition suitable for cleaning, bleaching, disinfecting, and / or sterilizing any object, article, and / or surface. Such compositions and formulations include, but are not limited to, liquid and / or solid compositions, including cleaning or detergent compositions (e.g., liquid, tablet, gel, stick, granule, and / or solid laundry cleaning or detergent compositions) and delicate fabric detergent compositions; hard surface cleaning compositions and formulations, such as those for glass, wood, ceramic, and metal countertops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile, garment enhancement cleaning or detergent compositions, garment additive cleaning compositions, and garment pre-spotter cleaning compositions; dishwashing compositions, including hand-wash or manual dishwashing compositions (e.g., “hand-wash” or “manual” dishwashing detergents) and automatic dishwashing compositions (e.g., “automatic dishwashing detergents”). The invention may also use single-dose unit forms, including but not limited to pills, tablets, gelcaps, or other single-dose units such as pre-measured powders or liquids.

[0050] Unless otherwise indicated, cleaning compositions or cleaning formulations as used herein include general-purpose or heavy-duty detergents in granular or powder form, particularly cleaning detergents; general-purpose detergents in liquid, granular, gel, solid, tablet, paste, or unit dosage forms, particularly so-called heavy-duty liquid (HDL) detergents or heavy-duty dry cleaning (HDD) detergent types; liquid fine fabric detergents; hand or manual dishwashing detergents, including those of high-foaming types; hand or manual dishwashing detergents, automatic dishwashing detergents, or dish or tableware detergents, including various tablet, powder, solid, granular, liquid, gel, and rinsing aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial handwashing types, cleaning sticks, mouthwash, denture cleaners, car wash shampoos, carpet shampoos, and bathroom cleaners; hair shampoos and / or hair rinsing agents for human and other animal use; shower gels and foam baths and metal cleaners; and cleaning aids such as bleach additives and “stain remover sticks” or pretreatment types. In some embodiments, the particulate composition is in a “compact” form; in some embodiments, the liquid composition is in a “concentrated” form.

[0051] Regarding compositions used in washing media intended for cleaning soiled or dirty objects, including certain fabric and / or non-fabric objects or articles, the terms "detergent composition" or "detergent formulation" are used. In some embodiments, the detergents disclosed herein comprise one or more variants of Bacillus subtilis protease described herein, and additionally comprise one or more surfactants, one or more transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleach, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme stabilizers, calcium, enzyme activators, antioxidants, and / or solubilizers. In some cases, the builder salt is a mixture of silicates and phosphates, preferably having more silicates (e.g., sodium metasilicate) than phosphates (e.g., sodium tripolyphosphate). Some embodiments relate to cleaning compositions or detergent compositions that do not contain any phosphates (e.g., phosphates or phosphate builders).

[0052] The phrases “one or more compositions that are substantially free of boron” or “one or more detergents that are substantially free of boron” refer to one or more compositions or detergents containing trace amounts of boron (e.g., less than about 1000 ppm (1 mg / kg or 1 mg / L equals 1 ppm), less than about 100 ppm, less than about 50 ppm, less than about 10 ppm, or less than about 5 ppm, or less than about 1 ppm), the boron of which may be derived from other compositions or detergent ingredients.

[0053] The term "bleaching" refers to treating a material (e.g., fabrics, clothing, pulp, etc.) or surface for a sufficiently long time and / or under suitable pH and / or temperature conditions to achieve whitening (i.e., brightening) and / or cleaning of said material. Examples of chemicals suitable for bleaching include, but are not limited to, ClO2, H2O2, peracids, NO2, etc. Bleaching agents also include enzymatic bleaching agents, such as perhydrolases and aryl esterases. Another embodiment relates to compositions comprising one or more Bacillus subtilis protease variants described herein and one or more perhydrolases, such as the perhydrolases described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0054] The term "washing performance" for a protease (e.g., one or more variants of the Bacillus subtilis protease described herein, or their recombinant polypeptides or active fragments) refers to the additional cleaning contribution of the one or more Bacillus subtilis protease variants described herein to the washing performance compared to a detergent without the addition of the one or more Bacillus subtilis protease variants described herein to the composition. Washing performance is compared under relevant washing conditions. In some testing systems, other relevant factors, such as detergent composition, sud concentration, water hardness, washing mechanics, time, pH, and / or temperature, can be controlled in a manner that mimics one or more conditions typical for household applications in certain market segments (e.g., hand washing or manual dishwashing, automatic dishwashing, tableware cleaning, countertop cleaning, fabric cleaning, etc.).

[0055] This article uses the phrase "relevant washing conditions" to indicate the conditions actually used in the home in the hand-washing, automatic dishwashing, or laundry detergent segments, specifically washing temperature, time, washing mechanics, foam concentration, detergent type, and water hardness.

[0056] The term "dishwashing" refers to both household and industrial dishwashing, and encompasses both automated dishwashing (e.g., washing with a dishwashing machine) and manual dishwashing (e.g., washing by hand).

[0057] The term "disinfection" refers to the removal of contaminants from surfaces, as well as the inhibition or killing of microorganisms on the surface of objects.

[0058] The term "compact" in this document for cleaning compositions is preferably reflected by density, and in relation to the composition by the amount of inorganic filler salt. Inorganic filler salt is a conventional component of detergent compositions in powder form. In conventional detergent compositions, filler salt is present in a basic amount, typically from about 17% to about 35% by weight of the total composition. In contrast, in compact compositions, filler salt is present in an amount not exceeding about 15% of the total composition. In some embodiments, filler salt is present in an amount not exceeding about 10%, or more preferably about 5%, by weight of the composition. In some embodiments, the inorganic filler salt is selected from alkali salts and alkaline earth metal salts of sulfates and chlorides. In some embodiments, the filler salt is sodium sulfate.

[0059] This document discloses one or more subtilisin protease variants that can be used in cleaning applications and methods, as well as in a variety of industrial applications. It also discloses one or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin protease variants. In some embodiments, the one or more subtilisin protease variants described herein can be used in cleaning applications and can be incorporated into cleaning compositions used in methods for cleaning items or surfaces in need.

[0060] In one embodiment, a Bacillus subtilis protease variant is provided, wherein the variant comprises one, two, three, four or more amino acid substitutions selected from the group consisting of: X039E, X099R, X126A, X127E and X128G, and further comprises one or more additional substitutions at one, two, three or more positions selected from the group consisting of: 74, 85, 116, 160, 179, 198, 200, 207, 211, 212, 242, 253 and 256, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0061] In another embodiment, a *Bacillus gibelio* subtilis protease variant is provided, the variant comprising one, two, three, four or more amino acid substitutions selected from the group consisting of: S039E, S099R, S126A, D127E and F128G, wherein the substitution comprises i) at least one substitution selected from the group consisting of S039E, S099R, S126A, D127E and F128G; ii) a substitution selected from S039E-S099R, S039E-S126A, S039E-D127E, S039E-F iii) Combinations of substitutions selected from S039E-S099R-S126A, S099R-D127E, S099R-F128G, S126A-D127E, S126A-F128G, and D127E-F128G; iv) Combinations of substitutions selected from S099R-S126A-D127E, S099R-S126A-F128G, S099R-D127E-F128G and S126A-D127E-F128G; Combinations of F128G substitutions; and combinations of S039E-S099R-S126A-D127E-F128G, wherein said variants further comprise one or more additional substitutions selected from the group consisting of: N074D, N085R, N116R, G160Q, R179Q, N198A / G / L / Q / R / S / T / V, Q200L, R207Q, M211E / L / N / Q, N212Q / S, N242D, N253P, and Q256E, wherein said amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0062] For clarity, *Bacillus gibrini* subtilis protease variants containing one, two, three, four, or more amino acid substitutions selected from the group consisting of S039E, S099R, S126A, D127E, and F128G refer to such variants, which include those in the following cases where the substitutions comprise i) at least one substitution selected from the group consisting of S039E, S099R, S126A, D127E, and F128G; ii) substitutions selected from S039E, S099R, S126A, D127E, and F128G. iii) Combinations of substitutions from S039E-S099R, S039E-S126A, S039E-D127E, S039E-F128G, S099R-S126A, S099R-D127E, S099R-F128G, S126A-D127E, S126A-F128G, and D127E-F128G; Combinations of substitutions for 039E-S099R-F128G, S039E-S126A-D127E, S039E-S126A-F128G, S039E-D127E-F128G, S099R-S126A-D127E, S099R-S126A-F128G, S099R-D127E-F128G, and S126A-D127E-F128G; iv) combinations selected from S039E-S099R- Combinations of substitutions of S126A-D127E, S039E-S099R-S126A-F128G, S039E-S099R-D127E-F128G, S039E-S126A-D127E-F128G, and S099R-S126A-D127E-F128G; and v) combinations of S039E-S099R-S126A-D127E-F128G, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0063] In another embodiment, a *Bacillus gibrini* subtilis protease variant is provided, wherein the variant comprises amino acid substitutions X039E-X099R-X126A-X127E-X128G, and further comprises one or more additional substitutions at one, two, three or more positions selected from the group consisting of: 74, 85, 116, 160, 179, 198, 200, 207, 211, 212, 242, 253 and 256, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1. In some embodiments herein, references to the substitutions X039E, X099R, X126A, X127E and X128G include S039E, S099R, S126A, D127E and F128G. In some embodiments, the variant exhibits improved performance (PI value ≥ 1.1) in one or both of the PAS-38 and French caramel pudding assays (as provided in Example 2), or improved stability in Tris-EDTA buffer compared to the parental / reference subtilisin having the amino acid sequence shown in SEQ ID NO: 2 (PI value ≥ 1.1), or improved performance (PI value ≥ 1.1) in one or both of the PAS-38 and French caramel pudding assays (as provided in Example 2), and also improved stability in Tris-EDTA buffer compared to the parental / reference subtilisin having the amino acid sequence shown in SEQ ID NO: 2 (PI value ≥ 1.1).

[0064] In another embodiment, a Bacillus subtilis protease variant is provided, wherein the variant comprises an amino acid substitution selected from one or more substitutions selected from X039E, X099R, X126A, X127E, and X128G, and further comprises one or more additional substitutions selected from the group consisting of: X074D, X085R, X116R, X160Q, X179Q, X198A / G / L / Q / R / S / T / V, X200L, X207Q, X211E / L / N / Q, X212Q / S, X242D, X253P, and X256E, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0065] In another embodiment, a Bacillus subtilis protease variant is provided, wherein the variant comprises an amino acid substitution selected from one or more substitutions selected from S039E, S099R, S126A, D127E, and F128G, and further comprises one or more additional substitutions selected from the group consisting of: N074D, N085R, N116R, G160Q, R179Q, N198A / G / L / Q / R / S / T / V, Q200L, R207Q, M211E / L / N / Q, N212Q / S, N242D, N253P, and Q256E, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0066] In another embodiment, a Bacillus subtilis protease variant is provided, wherein the variant comprises amino acid substitutions X039E-X074D-X099R-X126A-X127E-X128G, and further comprises one or more additional substitutions selected from the group consisting of: X085R, X116R, X160Q, X179Q, X198A / G / L / Q / R / S / T / V, X200L, X207Q, X211E / L / N / Q, X212Q / S, X242D, X253P, X256E, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1. In another embodiment, a Bacillus subtilis protease variant is provided, wherein the variant comprises amino acid substitutions S039E-N074D-S099R-S126A-D127E-F128G, and further comprises one or more additional substitutions selected from the group consisting of: N085R, N116R, G160Q, R179Q, N198A / G / L / Q / R / S / T / V, Q200L, R207Q, M211E / L / N / Q, N212Q / S, N242D, N253P, Q256E, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0067] In one embodiment, a subtilisin variant is provided, wherein the variant comprises one, two, three, four or more amino acid substitutions selected from the group consisting of X039E, X099R, X126A, X127E and X128G, and further comprises one or more additional substitutions, or combinations of one or more substitutions selected from the group consisting of X074D-X211L-X253P, X179Q-X211L-X253P, X074D-X253P, X085R-X160Q-X179Q-X211L-X212S-X253P, X179Q-X253P, X160Q-X179Q-X211L-X 212S-X253P, X179Q-X211L, X160Q-X179Q-X211L-X253P, 12S-X253P, X074D-X211L, X211L-X242D, X160Q-X179Q-X211L-X212S, D-X179Q-X211L-X253P, X160Q-X179Q-X211L, X160Q-X179Q-X253P, X074D -X200L-X211L, X074D-X160Q-X212S-X253P, X160Q-X179Q, X160Q-X179Q-X212S, X074D-X160Q-X253P, X074D-X160Q-X 179Q-X211L-X212S-X253P, X074D-X085R-X160Q-X179Q-X211L, X074D-X16 0Q-X211L-X212S-X253P, X074D-X085R-N116R-X200L-X256E, X074D-X160 Q-X179Q-X212S-X253P, X074D-X160Q-X211L-X212S, X074D-X160Q, X074D- X160Q-X179Q-X211L-X253P, X074D-X179Q-X211L, X074D-X160Q-X212S, 074D-X160Q-X211L, X074D-X160Q-X179Q-X253P, 9Q-X211L-X212S, X074D-X085R-X211L-X212S, X074D-X160Q-X179Q-X212 S, X074D-X160Q-X179Q-X211L, X074D-X211L-X256E,X179Q-X211L-X212S-X253P、X179Q-X211L-X212S、X074D-X085R-X179Q-X211L-X212S、X074D-X211L-X212S、X074D-X179Q-X211L-X212S、X074D-X211L-X242D、X074D-X200L-X211L-X256E、X074D-X200L-X211L-X242D-X256E、X074D-X200L、X074D-X211N、X074D-X211N-X212Q、X074D-X211N-X212Q-X256E、X074D-X211N-X256E、X074D-X211Q、X074D-X211Q-X212Q、X074D-X211Q-X212Q-X256E、X074D-X211Q-X256E、X074D-X198A-X211Q、X074D-X198A-X211Q-X212Q、X074D-X198A-X211Q-X256E、X074D-X198G-X211Q、X074D-X198G-X211Q-X212Q、X074D-X198G-X211Q-X256E、X074D-X198K-X211Q-X212Q、X074D-X198L-X211Q-X212Q、X074D-N198Q-X211Q-X212Q、X074D-X198R-X211Q-X212Q、X074D-X198T-X211Q-X212Q、X074D-X198V-X211Q-X212Q、X074D-X212Q、X074D-X212Q-X256E、X074D-X256E、X074D-X207Q、X074D-X207Q-X211N、X074D-X207Q-X211N-X212Q、X074D-X207Q-X211N-X212Q-X256E、X074D-X207Q-X211N-X256E、X074D-X207Q-X211Q、X074D-X207Q-X211Q-X212Q、X074D-X207Q-X211Q-X212Q-X256E、X074D-X207Q-X212Q、X074D-X207Q-X212Q-X256E、X074D-X207Q-X256E、X074D-X198S-X211Q、X074D-X198L-X211Q、X211E、X211Q、X212Q-X242D、X211Q-X212Q、X211E-X212Q-X242D、X198A-X211Q-X212Q, X074D-X198A-X211Q-X212Q, and X074D-X198A-X211Q-X212Q, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1. In one such embodiment, a subtilisin protease variant is provided, wherein the variant comprises amino acid substitutions X039E-X099R-X126A-X127E-X128G, and one or more substitutions, or a combination of one or more substitutions.

[0068] In another embodiment, a subtilisin protease variant is provided, wherein the variant comprises one, two, three, four or more amino acid substitutions selected from the group consisting of: S039E, S099R, S126A, D127E and F128G, or all of the substitutions S039E-S099R-S126A-D127E-F128G, and further comprises one or more additional substitutions, or combinations of one or more substitutions selected from the group consisting of: N074D-M211L-N253P, R179Q-M211L-N253P, N074D-N253P, N085R-G160Q-R179Q-M211L-N212S -N253P, R179Q-N253P, G160Q-R179Q-M211L-N212S-N253P, R179Q-M211L, G160Q-R179Q-M211L-N253P, G160Q-R179Q-N212S-N253P, N074D-M211L, M2 11L-N242D, G160Q-R179Q-M211L-N212S, N074D-R179Q-M211L-N253P, G16 0Q-R179Q-M211L, G160Q-R179Q-N253P, N074D-Q200L-M211L, N074D-G160Q -N212S-N253P, N074D-G160Q-M211L-N253P, G160Q-R179Q, G160Q-R179Q- N212S, N074D-G160Q-N253P, N074D-G160Q-R179Q-M211L-N212S-N253P, N0 74D-N085R-G160Q-R179Q-M211L, N074D-G160Q-M211L-N212S-N253P, N07 4D-N085R-N116R-Q200L-Q256E, N074D-G160Q-R179Q-N212S-N253P, N074D -G160Q-M211L-N212S, N074D-G160Q, N074D-G160Q-R179Q-M211L-N253P, N074D-R179Q-M211L, N074D-G160Q-N212S, N074D-G160Q-M211L, N074D-G1 60Q-R179Q-N253P, N074D, N074D-G160Q-R179Q-M211L-N212S, N074D-N085 R-M211L-N212S, N074D-G160Q-R179Q-N212S, N074D-G160Q-R179Q-M211L,N074D-M211L-Q256E、N074D-G160Q-R179Q、R179Q-M211L-N212S-N253P、R179Q-M211L-N212S、N074D-N085R-R179Q-M211L-N212S、N074D-M211L-N212S、N074D-R179Q-M211L-N212S、N074D-M211L-N242D、N074D-Q200L-M211L-Q256E、N074D-Q200L-M211L-N242D-Q256E、N074D-Q200L、N074D-M211N、N074D-M211N-N212Q、N074D-M211N-N212Q-Q256E、N074D-M211N-Q256E、N074D-M211Q、N074D-M211Q-N212Q、N074D-M211Q-N212Q-Q256E、N074D-M211Q-Q256E、N074D-N198A-M211Q、N074D-N198A-M211Q-N212Q、N074D-N198A-M211Q-Q256E、N074D-N198G-M211Q、N074D-N198G-M211Q-N212Q、N074D-N198G-M211Q-Q256E、N074D-N198K-M211Q-N212Q、N074D-N198L-M211Q-N212Q、N074D-N198Q-M211Q-N212Q、N074D-N198R-M211Q-N212Q、N074D-N198T-M211Q-N212Q、N074D-N198V-M211Q-N212Q、N074D-N212Q、N074D-N212Q-Q256E、N074D-Q256E、N074D-R207Q、N074D-R207Q-M211N、N074D-R207Q-M211N-N212Q、N074D-R207Q-M211N-N212Q-Q256E、N074D-R207Q-M211N-Q256E、N074D-R207Q-M211Q、N074D-R207Q-M211Q-N212Q、N074D-R207Q-M211Q-N212Q-Q256E、N074D-R207Q-N212Q、N074D-R207Q-N212Q-Q256E、N074D-R207Q-Q256E、N074D-N198S-M211Q、N074D-N198L-M211Q、M211E、M211Q、N212Q-N242D、M211Q-N212Q, M211E-N212Q-N242D, N198A-M211Q-N212Q, N074D-N198A-M211Q-N212Q, and N074D-N198A-M211Q-N212Q, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0069] In another embodiment, a *Bacillus gibelio* subtilis protease variant is provided, the variant comprising one, two, three, four or more amino acid substitutions selected from the group consisting of: S039E, S099R, S126A, D127E and F128G, wherein the substitution comprises i) at least one substitution selected from the group consisting of S039E, S099R, S126A, D127E and F128G; ii) a substitution selected from S039E-S099R, S039E-S126A, S039E-D127E, S039E-F128G, S099R-S126A, S099R-D127E, S099R-F128G, S126A-D127G. E) Combinations of substitutions from S126A-F128G and D127E-F128G; iii) Combinations of substitutions from S039E-S099R-S126A, S039E-S099R-D127E, S039E-S099R-F128G, S039E-S126A-D127E, S039E-S126A-F128G, S039E-D127E-F128G, S099R-S126A-D127E, S099R-S126A-F128G, S099R-D127E-F128G and S126A-D127E-F128G; iv) Combinations of substitutions from S039E-S099R-S126A-D Combinations of substitutions for 127E, S039E-S099R-S126A-F128G, S039E-S099R-D127E-F128G, S039E-S126A-D127E-F128G, and S099R-S126A-D127E-F128G; and v) combinations of S039E-S099R-S126A-D127E-F128G, and said variants further comprising one or more additional substitutions, or combinations of substitutions, selected from the group consisting of: N074D-M211L-N253P, R179Q-M211L-N253P, N074D-N253P, N085R-G160Q-R17 9Q-M211L-N212S-N253P, R179Q-N253P, G160Q-R179Q-M211L-N212S-N253P , R179Q-M211L, G160Q-R179Q-M211L-N253P, G160Q-R179Q-N212S-N253P, N 074D-M211L, M211L-N242D, G160Q-R179Q-M211L-N212S, N074D-R179Q-M21 1L-N253P, G160Q-R179Q-M211L, G160Q-R179Q-N253P, N074D-Q200L-M211L,N074D-G160Q-N212S-N253P、N074D-G160Q-M211L-N253P、G160Q-R179Q、G160Q-R179Q-N212S、N074D-G160Q-N253P、N074D-G160Q-R179Q-M211L-N212S-N253P、N074D-N085R-G160Q-R179Q-M211L、N074D-G160Q-M211L-N212S-N253P、N074D-N085R-N116R-Q200L-Q256E、N074D-G160Q-R179Q-N212S-N253P、N074D-G160Q-M211L-N212S、N074D-G160Q、N074D-G160Q-R179Q-M211L-N253P、N074D-R179Q-M211L、N074D-G160Q-N212S、N074D-G160Q-M211L、N074D-G160Q-R179Q-N253P、N074D、N074D-G160Q-R179Q-M211L-N212S、N074D-N085R-M211L-N212S、N074D-G160Q-R179Q-N212S、N074D-G160Q-R179Q-M211L、N074D-M211L-Q256E、N074D-G160Q-R179Q、R179Q-M211L-N212S-N253P、R179Q-M211L-N212S、N074D-N085R-R179Q-M211L-N212S、N074D-M211L-N212S、N074D-R179Q-M211L-N212S、N074D-M211L-N242D、N074D-Q200L-M211L-Q256E、N074D-Q200L-M211L-N242D-Q256E、N074D-Q200L、N074D-M211N、N074D-M211N-N212Q、N074D-M211N-N212Q-Q256E、N074D-M211N-Q256E、N074D-M211Q、N074D-M211Q-N212Q、N074D-M211Q-N212Q-Q256E、N074D-M211Q-Q256E、N074D-N198A-M211Q、N074D-N198A-M211Q-N212Q、N074D-N198A-M211Q-Q256E、N074D-N198G-M211Q、N074D-N198G-M211Q-N212Q、N074D-N198G-M211Q-Q256E, N074D-N198K-M211Q-N212Q, N074D-N198L-M211Q-N212Q, N074D -N198Q-M211Q-N212Q, N074D-N198R-M211Q-N212Q, N074D-N198T-M211Q-N212Q, N074D-N198V -M211Q-N212Q, N074D-N212Q, N074D-N212Q-Q256E, N074D-Q256E, N074D-R207Q, N074D-R207 Q-M211N, N074D-R207Q-M211N-N212Q, N074D-R207Q-M211N-N212Q-Q256E, N074D-R207Q-M211 N-Q256E, N074D-R207Q-M211Q, N074D-R207Q-M211Q-N212Q, N074D-R207Q-M211Q-N212Q-Q25 6E, N074D-R207Q-N212Q, N074D-R207Q-N212Q-Q256E, N074D-R207Q-Q256E, N074D-N198S-M21 1Q, N074D-N198L-M211Q, M211E, M211Q, N212Q-N242D, M211Q-N212Q, M211E-N212Q-N242D, N198A-M211Q-N212Q, N074D-N198A-M211Q-N212Q and N074D-N198A-M211Q-N212Q, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0070] Another embodiment relates to one or more subtilisin protease variants described herein, provided that one or more substitutions are not naturally occurring. Yet another embodiment relates to one or more subtilisin protease variants described herein, wherein said variant (i) is Bacillus giganteus BG46 subtilisin protease; (ii) is isolated; (iii) has proteolytic activity; or (iv) comprises a combination of (i) to (iii). Another embodiment relates to one or more Substantiae protease variants described herein, wherein said variants are derived from parental or reference polypeptides that (i) have 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 or 2; or (ii) have 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2. In yet another embodiment, the parental polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2. Even further embodiments involve one or more variants of the Bacillus subtilis protease described herein, wherein the variant comprises an amino acid sequence that (i) has 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1; (ii) has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1; or (iii) has 96%, 97%, 98%, 99%, or less than 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 or 2.

[0071] In some embodiments, the parent or variant molecule of Bacillus subtilis protease provided herein further comprises at least one, two, three or more additional substitutions selected from X012E / L / V, X021V, X025R, X037E, X039E / T, X041F, X043V, X044P, X060D, X074D, X078D, X079L, X084A, X087E, X097D, X099E, X101G, X012L, X107E, X115D, X117I, and X118N. , X122L, X127P, X142G, X145S, X149S, X154D, X156A, X160S, 0E, X205D, X208N, X209N, X211L / N / S, X212D / H / N, X222S, X228I, Examples of such combinations of one, two, three or more substitutions that can be combined with the Bacillus giganteus variants provided herein include, but are not limited to, X253D-X256E, X025R-X117I-X118N, X044P-X175N-X208N-X230H, X041F-X078D-X084A, X101G-X174A, and X021V-X17 7I, X021V-X142G-X188A, X021V-X122L-X222S, X122L-X253D, X021V-X177V-X228I, X021V-X039T-X122L-X177E, E-X209N-X222S, X021V-X122L-X222S-X247N, X021V-X122L, 039E-X074D-X087E-X253D, X021V-X039E-X074D-X087E-X253D, -X122L-X253D, X021V-X039E-X074D-X087E-X122L-X253D, X097D-X099E, X122L-X145S-X156A, X211N-X212D, X211L-X212D, X127P-X211L-X212D and X012L-X122L-X222S.

[0072] This disclosure includes variants of the subtilisin protease having one or more modifications on surface-exposed amino acids. The surface modifications of the enzyme variant can be used in detergent compositions by having a minimum performance index for detergent performance, enzyme stability in detergent compositions, and enzyme thermal stability, while simultaneously having at least one of these characteristics that are improved relative to the parental subtilisin protease. In some embodiments, the surface modification alters the hydrophobicity and / or charge of the amino acid at that location. Hydrophobicity can be determined using techniques known in the art, such as those described by White and Wimley (White, SH and Wimley, WC, (1999) Annu. Rev. Biophys. Biomol. Struct [Annual Review of Biophysics and Biomolecular Structure] 28:319-65). As used herein, “surface property” can refer to electrostatic charge and properties exhibited by the protein surface, such as hydrophobicity and hydrophilicity. In even further embodiments, one or more subtilisin protease variants described herein have one or more improved properties when compared to a reference subtilisin protease or parental subtilisin protease; wherein said improved property is selected from improved detergent cleaning performance, improved stability, and combinations thereof. In another embodiment, the parental subtilisin protease comprises the amino acid sequence of SEQ ID NO:1. In another embodiment, the parental subtilisin protease is a polypeptide having the amino acid sequence of SEQ ID NO:1. In yet another embodiment, the improved property is (i) improved detergent cleaning performance, wherein said variant has a cleaning PI of ≥1.1 for crème brûlée and / or egg stains; and / or (ii) improved stability, wherein said variant has a stability PI of ≥1.1. In yet another embodiment, the detergent cleaning performance is measured according to the cleaning performance in the ADW detergent assay of Example 2; and / or the stability is measured according to the stability assay of Example 2.

[0073] In the context of oxidation, chelating agents, denaturing agents, surfactants, and heat- and / or pH-stabilized proteases, the term "enhanced stability" or "improved stability" refers to a higher retention of proteolytic activity over time compared to a reference protease (e.g., wild-type or parental protease). Autolysis has been identified as a mode of loss of activity of Bacillus subtilis protease 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).

[0074] The terms "thermally stable," "thermostable," and "thermostability" for protease variants refer to the retention of a specified amount of enzymatic activity by the protease after exposure to altered temperatures for a given period of time under primary conditions (or "stress conditions") during proteolysis, hydrolysis, cleaning, or other processes. "Alternated temperature" encompasses both increases and decreases in temperature.

[0075] In some embodiments, the variant protease provided herein is available at a given time interval (e.g., at least about 5 minutes, at least about 20 minutes, at least about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, about 660 minutes, about 720 minutes, about 780 minutes, about 840 minutes, about 900 minutes, about 960 minutes, about 1020 minutes, about 1080 minutes). After exposure to temperatures of 40°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 58°C, 59°C, 60°C, 65°C, 70°C, 75°C, or 80°C, at least 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% of the proteolytic activity is retained. In one embodiment, by using the method described in Example 2, the performance index of the variant subtilisin provided herein is greater than 1 compared to the parent protease.

[0076] The subtilisin variants provided herein can be used to produce a variety of compositions, such as enzyme compositions and cleaning or detergent compositions. Enzyme compositions contain the subtilisin variants provided herein. Enzyme compositions can be in any form, such as granules, liquid formulations, or enzyme slurries.

[0077] Enzyme particles can be manufactured through methods such as rotary atomization, wet granulation, dry granulation, spray drying, disc granulation, extrusion, pot coating, spheroidization, drum granulation, fluidized bed agglomeration, high-shear granulation, fluidized bed spraying, crystallization, precipitation, emulsion gelation, rotary disc atomization, and other casting methods, as well as spheroidization processes. The core of the particle can be the particle itself or the core of a layered particle.

[0078] The core may contain one or more water-soluble agents or one or more water-dispersible agents, including but not limited to sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate and ammonium sulfate, citric acid, sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives, such as hydroxypropyl methylcellulose, carboxymethyl cellulose and hydroxyethyl cellulose), phosphates, calcium, protease inhibitors and combinations thereof. Suitable dispersants include, but are not limited to, clay, sugar pellets (a combination of sugar and starch; e.g., starch-sucrose sugar pellets-ASNP), talc, silicates, carboxymethyl cellulose, starch and combinations thereof.

[0079] In some embodiments, the core primarily comprises sodium sulfate. In some embodiments, the core is essentially composed of sodium sulfate. In certain embodiments, the core consists of sodium sulfate only.

[0080] In some embodiments, the core contains a subtilisin protease variant as provided herein. In other embodiments, the core contains one or more enzymes in addition to the protease. In other embodiments, the core is inert and does not contain any enzymes.

[0081] In some embodiments, the core is an enzyme powder, comprising a UFC containing the enzyme. The enzyme powder may be spray-dried and may optionally be blended with any water-soluble or water-dispersible agent listed herein. The enzyme may be or may include a protease to be stabilized, in which case the enzyme powder should further include a stabilizer.

[0082] In some embodiments, the core is coated with at least one coating. In a particular embodiment, the core is coated with at least two coatings. In another particular embodiment, the core is coated with at least three coatings. The materials used for one or more coatings may be suitable for use in cleaning compositions and / or detergent compositions (see, for example, US 20100124586, WO 9932595, and US5324649).

[0083] In some embodiments, the coating comprises one or more of the following materials: inorganic salts (e.g., sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, sugars (e.g., sucrose, lactose, glucose, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives, such as hydroxypropyl methylcellulose, carboxymethyl cellulose, and hydroxyethyl cellulose), clay, sugar pellets (a combination of sugar and starch), silicates, carboxymethyl cellulose, phosphates, starch (e.g., corn starch), fats, oils (e.g., rapeseed oil and paraffin oil), lipids, vinyl polymers, vinyl copolymers, polyvinyl alcohol (PVA), plasticizers (e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate, and water), anti-caking agents (e.g., talc, clay, amorphous silica, and titanium dioxide), and defoamers (e.g., FOAMBLAST). and EROL ) and talc. US 20100124586, WO9932595 and US 5324649 detail suitable components for coatings.

[0084] In some embodiments, the coating comprises sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin, and fructose). In some embodiments, the coating comprises polymers such as polyvinyl alcohol (PVA). Suitable PVAs for incorporating into one or more coatings of multilayer particles include partially hydrolyzed, fully hydrolyzed, and moderately hydrolyzed PVAs with low to high viscosity. In some embodiments, the coating comprises inorganic salts such as sodium sulfate.

[0085] In some embodiments, at least one coating is an enzyme coating. In some embodiments, the core is coated with at least two enzyme layers. In another embodiment, the core is coated with at least three or more enzyme layers.

[0086] In some embodiments, the enzyme is a protease in combination with one or more additional enzymes selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, aryl esterase, β-galactosidase, carrageenanase, catalase, cellobiase, cellulase, chondroitinase, keratinase, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucosylamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, ligninase. Lipases, lipoxygenases, mannanases, metalloproteinases, nucleases (e.g., DNases and / or RNases), oxidases, oxidoreductases, pectic acid lyases, pectin acetylesterases, pectinases, pentosanases, hydrolases, peroxidases, phenol oxidases, phosphatases, phospholipases, phytases, polygalacturonases, polyesterases, other proteases, amylopectinases, reductases, rhamnogalacturonases, β-glucanases, tannic acidases, transglutaminases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof. Typically, at least one enzyme coating contains at least one protease.

[0087] The enzyme list above is merely an example and does not imply exclusivity. Any enzyme can be used in the particles described herein, including wild-type enzymes, recombinant enzymes, and variant enzymes from bacterial, fungal, and yeast sources, as well as acidic, neutral, or alkaline enzymes.

[0088] Another embodiment relates to a method of cleaning a surface, wherein the method includes contacting the surface or article to be cleaned with an effective amount of one or more subtilisin protease variants as provided herein or a composition containing one or more subtilisin protease variants as provided herein. In some embodiments, the surface or article to be cleaned contains protein stains on the surface. In some embodiments, the surface or article to be cleaned contains protein stains or crème brûlée stains or egg stains. The term “stain” includes any type of dirt or grime on the surface of an article (e.g., a hard-surface article, such as tableware). In some embodiments, the stain is a protein stain. As used herein, “protein stain” is a stain or grime containing protein.

[0089] Another embodiment relates to a method for cleaning protein stains, the method comprising contacting the surface or article to be cleaned with an effective amount of one or more subtilisin variants as provided herein or a composition containing one or more subtilisin variants as provided herein.

[0090] Another embodiment relates to a method for cleaning stains from French caramel pudding, the method comprising contacting the surface or item to be cleaned with an effective amount of one or more subtilisin variants as provided herein or a composition containing one or more subtilisin variants as provided herein.

[0091] Another embodiment relates to a method for cleaning egg stains or egg yolk stains, the method comprising contacting the surface or item to be cleaned with an effective amount of one or more subtilisin protease variants as provided herein or a composition containing one or more such subtilisin protease variants.

[0092] In even further embodiments, one or more subtilisin variants used in the methods described herein comprise an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 or 2. In yet another embodiment, when compared with SEQ ID NO:2, one or more subtilisin variants used in the methods for cleaning French caramel pudding stains described herein have a French caramel pudding stain cleaning PI of ≥1.1. In yet another embodiment, when compared with SEQ ID NO:2, one or more subtilisin protease variants used in the method for cleaning French caramel pudding stains described herein have a French caramel pudding stain cleaning PI of ≥1.1, wherein the French caramel pudding stain cleaning performance of said variants is measured according to the French caramel pudding assay described in Example 2. Yet another embodiment relates to a method for cleaning French caramel pudding stains described herein, provided that one or more subtilisin proteases used in said method contain one or more non-naturally occurring substitutions. In yet another embodiment, when compared with SEQ ID NO:2, one or more subtilisin protease variants used in the method for cleaning egg yolk stains described herein have an egg yolk stain cleaning PI of ≥1.1. In yet another embodiment, when compared with SEQ ID NO:2, one or more subtilisin protease variants used in the method for cleaning egg yolk stains described herein have an egg yolk stain cleaning PI of ≥1.1, wherein the egg yolk stain cleaning performance of said variants is measured according to the egg yolk assay described in Example 2. Another embodiment relates to a method for cleaning egg yolk stains described herein, provided that one or more subtilisin proteases used in the method contain one or more non-naturally occurring substitutes. In further embodiments, one or more subtilisin protease variants used in the methods described herein are (i) isolated; (ii) have proteolytic activity; or (iii) comprise a combination of (i) and (ii).

[0093] In another embodiment, compared with the parental subtilisin having the amino acid sequence of SEQ ID NO:2, the variants provided herein comprise one or more variants having amino acid substitutions selected from the group consisting of those listed in Tables 3 and 4 that have a PI ≥ 1.1 in one or more cleanliness or stability assays (including clothing, BMI, egg, crème brûlée assays, or EDTA stability assays).

[0094] One or more Substantiium subtilis protease variants described herein can be subjected to various modifications, such as insertions, deletions, and / or substitutions (conserved or non-conserved) of one or more amino acids, including cases where such modifications substantially do not alter the enzymatic activity of the variant. Similarly, the nucleic acids of the present invention can also be modified, such as substitutions of one or more nucleotides in one or more codons, such that a particular codon encodes the same or different amino acids, resulting in silent changes (e.g., when the encoded amino acid is not altered by a nucleotide mutation) or non-silent changes; deletions of one or more nucleotides (or codons) in the sequence; additions or insertions of one or more nucleotides (or codons) in the sequence; and / or cleavages or truncations of one or more nucleotides (or codons) in the sequence. Many of these modifications in the nucleic acid sequence substantially do not alter the enzymatic activity of the resulting encoded peptidases compared to peptidases 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 optimal expression in an expression system (e.g., a bacterial expression system), while, if desired, the one or more codons still encode one or more of the same amino acids.

[0095] This document describes one or more isolated, non-naturally occurring, or recombinant polynucleotides comprising nucleic acid sequences encoding one or more *Bacillus subtilis* protease variants, or recombinant polypeptides or active fragments thereof described herein. The one or more nucleic acid sequences described herein can be used in the recombinant production (e.g., expression) of one or more *Bacillus subtilis* protease variants described herein, typically by expressing plasmid expression vectors containing sequences encoding one or more *Bacillus subtilis* protease variants or fragments thereof described herein. One embodiment provides nucleic acids encoding one or more *Bacillus subtilis* protease variants described herein, wherein said variants are mature forms with proteolytic activity. In some embodiments, one or more *Bacillus subtilis* protease variants described herein are recombinantly expressed using homologous propeptide sequences. In other embodiments, one or more *Bacillus subtilis* protease variants described herein are recombinantly expressed using heterologous propeptide sequences (e.g., propeptide sequences from *Bacillus tarda* (SEQ ID NO: 5)).

[0096] The one or more nucleic acid sequences described herein can be generated using any suitable synthesis, manipulation, and / or isolation techniques or combinations thereof. For example, the one or more polynucleotides described herein can be generated using standard nucleic acid synthesis techniques well known to those skilled in the art, such as solid-phase synthesis. In such techniques, fragments of up to 50 or more nucleotide bases are typically synthesized and then ligated (e.g., by enzymatic or chemical ligation methods) to substantially form any desired continuous nucleic acid sequence. The synthesis of the 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 classical phosphoramide methods (see, for example, Beaucage et al., Tetrahedron Letters 22:1859-69 (1981)), or methods typically practiced in automated synthesis methods as described in Mattes et al., EMBO J. 3:801-805 (1984). The one or more polynucleotides described herein can also be generated using an automated DNA synthesizer. Custom nucleic acids can be ordered from various commercial sources, such as ATUM (DNA 2.0), Newark, CA, USA; Life Tech (GeneArt), Carlsbad, CA, USA; GenScript, Ontario, Canada; Base Clear BV, Leiden, Netherlands; Integrated DNA Technologies, Skokie, IL, USA; Ginkgo Bioworks (Gen9), Boston, MA, USA; and Twist Bioscience, San Francisco, CA, USA. Other techniques and related principles used for the synthesis of nucleic acids are described, for example, by Itakura et al., Ann. Rev. Biochem. [Annals of Biochemistry] 53:323 (1984) and Itakura et al., Science 198:1056 (1984).

[0097] Recombinant DNA techniques for modifying nucleic acids are well known in the art, such as restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more polynucleotides described herein can also be obtained by screening cDNA libraries using one or more oligonucleotide probes, which can hybridize or PCR amplify the polynucleotide encoding one or more subtilisin variants, recombinant polypeptides, or active fragments thereof described herein. Procedures for screening and isolating cDNA clones and PCR amplification procedures are well known to those skilled in the art and are described in standard references known to those skilled in the art. One or more polynucleotides described herein can be obtained by altering the naturally occurring polynucleotide backbone (e.g., the polynucleotide backbone encoding one or more subtilisin variants or a reference subtilisin) using known mutagenesis procedures (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro recombination). A variety of methods suitable for generating modified polynucleotides described herein encoding one or more subtilisin variants are known in the art, including but not limited to site-saturation mutagenesis, scan mutagenesis, insertion mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution, and various other recombination methods.

[0098] Other embodiments involve one or more vectors comprising one or more subtilisin protease variants described herein (e.g., polynucleotides encoding one or more subtilisin protease 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.

[0099] Some embodiments involve one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein, the vectors comprising one or more nucleic acid or polynucleotide sequences described herein. Some of these recombinant cells are transformed or transfected using at least one such vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some of these cells comprise bacterial cells, including but not limited to Bacillus cells, such as Bacillus subtilis cells. Other embodiments involve recombinant cells (e.g., recombinant host cells) comprising one or more subtilisin proteases described herein.

[0100] In some embodiments, one or more vectors described herein are expression vectors or expression cassettes comprising one or more polynucleotide sequences described herein that are efficiently linked to one or more additional nucleic acid segments required for effective gene expression (e.g., promoters efficiently linked to one or more polynucleotide sequences described herein). The vector may include transcription terminators and / or selection genes (e.g., antibiotic resistance genes) capable of sustaining continuous culture of plasmid-infected host cells by growth in a medium containing antimicrobial agents.

[0101] 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, and pHP13 (see Harwood and Cutting, Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990)); suitable replication plasmids for Bacillus subtilis include those listed on page 92. (See also, Perego, “Integrational Vectors for Genetic Manipulations in Bacillus subtilis”; Sonenshein et al., [edit]; “Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics”, American Society for Microbiology, Washington, DC (1993), pp. 615-624; and p2JM103BBI).

[0102] To express and produce a target protein (e.g., one or more subtilisin variants described herein) in cells, one or more expression vectors containing one or more copies (and in some cases, multiple copies) of a polynucleotide encoding one or more subtilisin variants described herein are transformed into cells under conditions suitable for variant expression. 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 a host cell; however, in other embodiments, the plasmid vector containing the polynucleotide sequence encoding one or more subtilisin variants described herein remains an autonomous extrachromosomal element within the cell. Some embodiments provide extrachromosomal nucleic acid elements and introgressive nucleotide sequences integrated into the host cell genome. The vectors described herein can be used to produce 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 integration vector capable of integrating the polynucleotide encoding the variant into and optionally amplifying it in the host chromosome. Examples of integration sites are well known to those skilled in the art. In some embodiments, transcription of a multinucleotide encoding one or more subtilisin protease variants described herein is achieved via a promoter that is a wild-type promoter of the parental subtilisin protease. In some other embodiments, the promoter is heterologous to one or more subtilisin protease variants described herein but is functional in a host cell. Exemplary promoters for bacterial host cells include, but are not limited to, the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, and pHpaII promoters; the promoter for the *Bacillus stearothermophilus* maltose amylase gene; the *Bacillus amyloliquefaciens* (BAN) amylase gene; the *Bacillus subtilis* alkaline protease gene; the *Bacillus clausti* alkaline protease gene; the *Bacillus pumilis* xylosidase gene; *Bacillus thuringiensis* cryIIIA; and the *Bacillus licheniformis* α-amylase gene. Additional promoters include, but are not limited to, the A4 promoter, and the bacteriophage λPR or PL promoters, as well as the *Escherichia coli* lac, trp, or tac promoters.

[0103] One or more subtilisin variants described herein can be produced in host cells of any suitable microorganism, including bacteria and fungi. In some embodiments, one or more subtilisin variants described herein can be produced in Gram-positive bacteria. In some embodiments, the host cell is a species of Bacillus, Streptomyces, Escherichia, Aspergillus, Trichoderma, Pseudomonas, Corynebacterium, Saccharomyces, or Pichia. In some embodiments, one or more subtilisin variants described herein are produced by host cells of a Bacillus species. Examples of Bacillus host cells that can be used for the production of one or more Bacillus subtilis protease variants described herein include, but are not limited to: Bacillus licheniformis, Bacillus tarda, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus thermophilus, Bacillus alkaliphilus, Bacillus coagulans, Bacillus circulans, Bacillus brevis, Bacillus thuringiensis, Bacillus clausti, and Bacillus megaterium, as well as other organisms within the genus Bacillus. In some embodiments, Bacillus subtilis host cells are used to produce the variants described herein. USPN 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus host strains that can be used to produce one or more Bacillus subtilis protease variants described herein, but other suitable strains may be used.

[0104] Several bacterial strains that can be used to produce one or more subtilisin protease variants described herein include non-recombinant (i.e., wild-type) Bacillus strains, as well as variants of naturally occurring strains and / or recombinant strains. In some embodiments, the host strain is a recombinant strain in which a polynucleotide encoding one or more subtilisin protease variants described herein has been introduced into the host. In some embodiments, the host strain is a Bacillus subtilis host strain, particularly a recombinant Bacillus subtilis host strain. Many Bacillus subtilis strains are known, including but not limited to strains such as 1A6 (ATCC39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 to PB1758, PB3360, JH642, 1A243 (ATCC39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP211 (see, for example, Hoch et al., Genetics 73:215-228 (1973); see also, US 4,450,235; US 4,302,544; and EP 0134048). The use of Bacillus subtilis as an expression host cell is well known in the art (see, for example, 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)).

[0105] In some embodiments, the Bacillus host cell is a Bacillus species including at least one of the following genes with a mutation or deletion: 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, for example, Msadek et al., J. Bacteriol. [Journal of Bacteriology] 172:824-834 (1990); and Olmos et al., Mol. Gen. Genet. [Molecular and General Genetics] 253:562-567 (1997)). In some embodiments, the Bacillus host contains mutations or deletions in: scoC4 (see, e.g., Caldwell et al., J. Bacteriol. [Journal of Bacteriology] 183:7329-7340 (2001)); spoIIE (see, e.g., Arigoni et al., Mol. Microbiol. [Molecular Microbiology] 31:1407-1415 (1999)); and / or other genes of oppA or the opp operon (see, e.g., Perego et al., Mol. Microbiol. [Molecular Microbiology] 5:173-185 (1991)). In fact, any mutation in the opp operon that is expected to cause the same phenotype as the mutation in the oppA gene will be used in some embodiments of the altered Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, the modified Bacillus host cell strains that can be used to produce one or more Bacillus subtilis protease variants described herein are Bacillus host strains that already contain mutations of one or more of the genes described above. Alternatively, Bacillus host cells containing one or more mutations and / or one or more deletions of endogenous protease genes can be used. In some embodiments, the Bacillus host cells contain deletions of the aprE and nprE genes. In other embodiments, the Bacillus host cells contain deletions of five protease genes, while in other embodiments, the Bacillus host cells contain deletions of nine protease genes (see, for example, US 2005 / 0202535).

[0106] Transform host cells with one or more nucleic acid sequences encoding one or more subtilis protease variants described herein using any suitable method known in the art. Methods for introducing nucleic acids (e.g., DNA) into Bacillus or Escherichia coli cells using plasmid DNA constructs or vectors and for transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmid is subsequently isolated from E. coli cells and transformed into Bacillus cells. However, the use of an intervening microorganism such as E. coli is not necessary, and in some embodiments, the DNA construct or vector is introduced directly into the Bacillus host.

[0107] Exemplary methods for introducing one or more nucleic acid sequences described herein into Bacillus cells are described in, for example, Ferrari et al., “Genetics”, in Hardwood et al. (ed.), 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. [Fria Microbiology], 30:97 (1985); Chang et al., Mol. Gen. Genet. [Molecular and General Genetics] 168:11-115 (1979); Vorobjeva et al., FEMS Microbiol. Lett. [FEMS Microbiology Letters] 7:261-263 (1980); Smith et al., Appl. Env. Microbiol [Applied and Environmental Microbiology] 51:634 (1986); Fisher et al., Arch. Microbiol. [Archives of Microbiology], 139:213-217 (1981); and McDonald, J. Gen. Microbiol [Journal of Genetic Microbiology] 130:203 (1984). In fact, methods such as transformation (including protoplast transformation and transfection, transduction and protoplast fusion) are well known and suitable for use in this paper. Methods known in the art for transforming Bacillus cells include, for example, plasmid-labeled rescue transformation, which involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (see, Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol. 154:1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169:1205-1211 (1987)). In this method, the imported donor plasmid recombines with the homologous region of the resident “helper” plasmid during a process simulating chromosome transformation.

[0108] In addition to commonly used methods, in some embodiments, host cells are directly transformed with a DNA construct or vector containing nucleic acid encoding one or more Bacillus subtilis protease variants described herein (i.e., the intermediate cells are not used for amplification or otherwise treated before introduction into the host cells). Introducing the DNA construct or vector described herein into host cells includes those physical and chemical methods known in the art that introduce nucleic acid sequences (e.g., DNA sequences) into host cells without inserting them into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In another embodiment, the DNA construct or vector is co-transformed with a plasmid without inserting the plasmid. In a further embodiment, a selection marker is deleted from an altered Bacillus strain using methods known in the art (see Stahl et al., J. Bacteriol. [Journal of Bacteriology] 158:411-418 (1984); and Palmeros et al., Gene [Gene] 247:255-264 (2000)).

[0109] In some embodiments, the transformed cells are cultured in a conventional nutrient medium. Suitable specific culture conditions, such as temperature, pH, etc., are known to those skilled in the art and are described in detail in the scientific literature. Some embodiments provide cultures (e.g., cell cultures) containing one or more subtilis protease variants or nucleic acid sequences described herein.

[0110] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more subtilis protease variants described herein are cultured in a suitable nutrient medium under conditions that allow expression of the variants, and the resulting variants are subsequently recovered from the culture. In some embodiments, the variants produced by the cells are recovered from the culture medium by routine 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 by means of salt (e.g., ammonium sulfate), and purification by chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.).

[0111] In some embodiments, one or more subtilisin protease variants produced by recombinant host cells are secreted into a culture medium. A nucleic acid sequence encoding a purification-enhancing domain can be used to facilitate the purification of said variants. A vector or DNA construct comprising a polynucleotide sequence encoding one or more subtilisin protease variants described herein may further comprise a nucleic acid sequence encoding a purification-enhancing domain that promotes variant purification (see, for example, Kroll et al., DNA Cell Biol. 12:441-53 (1993)). Such purification-enhancing domains include, but are not limited to, metal chelate peptides, such as histidine-tryptophan modules that allow purification on immobilized metals (see Porath, Protein Expr. Purif. 3:263-281

[1992] ), protein A domains that allow purification on immobilized immunoglobulins, and domains utilized in the FLAGS extension / affinity purification system. It was also found that cleavable linker sequences such as factor XA or enterokinase (e.g., sequences available from Invitrogen, San Diego, California) between the purified domain and the heterologous protein can facilitate purification.

[0112] Various methods can be used to determine the production levels of one or more mature Bacillus subtilis protease variants described herein in host cells. Such methods include, but are not limited to, methods utilizing, for example, polyclonal or monoclonal antibodies specific to the protease. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescence activated cell sorting (FACS). These and other assays are well known in the art (see, for example, Maddox et al., J. Exp. Med. [Journal of Experimental Medicine] 158:1211 (1983)).

[0113] Some other embodiments provide methods for preparing or producing one or more mature subtilisin protease variants described herein. Mature subtilisin protease variants do not include a signal peptide or propeptide sequence. Some methods include preparing or producing one or more subtilisin protease variants described herein in recombinant bacterial host cells (e.g., Bacillus cells, e.g., Bacillus subtilis cells)). Other embodiments provide methods for producing one or more subtilisin protease variants described herein, wherein the method includes culturing a recombinant host cell containing a recombinant expression vector comprising a nucleic acid sequence encoding one or more subtilisin protease variants described herein under conditions favorable for producing the variants. Some such methods further include recovering the variants from the culture.

[0114] Further embodiments provide methods for producing one or more Bacillus subtilis protease variants described herein, wherein the methods include: (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 subtilis cells); and (b) culturing the cells in a culture medium under conditions favorable to the production of the variant encoded by the expression vector. Some such methods further include: (c) isolating the variant from the cells or from the culture medium.

[0115] Another embodiment relates to a method for improving the cleaning properties or stability of Bacillus griseus subtilis protease, the method comprising modifying Bacillus griseus subtilis protease to include one or more substitutions, or combinations of substitutions, as provided herein.

[0116] Unless otherwise indicated, all component or composition levels provided herein are given with reference to the activity level of the said component or composition and do not include impurities that may be present in commercially available sources, such as residual solvents or byproducts. Enzyme component weights are based on total active protein. Unless otherwise indicated, all percentages and ratios are by weight. Unless otherwise indicated, all percentages and ratios are calculated based on the total composition. The compositions described herein include cleaning compositions, such as detergent compositions. In the example detergent compositions, enzyme levels are expressed by pure enzymes on a weight basis of the total composition and, unless otherwise specified, detergent components are expressed by weight basis of the total composition.

[0117] In one embodiment, one or more subtilisin variants described herein can be used in cleaning applications, such as, but not limited to, cleaning tableware, tabletop items, fabrics, medical devices, and items with hard surfaces (e.g., tables, tabletops, walls, furniture items, floors, ceilings). In other embodiments, one or more subtilisin variants described herein can be used in disinfection applications, such as, but not limited to, disinfecting automatic dishwashing machines or washing machines.

[0118] Another embodiment relates to a composition comprising one or more variants of the subtilisin 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 laundry detergent compositions, automatic dishwashing (ADW) compositions, handwashing (manual) dishwashing compositions, hard surface cleaning compositions, eyeglass cleaning compositions, medical device cleaning compositions, disinfectant (e.g., odor or microbial) compositions, and personal care cleaning compositions. In still other embodiments, the composition is a laundry detergent composition, an ADW composition, or a handwashing (manual) dishwashing composition. Even still other embodiments relate to fabric cleaning compositions, while other embodiments relate 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 still other embodiments, the composition comprises one or more variants of the subtilisin described herein, as well as one or more excipients, auxiliary materials, and / or additional enzymes.

[0119] In another embodiment, this disclosure provides detergent compositions (e.g., ADW compositions) comprising a surfactant and at least one subtilis protease variant as provided herein. Such compositions may further comprise one or more excipients, auxiliary materials, and / or additional enzymes.

[0120] In yet another embodiment, the compositions described herein contain phosphates, are phosphate-free, contain boron, are boron-free, or combinations thereof. In other embodiments, the compositions are boron-free compositions. In some embodiments, the boron-free compositions are compositions without added borate stabilizers. In another embodiment, the boron-free compositions are compositions containing less than 5.5% boron. In yet another 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 another embodiment, the boron-free compositions are compositions containing less than 2.5% boron. In even further embodiments, 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 another embodiment, the boron-free compositions are compositions containing less than 0.5% boron. In other embodiments, the compositions are compositions containing no or substantially no enzyme stabilizers or peptide inhibitors.

[0121] In another embodiment, one or more compositions described herein are in the form of gels, tablets, powders, granules, solids, liquids, unit doses, and combinations thereof. In yet another embodiment, one or more compositions described herein are in the form of low-water tight formulations, low-water HDL or unit doses (UD), or high-water formulations or HDL. In some embodiments, the clean compositions described herein are in unit dosage forms. In other embodiments, the unit dosage forms are selected from pills, tablets, capsules, sac-like tablets, small capsules, sachets, multi-compartment sachets, and pre-measured powders or liquids. In some embodiments, the unit dosage form is designed to provide controlled release of the ingredient within a multi-compartment sachet (or other unit dosage form). Suitable unit doses and controlled release forms are described, for example, in EP 2100949, WO 02 / 102955, US 4,765,916, US4,972,017, and WO 04 / 111178. In some embodiments, the unit dosage form is a tablet or powder contained in a water-soluble film or sachet.

[0122] Exemplary laundry detergent compositions include, but are not limited to, liquid and powder laundry detergent compositions, for example. Exemplary hard surface cleaning compositions include, for example, compositions for cleaning hard surfaces such as non-tableware items, non-tabletop appliances, tables, tabletops, furniture items, walls, floors, and ceilings. Exemplary hard surface cleaning compositions are described, for example, in USPN 6,610,642, 6,376,450, and 6,376,450. Exemplary personal care compositions include, but are not limited to, compositions for cleaning dentures, teeth, hair, contact lenses, and skin. Exemplary components of such oral care compositions include, for example, those described in US 6,376,450.

[0123] In some embodiments, one or more subtilisin variants described herein are used for cleaning at low temperatures. In other embodiments, one or more compositions described herein are used for cleaning at low temperatures. In other embodiments, one or more compositions described herein contain an effective amount of one or more subtilisin variants described herein, which are useful or effective for cleaning surfaces requiring the removal of protein stains.

[0124] In some instances, auxiliary materials are incorporated, for example, to assist or enhance cleaning performance; to treat the substrate to be cleaned; or to alter the aesthetics of the cleaning composition, such as in the case of perfumes, colorants, dyes, etc. One embodiment relates to a composition comprising one or more auxiliary materials described herein and one or more variants of Bacillus subtilis protease. Another embodiment relates to a composition comprising one or more auxiliary materials described herein and one or more variants of Bacillus subtilis protease, wherein the auxiliary material is selected from: bleaching catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilizing systems), chelating agents, brighteners, dirt-releasing polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, fillers, photoactivators, fluorescent agents, fabric conditioning agents, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, and bactericides. Agents, fungicides, color enhancers, silver care agents, anti-dulling agents, anti-corrosion agents, alkaline sources, solubilizers, carriers, processing aids, pigments, pH control agents, surfactants, washing aids, chelating agents, dye transfer inhibitors, deposition aids, catalytic materials, bleaching activators, bleaching enhancers, hydrogen peroxide, hydrogen peroxide sources, pre-prepared peracids, polymer dispersants, clay stain removers / anti-redeposition agents, structural elasticizers, fabric softeners, carriers, water-soluble additives, processing aids, pigments and combinations thereof. Exemplary excipients and levels of use can be found in USPN 5,576,282, 6,306,812, 6,326,348, 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014, and 5,646,101. In embodiments where one or more cleaning excipients are incompatible with one or more Bacillus subtilis protease variants described herein, methods are used to keep the excipient and the one or more variants separate (i.e., not in contact with each other) until the combination of the two components is appropriate. Such separation methods include any suitable methods known in the art (e.g., capsules, encapsulation, tablets, physical separation, etc.).

[0125] Some embodiments relate to cleaning additive products comprising one or more Bacillus subtilis protease variants described herein. In some embodiments, the additive is encapsulated in a dosage form for addition to a cleaning process. In some embodiments, the additive is encapsulated in a dosage form for addition to a cleaning process in which a peroxide source is used and increased bleaching effect is desired.

[0126] Exemplary fillers or carriers for particulate compositions include, but are not limited to, various salts of sulfates, carbonates, and silicates; talc; and clay. Exemplary fillers or carriers for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols such as methanol, ethanol, propanol, and isopropanol). In some embodiments, the composition contains about 5% to about 90% of such fillers or carriers. Acidic fillers may be included in such compositions to lower the pH of the solution obtained in cleaning methods or applications.

[0127] In one embodiment, one or more cleaning compositions described herein comprise an effective amount of one or more Bacillus subtilis protease variants described herein, said variants being alone or in combination with one or more additional enzymes. Typically, the cleaning composition comprises at least about 0.0001 wt% to about 20 wt%, from about 0.0001 wt% to about 10 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.001 wt% to about 1 wt%, or from about 0.01 wt% to about 0.2 wt% of one or more proteases. In another embodiment, one or more cleaning compositions described herein comprise one or more protease / gram compositions in amounts ranging from about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 2 mg, about 0.01 to about 1 mg, about 0.05 to about 1 mg, about 0.5 to about 10 mg, about 0.5 to about 5 mg, about 0.5 to about 4 mg, about 0.5 to about 3 mg, about 0.5 to about 2 mg, about 0.5 to about 1 mg, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 4 mg, about 0.1 to about 3 mg, about 0.1 to about 2 mg, about 0.1 to about 2 mg, about 0.1 to about 1 mg, or about 0.1 to about 0.5 mg.

[0128] The cleaning compositions described herein are typically formulated such that, during use in aqueous cleaning operations, the wash water will have a pH from about 4.0 to about 11.5, or even from about 5.0 to about 11.5, or even from about 5.0 to about 8.0, or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from about 3.0 to about 9.0, or even from about 3 to about 5. Granular laundry products are typically formulated to have a pH from about 8 to about 11. In some embodiments, the cleaning compositions of the present invention may be formulated to have an alkaline pH under washing conditions, such as from about 8.0 to about 12.0, or from about 8.5 to about 11.0, or from about 9.0 to about 11.0. In some embodiments, the cleaning compositions of the present invention may be formulated to have a neutral pH under washing conditions, such as from about 5.0 to about 8.0, or from about 5.5 to about 8.0, or from about 6.0 to about 8.0, or from about 6.0 to about 7.5. In some embodiments, when the cleaning composition is dissolved in deionized water at a ratio of 1:100 (wt:wt) at 20°C, neutral pH conditions can be measured using a conventional pH meter. Techniques for controlling the pH at recommended usage levels include the use of buffer solutions, bases, acids, etc., and are well known to those skilled in the art.

[0129] In some embodiments, one or more subtilisin protease 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 cleaning. 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 protease variants described herein. Typically, the encapsulating material is water-soluble and / or water-dispersible. In some embodiments, the encapsulating material has a glass transition temperature (Tg) of 0°C or higher. Exemplary encapsulating materials include, but are not limited to: carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin, and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. In some embodiments, the encapsulating material is starch (see, for example, EP 0922499, US 4,977,252, US 5,354,559, and US 5,935,826). In some embodiments, the encapsulating material is microspheres made of plastics such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof. Exemplary commercial microspheres include, but are not limited to, those made of plastics such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, and mixtures thereof. (Stockviksverken, Sweden); and PM 6545, PM6550, PM 7220, PM 7228, and (PQ Corp., Valley Forge, PA)

[0130] Various washing conditions exist, including different detergent formulations, wash water volumes, wash water temperatures, and wash durations that may expose one or more of the Bacillus subtilis protease variants described herein. Low detergent concentration systems involve wash water containing less than about 800 ppm of detergent components. Medium detergent concentration systems involve wash water containing about 800 ppm to about 2000 ppm of detergent components. High detergent concentration systems involve wash water containing more than about 2000 ppm of detergent components. In some embodiments, the “cold water wash” of the present invention utilizes a “cold water detergent” suitable for washing at temperatures ranging from about 10°C to about 40°C, from about 20°C to about 30°C, or from about 15°C to about 25°C, and all other combinations thereof in the range of about 15°C to about 35°C or 10°C to 40°C.

[0131] Different geographical locations have different water hardness. Hardness is determined by the amount of calcium (Ca) in water. 2+ ) and magnesium (Mg 2+ The quantity of Ca is measured. It is usually expressed as particles per gallon (gpg) of a mixture. 2+ / Mg 2+ To describe water hardness. In the United States, most water is hard water, but the hardness varies. Medium-hard (60-120 ppm) to hard (121-181 ppm) water has a hardness of 60 to 181 ppm (ppm can be converted to particulate / US gallon by dividing ppm by 17.1).

[0132] water Pellet / gallon parts per million soft Less than 1.0 Less than 17 Slightly hard 1.0 to 3.5 17 to 60 medium hard 3.5 to 7.0 60 to 120 hard 7.0 to 10.5 120 to 180 Very hard Greater than 10.5 Greater than 180

[0133] Other embodiments relate to one or more cleaning compositions comprising, by weight of about 0.00001% to about 10% of one or more subtilisin variants described herein, and by weight of about 99.999% to about 90.0% of one or more auxiliary materials. In another embodiment, the cleaning composition comprises, by weight of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% of one or more subtilisin variants, and by weight of about 99.9999% to about 90.0%, about 99.999% to about 98%, or about 99.995% to about 99.5% of one or more auxiliary materials.

[0134] In other embodiments, the compositions described herein comprise one or more Bacillus subtilis protease variants described herein and one or more additional enzymes. The one or more additional enzymes are selected from acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, aryl esterases, β-galactosidases, carrageenanases, catalases, cellobiases, cellulases, chondroitinases, keratinases, endo-β-1,4-glucanases, endo-β-mannanases, esterases, exo-mannanases, galactanases, glucosylamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, lysozymes, etc. Mannanase, metalloproteinase, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectinase, pectin acetylesterase, pectinase, pentosanase, hydrolase, peroxidase, phenol oxidase, phosphatase, phospholipase, phytase, polygalacturonase, polyesterase, other proteases, amylopectinase, reductase, rhamnogalacturonase, β-glucanase, tannic acidase, transglutaminase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof. Some embodiments involve combinations (i.e., “mixtures”) of enzymes comprising conventional enzymes (like amylase, lipase, keratinase, mannanase, and / or cellulase) combined with one or more Bacillus subtilis protease variants and / or one or more other proteases described herein.

[0135] In another embodiment, one or more compositions described herein comprise one or more subtilisin variants described herein and one or more additional proteases. In one embodiment, the additional protease is a serine protease. In another embodiment, the additional protease is a metalloproteinase, fungal subtilisin, or alkaline microbial protease or trypsin-like protease. Suitable additional proteases include those of animal, plant, or microbial origin. In some embodiments, the additional protease is a microbial protease. In other embodiments, the additional protease is a chemically or genetically modified mutant. In another embodiment, the additional protease is an alkaline microbial protease or trypsin-like protease. In other embodiments, the additional protease does not contain an epitope that cross-reacts with the *Bacillus griseus* variant, as measured by antibody binding or other assays available in the art. Exemplary alkaline proteases include subtilisin derived from, for example, *Bacillus* (e.g., BPN', Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168), or fungal origin (e.g., those described in U.S. Patent No. 8,362,222).Other exemplary proteases include, but are not limited to, WO 92 / 21760, WO 95 / 23221, WO 2008 / 010925, WO 09 / 149200, WO 09 / 149144, WO 09 / 149145, WO 10 / 056640, WO 10 / 056653, WO 2010 / 0566356, WO 11 / 072099, WO 2011 / 13022, WO 11 / 140364, WO 12 / 151534, WO 2015 / 038792, WO 2015 / 089447, WO 2015 / 089441, WO 2017 / 215925 / US Publication No. 2008 / 0090747, US US 5,801,039, US 5,340,735, US 5,500,364, US 5,855,625, RE 34,606, US 5,955,340, US 5,700,676, US 6,312,936, US 6,482,628, US 8,530,219, US Provisional Application Nos. 62 / 180673 and 62 / 161077, and PCT Application Nos. PCT / US 2015 / 021813, PCT / US 2015 / 055900, PCT / US 2015 / 057497, PCT / US 2015 / 057492, PCT / US 2015 / 057512, PCT / US Those described in 2015 / 057526, PCT / US 2015 / 057520, PCT / US 2015 / 057502, PCT / US 2016 / 022282 and PCT / US 16 / 32514, and WO 1999014341, WO1999033960, WO 1999014342, WO 1999034003, WO 2007044993, WO 2009058303, WO2009058661, WO 2014071410, WO 2014194032, WO 2014194034, WO 2014194054, WO 2014 / 194117, EP Metalloproteinases described in WO 2017215925 and WO 2016203064. Exemplary additional proteases include, but are not limited to, trypsin (e.g., porcine or bovine origin) and Fusarium proteases described in WO 89 / 06270. Exemplary commercial proteases include, but are not limited to, those described in WO 89 / 06270. MAXACAL TM MAXAPEM TM , OXP, PURAMAX TM EXCELLASE TM PREFERENZ TM Proteases (e.g., P100, P110, P280, P300), EFFECTENZ TM Proteases (e.g., P1000, P1050, P2000), EXCELLENZ TM Proteases (e.g., P1000), and PURAFAST TM (DuPont / Danisco / Genencor); ULTRA Variants, 16L ULTRA DURAZYM TM , LIQUANASE PROGRESS and (Novozymes); BLAP TM and BLAP TM Variant (Henkel); LAVERGY TM PRO 104L (BASF), KAP (Alkaliophilic Bacillus subtilis protease (Kao Corporation)) and (AB Enzymes)

[0136] Another embodiment relates to a composition comprising one or more subtilisin variants and one or more lipases described herein. In some embodiments, the composition comprises lipase in amounts from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Exemplary lipases may be chemically or genetically modified mutants. Exemplary lipases include, but are not limited to, those of bacterial or fungal origin, such as *H. lanuginosa* lipase (see, for example, EP258068 and EP 305216), *T. lanuginosa* lipase (see, for example, WO 2014 / 059360 and WO 2015 / 010009), *Rhizomucor miehei* lipase (see, for example, EP238023), and *Candida* lipases such as *C. antarctica* lipase (e.g., *C. antarctica* lipase A or B) (see, for example, EP 258068). 214761), Pseudomonas lipases such as P. alcaligenes and P. pseudoalcaligenes lipases (see, for example, EP218272), P. cepacia lipase (see, for example, EP 331376), P. stutzeri lipase (see, for example, GB 1,372,034), P. fluorescens lipase, Bacillus lipases (e.g. Bacillus subtilis lipase (Dartois et al., Biochem. Biophys. Acta [Chinese Journal of Biochemistry and Biophysics] 1131:253-260 (1993)), Bacillus stearothermophilus lipase (see, for example, JP 64 / 744992), and Bacillus pumilus lipase (see, for example, WO 91 / 16422)).Exemplary cloned lipases include, but are not limited to, Penicillium camembertii lipase (see Yamaguchi et al., Gene, 103:61-67 (1991)); Geotricum candidum lipase (see Schimada et al., J. Biochem., 106:383-388 (1989)); and various Rhizopus lipases such as Rhizopus dellemar lipase (see Hass et al., Gene, 109:117-113 (1991)); Rhizopus niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 (1992)) and Rhizopus oryzae lipase. Other lipases, such as keratinases, may also be used in one or more of the compositions described herein, including, but not limited to, keratinases derived from *Pseudomonas mendocina* (see WO 88 / 09367) and / or *Fusarium solani* pisi (see WO 90 / 09446). Exemplary commercial lipases include, but are not limited to, M1 LIPASE. TM LUMAFAST TM LIPOMAX TM and PREFERENZ TM L100 (DuPont); and ULTRA (Novozymes); and LIPASE P TM Amano Pharmaceutical Co., Ltd.

[0137] Further embodiments relate to compositions comprising one or more Bacillus subtilis protease variants and one or more amylases described herein. In one embodiment, the composition comprises amylase in amounts from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any amylase suitable for use in alkaline solutions (e.g., α-amylase and / or β-amylase) may be included in such compositions. Exemplary amylases may be chemically or genetically modified mutants. Exemplary amylases include, but are not limited to, those of bacterial or fungal origin, such as those described in GB 1,296,839, WO 9100353, WO9402597, WO 94183314, WO 9510603, WO 9526397, WO 9535382, WO 9605295, WO 9623873, WO9623874, WO 9630481, WO 9710342, WO 9741213, WO 9743424, WO 9813481, WO 9826078, WO9902702, WO 9909183, WO 9919467, WO 9923211, WO 9929876, WO 9942567, WO 9943793, WO9943794, WO9946399, WO0029560, WO0060058, WO0060059, WO0060060, WO0114532, WO0134784, WO0164852, WO0166712, WO0188107, WO0196537, WO02092797, WO0210355, WO0231124, WO2004055178, WO2004113551, WO2005001064, WO2005003311, WO2005018336, WO2005019443, WO 2005066338, WO 2006002643, WO 2006012899, WO2006012902, WO 2006031554, WO 2006063594, WO 2006066594, WO 2006066596, WO2006136161, WO 2008000825, WO 2008088493, WO 2008092919, WO 2008101894, WO2008 / 112459, WO 2009061380, WO 2009061381, WO 2009100102, WO 2009140504, WO 2009149419、WO2010 / 059413、WO 2010088447、WO 2010091221、WO 2010104675、WO 2010115021、WO10115028、WO 2010117511、WO 2011076123、WO 2011076897、WO 2011080352, WO2011080353, WO 2011080354, WO 2011082425, WO 2011082429, WO 2011087836, WO2011098531, WO 2013063460, WO 2013184577, WO 2014099523、WO Amylases in WO2014164777, WO2015077126, and WO 2018184004. Exemplary commercial amylases include, but are not limited to, those listed in WO2014164777, WO2015077126, and WO 2018184004. STAINZYME STAINZYME STAINZYME and BAN TM (Novozymes); EFFECTENZ TM S 1000, POWERASE TM PREFERENZ TM S 100, PREFERENZ TM S 110, PREFERENZ TM S 210, EXCELLENZ TM S 2000 and P (DuPont). In some embodiments, the Bacillus griseus variants provided herein may be combined with one or more amylases and their variants, and combinations of said amylases and their variants, said one or more amylases being selected from the group consisting of: AA707, AA560, AAI10, BspAmy24, and CspAmy1.

[0138] Further embodiments relate to compositions comprising one or more Bacillus subtilis protease variants and one or more cellulases described herein. In one embodiment, the composition comprises cellulases in amounts from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any suitable cellulase may 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: WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. Provisional Application No. 62 / 296,678. Exemplary commercial cellulases include, but are not limited to, those of bacterial or fungal origin, such as those described in: WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. Provisional Application No. 62 / 296,678. Exemplary commercial cellulases include, but are not limited to, those of [missing information]. and PREMIUM (Novozymes); REVITALENZ TM 100. REVITALENZ TM 200 / 220, and 2000 (DuPont); and KAC-500 (B) TM (Kao Corporation). In some embodiments, cellulase is incorporated as a portion or fragment of a mature wild-type or variant cellulase (in which a portion of the N-terminus is missing) (see, for example, US 5,874,276).

[0139] Even further embodiments involve compositions comprising one or more Bacillus subtilis protease variants and one or more mannanases described herein. In one embodiment, the composition comprises mannanase in amounts from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Exemplary mannanases may be chemically or genetically modified mutants. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described below: WO2016 / 007929; USPN 6,566,114, 6,602,842, and 6,440,991; and U.S. Provisional Applications 62 / 251516, 62 / 278383, and 62 / 278387. Exemplary commercial mannanases include, but are not limited to, those of bacterial or fungal origin. (Novozymes) and EFFECTENZ TM M 1000, EFFECTENZ TM M 2000 M 100 and PURABRITE TM (DuPont)

[0140] Further embodiments involve compositions comprising one or more Bacillus subtilis protease variants described herein and one or more nucleases (e.g., DNases or RNases). In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition, of the nuclease. Exemplary nucleases include, but are not limited to, those described below: WO2015181287, WO 2015155350, WO 2016162556, WO 2017162836, WO 2017060475 (e.g., SEQ ID NO:21), WO 2018184816, WO 2018177936, WO 2018177938, WO2018 / 185269, WO2018185285, WO 2018177203, WO 2018184817, WO 2019084349, WO 2019084350, WO2019081721, WO 2018076800, WO 2018185267, WO 2018185280 and WO 2018206553. Other nucleases that can be used in combination with the Bacillus subtilis protease variants provided herein in the compositions and methods provided herein include those described below: Nijland R, Hall MJ, Burgess JG (2010) Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase [Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase] PLoS ONE [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.

[0141] Furthermore, other embodiments involve compositions comprising one or more Bacillus subtilis protease variants described herein, and one or more peroxidases and / or oxidases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% of peroxidase or oxidase by weight of the composition. The peroxidase may be used in combination with hydrogen peroxide or a source thereof (e.g., percarbonate, perborate, or persulfate), and the oxidase may be used in combination with oxygen. Peroxidases and oxidases, alone or in combination with synergists, are used for "solution bleaching" (i.e., preventing textile dyes from transferring from one dyed fabric to another when fabrics are washed together in a washing solution) (see, for example, WO 94 / 12621 and WO95 / 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.

[0142] Another embodiment relates to a composition comprising one or more Bacillus subtilis protease variants and one or more perhydrolysins described herein, such as the perhydrolysins described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0143] In yet another embodiment, one or more subtilisin variants described herein and one or more additional enzymes contained in one or more compositions described herein may each be independently varied to about 10% by weight of the composition, wherein the balance of the cleaning composition is one or more auxiliary materials.

[0144] In some embodiments, one or more compositions described herein can be used as detergent additives, wherein the additives are in solid or liquid form. Such additive products are designed to complement and / or enhance the performance of conventional detergent compositions and can be added at any stage of the cleaning process. In some embodiments, the density of the laundry detergent composition ranges from about 400 to about 1200 g / L, while in other embodiments, it ranges from about 500 to about 950 g / L as measured at 20°C.

[0145] Some embodiments relate to laundry detergent compositions comprising one or more Bacillus subtilis protease variants described herein and one or more adjuvants selected from: surfactants, enzyme stabilizers, detergent-building compounds, polymeric compounds, bleaches, additional enzymes, foam inhibitors, dispersants, calcium soap dispersants, soil suspending agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry composition further comprises a fabric softener.

[0146] Other embodiments relate to manual dishwashing compositions comprising one or more Bacillus subtilis protease variants described herein and one or more auxiliary materials selected from: surfactants, organic polymeric compounds, foaming agents, group II metal ions, solvents, water-soluble agents, and additional enzymes.

[0147] Other embodiments involve one or more compositions described herein, wherein said compositions are tightly granular fabric cleaning compositions for washing colored fabrics or providing softness by means of washing capacity, or heavy-duty liquid (HDL) fabric cleaning compositions. Exemplary fabric cleaning compositions and / or methods of preparation are described in USPN 6,610,642 and 6,376,450. Other exemplary cleaning compositions are described, for example, in USPN 6,605,458, 6,294,514, 5,929,022, 5,879,584, 5,691,297, 5,565,145, 5,574,005, 5,569,645, 5,565,422, 5,516,448, 5,489,392, and 5,486,303, 4,968,451, 4,597,898, 4,561,998, 4,550,862, 4,537,706, 4,515,707, and 4,515,705.

[0148] In some embodiments, the cleaning composition comprises acidified particles or an aminocarboxylic acid builder. Examples of aminocarboxylic acid builders include aminocarboxylic acids, their salts, and derivatives. In some embodiments, the aminocarboxylic acid builder is an aminopolycarboxylic acid builder, such as glycine-N,N-diacetic acid or having the general formula MOOC-CHR-N(CH2COOM)2 (where R is C). 1-12Alkyl groups (where M is an alkali metal) are derivatives. In some embodiments, the aminocarboxylic acid builder may be methylglycine diacetic acid (MGDA), GLDA (glutamic acid-N,N-diacetic acid), iminodisuccinic acid (IDS), carboxymethyl inulin and its salts and derivatives, aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SMMA ... Glutamic acid (SEGL), IDS (iminodiacetic acid), and their 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), as well as their alkali metal salts and derivatives. In some embodiments, the acidified particles have a weight geometric mean particle size from about 400 μm to about 1200 μm and a bulk density of at least 550 g / L. In some embodiments, the acidified particles contain at least about 5% detergent builder.

[0149] In some embodiments, the acidified particles may comprise any acid, including organic acids and mineral acids. Organic acids may have one or two carboxyl groups and, in some cases, up to 15 carbons, particularly up to 10 carbons, such as formic acid, acetic acid, propionic acid, decanoic acid, oxalic acid, succinic acid, adipic acid, maleic acid, fumaric acid, sebacic acid, malic acid, lactic acid, glycolic acid, tartaric acid, and glyoxylic acid hydrate. In some embodiments, the acid is citric acid. Mineral acids include hydrochloric acid and sulfuric acid. In some cases, the acidified particles are highly reactive particles containing high levels of aminocarboxylic acid builders. Sulfuric acid has also been found to further contribute to the stability of the final particles.

[0150] Other embodiments relate to cleaning compositions comprising one or more Bacillus subtilis protease variants and one or more surfactants and / or surfactant systems, wherein said surfactants are selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, facultative zwitterionic surfactants, semipolar nonionic surfactants, and mixtures thereof. In some embodiments, the surfactant is present at a level from about 0.1% to about 60% by weight of the cleaning composition, while in alternative embodiments the level is from about 1% to about 50%, and in yet another embodiment the level is from about 5% to about 40%.

[0151] In some embodiments, one or more compositions described herein comprise one or more detergent builders or builder systems. In one embodiment, the composition comprises a builder in an amount of 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 the composition. Exemplary builders include, but are not limited to, alkali metals; ammonium salts and alkanol ammonium salts of polyphosphates; alkali metal silicates; alkaline earth metals and alkali metal carbonates; aluminosilicates; polycarboxylate compounds; ether hydroxy polycarboxylates; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethyloxysuccinic acid; ammonium salts and substituted ammonium salts of polyacetic acid, such as ethylenediaminetetraacetic acid and hypozoxytriacetic acid; polycarboxylate salts, such as phenylhexacarboxylic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, phenyl-1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid; and soluble salts thereof. In some such compositions, the builder forms a water-soluble hardness ion complex (e.g., a chelating builder), such as citrate and polyphosphate, such as sodium tripolyphosphate, sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixtures of sodium tripolyphosphate and potassium tripolyphosphate. Exemplary detergent builders are described, for example, in EP 2100949. In some embodiments, the detergent builder comprises a phosphate builder and a non-phosphate builder. In some embodiments, the detergent builder is a phosphate builder. In some embodiments, the detergent builder is a non-phosphate builder. In some embodiments, the detergent builder comprises a mixture of phosphate and non-phosphate builders. Exemplary phosphate builders include, but are not limited to, monophosphates, diphosphates, tripolyphosphates, or oligophosphates, including alkali metal salts of these compounds, including sodium salts. In some embodiments, the detergent builder may be sodium tripolyphosphate (STPP). Additionally, the composition may contain carbonates and / or citrates. Other suitable non-phosphate builders include polycarboxylic acids and their partially or fully neutralized salts, homopolymers and copolymers of monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts. In some embodiments, the salts of the above compounds include ammonium salts and / or alkali metal salts, namely lithium salts, sodium salts, and potassium salts, including sodium salts. Suitable polycarboxylic acids include acyclic, alicyclic, heterocyclic, and aromatic carboxylic acids, wherein in some embodiments they may contain at least two carboxyl groups, which in each case are separated from each other, and in some cases are separated by no more than two carbon atoms.

[0152] In some embodiments, one or more compositions described herein comprise one or more chelating agents. In one embodiment, the composition comprises a chelating agent in amounts from about 0.1% to about 15% or from about 3% to about 10% by weight of the composition. Exemplary chelating agents include, but are not limited to, copper, iron, manganese, and mixtures thereof.

[0153] In some embodiments, one or more compositions described herein comprise one or more deposition aids. Exemplary deposition aids include, but are not limited to, polyethylene glycol; polypropylene glycol; polycarboxylates; fouling-releasing polymers, such as polyethylene terephthalate; clays, such as kaolinite, montmorillonite, magnesia, illite, bentonite, and hydrous kaolinite; and mixtures thereof.

[0154] In other embodiments, one or more compositions described herein comprise one or more anti-redeposition agents or nonionic surfactants (which can prevent the redeposition of dirt) (see, for example, EP 2100949). For example, in ADW compositions, nonionic surfactants can be used for surface modification purposes (specifically for sheets) to prevent film formation and staining and to improve gloss. These nonionic surfactants can also be used to prevent the redeposition of dirt. In some embodiments, the nonionic surfactant can be an ethoxylated nonionic surfactant, an epoxy-terminated poly(alkoxylated) alcohol, or an amine oxide surfactant.

[0155] In some embodiments, one or more compositions described herein comprise one or more dye transfer inhibitors. Exemplary polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone, polyvinylimidazole, and mixtures thereof. In one embodiment, the composition comprises a dye transfer inhibitor from about 0.0001% to about 10%, about 0.01% to about 5%, or about 0.1% to about 3% by weight of the composition.

[0156] In some embodiments, one or more compositions described herein comprise one or more silicates. Exemplary silicates include, but are not limited to, sodium silicate, such as sodium disilicate, sodium metasilicate, and crystalline folin silicate. In some embodiments, the silicate is present at a level of from about 1% to about 20% or from about 5% to about 15% by weight of the composition.

[0157] In some, yet still additional, embodiments, one or more compositions described herein comprise one or more dispersants. Exemplary water-soluble organic materials include, but are not limited to, homopolymerized or copolymerized acids or salts thereof, wherein polycarboxylic acids comprise at least two carboxyl radicals separated from each other by no more than two carbon atoms.

[0158] In some additional 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 oligosaccharides, polysaccharides, and inorganic divalent metal salts (including alkaline earth metal salts, such as calcium salts). In some embodiments, the enzymes used herein are stabilized by a water-soluble source of zinc(II), calcium(II), and / or magnesium(II) ions present in the finished compositions providing the enzyme with such ions, as well as other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and vanadium(IV)). Chlorides and sulfates may also be used in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described, for example, in WO 07 / 145964. In some embodiments, reversible protease inhibitors may also be used, for example, in boron-containing compounds (e.g., borates, 4-formylphenylboronic acid, and phenylboronic acid derivatives (e.g., those described in WO 96 / 41859)) and / or peptide aldehydes (e.g., as further described in WO2009 / 118375 and WO 2013004636).

[0159] As previously described (WO 199813458, WO 2011036153, US 20140228274), peptide aldehydes can be used as protease stabilizers in detergent formulations. Examples of peptide aldehyde stabilizers are peptide aldehydes, ketones, or halomethyl ketones, and can be “N-terminated,” for example having a urea group, carbamate, or urea moiety, or “double N-terminated,” for example having a carbonyl group, urea group, oxalamide, thiourea group, dithiooxalamide, or thiooxalamide moiety (EP 2358857 B1). The molar ratio of these inhibitors to proteases can be from 0.1:1 to 100:1, for example 0.5:1–50:1, 1:1–25:1, or 2:1–10:1. Other examples of protease stabilizers are benzophenone or aniline benzoate derivatives, which may contain a carboxyl group (US 7,968,508 B2). The molar ratio of these stabilizers to proteases is preferably in the range of 1:1 to 1000:1, particularly 1:1 to 500:1, especially preferably from 1:1 to 100:1, and most particularly preferably from 1:1 to 20:1.

[0160] In some embodiments, one or more compositions described herein comprise one or more bleaching agents, bleaching activators, and / or bleaching catalysts. In some embodiments, one or more compositions described herein comprise one or more inorganic and / or organic bleaching compounds. Exemplary inorganic bleaching agents include, but are not limited to, hydroperoxide salts, such as perborates, percarbonates, superphosphates, persulfates, and persilicates. In some embodiments, the inorganic hydroperoxide salt is an alkali metal salt. In some embodiments, the salt is an inorganic hydroperoxide salt that is a crystalline solid without additional protection, but in some other embodiments, the salt is coated. Bleaching activators are typically organic peracid precursors that enhance bleaching during the cleaning process at temperatures of 60°C and below. Exemplary bleaching activators comprise compounds that, under hydrolytic conditions, give an aliphatic peroxycarboxylic acid having from about 1 to about 10 carbon atoms or from about 2 to about 4 carbon atoms, and / or optionally substituted peroxybenzoic acid. Exemplary bleaching activators are described, for example, in EP 2100949. Exemplary bleaching catalysts include, but are not limited to, manganese triazacyclononane and related complexes, as well as cobalt, copper, manganese and iron complexes. Further exemplary bleaching catalysts are described, for example, in US 4,246,612; US 5,227,084; US 4,810,410; WO 99 / 06521; and EP 2100949.

[0161] In some embodiments, one or more compositions described herein comprise one or more catalytic metal complexes. In some embodiments, a metal-containing bleaching catalyst may be used. In some embodiments, the metal bleaching catalyst comprises a catalytic system comprising: a transition metal cation (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation) having defined bleaching catalytic activity, an auxiliary metal cation (e.g., zinc or aluminum cation) having little or no bleaching catalytic activity, and a chelate having defined stability constants for both the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts (see, for example, US 4,430,243). In some embodiments, one or more compositions described herein are catalyzed by means of manganese compounds. Such compounds and levels of use are described, for example, in US 5,576,282. In further embodiments, a cobalt bleaching catalyst may be used and included in one or more compositions described herein. Various cobalt bleaching catalysts are described, for example, in USPN 5,597,936 and 5,595,967.

[0162] In some other embodiments, one or more compositions described herein comprise transition metal complexes of multicyclic rigid ligands (MRLs). As a practical matter and not a limitation, in some embodiments, the compositions and cleaning methods described herein are adapted to provide active MRLs in concentrations of at least parts per hundred million, 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 in the washing solution. Exemplary MRLs include, but are not limited to, special ultra-rigid ligands with cross-linking bridging, such as 5,12-diethyl-1,5,8,12-tetraazabicyclo(6.6.2)hexadecane. Exemplary metallic MRLs are described, for example, in WO 2000 / 32601 and US 6,225,464.

[0163] In another embodiment, one or more compositions described herein comprise one or more metal care agents. In some embodiments, the composition comprises from about 0.1% to about 5% by weight of the composition of a metal care agent. Exemplary metal care agents include, for example, aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). Further exemplary metal care agents are described, for example, in EP 2100949, WO 94 / 26860, and WO 94 / 26859. In some compositions, the metal care agent is a zinc salt.

[0164] In some embodiments, the cleaning composition is a heavy-duty liquid (HDL) composition comprising one or more Bacillus subtilis protease variants described herein. The HDL liquid laundry detergent may comprise a cleaning surfactant (10%-40%) comprising anionic cleaning surfactants selected from the group consisting of linear, branched, or random chains, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof; and optionally nonionic surfactants selected from the group consisting of linear, branched, or random chains, substituted or unsubstituted alkyl alkoxylated alcohols, such as C8-C64. 18 Alkyl ethoxylated alcohols and / or C6-C 12 Alkylphenol alkoxylates, optionally wherein the weight ratio of anionic cleaning surfactant (hydrophilic index (HIc) from 6.0 to 9) to nonionic cleaning surfactant is greater than 1:1. Suitable cleaning surfactants also include cationic cleaning surfactants (selected from alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and / or mixtures thereof); zwitterionic and / or amphoteric cleaning surfactants (selected from alkanolamine sulfobetaine); amphoteric surfactants; semi-polar nonionic surfactants; and mixtures thereof.

[0165] In another embodiment, the cleaning composition is a liquid or gel detergent (not a unit dose) that may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70%, about 65%, about 55%, about 45%, or about 35% water by weight. Other types of liquids (including, but not limited to, alkanols, amines, glycols, ethers, and polyols) may be included in the aqueous liquid or gel. The aqueous liquid or gel detergent may contain from 0% to 30% organic solvent. The liquid or gel detergent may be non-aqueous.

[0166] The composition may optionally comprise a surface-enhancing polymer consisting of: an amphiphilic alkoxylated oleocleaning polymer selected from the group consisting of: alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkylimides (in the range of 0.05 wt% to 10 wt%); and / or a random graft polymer, which typically comprises a hydrophilic backbone containing monomers selected from the group consisting of: unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydrides, saturated polyols (e.g., glycerol) and mixtures thereof; and one or more hydrophobic side chains selected from the group consisting of: C4 .... 25 Alkyl groups, polypropylene, polybutene, saturated C2-C6 monocarboxylic acid vinyl esters, acrylic acid or methacrylic acid C1-C6 alkyl esters and mixtures thereof.

[0167] The composition may include additional polymers, such as dirt-releasing polymers comprising, for example, anionic-terminated polyesters, such as SRP1; polymers in random or block configurations comprising at least one monomer unit selected from sugars, dicarboxylic acids, polyols, and combinations thereof; polymers in random or block configurations based on polyethylene terephthalate and their copolymers, such as Repel-o-tex SF, SF-2, and SRP6; Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325; and Marloquest. SL; anti-redeposition polymers (0.1 wt% to 10 wt%, including, for example, carboxylate polymers, such as polymers containing at least one monomer selected from: acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesoconic acid, citraconic acid, methylene malonic acid and any mixture thereof; vinylpyrrolidone homopolymers; and / or polyethylene glycol having a molecular weight in the range of 500 to 100,000 Da); cellulose polymers (including, for example, alkyl cellulose; alkylalkoxyalkyl cellulose; carboxyl cellulose; alkylcarboxyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose; and mixtures thereof); and polymeric carboxyl esters (e.g., maleate / acrylate random copolymers or polyacrylate homopolymers).

[0168] The composition may further comprise saturated or unsaturated fatty acids, preferably saturated or unsaturated C. 12 -C 24 Fatty acids (0-10 wt%); deposition aids in random or block configurations (including, for example, polysaccharides, cellulose polymers, polydiallyl dimethyl ammonium halide (DADMAC)), copolymers of DADMAC with vinylpyrrolidone, acrylamide, imidazole, halogenated imidazolines and mixtures thereof; cationic guar gum; cationic cellulose, such as cationic hydroxyethyl cellulose; cationic starch; cationic polyacrylamide; and mixtures thereof.

[0169] The composition may further comprise dye transfer inhibitors, examples of which include manganese phthalocyanine, peroxidase, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone and polyvinylimidazole and / or mixtures thereof; chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA); diethylenetriaminepentamethylenephosphonic acid (DTPMP); hydroxyethanediphosphonic acid (HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methylglycine diacetic acid (MGDA); diethylenetriaminepentaacetic acid (DTPA); propylenediaminetetraacetic acid (PDT). A); 2-hydroxypyridine-N-oxide (HPNO); or methylglycine diacetic acid (MGDA); N,N-diacetic acid (N,N-dicarboxymethylglutamate tetrasodium salt (GLDA); nitrotriacetic acid (NTA); 4,5-dihydroxyisophenylsulfonic acid; citric acid and any salt thereof; N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and its derivatives.

[0170] The composition may further comprise a silicone-based or fatty acid-based foam inhibitor; an enzyme stabilizer; a color dye; calcium and magnesium cations; a visual signal transducer; an antifoaming agent (0.001 wt% to about 4.0 wt%); and / or a structural agent / thickener (0.01 wt% to 5 wt%), wherein the structural agent / thickener is selected from the group consisting of: diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based materials, ultrafine cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.

[0171] In some embodiments, the cleaning composition is a heavy-duty detergent (HDD) composition comprising one or more variants of the Bacillus subtilis protease described herein. The HDD powder laundry detergent may comprise a cleaning surfactant, including anionic cleaning surfactants (selected from linear, branched, or random, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof); and nonionic cleaning surfactants (selected from linear, branched, or random, substituted or unsubstituted C8-C...). 18 Alkyl ethoxylates and / or C6-C 12Alkylphenol alkoxylates); cationic cleaning surfactants (selected from alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and mixtures thereof); zwitterionic and / or amphoteric cleaning surfactants (selected from alkanolamine sulfobetaine); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof; detergent builders (phosphate-free detergent builders, such as zeolite builders, examples of which include zeolite A, zeolite X, zeolite P, and zeolite MAP in the range of 0 wt% to less than 10 wt%); phosphate detergent builders, Examples include sodium tripolyphosphate in the range of 0 to less than 10 wt%; citric acid, citrates, and hypozinotriacetic acid or their salts in the range of less than 15 wt%; silicates (sodium silicate, potassium silicate, sodium metasilicate, or layered silicates (SKS-6) in the range of 0 wt% to less than 10 wt%); carbonates (sodium carbonate and / or sodium bicarbonate in the range of 0 wt% to less than 10 wt%); and bleaching agents (photobleaching agents, such as zinc phthalocyanine sulfonate, aluminum phthalocyanine sulfonate, thallium dyes, and mixtures thereof); hydrophobic or hydrophilic bleaching activators (e.g., dodecanoyl). Oxybenzenesulfonates, decyloxybenzenesulfonates, decyloxybenzoic acid or their salts, 3,5,5-trimethylhexanoyloxybenzenesulfonates, tetraacetylethylenediamine-TAED, and nonanoyloxybenzenesulfonates-NOBS, nitrile quaternary ammonium salts, and mixtures thereof); hydrogen peroxide; hydrogen peroxide sources (inorganic hydrogen peroxide salts, such as mono- or tetrahydrated sodium salts of perborates, percarbonates, persulfates, superphosphates, or persilicates); pre-prepared hydrophilic and / or hydrophobic peracids (selected from percarboxylic acids and salts, percarbonates and salts, persulfates ... Amino acids and their salts, peroxymonosulfate and their salts, and mixtures thereof; and / or bleaching catalysts (e.g., imine bleaching enhancers, such as imine cations and polyions; imine zwitterions; modified amines; modified amine oxides; N-sulfonyl imides; N-phosphonyl imides; N-acyl imides; thiadiazole dioxide; perfluoroimides; cyclic glycoketones and mixtures thereof); metal-containing bleaching catalysts (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations and auxiliary metal cations (e.g., zinc or aluminum) and chelates (e.g., ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and their water-soluble salts).

[0172] The composition may further comprise additional detergent ingredients, including fragrance microcapsules, starch-encapsulated fragrance modifiers, enzyme stabilizers, toners, additional polymers (including fabric integrity and cationic polymers), dye-locking ingredients, fabric softeners, brighteners (e.g., CI fluorescent brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids, and / or cyclodextrins.

[0173] In some embodiments, the cleaning composition is an ADW detergent composition comprising one or more Bacillus subtilis protease variants described herein. The ADW detergent composition may comprise two or more nonionic surfactants selected from: ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-terminated poly(alkoxylated) alcohols, and amine oxide surfactants, present in an amount of 0-10% by weight; and a builder in the range of 5%-60% (by weight), said builder comprising: phosphates (monophosphates, diphosphates, triphosphates, or oligophosphates), sodium tripolyphosphate-STPP, or phosphate-free builders (amino acid-based compounds, such as MGDA (methylglycine-diacetic acid) and its salts and derivatives, GLDA). (Glutamic acid-N,N-diacetic acid) and its salts and derivatives, IDS (iminodisuccinic acid) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives and mixtures thereof, hyponitrotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and β-alanine diacetic acid (B-ADA) and their salts), homopolymers and copolymers of polycarboxylic acids and their partially or completely neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts, in the range of 0.5% to 50% (by weight); sulfonated / carboxylated polymers (to provide dimensional stability to the product), in the range of about 0.1% to about 50% (by weight); in the range of about 0% (by weight). Drying aids ranging from 0.1% to 10% (selected from polyesters, particularly anionic polyesters, polycarbonate-, polyurethane-, and / or polyurea-, polyorganosiloxane compounds, or their reactive cyclic carbonate and urea-type precursor compounds, optionally with additional monomers having 3-6 functional groups (particularly acid, alcohol, or ester functional groups) that favor polycondensation, for which polycondensation is favored); silicates (sodium silicate or potassium silicate, such as sodium disilicate, sodium metasilicate, and crystalline succinate) ranging from about 1% to about 20% by weight; inorganic bleaching agents (e.g., hydroperoxide salts such as perborate, percarbonate, superphosphate, persulfate, and persilicate). And organic bleaching agents (e.g., organic peroxy acids, including diacid and tetraacryl peroxides, especially disperoxydodecanoic acid, disperoxytetradecanoic acid, and disperoxyhexadecanoic acid); bleaching activators—organic peracid precursors, in the range of about 0.1% to about 10% by weight; bleaching catalysts (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn and Fe bispyridineamines and related complexes, and cobalt(III) pentamineacetate and related complexes); metal care agents in the range of about 0.1% to 5% by weight (selected from benzotriazole, metal salts and complexes, and silicates); in about 0.01% to 5%.0 mg active enzyme / g ADW detergent composition range of enzymes (acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, aryl esterase, β-galactosidase, carrageenanase, catalase, cellobiase, cellulase, chondroitinase, keratinase, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucosylamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipid peroxidation). Oxygenases, mannanases, nucleases, oxidases, oxidoreductases, pectic acid lyases, pectin acetylesterases, pectinases, pentosanases, peroxidases, phenol oxidases, phosphatases, phospholipidases, phytases, polyesterases, polygalacturonases, other proteases, amylopectinases, reductases, rhamnogalacturonases, β-glucanases, tannic acidases, transglutaminases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, and mixtures thereof; and enzyme stabilizer components (selected from oligosaccharides, polysaccharides, and inorganic divalent metal salts).

[0174] The table below provides specific exemplary ADW compositions.

[0175] Exemplary ADW Composition

[0176]

[0177] Further embodiments relate to compositions and methods for treating fabrics (e.g., desizing textiles) using one or more of the Bacillus subtilis protease variants described herein. Fabric treatment methods are well known in the art (see, for example, US 6,077,316). For example, the hand feel and appearance of a fabric can be improved by including contacting the fabric with a solution of the variants described herein. The fabric can be treated with the solution under pressure.

[0178] One or more of the Bacillus subtilis protease variants described herein can be applied during or after weaving of textiles, during the desizing stage, or in one or more other fabric processing steps. During weaving of textiles, the yarn is exposed to considerable mechanical strain. Before weaving on a machine loom, the warp yarns are typically coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. One or more of the Bacillus subtilis protease variants described herein can be applied during or after weaving to remove the sizing starch or starch derivatives. After weaving, the variants can be used before further processing of the fabric to remove the sizing coating to ensure uniform and wash-resistant results. One or more Bacillus subtilis protease variants described herein can be used alone or in combination with other desizing chemicals and / or desizing enzymes as detergent additives (e.g., in aqueous compositions) to desizing fabrics (including cotton-containing fabrics). Amylases can also be used in combination with Bacillus subtilis protease variants in compositions and methods for producing a stonewashed appearance on indigo-dyed denim fabrics and garments. For garment production, fabrics can be cut and sewn into garments or apparel, which are then finished. In particular, different enzymatic finishing methods have been developed for denim production. The finishing of denim garments typically begins with an enzymatic desizing step, in which the garment is subjected to proteolytic enzymes to provide softness to the fabric and make the cotton more suitable for subsequent enzymatic finishing steps. One or more Bacillus subtilis protease variants described herein can be used in methods for finishing denim garments (e.g., “biosanding methods”), enzymatic desizing and providing softness to the fabric, and / or finishing methods.

[0179] This disclosure also provides a method for cleaning the surface of an article, the method comprising contacting the article with at least one variant of the subtilisin protease (or a composition comprising this subtilisin protease variant) provided herein. In some embodiments, the article may have, for example, protein stains on its surface. In some embodiments, protein stains may comprise egg or egg-based stains, such as crème brûlée, or other protein-containing substances. Example

[0180] Example 1. A variant of Bacillus subtilis protease comprising one, two, three, four or more amino acid substitutions selected from the group consisting of: X039E, X099R, X126A, X127E and X128G, and further comprising one or more additional substitutions at one, two, three or more positions selected from the group consisting of: 74, 85, 116, 160, 179, 198, 200, 207, 211, 212, 242, 253 and 256, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0181] Example 2. A variant of Bacillus subtilis protease as described in Example 1, wherein the variant comprises the following amino acid substitutions.

[0182] i) Select one or more substitutions from the group consisting of X039E, X099R, X126A, X127E and X128G;

[0183] ii) Combinations of substitutions selected from X039E-X099R, X039E-X126A, X039E-X127E, X039E-X128G, X099R-X126A, X099R-X127E, X099R-X128G, X126A-X127E, X126A-X128G, and X127E-X128G;

[0184] iii) Combinations of substitutions selected from X039E-X099R-X126A, X039E-X099R-X127E, X039E-X099R-X128G, X039E-X126A-X127E, X039E-X126A-X128G, X039E-X127E-X128G, X099R-X126A-X127E, X099R-X126A-X128G, X099R-X127E-X128G, and X126A-X127E-X128G;

[0185] iv) Combinations of substitutions selected from X039E-X099R-X126A-X127E, X039E-X099R-X126A-X128G, X039E-X099R-X127E-X128G, X039E-X126A-X127E-X128G, and X099R-X126A-X127E-X128G; and

[0186] The combination of v)X039E-X099R-X126A-X127E-X128G.

[0187] Example 3. A variant of Bacillus subtilis protease as described in Example 1 or 2, wherein one, two, three, or more additional substitutions are selected from the group consisting of: X074D, X085R, X116R, X160Q, X179Q, X198A / G / L / Q / R / S / T / V, X200L, X207Q, X211E / L / N / Q, X212Q / S, X242D, X253P, and X256E, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0188] Example 4. A *Bacillus gibelio* subtilis protease variant as described in any one of Examples 1-3, wherein the one or more additional substitutions comprise a combination of one or more substitutions selected from the group consisting of: X074D-X211L-X253P, X179Q-X211L-X253P, X074D-X253P, X085R-X160Q-X179Q-X211L-X212S-X253P, X179Q-X253P, X160Q-X179Q-X211L-X212S-X253P, X179Q-X211L, X160Q-X179Q-X211L-X253P, X160Q-X179Q-X211L-X253P, X160Q-X179Q-X211L-X253P, X160Q-X179Q-X21 2S-X253P, X074D-X211L, X211L-X242D, X160Q-X179Q-X211L-X212S, -X179Q-X211L-X253P, X160Q-X179Q-X211L, X160Q-X179Q-X253P, X074D-X 200L-X211L, X074D-X160Q-X212S-X253P, 0Q-X179Q, X160Q-X179Q-X212S, X074D-X160Q-X253P, X211L-X212S-X253P, X074D-X085R-X160Q-X179Q-X211L, X074D-X160Q-X2 11L-X212S-X253P, X074D-X085R-N116R-X200L-X256E, X074D-X160Q-X179 Q-X212S-X253P, X074D-X160Q-X211L-X212S, X074D-X160Q, X074D-X160Q- X179Q-X211L-X253P, X074D-X179Q-X211L, X074D-X160Q-X212S, X074D-X16 0Q-X211L, X074D-X160Q-X179Q-X253P, X074D, -X212S, X074D-X085R-X211L-X212S, X074D-X160Q-X179Q-X212S, 160Q-X179Q-X211L, X074D-X211L-X256E, X074D-X160Q-X179Q, X179Q-X21 1L-X212S-X253P, X179Q-X211L-X212S,X074D-X211L-X212S、X074D-X179Q-X211L-X212S、X074D-X211L-X242D、X074D-X200L-X211L-X256E、X074D-X200L-X211L-X242D-X256E、X074D-X200L、X074D-X211N、X074D-X211N-X212Q、X074D-X211N-X212Q-X256E、X074D-X211N-X256E、X074D-X211Q、X074D-X211Q-X212Q、X074D-X211Q-X212Q-X256E、X074D-X211Q-X256E、X074D-X198A-X211Q、X074D-X198A-X211Q-X212Q、X074D-X198A-X211Q-X256E、X074D-X198G-X211Q、X074D-X198G-X211Q-X212Q、X074D-X198G-X211Q-X256E、X074D-X198K-X211Q-X212Q、X074D-X198L-X211Q-X212Q、X074D-N198Q-X211Q-X212Q、X074D-X198R-X211Q-X212Q、X074D-X198T-X211Q-X212Q、X074D-X198V-X211Q-X212Q、X074D-X212Q-X256E、X074D-X256E、X074D-X207Q、X074D-X207Q-X211N、X074D-X207Q-X211N-X212Q、X074D-X207Q-X211N-X212Q-X256E、X074D-X207Q-X211N-X256E、X074D-X207Q-X211Q、X074D-X207Q-X211Q-X212Q、X074D-X207Q-X211Q-X212Q-X256E、X074D-X207Q-X212Q、X074D-X207Q-X212Q-X256E、X074D-X207Q-X256E、X074D-X198S-X211Q、X074D-X198L-X211Q、X211E、X211Q、X212Q-X242D、X211Q-X212Q、X211E-X212Q-X242D、X198A-X211Q-X212Q、X074D-X198A-X211Q-X212Q and X074D-X198A-X211Q-X212Q, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:1.

[0189] Example 5. A variant of Bacillus subtilis protease as described in any of the preceding examples, wherein the variant is derived from a parent polypeptide or reference polypeptide having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:1 or 2.

[0190] Example 6. A variant of Bacillus subtilis protease as described in any of the preceding examples, wherein the variant comprises an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with SEQ ID NO:1 or 2.

[0191] Example 7. A variant of Bacillus subtilis protease as described in any of the preceding examples, wherein the variant has one or more improved properties when compared with the parent or reference Bacillus subtilis protease; wherein the improved properties are selected from improved detergent cleaning performance, improved stability, and combinations thereof.

[0192] Example 8. A variant of Bacillus subtilis protease as described in Example 7, wherein the improved properties are

[0193] (i) Improved detergent cleaning performance, wherein said variant has a cleaning PI of ≥1.1 for French caramel pudding stains and / or egg stains compared to Bacillus subtilis protease having the amino acid sequence of SEQ ID NO:2; and / or

[0194] (ii) Improved stability, wherein the variant has a stability PI of ≥1.1 compared to the subtilisin having the amino acid sequence of SEQ ID NO:2.

[0195] Example 9. A variant of Bacillus subtilis protease as described in any one of Examples 7 or 8, wherein...

[0196] (i) The cleaning performance of the detergent was measured according to the cleaning performance in the ADW detergent test of Example 2; and / or

[0197] (ii) Stability was measured according to the stability determination in Example 2.

[0198] Example 10. An enzyme composition comprising one or more Bacillus subtilis protease variants as described in any of the preceding examples.

[0199] Example 11. An enzyme composition as described in Example 9, wherein the composition is a granule, liquid formulation, or slurry.

[0200] Example 12. An enzyme composition comprising one or more Bacillus subtilis protease variants as described in any of the preceding examples and further comprising at least one additional enzyme selected from the group consisting of: acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, aryl esterases, β-galactosidases, carrageenanases, catalases, cellobiases, cellulases, chondroitinases, keratinases, endo-β-1,4-glucanases, endo-β-mannanases, esterases, exo-mannanases, galactanases, glucosylamylases, hemicellulases, and clear enzymes. Hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectic acid lyase, pectin acetylesterase, pectinase, pentosanase, hydrolase, peroxidase, phenol oxidase, phosphatase, phospholipase, phytase, polygalacturonase, polyesterase, other proteases, amylopectinase, reductase, rhamnogalacturonase, β-glucanase, tannic acidase, transglutaminase, xylan acetylesterase, xylanase, xyglucanase, xylosidase, and any combination or mixture thereof.

[0201] Example 13. An enzyme composition as described in Example 12, wherein one or more enzymes comprise amylases and variants thereof, and combinations of said amylases and variants thereof, said amylases being selected from the group consisting of: AA707, AA560, AAI10, BspAmy24 and CspAmy1.

[0202] Example 14. A method for removing protein stains or dirt from a surface, the method comprising contacting the surface with an effective amount of a Bacillus subtilis protease variant as described in any one of Examples 1-9 or an enzyme composition as described in any one of Examples 10-13.

[0203] Example 15. The method as described in Example 14, wherein the protein stain or dirt comprises egg.

[0204] Example 16. A nucleic acid encoding a Bacillus subtilis protease variant as described in any one of Examples 1-9.

[0205] Example 17. A host cell comprising the nucleic acid as described in Example 16.

[0206] The following examples are provided to demonstrate and illustrate certain preferred embodiments and aspects of this disclosure, and should not be construed as limiting.

[0207] Example

[0208] Example 1

[0209] Expression of BG46 Bacillus subtilis protease variant

[0210] SEQ ID NO:1 provides for Bacillus subtilis protease (BG46) of wild-type Bacillus subtilis Bgi02446. In this study, a BG46 Bacillus subtilis protease variant (SEQ ID NO:2) with substitutions of S039E, S099R, S126A, D127E, and F128G was used as a starting point in the engineered variant with further substitutions and was designated BG46+S039E-S099R-S126A-D127E-F128G. In some studies, the prepared BG46 variant contained a subset of substitutions of S039E, S099R, S126A, D127E, and F128G. All BG46 Bacillus subtilis protease variants were expressed using a DNA fragment comprising, in sequence: a 5' AprE flanking region containing a variant of the Bacillus subtilis rrnIp2 promoter sequence (SEQ ID NO:3) (the Bacillus subtilis rrnIp2 promoter and the engineered variant are more fully described in patent application No. 62 / 772363, filed November 28, 2018); a nucleotide sequence encoding the aprE signal peptide sequence (SEQ ID NO:4); a nucleotide sequence encoding the Bacillus tarda propeptide (SEQ ID NO:5); a sequence corresponding to the gene encoding the mature BG46 Bacillus subtilis protease; a BPN' terminator (SEQ ID NO:6); and a 3' AprE flanking sequence comprising the kanamycin gene expression cassette (SEQ ID NO:7). This DNA fragment was assembled using standard molecular biotechnology. The linear DNA containing the expression cassette was used to transform competent Bacillus subtilis cells of suitable strains.

[0211] The conversion mixture was plated onto LA plates containing 1.6% skim milk and 1.8 ppm kanamycin and incubated overnight at 37°C. Single colonies were picked and grown in Luria broth at 37°C under antibiotic selection.

[0212] For protein expression experiments, transformed cells were grown for 3 days in 96-well MTP culture media (a semi-limited medium enriched with MOP buffer, using urea as the primary nitrogen source, glucose as the primary carbon source, supplemented with 1% soybean peptone for robust cell growth, and containing antibiotic selection) at 32°C, 300 rpm, and 80% humidity in a shaking incubator. After centrifugation and filtration, the clarified culture supernatant containing the target protease was used for assays.

[0213] Example 2

[0214] Measurement

[0215] Protein identification

[0216] The concentration of the BG46 Bacillus subtilis protease variant in the culture supernatant was determined by UHPLC using a Zorbax 300SB-C3 column with a linear gradient of 0.1% trifluoroacetic acid (buffer A) and 0.07% trifluoroacetic acid in acetonitrile (buffer B), and detection at 220 nm. The culture supernatant was diluted with 10 mM NaCl, 0.1 mM CaCl2, and 0.005% Tween 80 before loading onto the column. The protein concentration of the sample was calculated using a standard curve of the purified parental enzyme.

[0217] Protease activity

[0218] The protease activity of the BG46 Bacillus subtilis protease variant was tested by measuring the hydrolysis of the AAPF-pNA synthetic peptide substrate.

[0219] For the AAPF assay, the reagent solution used was: 100 mM Tris pH 8.6, 10 mM CalCl2, 0.005% The working solution was prepared using Tris / Ca buffer and 160 mM suc-AAPF-pNA stock solution in DMSO (Sigma-Aldrich: S-7388). To prepare the working solution, 1 mL of suc-AAPF-pNA stock solution was added to 100 mL of Tris / Ca buffer and mixed. The enzyme sample was added to a microtiter plate (MTP) containing 1 mg / mL suc-AAPF-pNA working solution, and the activity was measured kinetically at 405 nm over 3–5 min at room temperature using a SpectraMax plate reader. Protease activity was expressed as mOD / min.

[0220] Tris-EDTA Stability determination

[0221] The stability of the BG46 subtilis protease variant described herein was measured as follows: the variant was diluted in stress buffer and its proteolytic activity was measured before and after the heating incubation step using AAPF as described above. The temperature and duration of the heating incubation step were selected such that the reference protease showed approximately 15%–30% residual activity. Samples were incubated at 57°C for 5 min in a 384-well thermal cycler. Stability was measured under Tris-EDTA (50 mM Tris pH 9; 5 mM EDTA; 0.005% Tween 80) buffer. The stability PI was obtained by dividing the residual activity of the subtilis protease variant by the residual activity of the parent protease BG46-S039E-S099R-S126A-D127E-F128G. Alternatively, stability results were calculated as a percentage (%) of residual activity for each enzyme sample, calculated by taking the ratio of mOD / min under stress conditions to that under no stress conditions and multiplying by 100.

[0222] Automatic dishwashing and cleaning measurement

[0223] French Caramel Pudding Stains: As described in this article, the cleaning performance of a BG46 Bacillus subtilis protease variant on French caramel pudding stains was tested using a custom-ordered melamine dishwasher monitor (tile) prepared by CFT (in Vlaardingen, Netherlands) and labeled DM10c. The DM10c tiles used in this study were prepared using the same stain, but not at 150°C; instead, they were baked at 140°C for 2 hours. The same stain was used to prepare the commercially available DM10 monitor (a product from Debic.com).

[0224] A DM10c melamine brick was used as a cap and pressed firmly onto a microtiter plate (MTP). A 3 g / L ADW detergent solution was adjusted to a water hardness of 374 ppm, and each enzyme sample was added to the MTP before attaching the melamine brick cap. The volumetric capacity of the MTP and therefore the volume of solution added thereto can vary, but a minimum volume of solution should be added to the MTP to allow contact between the solution and the stained surface. In this example, 300 μL of enzyme-containing detergent was added to each well of an aluminum 96-well MTP. Unless otherwise specified, the MTP was incubated in an Inforrs thermal shaker at 40°C and 250 rpm for 45 min. After incubation, the brick was removed from the MTP, rinsed briefly with tap water, and air-dried.

[0225] Stain removal was quantified by photographing these boards and measuring the RGB values ​​from each stained area using custom software. The percentage of stain removal (%SRI) of the washed bricks was calculated using the RGB values ​​in the following formula:

[0226] %SRI=(ΔE / ΔE) 初始 )*100

[0227] Where ΔE=SQR((R 之后 -R 之前 ) 2 +(G 之后 -G 之前 ) 2 +(B 之后 -B 之前 ) 2 )

[0228] Where ΔE 初始 =SQR((R 白色 -R 之前 ) 2 +(G 白色 -G 之前 ) 2 +(B 白色 -B 之前 ) 2 )

[0229] Cleanliness performance (hereinafter referred to as "cleanliness minus blank") is obtained by subtracting the value of the blank control (enzyme-free) from each sample value. For each condition and BG46 Bacillus subtilis protease variant, the performance index (PI) is calculated by dividing the cleanliness minus blank by the cleanliness of the same concentration of parental protease. The value of the parental protease PI is determined from a standard curve of the parental protease included in the test and fitted to a Langmuir fit or a Hill sigmoid fit.

[0230] Egg Yolk Staining: The cleaning performance of the BG46 Bacillus subtilis protease variant on egg yolk microsamples (PAS-38, Center for Testmaterials BV, Vladivostok, Netherlands) was measured on pre-washed or unwashed samples. To prepare the washed PAS38 samples, 180 μl of 10 mM CAPS buffer (pH 11) was added to the MTP containing the PAS38 microsamples. The plates were sealed and incubated in an iEMS incubator at 60 °C and 1100 rpm for 30 min with shaking. After this incubation, the buffer was removed, and the samples were rinsed with deionized water to remove any residual buffer. The plates were then air-dried before use for performance assays. Before adding the enzyme, the microsample plates were packed with a 3 g / L ADW detergent solution with a water hardness of 374 ppm, resulting in a final enzyme concentration between 0.05 and 10 ppm.

[0231] After incubating PAS-38 samples with detergent and enzyme at 40°C for 30 minutes, aliquots were transferred to empty MTP plates, and absorbance was read at 405 nm using a SpectraMax plate reader. Absorbance results were obtained by subtracting the value of the blank control (enzyme-free) from each sample value (hereinafter referred to as "Absorbance minus blank"). For each condition and BG46 Bacillus subtilis protease variant, the performance index (PI) was calculated by dividing the absorbance minus blank by the absorbance of the same concentration of the parent protease BG46+S039E-S099R-S126A-D127E-F128G (SEQ ID NO:2).

[0232] Detergent

[0233] Various detergent formulations were used, as listed below. Automatic dishwashing (ADW) cleaning tests were conducted using the following detergents at final concentrations shown in parentheses: GSM-B detergent (3 g / L) (enzyme-free GSM-B phosphate-free ADW detergent, from WFK Testgewebe GmbH, Brüggen, Germany) www.testgewebe.de Purchase (Table 1 shows the composition) and MGDA detergent (3g / L) (Table 2 shows the composition).

[0234]

[0235]

[0236] Example 3

[0237] Automatic dishwashing performance and stability of BG46 Bacillus subtilis protease variant

[0238] In most cases, the Bacillus gibsiensis Bgi02446 subtilis protease variant (BG46) (SEQ ID NO:2) with substitutions S039E-S099R-S126A-D127E-F128G was used as the parent for evaluating additional substitutions, while in some cases, the wild-type parent of the Bacillus gibsiensis Bgi02446 subtilis protease variant (BG46) was used as the reference enzyme. Example 1 describes the expression of these proteins. The ADW cleaning performance of these BG46 subtilis protease variants on egg (PAS-38) and crème brûlée (DM10c) technical stains, and the stability (Tris / EDTA) of the variants, were measured using the detergents and assays described in Example 2, and the results are reported in Tables 3, 4, 5, and 6. Cleanliness benefits and stability are expressed as PI values ​​relative to the parental enzymes BG46+S039E-S099R-S126A-D127E-F128G in Tables 3, 4, and 6. Table 5 shows data for variants compared to the wild-type parent BG46, where cleanliness benefits are expressed as PI values ​​and stability as a percentage of residual activity.

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] Although this disclosure has been described in conjunction with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Therefore, this disclosure is intended to cover all such alternatives, modifications, and variations falling within the spirit and broad scope of the appended claims.

[0248] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the same extent as if each individual publication, patent, or patent application were expressly and individually incorporated herein by reference. Furthermore, any reference or designation of any reference in this application should not be construed as an admission that such reference is available as prior art to this disclosure. The use of section headings should not be construed as a necessary limitation.

Claims

1. A variant of Bacillus gibsonii subtilis protease, which differs from the parental subtilis protease in that the amino acid substitutions S039E-S099R-S126A-D127E-F128G and N074D-N198A-M211Q-N212Q are used, wherein the amino acid sequence of the parental subtilis protease is as shown in SEQ ID NO:1 or 2.

2. The subtilisin variant of claim 1, wherein the variant has one or more improved properties when compared with parental subtilisin or reference subtilisin; wherein the improved properties are selected from improved detergent cleaning performance, improved stability, and combinations thereof.

3. An enzyme composition comprising one or more variants of Bacillus subtilis protease as described in any of the preceding claims.

4. The enzyme composition of claim 3, wherein the composition is enzyme particles.

5. The enzyme composition of any one of claims 3 or 4, further comprising one or more other enzymes selected from: acyltransferases, amylases, α-galactosidases, arabinogalactanases, arabinosidases, β-galactosidases, carrageenanases, chondroitinases, esterases, exomannanases, galactanases, hemicellulases, hyaluronidases, ligninases, lipases, lipoxygenases, nucleases, oxidases, pectic acid lyases, pentosanases, hydrolases, phosphatases, phospholipases, polyesterases, polygalacturonases, reductases, rhamnogalacturonases, cellulases, transglutaminases, and xylosidases, additional proteases, and combinations thereof.

6. The enzyme composition of claim 5, wherein the oxidase is an oxidoreductase.

7. The enzyme composition of claim 5, wherein the oxidase is a peroxidase.

8. The enzyme composition of claim 5, wherein the reductase is an oxidoreductase.

9. The enzyme composition of claim 5, wherein the amylase is α-amylase, β-amylase, glucosylamylase, or amylopectin.

10. The enzyme composition of claim 5, wherein the esterase is an aryl esterase, lipase, keratinase, pectin acetylesterase, pectinase, tannic acidase, or xylan acetylesterase.

11. The enzyme composition of claim 5, wherein the additional protease is a keratinase.

12. The enzyme composition of claim 11, wherein the keratinase is a metalloproteinase.

13. The enzyme composition of claim 5, wherein the phosphatase is phytase.

14. The enzyme composition of claim 7, wherein the peroxidase is catalase.

15. The enzyme composition of claim 5, wherein the oxidase is laccase or phenol oxidase.

16. The enzyme composition of claim 5, wherein the cellulase is β-glucanase or xylanase.

17. The enzyme composition of claim 5, wherein the mannanase is an endo-β-mannanase or an exo-β-mannanase.

18. The enzyme composition of claim 5, wherein the hemicellulase is a mannanase.

19. The enzyme composition of claim 5, wherein the one or more enzymes comprise an amylase selected from the group consisting of: AA707, AA560, AAI10, BspAmy24, and CspAmy1.

20. A polynucleotide comprising a nucleic acid sequence encoding a variant as claimed in claim 1 or 2.

21. The polynucleotide of claim 20, wherein the nucleic acid sequence is effectively linked to a promoter.

22. The polynucleotide of claim 20, wherein the polynucleotide is isolated.

23. An expression vector or expression cassette comprising the polynucleotide as described in any one of claims 20-22.

24. A recombinant host cell comprising the polynucleotide as described in any one of claims 20-22 or the expression vector or expression cassette as described in claim 23.

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