Protease variants and polynucleotides encoding same

Abstract

The present invention relates to protease variants. The invention also relates to polynucleotides encoding the variants, nucleic acid constructs, vectors and host cells comprising the polynucleotides, detergent compositions comprising the variants, and the use of the variants in cleaning processes.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202380081615.9, filed on December 4, 2023, with a priority date of December 5, 2022, entitled "Protein Variant and Polynucleotide Encoding Thereof".

[0002] References to sequence lists

[0003] This application contains a sequence list in computer-readable form, which is incorporated herein by reference. Technical Field

[0004] This invention relates to protease variants. It also relates to polynucleotides encoding these variants, nucleic acid constructs, vectors, and host cells containing these polynucleotides, detergent compositions containing these variants, and the use of these variants in cleaning processes. Background Technology

[0005] Enzymes have been used in detergent compositions for decades in the detergent industry. Enzymes used in such compositions include proteases, lipases, amylases, cellulases, mannanases, and other enzymes or mixtures thereof. Commercially, proteases are the most important enzymes.

[0006] An increasing number of commercially available proteases used in products such as laundry and dishwashing detergents are engineered variants of naturally occurring wild-type proteases. Protease variants with alterations relative to the parent protease have been described in the art, resulting in improvements such as better washing performance, thermal stability, storage stability, and catalytic activity.

[0007] However, several factors make further improvements to proteases advantageous. For example, washing conditions (such as temperature and pH) tend to vary over time and differ across different countries or regions worldwide. Additionally, many stains remain difficult to remove completely under conventional washing conditions. Therefore, new protease variants with improved washing performance under various conditions remain commercially relevant. Furthermore, there is a need for new protease variants with improved storage stability to maintain sufficient proteolytic activity after storage in detergent compositions for a period of time (during the manufacturing and distribution of the detergent composition and / or after storage before end-consumer use), thereby preserving the washing performance of the protease variant. Moreover, there is a need for new protease variants exhibiting improved mildness towards concomitant enzymes (i.e., other enzymes present in the detergent composition) to avoid unnecessary proteolytic degradation and maintain the enzymatic activity of such enzymes. Finally, there is a need for new protease variants with improved stain removal rates, as they act more quickly and are therefore particularly suitable for cleaning methods involving shorter wash cycles, such as laundry and dishwashing methods. Summary of the Invention

[0008] This invention relates to protease variants with improved properties. The variants of this invention exhibit improved washing performance and improved storage stability under several different temperature and pH conditions. The variants of this invention also provide improved mildness to accompanying enzymes, which expands the compatibility of the variants of this invention with other enzymes and reduces the need for co-formulation with protease inhibitors. The variants of this invention also exhibit improved stain removal rates, making them particularly suitable for cleaning methods with short wash cycles.

[0009] Variants of the present invention are particularly suitable for high-pH liquid detergents and laundry soap bars with a pH of 10 or higher. In high-pH liquid detergents, variants of the present invention exhibit improved washing performance and improved storage stability. When included in laundry soap bars, variants of the present invention provide improved removal of protein stains and reduce the need for stabilizers, which further reduces the production costs associated with the manufacture of laundry soap bars.

[0010] In a first aspect, the present invention relates to a variant of a parent protease, wherein the variant comprises substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprises substitutions at at least three, for example at least four, at least five, at least six, at least seven, at least eight, or nine positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein the position numbers are based on the numbering of SEQ ID NO:1; wherein the variants have a TM score of at least 0.80 but less than 1.0 compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold; and wherein the variant has protease activity.

[0011] In a second aspect, the present invention relates to a variant of a parent protease, wherein the variant comprises substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprises substitutions at at least three positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein the position numbering is based on the numbering of SEQ ID NO:1; wherein the variant has at least 60% but less than 100% sequence identity with the parent protease; and wherein the variant has protease activity.

[0012] In a third aspect, the present invention relates to a polynucleotide that encodes a variant described in the first or second aspect.

[0013] In a fourth aspect, the present invention relates to a nucleic acid construct or expression vector comprising the polynucleotides described in the third aspect.

[0014] In a fifth aspect, the present invention relates to a recombinant host cell that contains a nucleic acid construct or expression vector according to the fourth aspect in its genome.

[0015] In a sixth aspect, the present invention relates to a method for obtaining a variant according to a first or second aspect, the method comprising (a) introducing a parental protease substitution at positions 95 and 209 corresponding to SEQ ID NO:1, and further introducing the substitution at at least three, for example at least four, at least five, at least six, at least seven, at least eight or nine, positions corresponding to any one of SEQ ID NO:1, 42, 74, 199, 200, 203, 253, 255 and 256; wherein the position numbering is based on the numbering of SEQ ID NO:1; wherein the variant has protease activity; and (b) recovering the variant.

[0016] In a seventh aspect, the present invention relates to a method for producing a variant as described in the first or second aspect, the method comprising (a) culturing a recombinant host cell as described in the fifth aspect under conditions suitable for expressing the variant; and (b) recovering the variant.

[0017] In an eighth aspect, the present invention relates to a detergent composition comprising a variant described in the first or second aspect.

[0018] In a ninth aspect, the present invention relates to a method for cleaning an object, the method comprising contacting the object with the detergent composition described in the eighth aspect under conditions suitable for cleaning the object.

[0019] In a tenth aspect, the present invention relates to the use of the detergent composition described in the first or second aspect, or the eighth aspect, in a cleaning process. Attached Figure Description

[0020] Figure 1 This is a comparison of the peptides in SEQ ID NO:1 and SEQ ID NO:2.

[0021] Figure 2 This is a comparison of the peptides in SEQ ID NO:1 and SEQ ID NO:3.

[0022] Figure 3 This is a comparison of the peptides in SEQ ID NO:1 and SEQ ID NO:4.

[0023] Figure 4 This is a comparison of the peptides in SEQ ID NO:1 and SEQ ID NO:5.

[0024] Figure 5 This is a comparison of the peptides in SEQ ID NO:1 and SEQ ID NO:6.

[0025] Figure 6 This is a comparison of the peptides in SEQ ID NO:1 and SEQ ID NO:7.

[0026] Figure 7 This is an overview of the corresponding substitutions in SEQ ID NO:1-7.

[0027] Sequence Overview

[0028] SEQ ID NO:1 is Savinase (Savinase®), a subtilis protease from Bacillus lentus.

[0029] SEQ ID NO:2 is BPN', a subtilisin from Bacillus amyloliquefaciens.

[0030] SEQ ID NO:3 is Carlsberg (Alcalase®), a subtilis protease from Bacillus licheniformis.

[0031] SEQ ID NO:4 is a protease from Bacillus gibsonii.

[0032] SEQ ID NO:5 is a protease from Bacillus giganteus.

[0033] SEQ ID NO:6 is a protease from Bacillus sp. TY-145.

[0034] SEQ ID NO:7 is a protease from keratin-degrading Actinomadura keratinilytica.

[0035] SEQ ID NO:8 is a stable variant of SEQ ID NO:1 with the following substitutions: S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E.

[0036] SEQ ID NO:9 is SEQ ID NO:1 with the following substitutions: S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W and L256E.

[0037] SEQ ID NO:10 is SEQ ID NO:3 with the following substitutions: A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S and S258P.

[0038] SEQ ID NO:11 is SEQ ID NO:3 with the following substitutions: P9E, N43R, V204I, Y205L, Y208W, S258D, F260W and Y261E.

[0039] SEQ ID NO:12 is SEQ ID NO:3 with the following substitutions: P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W and Y261E.

[0040] SEQ ID NO:13 is SEQ ID NO:5 with the following substitutions: T9E, N42R, N74D, V199I, Q200L, Y203W, N253D, S255W and Q256E.

[0041] SEQ ID NO:14 is SEQ ID NO:5 with the following substitutions: T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D and S255W.

[0042] definition

[0043] Based on this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural indicators unless the context explicitly indicates otherwise.

[0044] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] Protease: The term "protease" refers to an enzyme that catalyzes the hydrolysis of peptide bonds and possesses peptidase activity (EC 3.4; also known as proteolytic activity or protease activity). The EC 3.4 group comprises several subgroups, including EC 3.4.21 (serine endopeptidase), which further contains several subgroups, including EC 3.4.21.62 (subtilisin). For the purposes of this invention, protease activity can be determined according to protease activity assay I or protease activity assay II described in the examples herein.

[0046] AlphaFold structure prediction: AlphaFold is a computational method for predicting the three-dimensional structure of peptides based on their amino acid sequences (Jumper et al., Highly accurate protein structure prediction with AlphaFold. Nature, 2021). The predicted structures of millions of peptides in the UniProt database are already available in the AlphaFold protein structure database, using the AlphaFold monomer v2.0 model (Varadi et al., AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research, 2021). In the AlphaFold protein structure database, the three-dimensional structure of a peptide can be obtained by searching for its UniProt accession number.

[0047] In addition to many publicly available 3D structures, the code can be used to reproduce and predict the structures of novel peptides in source code repositories, such as notebooks / AlphaFold.ipynb at deepmind / alphafold / Github.com. It can also be found at sokrypton / ColabFold / Github.com using version v1.5.2 or later (AlphaFold2.ipynb). For technical details, see Jumper et al. (see above).

[0048] AlphaFold generates per-residue estimates of its confidence level on a scale of 0 to 100. This confidence level metric is called pLDDT and corresponds to the model's prediction score on the lDDT-Cα index. It is stored in the B-factor field of the downloadable mmCIF and PDB files (although, unlike the B-factor, a higher pLDDT score is better). Regions with pLDDT scores above 90 are expected to be modeled with high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with pLDDT scores between 70 and 90 are expected to be well-modeled, corresponding to generally good main-chain predictions.

[0049] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks intron sequences that can be present in the corresponding genomic DNA. The initial primary RNA transcript is the precursor of mRNA, which is processed through a series of steps (including splicing) to become mature, spliced ​​mRNA.

[0050] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly defines the amino acid sequence of a variant. The boundaries of a coding sequence are typically defined by an open reading frame (OPF), which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0051] Control Sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of polynucleotides in a particular organism, either in vivo or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from a different gene) for the polynucleotide encoding a variant, and is native or heterologous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided with adapters for the purpose of introducing specific restriction sites that facilitate the connection of the control sequence to the coding region of the polynucleotide encoding the variant.

[0052] Expression: The term “expression” includes any steps involved in variant generation, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0053] Expression vector: An expression vector is a linear or circular DNA construct containing a DNA sequence that encodes a variant, operatively linked to a suitable control sequence that can influence the expression of the DNA in a suitable host. Such control sequences may include promoters that influence transcription, optional operon sequences that control transcription, sequences encoding suitable ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0054] Extension: The term "extension" refers to the addition of one or more amino acids to the amino and / or carboxyl termini of a variant, wherein the "extended" variant has protease activity.

[0055] Fragment: The term "fragment" refers to a variant in which one or more amino acids are missing from the amino and / or carboxyl terminus; wherein the fragment has protease activity.

[0056] Fusion polypeptide: The term "fusion polypeptide" is a polypeptide in which one polypeptide is fused to the N-terminus and / or C-terminus of a variant of the present invention. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide with a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences of the polypeptides such that they conform to reading frames, and that the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intronomer technology, wherein the fusion polypeptide is generated post-translational (Cooper et al., 1993, EMBO J. [Journal of the European Society for Molecular Biology] 12: 2575-2583; Dawson et al., 1994, Science [Science] 266: 776-779). Fusion polypeptides may further include a cleavage site between the two polypeptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology and Biotechnology] 3: 568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63: 3488-3493; Ward et al., 1995, Biotechnology [Biotechnology] 13: 498-503; and Contreras et al., 1991, Biotechnology [Biotechnology] 9: 378-381; Eaton et al., 1986, Biochemistry [Biochemistry] 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0057] Heterogeneous: For host cells, the term "heterogeneous" means that the polypeptide or nucleic acid is not naturally present in the host cell. For polypeptides or nucleic acids, the term "heterogeneous" means that the control sequence (e.g., promoter) of the polypeptide or nucleic acid is not naturally associated with that polypeptide or nucleic acid; that is, the control sequence comes from a gene other than the gene encoding the mature polypeptide.

[0058] Host strain or host cell: A “host strain” or “host cell” refers to an organism in which an expression vector, bacteriophage, virus, or other DNA construct (including polynucleotides encoding variants) has been introduced. An exemplary host strain is a microbial cell (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a target polypeptide and / or fermentable sugar. The term “host cell” includes protoplasts produced by cells.

[0059] Improved properties: The term "improved properties" refers to characteristics associated with variants that are improved relative to their parents. Such improved properties include, but are not limited to: catalytic efficiency, catalytic rate, chemical stability, mildness, oxidative stability, pH activity, pH stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, thermal stability, and washability.

[0060] Introduction: In the context of inserting a nucleic acid sequence into a cell, the term “introduction” means “transfection,” “conversion,” or “transduction,” as is known in the art.

[0061] Isolated: The term "isolated" means a variant, nucleic acid, cell, or other specified material or component isolated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.). Therefore, the isolated polypeptide, nucleic acid, cell, or other material exists in a form not found in nature. Isolated polypeptides include, but are not limited to, culture media containing secreted variants expressed in host cells.

[0062] Mature peptide: The term “mature peptide” refers to a peptide that has been processed at the N-terminus and / or C-terminus (e.g., removal of the signal peptide) to be in its mature form.

[0063] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to the polynucleotide that encodes a mature polypeptide with protease activity.

[0064] Mutant: The term “mutant” refers to a polynucleotide that encodes a variant.

[0065] Natural: The term "natural" refers to nucleic acids or polypeptides that are naturally present in host cells.

[0066] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of this invention cover nucleotide sequences encoding specific amino acid sequences. Unless otherwise stated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0067] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a way that does not originally exist in nature to contain a segment of nucleic acid or is synthesized and contains one or more control sequences operatively linked to the nucleic acid sequence.

[0068] Operationally linked: The term "operationally linked" means that the specified components are in a relationship that allows them to function in the intended manner (including but not limited to juxtaposition). For example, a regulatory sequence is operationally linked to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence.

[0069] Parent or parental protease: The term “parent” or “parental protease” refers to a protease that has been modified to produce a protease variant of the present invention.

[0070] Purified: The term "purified" means nucleic acids, variants, or cells that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., in electrophoretic gels, chromatographic eluates, and / or media subjected to density gradient centrifugation, where purified variants or nucleic acids form discrete bands). Purified nucleic acids or variants 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., weight percentage or molar percentage). In a relevant sense, a composition is enriched with the molecule when the concentration of the molecule increases significantly after the application of purification or enrichment techniques. The term “enrichment” refers to the presence of a compound, variant, cell, nucleic acid, amino acid, or other specified material or component in a composition at a relative or absolute concentration higher than that of the starting composition.

[0071] In one respect, as used herein, the term "purified" means that the variant or cells are substantially free of components (especially insoluble components) from the producing organism. In other respects, the term "purified" means that the variant is substantially free of insoluble components (especially insoluble components) from the natural organism from which it was obtained. In one respect, the variant is separated from some soluble components of the organism from which it was recovered and the culture medium. The variant can be purified (i.e., separated) by one or more of the following unit operation methods: filtration, precipitation, or chromatography.

[0072] Accordingly, the variant can be purified such that only small amounts of other proteins, particularly other polypeptides, are present. As used herein, the term "purified" can mean the removal of other components, particularly other proteins and most particularly other enzymes, present in the cells from which the polypeptide originates. The variant can be "substantially pure," meaning it does not contain other components from the organism that produced it (e.g., the host organism used to recombinantly produce the variant). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the formulation. In another aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the formulation. As used herein, "substantially pure polypeptide" can mean a polypeptide formulation containing, by weight, at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of other polypeptide material naturally or recombinantly associated with the polypeptide.

[0073] Therefore, it is preferred that the substantially pure variants, based on the weight of the total polypeptide material present in the formulation, are at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure. The variants of the present invention are preferably in a substantially pure form (i.e., the formulation is substantially free of other polypeptide materials associated with it, either naturally or recombinantly). This can be accomplished, for example, by preparing the variants using well-known recombinant methods or by classical purification methods.

[0074] Recombination: The term "recombination," used in its conventional sense, refers to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a sequence group different from that found in nature. The term recombination refers to cells, nucleic acids, variants, or vectors that have been modified from their natural state. Thus, for example, recombinant cells express genes not found in their natural (non-recombinant) forms, or express natural genes at different levels or under different conditions compared to those found in nature. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0075] Recovery: The term "recovery" refers to the removal of peptides from at least one fermentation broth component selected from a list of cells, nucleic acids, or other specified materials, for example, by means of: harvesting peptides from whole or cell-free fermentation broths by means of peptide crystallization, by means of filtration (e.g., deep filtration (using filter aids or packed filter media, cloth filtration in a box filter, rotary drum filtration, drum filtration, rotary vacuum drum filtration, candle filter, horizontal leaf filter, or the like, sheet or pad filtration in a frame or modular apparatus) or membrane filtration (using plate filtration, modular filtration, candle filtration, microfiltration, crossflow, dynamic crossflow, or ultrafiltration in dead-end operation)), or by means of centrifugation (using a horizontal centrifuge, disc stack centrifuge, hyrdo cyclone, or the like), or by means of precipitating peptides and using relevant solid-liquid separation methods to harvest peptides from broth media by means of particle size fractionation. Recovery encompasses the isolation and / or purification of peptides.

[0076] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0077] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity". This algorithm is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0078] (identical residues × 100) / (alignment length - total number of vacancies in the alignment)

[0079] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the sequence identity between two polynucleotide sequences as the output of "longest identity," as implemented by the Needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4). For the Needle program to report the longest identity, the non-simplified option must be specified in the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0080] (Identical deoxyribonucleotides × 100) / (Alignment length – Total number of vacancies in the alignment)

[0081] Signal peptide: A signal peptide is an amino acid sequence that attaches to the N-terminal portion of a protein and promotes its secretion outside the cell. The mature form of extracellular proteins lacks a signal peptide, which is cleaved during the secretion process.

[0082] Structural similarity: The degree of association between two amino acid sequences is usually described by the parameter "sequence identity". However, since the biological function of a polypeptide is defined by its three-dimensional structure rather than its amino acid sequence, a better way to assess the functional relationship between polypeptides is to compare their three-dimensional structures. Therefore, for the purposes of this invention, the degree of association between the three-dimensional structures of two polypeptides is described by the parameter "structural similarity".

[0083] The three-dimensional structure of any polypeptide can be obtained experimentally, for example by X-ray crystallography or using computational methods such as AlphaFold (see above). The structural similarity between the three-dimensional structures can then be determined by a TM score, which is calculated using the following general formula (Zhang and Skolnick, Proteins 57:702–710, 2004):

[0084]

[0085] Where L N It is the length of the natural structure, L T It is the length of the residues compared with the template structure, d i d is the distance between residues in the i-th pair, and d0 is the scale of the normalized matching difference. “Max” represents the maximum value after optimal spatial superposition.

[0086] For the purposes of this invention, L N Always referencing the protein length indicates that using a fixed reference length L prevents artificially large TM scores in substructure alignment.

[0087]

[0088] Before the TM score can be calculated, structural alignment of the three-dimensional structures of the two peptides is necessary. This is achieved by optimizing the structural overlap algorithm, and several methods are available, such as CEalign (Shindyalov and Bourne, ProteinEng., 11, 739-747, 1998), DALI (Holm and Sander, Trends Biochem., 20, 478-480, 1995), or TM-align (Nucleic Acids Res. 33:2302-2309, 2005).

[0089] For the purposes of this invention, TM-align is applied. For convenience, the TM score is integrated into the TM-align software, which is available from the authors' website. The version of TM-align is preferably the latest version from August 22, 2019, or later, and the TM score between the reference protein and the query protein is determined by running the following command:

[0090] TMalign<query.pdb><reference.pdb> -L<length of reference>

[0091] in<query.pdb> It is the name of the PDB file containing the coordinates of the queried peptide.<reference.pdb> This is the name of the PDB file containing the coordinates of the reference peptide. The TM score is calculated and reported in the output, along with several other parameters from the alignment.

[0092] The maximum TM score is 1, for example 1.0, corresponding to the same three-dimensional structure.

[0093] Subsequence: The term "subsequence" refers to a polynucleotide whose 5' and / or 3' ends are missing one or more nucleotides from the coding sequence of a mature polypeptide; wherein the subsequence encodes a fragment with protease activity.

[0094] Variants: The term "variant" refers to a polypeptide that has protease activity and contains substitutions, insertions (including extensions), and / or deletions (e.g., truncations) at one or more positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, especially 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0095] Wild-type: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences).

[0096] Variant Naming Conventions

[0097] For the purposes of this invention, the polypeptide disclosed in SEQ ID NO:1 was used to determine the corresponding amino acid positions in another protease. The amino acid sequence of the other protease was aligned with the polypeptide disclosed in SEQ ID NO:1, and based on this alignment, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) was used to determine the amino acid position numbers corresponding to any amino acid residues in the polypeptide disclosed in SEQ ID NO:1. This algorithm was implemented by the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 5.0.0 or later). The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0098] The comparison of SEQ ID NO:1 with SEQ ID NO:2, 3, 4, 5, 6 and 7 is as follows: Figures 1-6 supply. Figure 7 An overview of the corresponding substitutions in SEQ ID NO: 1-7 is provided.

[0099] In describing variations of the invention, the nomenclature described below has been adjusted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0100] replace: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of Thr at position 226 with Ala is represented as "T226A". Multiple mutations are separated by a plus sign ("+") or a comma, for example, "G205R+S411F" or "G205R,S411F" represents the substitution of Gly (G) and Ser (S) at positions 205 and 411 with Arg (R) and Phe (F), respectively. Since the amino acid residue at a given position varies from parent to parent, the amino acid to be substituted can be represented by X, for example, "X226A".

[0101] Missing: For amino acid deletions, use the following nomenclature: original amino acid, position, * Accordingly, the deletion of the amino acid Gly at position 195 is represented as "Gly195*". Multiple deletions are separated by a plus sign ("+") or a comma, such as "G195*+S411*" or "G195*,S411*". Since the amino acid residue at a given position varies from parent to parent, the amino acid to be deleted can be represented by X, such as "X195*".

[0102] insert: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, inserting Lys after the amino acid Gly at position 195 is represented as "G195GK". Since the amino acid residue at a given position varies from parent to parent, inserting lysine after the amino acid at position 195 can be represented as "X195*".

[0103] The insertion of multiple amino acids is represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, inserting Lys and Ala after the amino acid Gly at position 195 is represented as "G195GKA". In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding the inserted amino acid residues. In the above example, the sequence would therefore be:

[0104] <![CDATA[ Parent: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G G - K - A

[0105] Alternatively, inserting an amino acid residue (e.g., lysine) after the amino acid at position 195 can be represented as "195aK", and inserting two or more additional amino acid residues (e.g., Lys and Ala) after the amino acid at position 195 can be represented as "195aK,195bA".

[0106] Multiple changes: Variations containing multiple changes are separated by a plus sign ("+"), for example, "R170Y+G195E" means replacing Arg and Gly at positions 170 and 195 with Tyr and Glu respectively.

[0107] Different changes: Where different changes can be introduced at a single location, these changes are separated by commas. For example, "R170Y,E" means that Arg at location 170 is replaced by Tyr or Glu. Therefore, "Y167G,A+R170G,A" represents the following variants: "Y167G+R170G", "Y167G+R170A", "Y167A+R170G", and "Y167A+R170A". Detailed Implementation

[0108] This invention relates to protease variants with improved properties. The variants of this invention exhibit improved washing performance and improved storage stability under several different temperature and pH conditions. The variants of this invention also provide improved mildness to accompanying enzymes, which expands the compatibility of the variants of this invention with other enzymes and reduces the need for co-formulation with protease inhibitors. The variants of this invention also exhibit improved stain removal rates, making them particularly suitable for cleaning methods with short wash cycles.

[0109] Variants of the present invention are particularly suitable for high-pH liquid detergents and laundry soap bars with a pH of 10 or higher. In high-pH liquid detergents, variants of the present invention exhibit improved washing performance and improved storage stability. When included in laundry soap bars, variants of the present invention provide improved removal of protein stains and reduce the need for stabilizers, which further reduces the production costs associated with the manufacture of laundry soap bars.

[0110] The present invention also relates to polynucleotides encoding variants of the invention, nucleic acid constructs and expression vectors containing such polynucleotides, recombinant host cell expression variants of the invention, methods for obtaining variants of the invention, methods for generating variants of the invention, detergent compositions containing variants of the invention, and uses of variants of the invention.

[0111] variants

[0112] In a first aspect, the present invention relates to variants of parental proteases, wherein these variants comprise substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprise substitutions at at least three, for example at least four, at least five, at least six, at least seven, at least eight, or nine positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein the position numbering is based on SEQ ID NO:1. The variants are numbered NO:1; wherein, compared to the three-dimensional structure of the parent protease, these variants have a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999 but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold; and wherein these variants possess protease activity.

[0113] In the examples, the variants have a TM score of at least 0.90 compared to the three-dimensional structure of the parent protease, such as at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, where the three-dimensional structure is calculated using AlphaFold.

[0114] In the examples, the variants have a TM score of at least 0.95 compared to the three-dimensional structure of the parent protease, such as at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, where the three-dimensional structure is calculated using AlphaFold.

[0115] In the embodiments, the variants have a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0116] In the examples, the variants have a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, where the three-dimensional structure is calculated using AlphaFold.

[0117] In the embodiments, the parent protease is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. In a preferred embodiment, the parent protease is SEQ ID NO:1.

[0118] In another embodiment, the parent protease is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0119] In another embodiment, the parent protease is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0120] In one embodiment, the parent protease is the same as the protease except that it does not have the following: substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and substitutions at at least three positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0121] In another embodiment, the parent is SEQ ID NO:1, and the variant has a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1, but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:1, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:1, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1, wherein the three-dimensional structure is calculated using AlphaFold.

[0122] In another embodiment, the parent is SEQ ID NO:2, and the variant has a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:2, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:2, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold.

[0123] In another embodiment, the parent is SEQ ID NO:3, and the variant has a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:3, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:3, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold.

[0124] In another embodiment, the parent is SEQ ID NO:4, and the variant has a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:4, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:4, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold.

[0125] In another embodiment, the parent is SEQ ID NO:5, and the variant has a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:5, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:5, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold.

[0126] In another embodiment, the parent is SEQ ID NO:6, and the variant has a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:6, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:6, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold.

[0127] In another embodiment, the parent is SEQ ID NO:7, and the variant has a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:7, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:7, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold.

[0128] For some parental proteases, the three-dimensional structures are publicly available. The three-dimensional structure of SEQ ID NO:1 (Savinase®) is available under UniProt accession number P29600 or alternatively under PDB accession number 1SVN. The three-dimensional structure of SEQ ID NO:2 (BPN') is available under UniProt accession number P00782. The three-dimensional structure of SEQ ID NO:3 (Alcalase®) is available under UniProt accession number P00780.

[0129] In one embodiment, the parent is SEQ ID NO:1, and the variant has a TM score of at least 0.80 compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0. Preferably, the parent is SEQ ID NO:1, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600. Most preferably, the parent is SEQ ID NO:1, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600.

[0130] In one embodiment, the parent is SEQ ID NO:2, and the variant has a TM score of at least 0.80 compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0. Preferably, the parent is SEQ ID NO:2, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782. Most preferably, the parent is SEQ ID NO:2, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782.

[0131] In one embodiment, the parent is SEQ ID NO:3, and the variant has a TM score of at least 0.80 compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0. Preferably, the parent is SEQ ID NO:3, and the variant has a TM score of at least 0.980, such as at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780. Most preferably, the parent is SEQ ID NO:3, and the variant has a TM score of at least 0.990, such as at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780.

[0132] In a second aspect, the present invention relates to variants of a parent protease, wherein these variants comprise substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprise substitutions at at least three, such as at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any one of SEQ ID NO:1; wherein the position numbering is based on the numbering of SEQ ID NO:1; wherein these variants have at least 60% sequence identity with the parent protease, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity.

[0133] In the embodiments, the parent protease is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. In a preferred embodiment, the parent protease is SEQ ID NO:1.

[0134] In another embodiment, the parent protease is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0135] In another embodiment, the parent protease is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0136] In one embodiment, the parent protease is the same as the protease except that it does not have the following: substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and substitutions at at least three positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0137] In another embodiment, the parent is SEQ ID NO:1, and the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:1.

[0138] In another embodiment, the parent is SEQ ID NO:2, and the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:2.

[0139] In another embodiment, the parent is SEQ ID NO:3, and the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:3.

[0140] In another embodiment, the parent is SEQ ID NO:4, and the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:4.

[0141] In another embodiment, the parent is SEQ ID NO:5, and the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:5.

[0142] In another embodiment, the parent is SEQ ID NO:6, and the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:6.

[0143] In another embodiment, the parent is SEQ ID NO:7, and the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO:7.

[0144] In one aspect, the number of substitutions in the variants of the invention is 5-30, for example 5-25, 5-20, 5-15, and 5-10, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 substitutions. In a preferred embodiment, the number of substitutions in the variants of the invention is 5-11, such as 5, 6, 7, 8, 9, 10, or 11 substitutions.

[0145] On the other hand, the variant includes substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further includes at least three substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0146] On the other hand, the variant includes substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further includes at least four substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0147] On the other hand, the variant includes substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further includes at least five substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0148] On the other hand, the variant includes substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further includes at least six substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0149] On the other hand, the variant includes substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further includes at least seven substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0150] On the other hand, the variant includes substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further includes at least eight substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0151] On the other hand, the variant includes substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further includes nine substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to each of SEQ ID NO:1.

[0152] Variations of the invention include a substitution at position 95 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 95 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Asp.

[0153] Variations of the invention include a substitution at position 209 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 209 corresponding to SEQ ID NO:1 is substituted with Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably Lys.

[0154] Variations of the invention may include a substitution at position 9 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 9 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Trp, Tyr, or Val, preferably Glu.

[0155] Variations of the invention may include a substitution at position 42 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 42 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Cys, Gln, Glu, His, Ile, Leu, Met, Phe, Pro, Ser, Trp, Tyr, or Val, preferably Arg.

[0156] Variations of the invention may include a substitution at position 74 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 74 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, preferably Asp.

[0157] Variations of the invention may include a substitution at position 199 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 199 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably Ile.

[0158] Variations of the invention may include a substitution at position 200 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 200 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, or Val, preferably Leu.

[0159] Variations of the invention may include a substitution at position 203 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 203 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, preferably Trp.

[0160] Variations of the invention may include a substitution at position 253 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 253 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Trp, Tyr, or Val, preferably Asp.

[0161] Variations of the invention may include a substitution at position 255 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 255 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Thr, Trp, Tyr, or Val, preferably Trp.

[0162] Variations of the invention may include a substitution at position 256 corresponding to SEQ ID NO:1. In one aspect, the amino acid at position 256 corresponding to SEQ ID NO:1 is substituted with Ala, Arg, Asn, Cys, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, preferably Glu.

[0163] In one aspect, variations of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprise at least three, for example at least four, at least five, at least six, at least seven, at least eight or nine, substitutions selected from the group consisting of S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W and L256E of SEQ ID NO:1.

[0164] In one aspect, variations of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprise at least four substitutions selected from the group consisting of S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0165] In one aspect, variations of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprise at least five substitutions selected from the group consisting of S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0166] In one aspect, variations of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprise at least six substitutions selected from the group consisting of S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0167] In one aspect, variations of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprise at least seven substitutions selected from the group consisting of S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E corresponding to SEQ ID NO:1.

[0168] In one aspect, variations of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprise at least eight substitutions selected from the group consisting of S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0169] In one aspect, variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprise nine substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0170] In one aspect, the invention relates to variations of SEQ ID NO:1, which comprise substitutions for X95D (e.g., G95D) and X209K (e.g., A209K), and further comprise at least three, for example at least four, at least five, at least six, at least seven, at least eight, or nine substitutions selected from the group consisting of: X9E (e.g., S9E), X42R (e.g., N42R), X74D (e.g., N74D), X199I (e.g., V199I), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., S253D), X255W (e.g., N255W), and X256E (e.g., L256E); wherein the position numbering is based on the number of SEQ ID NO:1; wherein these variations are related to SEQ ID NO:1. NO:1 has at least 60% sequence identity, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In preferred embodiments, the variants comprise or consist of SEQ ID NO:1 having the following substitutions: S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.

[0171] In one aspect, the invention relates to variations of SEQ ID NO:2, which comprise substitutions for X97D (e.g., G97D) and X215K (e.g., G215K), and further comprise at least three, for example at least four, at least five, at least six, or seven substitutions selected from the group consisting of: X9E (e.g., S9E), X43R (e.g., K43R), X76D (e.g., N76D), X206L (e.g., Q206L), X209W (e.g., L209W), X261W (e.g., F261W), and X262E (e.g., Y262E); wherein the position numbering is based on the numbering of SEQ ID NO:2; wherein these variations are related to SEQ ID NO:2. NO:2 has at least 60% sequence identity, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In preferred embodiments, the variants comprise or consist of SEQ ID NO:2 having the following substitutions: S9E, K43R, N76D, G97D, Q206L, L209W, G215K, F261W, and Y262E.

[0172] In one aspect, the invention relates to variations of SEQ ID NO:3, which comprise substitutions for X96D (e.g., N96D) and X214K (e.g., A214K), and further comprise at least three, for example at least four, at least five, at least six, at least seven, or eight substitutions selected from the group consisting of: X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261E (e.g., Y261E); wherein the position numbering is based on the numbering of SEQ ID NO:3; wherein these variations are related to SEQ ID NO:3. NO:3 has at least 60% sequence identity, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In preferred embodiments, the variants comprise or consist of SEQ ID NO:3 having the following substitutions: P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E.

[0173] In one aspect, the invention relates to variations of SEQ ID NO:4, which comprise substitutions for X95D (e.g., G95D) and X209K (e.g., A209K), and further comprise at least three, for example at least four, at least five, at least six, at least seven, or eight substitutions selected from the group consisting of: X9E (e.g., T9E), X42R (e.g., T42R), X74D (e.g., N74D), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., N253D), X255W (e.g., S255W), and X256E (e.g., Q256E); wherein the position numbering is based on the numbering of SEQ ID NO:4; wherein these variations are related to SEQ ID NO:4. NO:4 has at least 60% sequence identity, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In preferred embodiments, the variants comprise or consist of SEQ ID NO:4 with or composed of the following substitutions: T9E, T42R, N74D, G95D, Q200L, Y203W, A209K, N253D, S255W, and Q256E.

[0174] In one aspect, the invention relates to variations of SEQ ID NO:5, which comprise substitutions for X95D (e.g., G95D) and X209K (e.g., V209K), and further comprise at least three, for example at least four, at least five, at least six, at least seven, at least eight, or nine substitutions selected from the group consisting of: X9E (e.g., T9E), X42R (e.g., N42R), X74D (e.g., N74D), X199I (e.g., V199I), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., N253D), X255W (e.g., S255W), and X256E (e.g., Q256E); wherein the position numbering is based on the numbering of SEQ ID NO:5; wherein these variations are related to SEQ ID NO:5. NO:5 has at least 60% sequence identity, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:5 having the following substitutions: T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D, S255W, and Q256E.

[0175] In one aspect, the invention relates to variants of SEQ ID NO:6, which comprise substitutions for X107D (e.g., G107D) and X245K (e.g., N245K), and further comprise at least three, for example at least four, at least five, at least six, at least seven, or eight substitutions selected from the group consisting of: X12E (e.g., K12E), X44R (e.g., D44R), X86D (e.g., S86D), X235I (e.g., V235I), X236L (e.g., E236L), X297D (e.g., T297D), X299W (e.g., D299W), and X300E (e.g., D300E); wherein the position numbering is based on the numbering of SEQ ID NO:6; wherein these variants are related to SEQ ID NO:6. NO:6 has at least 60% sequence identity, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In preferred embodiments, the variants comprise or consist of SEQ ID NO:6 having the following substitutions: K12E, D44R, S86D, G107D, V235I, E236L, N245K, T297D, D299W, and D300E.

[0176] In one aspect, the present invention relates to variants of SEQ ID NO:7 comprising substitutions of X99D (e.g., N99D) and X215K (e.g., N215K), and further comprising at least three, for example at least four, at least five, or six substitutions selected from the group consisting of: X12E (e.g., D12E), X51R (e.g., G51R), X206L (e.g., T206L), X266D (e.g., T266D), X268W (e.g., N268W), and X269E (e.g., L269E); wherein the position numbering is based on the numbering of SEQ ID NO:7; wherein these variants have at least 60% sequence identity with SEQ ID NO:7, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:7 with the following substitutions: D12E, G51R, N99D, T206L, N215K, T266D, N268W, and L269E.

[0177] In one aspect, the invention relates to variants of SEQ ID NO:7 comprising substitutions for X99D (e.g., N99D) and X215K (e.g., N215K), and further comprising substitutions for X12E (e.g., D12E), X51R (e.g., G51R), and X206L (e.g., T206L); wherein the position numbering is based on the number of SEQ ID NO:7; wherein these variants have at least 60% sequence identity with SEQ ID NO:7, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In a preferred embodiment, the variant comprises or is composed of SEQ ID NO:7 having substitutions for D12E, G51R, N99D, T206L, and N215K.

[0178] In an alternative aspect, the present invention relates to SEQ ID. Variations of NO:3, comprising substitutions for X96D (e.g., N96D) or X214K (e.g., A214K), preferably X96D (e.g., N96D) and X214K (e.g., A214K), and further comprising at least one, for example at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten substitutions selected from the group consisting of: X68S (e.g., A68S), X77N (e.g., T77N), X78I (e.g., T78I), X127S (e.g., G127S), X128P (e.g., A128P), X165Q (e.g., G165Q), X184Q (e.g., N184Q), X202V (e.g., A202V), X217S (e.g., N217S), and X258P (e.g., S258P); wherein the position number is based on SEQ ID The variants are numbered NO:3; wherein these variants have at least 60% sequence identity with SEQ ID NO:3, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In one embodiment, the variants comprise substitutions for N96D and A214K, and further comprise at least one, for example at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten substitutions selected from the group consisting of: A68S, T77N, T78I, G127S, A128P, G165Q, N184Q, A202V, N217S, and S258P. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:3 having the following substitutions: A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S, and S258P. In a more preferred embodiment, the variant further comprises at least one, for example at least two, at least three, at least four, at least five, at least six, at least seven, or eight substitutions selected from the group consisting of: X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261E (e.g., Y261E).In even more preferred embodiments, the variant further comprises at least one, for example at least two, at least three, at least four, at least five, at least six, at least seven, or eight substitutions selected from the group consisting of: P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261E. In the most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 having the following substitutions: P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258P, F260W, and Y261E. In the most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the following substitutions: P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258D, F260W, and Y261E.

[0179] In alternative aspects, the present invention relates to variations of SEQ ID NO:3, which include substitutions for X214K (e.g., A214K) and further include at least one, for example at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or fourteen substitutions selected from the group consisting of: X9T (e.g., P9T), X17H (e.g., Q17H), X77N (e.g., T77N), X78I (e.g., The variants are X78I, X96D (e.g., N96D), X103F (e.g., Y103F), X127T (e.g., G127T), X128K (e.g., A128K), X129Q (e.g., S129Q), X165Q (e.g., G165Q), X184Q (e.g., N184Q), X202V (e.g., A202V), X203E (e.g., G203E), and X258P (e.g., S258P); wherein the position numbering is based on the number of SEQ ID NO:3; wherein these variants have at least 60% sequence identity with SEQ ID NO:3, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein these variants have protease activity. In one embodiment, the variant includes a substitution for A214K, and further includes at least one of the following substitutions selected from the group consisting of, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or fourteen: P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, and S258P. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the following substitutions: P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, A214K, and S258P.In a more preferred embodiment, the variant further comprises at least one, such as at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of: X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261E (e.g., Y261E). In an even more preferred embodiment, the variant further comprises at least one, such as at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of: P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261E. In the most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the following substitutions: P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261E. In the most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the following substitutions: P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E. In the most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the following substitutions: P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261E. In the most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the following substitutions: P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E.

[0180] Variations of the invention may include additional substitutions at one or more other positions corresponding to positions 60 (e.g., N60D), 97 (e.g., S97E), 99 (e.g., S99E), 116 (e.g., G116N), and 246 (e.g., N246L) of SEQ ID NO:1. Preferably, the variation includes one or more additional substitutions selected from the group consisting of substitutions corresponding to N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L of SEQ ID NO:1.

[0181] Variations of the invention may further include extensions of one or more amino acids at the N-terminus and / or C-terminus.

[0182] Alternatively, variations of the invention may further comprise truncation of one or more amino acids at the N-terminus and / or C-terminus.

[0183] The amino acid alterations introduced to provide the variants according to the invention can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine fragment, an antigenic epitope, or a binding domain.

[0184] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, *The Proteins*, Academic Press, New York. Common substitutes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0185] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, amino acid alterations can improve the thermal stability of the peptide, change its substrate specificity, or change its optimal pH.

[0186] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the protease activity of the resulting molecule is tested to identify amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutagenesis of the amino acids at the putative contact sites. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from alignments with related peptides, and / or from sequence homology and conserved catalytic mechanisms with related peptides or peptide / protein families from a common ancestor (typically possessing similar three-dimensional structures, functions, and significant sequence similarities). Alternatively or concurrently, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of peptides. See, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

[0187] In one aspect, the variants of the present invention exhibit improved stability under storage conditions (i.e., improved storage stability) compared to a reference protease. In one embodiment, the improvement in storage stability is at least 5%, for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or higher.

[0188] In some embodiments, the reference protease is the parent protease. In one embodiment, the parent protease is the same as the protease except that it has the following: substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and substitutions at at least three positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:6. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:7.

[0189] In some embodiments, the variant has improved storage stability compared to proteases that are otherwise identical except for the absence of substitutions at positions 95 and 209 corresponding to SEQ ID NO:1. In one embodiment, the improvement in storage stability is at least 5%, for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500%. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:8. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:11. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:13.

[0190] In one embodiment, the variant has improved storage stability in liquid detergents with a pH of 8-14, preferably 9-13, and most preferably 10-12.

[0191] In one embodiment, the variant has improved storage stability in liquid detergents with a pH of 7-11, preferably 7-10, and most preferably 8-10.

[0192] In one embodiment, the variant exhibits improved storage stability at temperatures ranging from 10°C to 40°C, preferably from 10°C to 30°C, and most preferably from 15°C to 25°C.

[0193] In one embodiment, the variant exhibits improved storage stability at temperatures ranging from 10°C to 60°C, more preferably from 30°C to 55°C, and most preferably from 45°C to 55°C.

[0194] In a preferred embodiment, the variant has improved storage stability in liquid detergents, as determined according to Example 7 herein.

[0195] In preferred embodiments, the variants have improved storage stability in liquid detergents, preferably in standard O detergents at pH 8-10 or preferably in standard B detergents at pH 7-8, as determined according to Example 11 herein.

[0196] In one aspect, the variants of the present invention have improved washing performance compared to the reference parent protease. In one embodiment, the washing performance is improved by at least 5%, for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.

[0197] In some embodiments, the reference protease is the parent protease. In one embodiment, the parent protease is the same as the protease except that it does not have the following: substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and substitutions at at least three positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In one embodiment, the parent is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:6. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:7.

[0198] In some embodiments, the variant has improved washing performance compared to a protease that is identical in all respects except for the absence of substitutions at positions 95 and 209 corresponding to SEQ ID NO:1. In one embodiment, the washing performance is improved by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500%. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:8. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:11. In a preferred embodiment, the variant has improved washing performance compared to SEQ ID NO:13.

[0199] In one embodiment, the variant has improved washing performance in liquid detergents with a pH of 8-14, preferably 9-13, and most preferably 10-12.

[0200] In one embodiment, the variant has improved washing performance in liquid detergents with a pH of 7-11, preferably 7-10, and most preferably 8-10.

[0201] In one embodiment, the variant exhibits improved washing performance at washing temperatures of 10°C-60°C, preferably 10°C-40°C, more preferably 10°C-30°C, and most preferably 15°C-25°C.

[0202] In one embodiment, the variant has improved washing performance, as determined in Examples 1, 2, 3, 4, 5, 6, or 10 herein. In a preferred embodiment, the variant has improved washing performance in a liquid detergent, as determined according to Example 1 herein. In a preferred embodiment, the variant has improved washing performance in a liquid detergent, as determined according to Example 2 herein. In a preferred embodiment, the variant has improved washing performance in a liquid detergent, as determined according to Example 3 herein. In a preferred embodiment, the variant has improved washing performance in a liquid detergent, as determined according to Example 4 herein. In a preferred embodiment, the variant has improved washing performance in a liquid detergent, as determined according to Example 5 herein. In a preferred embodiment, the variant has improved washing performance in a liquid detergent, as determined according to Example 6 herein.

[0203] In preferred embodiments, the variants have improved washing performance in liquid detergents, preferably in standard O detergents with a pH of 8-10 or preferably in standard B detergents with a pH of 7-8, as determined according to Example 10 herein.

[0204] In one aspect, the variants of the present invention have improved mildness compared to a reference protease. In the context of the present invention, improved mildness means that the protease variant is less aggressive towards other enzymes (also known as concomitant enzymes or secondary enzymes) in the detergent matrix, thereby providing improved residual activity of the concomitant enzymes after being stored in a detergent composition with the protease variant of the present invention. In one embodiment, the mildness improvement is at least 5%, for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or higher.

[0205] In some embodiments, the reference protease is the parent protease. In one embodiment, the parent protease is the same as the protease except that it does not have the following: substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and substitutions at at least three positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In one embodiment, the parent is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:6. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:7.

[0206] In some embodiments, the variant has improved mildness compared to proteases that are otherwise identical except for the absence of substitutions at positions 95 and 209 corresponding to SEQ ID NO:1. In one embodiment, the mildness improvement is at least 5%, for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500%. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:8. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:11. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:13.

[0207] In some embodiments, the variant provides improved residual activity of the concomitant enzyme. In a preferred embodiment, the residual activity of the concomitant enzyme is improved by at least 5%, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or higher. In one embodiment, the concomitant enzyme is selected from the group consisting of: amylase (e.g., α-amylase), arabinase, glycosylase, cellulase (e.g., endoglucanase), keratinase, DNase, galactanase, halogenated peroxyase, lipase, mannanase, oxidase (e.g., laccase or peroxidase), pectinase, pectin lyase, protease, xylanase, xanthan gumase, or xyloglucanase. In a preferred embodiment, the concomitant enzyme is α-amylase. In a preferred embodiment, the residual activity is determined according to Example 8 herein.

[0208] In one embodiment, the variant provides improved residual activity of the accompanying enzyme (preferably α-amylase) after storage in a liquid detergent at pH 8-14, preferably pH 9-13, and most preferably pH 10-12.

[0209] In one embodiment, when stored in a liquid detergent at a temperature of 10°C-60°C, preferably 15°C-50°C, most preferably 20°C-40°C, this variant provides improved residual activity of the accompanying enzyme (preferably α-amylase) after storage with the protease variant of the present invention.

[0210] In a preferred embodiment, the variant has improved mildness in liquid detergents, as determined according to Example 8 herein.

[0211] In a preferred embodiment, the variant provides improved residual activity of α-amylase in the liquid detergent, as determined according to Example 8 herein.

[0212] In a preferred embodiment, the variant has improved mildness in standard O liquid detergent.

[0213] In a preferred embodiment, the variant provides improved residual activity of α-amylase in standard O liquid detergent.

[0214] In one aspect, the variants of the present invention have improved stain removal rates compared to a reference protease. In the context of the present invention, improved stain removal rate means that, within a given time range, the variant is relatively faster in removing proteinaceous stains compared to the parent protease. In one embodiment, the stain removal rate is improved by at least 5%, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or higher.

[0215] In some embodiments, the reference protease is the parent protease. In one embodiment, the parent protease is the same as the protease except that it has the following: substitutions at positions 95 and 209 of SEQ ID NO:1, and substitutions at at least three positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:3. In a preferred embodiment, the variant has an improved stain removal rate compared to SEQ ID NO:4. In a preferred embodiment, the variant has an improved stain removal rate compared to SEQ ID NO:5. In a preferred embodiment, the variant has an improved stain removal rate compared to SEQ ID NO:6. In a preferred embodiment, the variant has an improved stain removal rate compared to SEQ ID NO:7.

[0216] In some embodiments, the variant has improved stain removal rate compared to a protease that is otherwise identical except for the absence of substitutions at positions 95 and 209 corresponding to SEQ ID NO:1. In one embodiment, the stain removal rate is improved by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500%. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:8. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:11. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:13.

[0217] In one embodiment, the variant exhibits improved stain removal at washing temperatures of 10°C-60°C, more preferably 15°C-50°C, and most preferably 20°C-40°C.

[0218] In a preferred embodiment, the variant has improved stain removal efficiency in liquid detergents, as determined according to Example 9 herein.

[0219] Variants of the present invention may be fusion peptides comprising variants of the present invention.

[0220] In one respect, the variants of the invention are separate.

[0221] On the other hand, the variant of the invention is purified.

[0222] Parental protease

[0223] The parent protease can be a polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0224] On the one hand, the parent protease is a polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0225] On the one hand, the parent protease is a polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0226] In one aspect, the parent has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:1, and the parent possesses protease activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:1 by up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect, the parent comprises or is composed of the polypeptide of SEQ ID NO:1.

[0227] In one aspect, the parent has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:2, and the parent has protease activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:2 by up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect, the parent comprises or is composed of the polypeptide of SEQ ID NO:2.

[0228] In one aspect, the parent has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:3, and the parent possesses protease activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:3 by up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect, the parent comprises or is composed of the polypeptide of SEQ ID NO:3.

[0229] In one aspect, the parent has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:4, and the parent possesses protease activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:4 by up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect, the parent comprises or is composed of the polypeptide of SEQ ID NO:4.

[0230] In one aspect, the parent has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:5, and the parent possesses protease activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:5 by up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect, the parent comprises or is composed of the polypeptide of SEQ ID NO:5.

[0231] In one aspect, the parent has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:5, and the parent possesses protease activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:5 by up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect, the parent comprises or is composed of the polypeptide of SEQ ID NO:5.

[0232] In one aspect, the parent has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:6, and the parent possesses protease activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:6 by up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect, the parent comprises or is composed of the polypeptide of SEQ ID NO:6.

[0233] In one aspect, the parent has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:7, and the parent possesses protease activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO:7 by up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect, the parent comprises or is composed of the polypeptide of SEQ ID NO:7.

[0234] The parent peptide can be a fusion peptide or a cleavable fusion peptide. Fusion peptides are produced by fusing a polynucleotide encoding another peptide with the polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking the coding sequences of the peptides such that they conform to reading frames, and that the expression of the fusion peptide is under the control of the same promoter and terminator. Fusion peptides can also be constructed using intron technology, where the fusion peptide is generated post-translational (Cooper et al., 1993, EMBO J. [Journal of the European Society for Molecular Biology] 12: 2575-2583; Dawson et al., 1994, Science [Science] 266: 776-779).

[0235] The fusion peptide may further include a cleavage site between the two peptides. This site is cleaved during the secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology and Biotechnology] 3: 568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63: 3488-3493; Ward et al., 1995, Biotechnology [Biotechnology] 13: 498-503; and Contreras et al., 1991, Biotechnology [Biotechnology] 9: 378-381; Eaton et al., 1986, Biochemistry [Biochemistry] 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0236] Parental lines can be obtained from any genus of microorganisms. For the purposes of this invention, as used herein in conjunction with a given source, the term "obtained from" should mean that a parental line encoded by a polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, the parental line is extracellularly secreted.

[0237] In one aspect, the parent is a Bacillus retarder protease, for example, the protease of SEQ ID NO:1. In one aspect, the parent is a Bacillus amylolyticus protease, for example, the protease of SEQ ID NO:2. In one aspect, the parent is a Bacillus licheniformis protease, for example, the protease of SEQ ID NO:3. In one aspect, the parent is a Bacillus giganteus protease, for example, the protease of SEQ ID NO:4. In one aspect, the parent is a Bacillus giganteus protease, for example, the protease of SEQ ID NO:5. In one aspect, the parent is a Bacillus species TY145 protease, for example, the protease of SEQ ID NO:6. In one aspect, the parent is a keratin-degrading Actinobacter madura protease, for example, the protease of SEQ ID NO:7.

[0238] Preparation of variants

[0239] The present invention also relates to methods for obtaining variants having protease activity, the methods comprising: (a) introducing a parent protease substitution at positions 95 and 209 corresponding to SEQ ID NO:1, and further introducing the substitution at at least three, for example at least four, at least five, at least six, at least seven, at least eight or nine, positions corresponding to any one of SEQ ID NO:1, wherein the variant has protease activity; and (b) recovering the variant.

[0240] Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0241] Site-directed mutagenesis is a technique that introduces one or more mutations at one or more designated sites in a polynucleotide encoding a parent.

[0242] Site-directed mutagenesis in vitro can be achieved through PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis in vitro can also be performed via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent, followed by ligation of the mutated oligonucleotide into the polynucleotide. Typically, the restriction enzymes digesting the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. [Nucleic Acid Research] 18: 7349-4966.

[0243] In vivo site-directed mutagenesis can also be achieved using methods known in the art. See, for example, US 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, FungalGenet. Newslett. 43: 15-16.

[0244] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0245] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode target peptides. Gene synthesis can be performed using several techniques, such as the multi-channel microchip-based technique described by Tian et al., 2004, Nature [Nature] 432: 1050-1054, and similar techniques for synthesizing and assembling oligonucleotides on optically programmable microfluidic chips.

[0246] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, one or more amino acid substitutions, deletions, and / or insertions can be made and tested, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0247] Mutagenesis / recombination methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenesis-encoded peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenesis-encoded DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0248] Semi-synthetic gene construction is achieved through a combination of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes a combination of the process of synthesizing polynucleotide fragments with PCR technology. Therefore, defined regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can be amplified using error-prone or non-error-prone PCR. The polynucleotide subsequence can then be shuffled.

[0249] Polynucleotides

[0250] The present invention also relates to polynucleotides encoding variants of the invention.

[0251] Polynucleotides can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or combinations thereof.

[0252] On the one hand, polynucleotides are isolated.

[0253] On the other hand, the polynucleotides are purified.

[0254] Nucleic acid constructs

[0255] The present invention also relates to a nucleic acid construct comprising a polynucleotide encoding a variant of the invention, operatively linked to one or more control sequences, which in turn guide the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0256] Polynucleotides can be manipulated in a variety of ways to provide expression of variants. Depending on the expression vector, manipulation of the polynucleotide before insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0257] promoter

[0258] The control sequence can be a promoter, i.e., a polynucleotide recognized by the host cell to express a polynucleotide encoding a variant of the present invention. The promoter contains a transcriptional control sequence that mediates the expression of the variant. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutant promoters, truncated promoters, and heterozygous promoters, and can be a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0259] Examples of suitable promoters for guiding the transcription of polynucleotides in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab, New York; Davis et al., 2012, Basic Methods in Molecular Biology, Elsevie; and Song et al., 2016, PLOS One, 11(7): e0158447.

[0260] Examples of suitable promoters for guiding the transcription of polynucleotides in filamentous fungal host cells are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as those described in: Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0261] Examples of useful promoters for expression in yeast hosts are described in: Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae) and Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0262] Termination

[0263] The control sequence can also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operatively linked to the 3' end of a polynucleotide encoding the variant. Any terminator that is functional in the host cell can be used in this invention.

[0264] Preferred terminators for bacterial host cells can be obtained from the genes of Bacillus clausii alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0265] Preferred terminators for filamentous fungal host cells can be obtained from species of the genera *Aspergillus* or *Trichoderma*, such as genes obtained from *Aspergillus niger* glucosidase, *Trichoderma reesei* β-glucosidase, *Trichoderma reesei* cellobiase I, and *Trichoderma reesei* endoglucanase I, as described in the following terminators: Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, *Fungal Biology*.

[0266] Preferred terminators for yeast host cells can be obtained from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast [Yeast] 8: 423-488.

[0267] mRNA stabilizers

[0268] Control sequences can also be mRNA stabilizer regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of that gene.

[0269] Examples of suitable mRNA stabilizer regions were obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. [Journal of Bacteriology] 177: 3465-3471).

[0270] Examples of mRNA stabilizer regions in fungal cells are described in Geisberg et al., 2014, Cell [Cell] 156(4): 812-824 and Morozov et al., 2006, Eukaryotic Cell [Eukaryotic Cell] 5(11): 1838-1846.

[0271] Leader sequence

[0272] The control sequence can also be a leader sequence, which is the untranslated region of mRNA that is important for translation in the host cell. This leader sequence is operatively linked to the 5' end of the multinucleotide encoding the variant. Any leader sequence that is functional in the host cell can be used.

[0273] The appropriate leader sequence for bacterial host cells is described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059 and Kaberdin and Bläsi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.

[0274] Preferred leader sequences for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triphosphate isomerase.

[0275] Suitable leader sequences for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0276] polyadenylation sequence

[0277] The control sequence can also be a polyadenylation sequence, i.e., a sequence operatively linked to the 3' end of a polynucleotide and recognized by the host cell during transcription as a signal to add polyadenylation residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

[0278] Preferred polyadenylated sequences for filamentous fungal host cells are obtained from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0279] Useful polyadenylation sequences in yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. [Molecular Cell Biology] 15: 5983-5990.

[0280] signal peptide

[0281] The control sequence can also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant and directs the variant's entry into the cell's secretory pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked within the translation reading frame to a segment encoding the variant's coding sequence. Alternatively, the 5' end of the coding sequence may contain a foreign signal peptide coding sequence relative to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a foreign signal peptide coding sequence may be required. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance the variant's secretion. However, any signal peptide coding sequence that directs the expressed variant into the host cell's secretory pathway can be used.

[0282] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the following genes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Humicolainsolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic protease, as described by Xu et al., 2018, Biotechnology Letters 40: 949-955.

[0283] Useful signal peptides in yeast host cells are obtained from the genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Sequences encoding other useful signal peptides are described above by Romanos et al., 1992.

[0284] propeptide

[0285] The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the variant. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active variant by catalytic or autocatalytic cleavage of the propeptide progenitor. Propeptide-coding sequences can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizopus miltiorrhiza aspartic protease, and Saccharomyces cerevisiae α-factor.

[0286] In the presence of both the signal peptide and the propeptide sequence, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant, and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0287] Regulation sequence

[0288] It is also desirable to add regulatory sequences that modulate variant expression relative to the growth of the host cell. Examples of regulatory sequences are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 systems can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter and *Aspergillus oryzae* glucosylamylase promoter, and the *Trichoderma reesei* cellobiose hydrolase I and cellobiose hydrolase II promoters can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals.

[0289] transcription factors

[0290] Control sequences can also be transcription factors, which are polynucleotides encoding polynucleotide-specific DNA-binding polypeptides that control the rate of transcription of genetic information from DNA to mRNA by binding to specific polynucleotide sequences. Transcription factors can function alone and / or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by containing at least one DNA-binding domain, which is typically attached to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of a target protein directly (i.e., by binding to their promoter to activate the transcription of a gene encoding the target protein) or indirectly (i.e., by binding to the promoter of another transcription factor that regulates the transcription of a gene encoding the target protein) or indirectly (e.g., by binding to the promoter of another transcription factor that regulates the transcription of a gene encoding the target protein). Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52: 7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.

[0291] expression carrier

[0292] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more suitable restriction sites to allow insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is positioned in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0293] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0294] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with the chromosome in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids collectively containing the total DNA of the host cell genome to be introduced, or transposons can be used.

[0295] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, or prototrophic auxotrophic traits, etc.

[0296] The vector preferably contains at least one element that allows the vector to integrate into the genome of the host cell or to replicate autonomously in the cell independently of the genome.

[0297] In order to integrate into the host cell genome, the vector may depend on a polynucleotide sequence encoding a polypeptide or any other element of the vector used for integration into the genome via homologous recombination (such as homologous directed repair (HDR)) or non-homologous recombination (such as non-homologous end joining (NHEJ)).

[0298] For autonomous replication, the vector may further include an origin of replication, which enables the vector to replicate autonomously in the host cell discussed. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0299] More than one copy of the polynucleotide of the present invention can be inserted into host cells to enhance polypeptide production. For example, two, three, four, five, or more copies can be inserted into host cells. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selective marker gene along with the polynucleotide, wherein cells containing the amplified copy of the selective marker gene and thus additional copies of the polynucleotide can be selected by culturing cells in the presence of a suitable selective agent.

[0300] host cells

[0301] The present invention also relates to recombinant host cells containing polynucleotides of the invention operatively linked to one or more control sequences that direct the generation of variants of the invention.

[0302] A construct or vector containing a polynucleotide is introduced into a host cell, such that the construct or vector is maintained as a chromosomal integrator or as a self-replicating extrachromosomal vector, as previously described. The choice of host cell will depend largely on the gene encoding the variant and its origin. The recombinant host cell may contain a single copy or at least two copies, such as three, four, five or more copies of the polynucleotide of the present invention.

[0303] The host cell can be any cell that can be used for recombination to generate variants of the invention, such as prokaryotic cells or fungal cells.

[0304] The host cell can be any microbial cell that can be used to recombinantly generate the polypeptides of the present invention, such as prokaryotic cells or fungal cells.

[0305] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0306] The bacterial host cell can be any Bacillus genus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus tarda, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In the embodiments, the Bacillus cells are Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis cells.

[0307] For the purposes of this invention, Bacillus species should be defined as described in Patel and Gupta, 2020, Int. J.Syst.Evol.Microbiol. [International Journal of Systematic and Evolutionary Microbiology] 70: 406-438.

[0308] The bacterial host cell can also be any streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus.

[0309] The bacterial host cell can also be any Streptomyces cell, including but not limited to: Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0310] Methods for introducing DNA into prokaryotic host cells are well known in the art, and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, and transduction, wherein the DNA is introduced as a linearized or circular polynucleotide. Those skilled in the art will be able to readily determine, for example, a suitable method for introducing DNA into a given prokaryotic cell based on the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018, BMC Microbiology 18:56; Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294; Choi et al., 2006, J. Microbiol. Methods 64: 391-397; and Donald et al., 2013, J. Bacteriol. 195(11): 2612-2620.

[0311] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi (as defined in the following literature: Hawksworth et al., Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0312] Fungal cells can be transformed via processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods, and shock wave-mediated transformation (as reviewed in Li et al., 2017, Microbial Cell Factories, 16: 168), as well as procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 1470-1474; Christensen et al., 1988, Bio / Technology, 6: 1419-1422; and Lubertozzi and Keasling, 2009, Biotechn. Advances, 27: 53-75. However, any method known in the art for introducing DNA into fungal host cells can be used, and the DNA can be introduced as linearized or circular polynucleotides.

[0313] The host cell for fungi can be a yeast cell. As used herein, “yeast” includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeasts belonging to the class Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0314] Yeast host cells can be cells from the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Schizosaccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Saccharomyces carlsbergensis*, *Saccharomyces diastaticus*, *Saccharomyces douglasii*, *Saccharomyces kluyveri*, *Saccharomyces norbensis*, *Saccharomyces oviformis*, or *Yarrowia lipolytica*. In a preferred embodiment, the yeast host cell is a cell of the genera *Pichia* or *Komagataella*, such as *Pichia pastoris* cells (*Komagataella phaffii*).

[0315] The host cell for fungi can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms within the phylum Eumycota and the subphylum Oomycetes (as defined by Hawksworth et al., 1995, ibid.). Filamentous fungi are generally characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, the vegetative growth of yeasts (such as Saccharomyces cerevisiae) occurs through budding of single-celled cells, and carbon metabolism can be fermentative.

[0316] The host cells of filamentous fungi can be *Acremonium*, *Aspergillus*, *Aureobasidium*, *Bjerkandera*, *Ceriporiopsis*, *Chrysosporium*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Humicola*, *Magnaporthe*, *Mucor*, *Myceliophthora*, and *Neurospora*. Cells of the genera *Neocallimastix*, *Neurospora*, *Paecilomyces*, *Penicillium*, *Phanerochaete*, *Phlebia*, *Piromyces*, *Pleurotus*, *Schizophyllum*, *Talaromyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trametes*, or *Trichoderma* are used. In a preferred embodiment, the filamentous fungal host cells are *Aspergillus*, *Trichoderma*, or *Fusarium* cells. In another preferred embodiment, the filamentous fungal host cells are *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, or *Fusarium venenatum* cells.

[0317] For example, the host cells of filamentous fungi can be *Aspergillus awamori*, *Aspergillus foetidus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus niger*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis aneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, *Chrysosporium inops*, and *Chrysosporium horneri*. *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium pannicola*, *Chrysosporium queenslandicum*, *Chrysosporium tropicum*, *Chrysosporium zonatum*, *Coprinus cinereus*, *Coriolus hirsutus*, *Fusarium bactridioides*, *Fusarium cerealis*, *Fusarium crookwellense*, *Fusarium culmorum*, *Fusarium graminearum*, *Fusarium graminum*, *Fusarium heterosporum*, *Fusarium scabra* Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium peltataFusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium sporotrichioides, Fusarium sporotrichioides, Fusarium sulphureum, Mucor miehei, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebiaradiata, Pleurotus eryngii, Talaromyces emersonii, Thievaria terrestris, Trametes villosa, Trametes versicolor, Trichoderma Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0318] On the one hand, the host cell is isolated.

[0319] On the other hand, the host cell is purified.

[0320] Generation method

[0321] The present invention also relates to methods for generating variants of the invention, the methods comprising (a) culturing recombinant host cells of the invention under conditions favorable to generating the variant; and optionally (b) recovering the variant.

[0322] Host cells are cultured in a nutrient medium suitable for generating the variant using methods known in the art. For example, cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-batch fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for variant expression and / or isolation in a laboratory or industrial fermenter. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0323] Variances can be detected using methods known in the art that are specific to the variant, including but not limited to enzyme assays that use specific antibodies, enzyme product formation, enzyme substrate disappearance, or determine the relative or specific activity of the variant.

[0324] Variants can be recovered from the culture medium using methods known in the art, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the entire fermentation broth is recovered. In another aspect, cell-free fermentation broth containing peptides is recovered.

[0325] Variants can be purified using a variety of procedures known in the art to obtain substantially pure variants and / or fragments (see, for example, Wingfield, 2015, Current Protocols in Protein Science; 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0326] In terms of alternatives, variants are not recycled.

[0327] protease particles

[0328] The present invention also relates to enzyme particles / granules comprising variations of the present invention. In embodiments, the particles comprise a core and optionally one or more coatings (outer layers) surrounding the core.

[0329] The core diameter (measured as equivalent sphere diameter (volume-average particle size)) can be 20–2000 µm, particularly 50–1500 µm, 100–1500 µm, or 250–1200 µm. The core diameter as an equivalent sphere diameter can be determined using laser diffraction methods such as those using the Malvern Mastersizer and / or those described under ISO 13320 (2020).

[0330] In the embodiments, the core includes variations of the invention.

[0331] The core may include other materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0332] The core may include binders, such as synthetic polymers, waxes, fats, or carbohydrates.

[0333] The core, typically as a homogeneous blend, may include salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components.

[0334] The core may include inert particles into which variants are adsorbed or applied (e.g., by fluidized bed coating) to a surface.

[0335] The diameter of the core can be 20-2000 µm, especially 50-1500 µm, 100-1500 µm or 250-1200 µm.

[0336] The core may be surrounded by at least one coating, for example, to improve storage stability, reduce dust formation during handling, or to color the particles. Optional coatings may include salt coatings or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0337] The coating may be applied at a rate of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%) of the core weight. This amount may be at most 100%, 70%, 50%, 40%, or 30%.

[0338] The coating is preferably at least 0.1 µm thick, particularly at least 0.5 µm, at least 1 µm, or at least 5 µm thick. In some embodiments, the coating thickness is less than 100 µm, such as less than 60 µm or less than 40 µm.

[0339] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating with very few or no pores, such that the core unit has very few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0340] The coating may further contain other materials as known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers and / or adhesives, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0341] Salt coatings may contain at least 60% salt by weight, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% salt by weight.

[0342] To provide acceptable protection, the salt coating is preferably at least 0.1 µm thick, for example at least 0.5 µm, at least 1 µm, at least 2 µm, at least 4 µm, at least 5 µm, or at least 8 µm. In particular embodiments, the thickness of the salt coating is less than 100 µm, such as less than 60 µm or less than 40 µm.

[0343] Salt can be added from a salt solution (where the salt is completely dissolved) or from a salt suspension (where the fine particles are less than 50 µm, for example less than 10 µm or less than 5 μm).

[0344] Salt coatings may contain a single salt or a mixture of two or more salts. The salts may be water-soluble, particularly having a solubility of at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g / 100 g of water, for example at least 1 g / 100 g of water, for example at least 5 g / 100 g of water.

[0345] Salts can be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides, or carbonates, or salts of simple organic acids (less than 10 carbon atoms, such as 6 or fewer carbon atoms), such as citrates, malonates, or acetates. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or first transition metal ions, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonic acid, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate, or salts of simple organic acids such as citrate, malonate or acetate can be used.

[0346] The salt in the coating may have a constant humidity of 60% or more, particularly 70%, 80% or more or 85% or more at 20°C, or it may be another hydrated form of such salt (e.g., anhydrous form). Salt coatings may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0347] A specific example of a suitable salt is NaCl (CH4). 20°C = 76%), Na2CO3 (CH 20°C = 92%), NaNO3 (CH 20°C =73%), Na2HPO4 (CH 20°C = 95%), Na3PO4 (CH 25°C = 92%), NH4Cl (CH 20°C = 79.5%), (NH4)2HPO4 (CH 20°C= 93.0%), NH4H2PO4 (CH 20°C = 93.1%), (NH4)2SO4 (CH 20°C = 81.1%), KCl (CH 20°C =85%), K2HPO4 (CH 20°C = 92%), KH2PO4 (CH 20°C = 96.5%), KNO3 (CH 20°C = 93.5%), Na2SO4 (CH 20°C =93%), K2SO4 (CH 20°C = 98%), KHSO4 (CH 20°C = 86%), MgSO4 (CH 20°C = 90%), ZnSO4 (CH 20°C = 90%) and sodium citrate (CH 25°C = 86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0348] Salts can be in anhydrous form, or they can be hydrated salts, i.e., crystalline salt hydrates with bound water for crystallization, such as those described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na₂SO₄), anhydrous magnesium sulfate (MgSO₄), and magnesium sulfate heptahydrate (MgSO₄). . 7H2O), zinc sulfate heptahydrate (ZnSO4) . 7H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4) . 7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0349] Preferably, the salt is used as a salt solution, for example, in a fluidized bed.

[0350] Coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols containing 12 to 20 carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591.

[0351] The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0352] The core can be prepared by blends of granulated components, for example by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, granulation, spheronization, particle size reduction, drum granulation, and / or high-shear granulation.

[0353] Methods for preparing the core can be found in *Handbook of Powder Technology*; CE Capes, *Particle Size Enlargement*; Volume 1; 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, such as:

[0354] (a) Spray-dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets, which are dried as they descend along the drying tower to form enzyme-containing particulate material. This method can produce very small particles (Michael S. Showell (ed.); Powdered detergents; SurfactantScience Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0355] (b) Layered products in which an enzyme is coated in layers around a pre-formed inert core particle, wherein the enzyme-containing solution is typically atomized in a fluidized bed apparatus, in which the pre-formed core particle is fluidized and the enzyme-containing solution adheres to the core particle and is dried until a dry enzyme layer remains on the surface of the core particle. If useful core particles of the desired size can be found, particles of the desired size can be obtained in this manner. This type of product is described, for example, in WO 97 / 23606.

[0356] (c) Absorption of the core particle, wherein the enzyme is absorbed not by coating the variant in layers around the core, but by absorbing it on and / or within the surface of the core. Such a method is described in WO 97 / 39116.

[0357] (d) Extruded or pelletized products, wherein a variant-containing paste is pressed into pellets or extruded under pressure through small openings and cut into particles, which are then dried. Such particles are typically of considerable size because the material with the extrusion openings (usually a flat plate with perforations) limits the pressure drop allowed through the extrusion openings. Furthermore, when using small openings, the very high extrusion pressure increases the heat generated in the enzyme paste, which is detrimental to the enzyme (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0358] (e) Spray-granulated products, wherein a variant-containing powder is suspended in molten wax, and the suspension is sprayed (e.g., via a rotary sprayer) into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one in which the variant is uniformly distributed throughout the inert material rather than concentrated on its surface. This technique is described in US 4,016,040 and US 4,713,245.

[0359] (f) A mixer-granulated product in which a variant liquid is added to a dry powder composition of conventional granulation components. The liquid and powder are mixed in a suitable ratio, and because the water content of the liquid is absorbed by the dry powder, the dry powder components begin to adhere and aggregate, and the particles accumulate to form enzyme-containing granules. Such methods are described in US 4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO 90 / 09428. In certain aspects of this process, various high-shear mixers can be used as granulators. Granules consisting of variants, fillers, and binders are mixed with cellulose fibers to reinforce the granules, thereby producing so-called T-granulates. The reinforced granules are more robust and release less enzyme dust.

[0360] (g) Particle size reduction, in which core particles are generated by grinding or crushing larger enzyme-containing particles, pellets, flat sheets, briquettes, etc. The desired core particle fraction is obtained by sieving the grinding or crushing product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0361] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending microparticles in an airflow and spraying liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets become wetted and sticky. The sticky particles collide with and adhere to other particles to form granules.

[0362] (i) These cores can be dried, for example, in a fluidized bed dryer. Those skilled in the art can use other known methods for drying pellets in the feed or enzyme industry. Drying is preferably carried out at a product temperature of 25°C to 90°C. For some enzymes, it is important that the cores containing the variants contain a small amount of water before salt coating. If water-sensitive enzymes are salt-coated before removing excess water, the excess water will be trapped in the core and may negatively affect enzyme activity. After drying, these cores preferably contain 0.1%–10% w / w water.

[0363] Dust-free particles may be generated, for example, as disclosed in US 4,106,991 and US 4,661,452, and may optionally be coated by methods known in the art.

[0364] The particle may further contain one or more additional enzymes. Each enzyme will then be present in more particles, ensuring a more uniform distribution of the enzymes and also reducing the physical separation of different enzymes due to the different particle sizes. The method for generating multi-enzyme co-particles is disclosed in ip.com disclosure IPCOM000200739D.

[0365] Another example of enzyme formulation using co-particles is disclosed in WO 2013 / 188331.

[0366] The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238216.

[0367] In embodiments, the particles further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0368] Liquid preparations

[0369] The present invention also relates to liquid compositions comprising variations of the present invention. The compositions may comprise enzyme stabilizers (examples of which include polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives such as aromatic borate esters, or phenyl boric acid derivatives such as 4-formylphenylboronic acid).

[0370] In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfate, carbonate, and silicate, as well as talc, clay, etc. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, these compositions contain about 5% to about 90% of such materials.

[0371] In one aspect, the liquid formulation contains 20%-80% w / w polyol. In one embodiment, the liquid formulation contains 0.001%-2% w / w preservative.

[0372] In another embodiment, the present invention relates to a liquid preparation comprising:

[0373] (A) Variations of the invention, 0.001%-25% w / w;

[0374] (B) 20%-80% w / w polyols;

[0375] (C) Optionally 0.001%-2% w / w preservative; and

[0376] (D) Water.

[0377] In another embodiment, the present invention relates to a liquid preparation comprising:

[0378] (A) Variations of the invention, 0.001%-25% w / w;

[0379] (B) 0.001%-2% w / w preservative;

[0380] (C) Optionally 20%-80% w / w polyols; and

[0381] (D) Water.

[0382] In another embodiment, the liquid formulation comprises one or more formulations, such as formulations selected from the group consisting of: polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate, and phosphate, preferably selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0383] In another embodiment, the liquid formulation comprises 20%-80% polyols (i.e., the total amount of polyols), such as 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols. In one embodiment, the liquid formulation comprises 20%-80% polyols, such as 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols, wherein the polyols are selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the total amount of polyol), such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0384] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof. In one embodiment, the liquid formulation contains 0.02%-1.5% w / w preservative, such as 0.05%-1% w / w or 0.1%-0.5% w / w preservative. In one embodiment, the liquid formulation contains 0.001%-2% w / w preservative (i.e., the total amount of preservative), such as 0.02%-1.5% w / w, 0.05%-1% w / w, or 0.1%-0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0385] In another embodiment, the liquid formulation further comprises one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranoside, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannoside, β-mannoside (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0386] Fermentation broth preparations or cell compositions

[0387] The present invention also relates to fermentation broth formulations or cell compositions comprising variants of the invention. The fermentation broth formulations or cell compositions further comprise additional components used in the fermentation process, such as cells (including host cells containing genes encoding variants of the invention, these host cells being used to produce the desired variant), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killing whole culture medium containing organic acids, killed cells and / or cell debris, and a culture medium.

[0388] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., expression of enzymes by the host cell) and secretion of proteins into the cell culture medium. Fermentation broth may contain unfractionated or fractionated contents of fermentation material derived at the end of fermentation. Typically, fermentation broth is unfractionated and contains used culture medium and cell debris remaining after, for example, removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, fermentation broth contains used cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0389] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component (an organic acid containing at least one to five carbons and / or its salt) and a second organic acid component (an organic acid containing at least six or more carbons and / or its salt). In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, its salt, or a mixture of two or more of the foregoing.

[0390] In one aspect, the composition contains one or more organic acids and optionally further contains killed cells and / or cell debris. In some embodiments, these killed cells and / or cell debris are removed from the cell-killing whole culture medium to provide a composition free of these components.

[0391] Fermentation broth formulations or cell compositions may further contain preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.

[0392] The cell-killing whole culture or cell composition may contain ungraded contents of fermentation material derived at the end of fermentation. Typically, the cell-killing whole culture or cell composition contains used culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions to allow protein synthesis. In some embodiments, the cell-killing whole culture or cell composition contains used cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeate and / or lyse the microbial cells present in the cell-killing whole culture or cell composition.

[0393] The whole culture medium or cell composition described herein is typically a liquid, but may contain insoluble components such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

[0394] The whole culture medium formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0395] Detergent composition

[0396] The present invention also relates to compositions comprising variations of the present invention, such as detergent compositions or cleaning compositions.

[0397] The present invention also relates to compositions comprising variations of the invention and further comprising: one or more detergent components; and / or one or more additional enzymes. In preferred embodiments, the composition is a detergent composition comprising one or more detergent components, particularly one or more non-naturally occurring detergent components.

[0398] The present invention also relates to compositions comprising variations of the invention and further comprising one or more additional enzymes selected from the group consisting of: amylase (e.g., α-amylase), catalase, cellulase (e.g., endoglucanase), keratinase, DNase, halogenated peroxidase, lipase, mannanase, pectinase, pectin lyase, peroxidase, protease, xanthan gumase, licheninase, and xyloglucanase, or any mixture thereof.

[0399] Detergent compositions may be in the form of, for example, strips, uniform tablets, tablets having two or more layers, bags having one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or concentrated liquids.

[0400] In a preferred embodiment, the detergent composition is a liquid composition.

[0401] In a preferred embodiment, the detergent composition is a powder composition.

[0402] In a preferred embodiment, the detergent composition is a laundry soap bar.

[0403] The present invention also relates to the use of the compositions of the invention in cleaning processes, such as laundry or hard surface cleaning, such as dishwashing.

[0404] The selection of additional components of the detergent composition is within the skill of a person skilled in the art and includes conventional ingredients, including exemplary, non-limiting components listed below. For fabric care, the selection of components may include considerations such as the type of fabric to be cleaned, the type and / or extent of soiling, the temperature at which cleaning is performed, and the formulation of the detergent product.

[0405] In particular embodiments, the detergent composition comprises variations of the invention and one or more non-naturally occurring detergent components, such as surfactants, water-soluble additives, builders, co-builders, chelating agents or chelating agents, bleaching systems or bleaching components, polymers, fabric toners, fabric conditioning agents, foaming agents, defoaming agents, dispersants, dye transfer inhibitors, fluorescent brighteners, fragrances, optical brighteners, bactericides, fungicides, dirt suspending agents, dirt-releasing polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0406] In one embodiment, a variant of the invention may be added to a detergent composition in amounts corresponding to: 0.01-200 mg enzyme protein / L detergent solution, preferably 0.05-50 mg enzyme protein / L detergent solution, particularly 0.1-10 mg enzyme protein / L detergent solution.

[0407] Automatic dishwashing (ADW) compositions may, for example, include 0.001%-30%, such as 0.01%-20%, such as 0.1%-15%, such as 0.5%-10% of enzyme protein by weight of the composition.

[0408] The granular composition for laundry washing may, for example, include 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% of enzyme protein by weight of the composition.

[0409] Liquid compositions for washing clothes may include, for example, 0.0001%-10%, such as 0.001%-7%, such as 0.1%-5% of enzyme protein by weight of the composition.

[0410] The enzyme (e.g., variants of the invention) can be stabilized using conventional stabilizers, such as polyols like propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives like aromatic borate esters, or phenyl boric acid derivatives like 4-formylphenylboronic acid. The composition can be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708, or the variants according to the invention can be stabilized using peptide aldehydes or ketones as described in WO 2005 / 105826 and WO 2009 / 118375.

[0411] Variations of the present invention can be formulated in liquid laundry compositions, such as liquid laundry compositions comprising:

[0412] a) At least 0.01 mg of the active variant per liter of detergent,

[0413] b) At least one surfactant, ranging from 2 wt% to 60 wt%.

[0414] c) At least one detergent builder, ranging from 5 wt% to 50 wt%.

[0415] Detergent compositions can be formulated into granular detergents for washing clothes. Such detergents may contain:

[0416] a) At least 0.01 mg of active protease variant / gram composition

[0417] b) Preferably, 5 wt% to 50 wt% of anionic surfactant,

[0418] c) Preferably, 1 wt% to 8 wt% of a nonionic surfactant,

[0419] d) Preferably, 5 wt% to 40 wt% of a detergent builder, such as a carbonate, zeolite, phosphate detergent builder, calcium chelating detergent builder, or complexing agent.

[0420] Although the components mentioned below are classified under a general heading according to their specific functionality, this is not to be construed as limiting, as will be understood by those skilled in the art, the components may contain additional functionality.

[0421] surfactants

[0422] Detergent compositions may contain one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or mixtures thereof. In particular embodiments, the detergent composition comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. The one or more surfactants are typically present at levels ranging from about 0.1% to 60% by weight, for example, from about 1% to about 40%, or from about 3% to about 20%, or from about 3% to about 10%. The one or more surfactants are selected based on the desired cleaning application, and the surfactants include any one or more conventional surfactants known in the art. Any surfactant known in the art for use in detergents may be used. Surfactants reduce the surface tension in the detergent, which causes the stains being cleaned to be lifted and dispersed, and then washed away.

[0423] When included therein, detergents typically contain from about 1% to about 40% by weight, for example from about 5% to about 30%, including from about 5% to about 15% or from about 20% to about 25% of anionic surfactants. Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates, and disulfonates, alkyl sulfates (AS) (e.g., sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), and primary alcohol sulfates (PAS). Alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl sulfonate (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or soaps, and combinations thereof.

[0424] When included therein, detergents typically contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0425] When included therein, detergents typically contain from about 0.2% to about 40% by weight (e.g. from about 0.5% to about 30%, particularly from about 1% to about 20%, from about 3% to about 10%, for example from about 3% to about 5% or from about 8% to about 12%) of nonionic surfactants. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), N-acyl N-alkyl derivatives of polyhydroxyalkyl fatty acid amides or glucosamines (glucosamide GA, or fatty acid glucosamide FAGA), as well as products available under the trademarks SPAN and TWEEN, and combinations thereof.

[0426] When included therein, the detergent will typically contain about 0% to about 10% by weight of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs), such as alkyl dimethyl amine oxides, N-(cocoylalkyl)-N,N-dimethyl amine oxides and N-(butter-alkyl)-N,N-bis(2-hydroxyethyl) amine oxides, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides and combinations thereof.

[0427] When included, the detergent will typically contain about 0% to about 10% by weight of a facultative zwitterionic surfactant. Non-limiting examples of facultative zwitterionic surfactants include betaine, alkyl dimethyl betaine, sulfobetaine, and combinations thereof.

[0428] Builders and co-builders

[0429] Detergent compositions may contain about 0% to 65% (e.g., about 5% to about 45%) of detergent builders or co-builders, or mixtures thereof, by weight. In dishwashing detergents, the level of builders is typically 40% to 65%, particularly 50% to 65%. Builders and chelating agents soften wash water, for example, by removing metal ions from the liquid. Builders and / or co-builders may be chelating agents, particularly those that form water-soluble complexes with Ca and Mg. Any builders and / or co-builders known in the art for use in laundry detergents may be used. Non-limiting examples of detergent builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethanol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2'-meta-aminotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.

[0430] The detergent composition may also contain 0-20% by weight, for example, about 5% to about 10%, of a detergent co-adjuvant or a mixture thereof. The detergent composition may include a co-adjuvant alone or in combination with a builder (e.g., a zeolite builder). Non-limiting examples of co-adjuvants include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers of (acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents (such as aminocarboxylates, aminopolycarboxylates, and phosphonates), and alkylsuccinic acids or alkenylsuccinic acids. Other specific examples include 2,2',2”-nitrotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta-(methylenephosphonic acid) (DTPMPA or DTMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-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 (SEGL), 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), N-(2-hydroxyethyl)-ethylenediamine-N,N',N'-triacetic acid (HEDTA), diethanolamine glycine (DEG), diethylenetriaminepenta (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations thereof and salts thereof. Further exemplary builders and / or co-builders are described in, for example, WO In 2009 / 102854 and US 5,977,053.

[0431] Variations of the present invention can also be formulated in dishwashing compositions, preferably automatic dishwashing (ADW) compositions, which comprise:

[0432] a) At least 0.01 mg of the active protease variant according to the invention, and

[0433] b) 10 wt%-50 wt% of a detergent builder, preferably selected from citric acid, methylglycine-N,N-diacetic acid (MGDA), and / or glutamic acid-N,N-diacetic acid (GLDA), and mixtures thereof, and

[0434] c) At least one bleaching component.

[0435] bleaching system

[0436] Detergents may contain 0-50% by weight, such as about 0.1% to about 25%, of a bleaching system. Bleaching systems remove fading often caused by oxidation, and many bleaches also have strong bactericidal properties and are used for disinfection and sterilization. Any bleaching system known in the art for use in laundry detergents can be used. Suitable bleaching system components include bleaching catalysts, photobleaching agents, bleaching activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, pre-formed peracids, and mixtures thereof. Suitable pre-formed peracids include, but are not limited to: peroxycarboxylic acids and their salts, percarbonate and their salts, perimidic acid and its salts, peroxymonosulfate and its salts (e.g., potassium peroxymonosulfate (Oxone(R))), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems combined with peracids to form bleaching activators, which may contain, for example, inorganic salts, including alkali metal salts such as sodium salts of perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, superphosphates, and persilicates.

[0437] The term bleaching activator, as used herein, refers to a compound that reacts with a peroxide bleaching agent (such as hydrogen peroxide) to form a peracid. The resulting peracid constitutes the activated bleaching agent. Suitable bleaching activators as used herein include those belonging to the classes of esters, amides, imides, or acid anhydrides. Suitable examples are tetraacetyl ethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzenesulfonate (ISONOBS), disperoxylauric acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)benzenesulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A particular family of desired bleaching activators is disclosed in EP 624154, and a particularly preferred family is triethyl acetylacetate (ATC). ATC, or short-chain triglycerides like glyceryl acetate, has the advantage of being environmentally friendly because it ultimately degrades into citric acid and alcohol. Furthermore, triethyl acetylcitrate and glyceryl triacetate exhibit good hydrolytic stability in the product during storage and are highly effective bleaching activators. Finally, ATC provides excellent washing aid properties for laundry detergent additives. Alternatively, the bleaching system may contain peracids of the type such as amides, imides, or sulfones. The bleaching system may also contain peracids, such as 6-(phthalimide)percapanoic acid (PAP). The bleaching system may also include bleaching catalysts or accelerators.

[0438] Some non-limiting examples of bleaching catalysts that can be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese collagen, cobalt-amine catalysts, and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), especially Me3-TACN, such as the binuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2,2',2''-metaminotris(ethane-1,2-diylazalkylidene-κN-methylidene)triphenol-κ3O]manganese(III). These bleaching catalysts can also be other metal compounds, such as iron or cobalt complexes.

[0439] In some embodiments, the bleaching component may be an organic catalyst selected from the group consisting of organic catalysts having the following formula:

[0440]

[0441] (iii) and mixtures thereof; wherein each R1 is independently a branched alkyl group containing 9 to 24 carbons or a straight-chain alkyl group containing 11 to 24 carbons, preferably, each R1 is independently a branched alkyl group containing 9 to 18 carbons or a straight-chain alkyl group containing 11 to 18 carbons, more preferably, each R1 is independently selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isononyl, isodecyl, isothidecyl, isotriadecyl, and isopentadecyl. Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242. Suitable photobleaching agents may be, for example, sulfonated zinc phthalocyanine.

[0442] Water-soluble additives

[0443] Water-soluble additives are compounds that dissolve hydrophobic compounds (or conversely, polar substances in nonpolar environments) in aqueous solutions. Typically, water-soluble additives exhibit both hydrophilic and hydrophobic characteristics (so-called amphiphilic properties, as known from surfactants); however, the molecular structure of water-soluble additives generally does not favor spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler, 2007, Current Opinion in Colloid & Interface Science 12:121-128. Water-soluble additives do not exhibit the critical concentrations (above which self-aggregation occurs) seen in surfactants and lipids that form micelles, thin layers, or other well-defined meso-phases. Instead, many water-soluble additives exhibit a continuous type of aggregation process in which the size of the aggregates increases with increasing concentration. However, many water-soluble additives alter the phase behavior, stability, and colloidal properties of systems containing substances with both polar and nonpolar characteristics, including mixtures of water, oils, surfactants, and polymers. Water-soluble adjuvants are commonly used in various industries, from pharmaceuticals and personal care to food and technical applications. The use of water-soluble adjuvants in detergent compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0444] Detergents may contain 0-5% by weight, such as about 0.5% to about 5%, or about 3% to about 5%, of a water-soluble adjuvant. Any water-soluble adjuvant known in the art for use in detergents may be used. Non-limiting examples of water-soluble adjuvants include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols, and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthoate, sodium ethylhexyl sulfate, and combinations thereof.

[0445] polymer

[0446] The detergent may contain 0-10% (e.g., 0.5%-5%, 2%-5%, 0.5%-2%, or 0.2%-1%) of a polymer by weight. Any polymer known in the art for use in detergents may be used. The polymer may function as a co-agent as mentioned above, or may provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, grease removal, and / or defoaming properties. Some polymers may have more than one of the properties mentioned above and / or more than one of the motifs mentioned below. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylate esters such as PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicone, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(ethylene oxyterephthalate) (PET-POET), PVP, poly(vinylimidazolium) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazolium (PVPVI). Other exemplary polymers include sulfonated polycarboxylate esters, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the polymers mentioned above are also considered.

[0447] Fabric colorant

[0448] The detergent compositions of the present invention may further include fabric colorants, such as dyes or pigments, which, when formulated in the detergent composition, can deposit on the fabric when the fabric comes into contact with a washing liquid containing the detergent composition, and thus alter the color of the fabric through absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. Conversely, fabric colorants alter the color of the surface when they absorb at least a portion of the visible spectrum. Suitable fabric colorants include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of dyes falling into the Color Index (CI) classification: Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, or mixtures thereof, such as those described in WO 2005 / 003274, WO 2005 / 003275, WO 2005 / 003276 and EP 1876226 (incorporated herein by reference). The detergent composition preferably comprises from about 0.00003 wt.% to about 0.2 wt.%, from about 0.00008 wt.% to about 0.05 wt.%, or even from about 0.0001 wt.% to about 0.04 wt.% of a fabric toner. The composition may contain from 0.0001 wt% to 0.2 wt.% of a fabric toner, which may be particularly preferred when the composition is in the form of unit dose pouches. Suitable toners are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0449] Other enzymes

[0450] Detergent additives or detergent compositions comprising variations of the present invention may contain one or more enzymes, such as amylase (e.g., α-amylase), arabinase, glycosylase, cellulase (e.g., endoglucanase), keratinase, DNase, galactanase, halogenated peroxidase, lipase, mannanase, oxidase (e.g., laccase and / or peroxidase), pectinase, pectin lyase, protease, xylanase, xanthan gumase, or xyloglucanase.

[0451] The properties of the selected one or more enzymes should be compatible with the selected detergent (e.g., optimal pH, compatibility with other enzyme or non-enzyme components, etc.).

[0452] Cellulase

[0453] The term "cellulase" refers to one or more (e.g., several) enzymes that hydrolyze cellulose materials. The term "cellulase" and the expression "polypeptide with cellulase activity" are used interchangeably. Cellulases can be selected from the group consisting of cellulases belonging to GH5, GH44, GH45, EC 3.2.1.4, EC 3.2.1.21, EC 3.2.1.91, and EC 3.2.1.172. Such enzymes include one or more endoglucanases (e.g., EC 3.2.1.4), one or more cellobiases, one or more β-glucosidases, or combinations thereof.

[0454] Suitable cellulases include single-component and mixtures of enzymes derived from bacteria or fungi. Chemically modified mutants or protein-engineered mutants are also considered. Cellulases can be, for example, single-component endo-1,4-β-glucanases (also known as endoglucans) or mixtures of single-component endo-1,4-β-glucanases.

[0455] Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Pyrophyllus*, *Fusarium*, *Fusarium*, *Fusarium*, *Trichoderma*, and *Cladosporium*. Exemplary cellulases include fungal cellulases from *Pyrophyllus* (US 4,435,307) or from the genus *Trichoderma* (e.g., *Trichoderma reesei* or *Trichoderma viride*). Other suitable cellulases are from the genus *Fusarium*, such as *Fusarium terreum* as described in WO 96 / 29397 or fungal cellulases produced by *Pyrophyllus thermophilus* and *Fusarium oxysporum* disclosed in US 5,648,263, US 5,691,178, US 5,776,757, WO 89 / 09259, and WO 91 / 17244. Cellulases from the genus *Bacillus* are also relevant, as described in WO 02 / 099091 and JP 2000210081. Suitable cellulases are alkaline or neutral cellulases that provide nursing benefits. Examples of cellulases are described in EP 0 495257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are those cellulase variants described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, and WO 98 / 12307.

[0456] Other cellulases are endoglucanases having the following sequence, which has at least 97% identity with the amino acid sequence at positions 1 to 773 of SEQ ID NO: 2 of WO 2002 / 099091; or family 44 xyloglucanases having the following sequence, which has at least 60% identity with positions 40-559 of SEQ ID NO: 2 of WO 2001 / 062903.

[0457] Another suitable group of cellulases includes a stable linker between the core and the CBM. Of particular use are cellulases that have at least 80% identity with SEQ ID NO: 397, SEQ ID NO: 398 or SEQ ID NO: 399 of WO2023 / 061928.

[0458] Commercially available cellulases include Carezyme®, Carezyme® Premium, Celluzyme®, Carezyme Elite®, Celluclean®, Celluclast®, Endolase®, Renozyme®, Whitezyme®, Celluclean® Classic, and Cellusoft® (Novozymes A / S); Puradax®, Puradax HA, Puradax EG, Revitarenz 1000, Revitarenz 200, and Revitarenz 2000 (DuPont Industrial Biosciences); KAC-500(B)™ (Kao Corporation); and Biotouch DCL and Biotouch FLX1 (AB Enzymes).

[0459] Two basic methods for measuring cellulase activity include: (1) measuring total cellulase activity, and (2) measuring individual cellulase activity (endoglucanase, cellobiase, and β-glucosidase), as described in Zhang et al., 2006, Biotechnology Advances 24: 452-481. Insoluble substrates, including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc., can be used to measure total cellulase activity. The most common assay for total cellulase activity is the filter paper assay using Whatman No. 1 filter paper as the substrate. This assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59: 257-68).

[0460] protease

[0461] The composition may contain one or more additional proteases, including those of bacterial, fungal, plant, viral, or animal origin, such as those of plant or microbial origin. Microbial origin is preferred. This includes chemically modified mutants or protein-engineered mutants. It may be an alkaline protease, such as a serine protease or a metalloproteinase. Serine proteases may be, for example, from the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteinases may be, for example, thermophilic bacterial proteases from family M4 or other metalloproteinases, such as those from the M5, M7, or M8 families.

[0462] Examples of metalloproteinases are neutral metalloproteinases as described in WO 2007 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0463] Suitable commercially available proteases include those sold under the following trade names: Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, Esperase®, Progress® Excel, Progress® Key, and Progress® Uno (Novozymes), and those sold under the following trade names: Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Ox®, PurafectOxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Opticlean®, Optimase®, and Preferenz®. P200, Preferenz® P300 and Preferenz® P400 (DuPont / International Flavors & Fragrances, Inc. (IFF)), Axapem™ (Gist-Brocades NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (Alkaliophilic Bacillus subtilis protease) from Kao Corporation.

[0464] Lipase and keratinase

[0465] Suitable lipases and keratins include those derived from bacteria or fungi. This includes chemically modified mutant enzymes or protein-engineered mutant enzymes. Examples include lipases from the genus *Thermomyces*, such as *T. lanuginosus* (formerly named *Pyrophyllus lanuginosus*) as described in EP 258068 and EP 305216; keratinases from the genus *Pyrophyllus*, such as *Pyrophyllus specificus* (WO 96 / 13580); and lipases from strains of the genus *Pseudomonas* (some of which are now renamed *Burkholderia*), such as *P. alcaligenes* or *P. pseudoalcaligenes* (EP218272), *P. cepacia* (EP 331376), *P. SD705* (WO 95 / 06720 and WO 96 / 27002), and *P. wisconsinii*. wisconsinensis (WO 96 / 12012); GDSL-type Streptomyces lipase (WO 2010 / 065455); keratinase from Magnaporthe grisea (WO2010 / 107560); keratinase from Pseudomonas mendocina (US 5,389,536); lipase from Thermobifida fusca (WO 2011 / 084412); lipase from Geobacillus stearothermophilus (WO 2011 / 084417); lipase from Bacillus subtilis (WO 2011 / 084599); and lipase from Streptomyces griseus (WO 96 / 12012). Lipases of *Streptomyces pristinaespiralis* (WO 2012 / 137147).

[0466] Other examples are lipase variants, such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 2007 / 87508, and WO 2009 / 109500.

[0467] Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes), Lumafast (Genencor), and Lipomax (Gist Brocardes).

[0468] Other examples include lipases sometimes called acyltransferases or perhydrolases, such as acyltransferases homologous to Candida antarctica lipase A (WO 2010 / 111143), acyltransferases from Mycobacterium smegmatis (WO 2005 / 056782), perhydrolases from the CE 7 family (WO 2009 / 067279), and variants of Mycobacterium smegmatis perhydrolases (especially the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd) (WO 2010 / 100028).

[0469] amylase

[0470] Suitable amylases that can be used with variants of the present invention may be α-amylases or glucosylamylases and may be of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Amylases include, for example, α-amylases obtained from specific strains of the genus Bacillus, such as Bacillus licheniformis (described in more detail in GB 1,296,839).

[0471] Suitable amylases include the amylase having SEQ ID NO:2 in WO 95 / 10603 or a variant thereof having 90% sequence identity with SEQ ID NO:3. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and SEQ ID NO:4 in WO 99 / 19467, such variants having substituted positions at one or more of the following locations: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444.

[0472] Suitable amylases include the amylase having SEQ ID NO:6 in WO 02 / 10355 or its variants having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those with deletions at positions 181 and 182 and substitutions at position 193.

[0473] Other suitable amylases are hybrid α-amylases comprising residues 1-33 of the α-amylase derived from *Bacillus amylolyticus* as shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of the α-amylase from *Bacillus licheniformis* as shown in SEQ ID NO:4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid α-amylase are those with substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid α-amylase containing residues 1-33 of the α-amylase derived from Bacillus amylolyticus as shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO:4 is those having the following substitutions:

[0474] M197T;

[0475] H156Y+A181T+N190F+A209V+Q264S; or

[0476] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0477] Other suitable amylases are those having the sequence of SEQ ID NO:6 in WO 99 / 19467 or variants thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those with substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those with deletions at positions R181 and G182, or positions H183 and G184.

[0478] Other amylases that may be used are those of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:7 of WO 96 / 23873, or variants thereof having 90% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7. Preferred variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7 are those with substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476, numbered using SEQ ID 2 of WO 96 / 23873. More preferred variants are those with deletions at two positions selected from 181, 182, 183, and 184 (e.g., 181 and 182, 182 and 183, or positions 183 and 184). The most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7 are those with deletions at positions 183 and 184 and substitutions at one or more positions 140, 195, 206, 243, 260, 304, and 476.

[0479] Other amylases that may be used are those having SEQ ID NO:2 of WO 2008 / 153815, SEQ ID NO:10 of WO 01 / 66712, or a variant thereof having 90% sequence identity with SEQ ID NO:2 of WO 2008 / 153815, or a variant thereof having 90% sequence identity with SEQ ID NO:10 of WO 01 / 66712. Preferred variants of SEQ ID NO:10 of WO 01 / 66712 are those having substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0480] Another suitable amylase is the amylase of SEQ ID NO:2 with WO 2009 / 061380 or a variant thereof having 90% sequence identity with SEQ ID NO:2. Preferred variants of SEQ ID NO:2 are those with C-terminal truncation, and / or substitution, deletion, or insertion at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO:2 are those having substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or those having deletions at positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variant of SEQ ID NO:2 is those having the following substitutions:

[0481] N128C+K178L+T182G+Y305R+G475K;

[0482] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0483] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0484] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K,

[0485] These variants are C-terminated and optionally further include a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0486] Another suitable amylase is the amylase of SEQ ID NO:1 having WO 2013 / 184577 or a variant thereof having 90% sequence identity with SEQ ID NO:1. Preferred variants of SEQ ID NO:1 are those with substitutions, deletions or insertions at one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO:1 are those having substitutions at one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K, and G477K, and / or those having deletions at positions R178 and / or S179 or T180 and / or G181. The most preferred amylase variant of SEQ ID NO:1 comprises the following substitutions:

[0487] E187P+I203Y+G476K

[0488] E187P+I203Y+R458N+T459S+D460T+G476K

[0489] And optionally further include a substitution at position 241 and / or a deletion at positions 178 and / or 179.

[0490] Another suitable amylase is the amylase of SEQ ID NO:1 having WO 2010 / 104675 or a variant thereof having 90% sequence identity with SEQ ID NO:1. Preferred variants of SEQ ID NO:1 are those with substitutions, deletions, or insertions at one or more of the following positions: N21, D97, V128, K177, R179, S180, I181, G182, M200, L204, E242, G477, and G478.

[0491] More preferred variants of SEQ ID NO:1 are those with substitutions at one or more of the following positions: N21D, D97N, V128I, K177L, M200L, L204YF, E242QA, G477K, and G478K, and / or those with deletions at positions R179 and / or S180 or I181 and / or G182. The most preferred amylase variant of SEQ ID NO:1 comprises a substitution of N21D+D97N+V128I, and optionally further comprises a substitution at position 200 and / or a deletion at positions 180 and / or 181.

[0492] Other suitable amylases are α-amylases having SEQ ID NO:12 in WO 01 / 66712 or variants having at least 90% sequence identity with SEQ ID NO:12. Preferred amylase variants are those having substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO:12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions for R118K, N195F, R320K, and R458K, as well as variants having additional substitutions at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with the most preferred being variants having additional substitutions at all of these positions.

[0493] Other examples are those amylase variants described in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087. Commercially available amylases are Duramyl™, Termomyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, and BAN™ (from Novozymes), as well as Rapidase™, Purastar™ / Effectenz™, Powerase, Preferenz S1000, Preferenz S100, and Preferenz S110 (from Genentech International / DuPont).

[0494] Peroxidase / oxidase

[0495] Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, such as those from *Coprinus spp.*, and their variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0496] Commercially available peroxidases include Guardzyme™ (Novozymes).

[0497] auxiliary materials

[0498] Any detergent component known in the art for use in laundry detergents may also be used. Other optional detergent components include corrosion inhibitors, shrinkage inhibitors, anti-fouling redeposition agents, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegrant agents, dyes, enzyme stabilizers (including orthoboric acid, borates, CMC, and / or polyols, such as propylene glycol), fabric conditioners (including clays), fillers / processing aids, optical brighteners / brighteners, foaming agents, foam (foam) regulators, fragrances, soil suspending agents, softeners, defoaming agents, rust inhibitors, and wicking agents, used alone or in combination. Any ingredient known in the art for use in laundry detergents may be used. The selection of such ingredients is entirely within the skill of a person skilled in the art.

[0499] Dispersant: The detergent compositions of the present invention may also contain dispersants. In particular, powdered detergents may contain dispersants. Suitable water-soluble organic materials include homopolymeric or copolymeric acids or salts thereof, wherein the polycarboxylic acid contains at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker, 1997.

[0500] Dye transfer inhibitors: The detergent compositions of the present invention may further include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone, and polyvinylimidazole or mixtures thereof. When present in the subject composition, the dye transfer inhibitor can be present at levels of about 0.0001% to about 10%, about 0.01% to about 5%, or even about 0.1% to about 3% by weight of the composition.

[0501] Fluorescent whitening agent:The detergent compositions of the present invention will preferably also contain additional components that can color the article being cleaned, such as optical brighteners or fluorescent whitening agents. When present, the level of the brightening agent is preferably from about 0.01% to about 0.5%. Any suitable optical brightener used in laundry detergent compositions can be used in the compositions of the present invention. The most commonly used optical brighteners are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-bistyryl derivatives. Examples of diaminostilbene-sulfonic acid derivative-type fluorescent whitening agents include the following sodium salts: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2,4-diphenylamino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazine- 6-(2-phenyl-2,1,3-triazol-2-yl)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, and 2-(stilbene-4"-naphthalene-1.,2':4,5)-1,2,3-triazine-2"-sulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styrene)disulfonate. A further preferred fluorescent brightener is Parawhite KX, commercially available and supplied by Paramount Minerals and Chemicals in Mumbai, India. Other fluorescent agents suitable for use in this invention include 1,3-diarylpyrazoline and 7-alkylaminocoumarin. Suitable fluorescent brightener levels range from about 0.01 wt.%, from 0.05 wt.%, from about 0.1 wt.%, or even from a lower level of about 0.2 wt.% to a higher level of 0.5 wt.%, or even 0.75 wt.%.

[0502] Fouling-releasing polymers:The detergent compositions of the present invention may also include one or more dirt-releasing polymers that help remove dirt from fabrics, such as cotton and polyester-based fabrics, particularly hydrophobic dirt from polyester-based fabrics. Dirt-releasing polymers may be, for example, polymers based on nonionic or anionic terephthalic acid, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, PowderedDetergents, Surfactant Science Series, Volume 71, Chapter 7, Marcel Decker. Another type of dirt-releasing polymer is an amphiphilic alkoxylated grease-cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to that core structure. The core structure may comprise a polyalkylimide structure or a polyalkanolamine structure, as detailed in WO 2009 / 087523 (incorporated hereby by reference). Furthermore, random graft copolymers are suitable dirt-releasing polymers. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856, and WO 2006 / 113314 (which are hereby incorporated by reference). Other fouling-releasing polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, as described in EP 1867808 or WO 03 / 040279 (both of which are incorporated herein by reference). Suitable cellulose polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulose polymers include anionic modified cellulose, nonionic modified cellulose, cationic modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulose polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, ester carboxymethylcellulose, and mixtures thereof.

[0503] Anti-redeposition agent: The detergent compositions of the present invention may further include one or more anti-redeposition agents, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulose-based polymers described above under the category of dirt-releasing polymers may also function as anti-redeposition agents.

[0504] Other suitable auxiliary materials Including but not limited to shrink-proof agents, wrinkle-resistant agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, water-soluble additives, fragrances, pigments, defoamers, solvents, and structural agents and / or structural elastic agents used in liquid detergents.

[0505] Detergent product formulation

[0506] One or more detergent enzymes (i.e., variations of the invention and optionally one or more additional enzymes) can be included in a detergent composition by adding a single additive containing one or more enzymes, or by adding a combination additive containing all of these enzymes. Detergent additives containing one or more enzymes can be formulated as, for example, granules, liquids, slurries, etc. Preferred detergent additive formulations include granules, particularly dust-free granules; liquids, particularly stabilized liquids; or slurries.

[0507] The detergent compositions of the present invention can be in any suitable form, such as strips, homogeneous tablets, tablets having two or more layers, pouches having one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or concentrated liquids. Various detergent formulations exist, such as layers (same or different phases), pouches, and forms for machine dispensing units.

[0508] The bag can be configured as a single or multiple chambers. It can have any form, shape, and material suitable for preserving the composition, for example, preventing the composition from being released from the bag before contact with water. The bag is made of a water-soluble membrane containing an internal volume. This internal volume can be divided into chambers of the bag. Preferred membranes are polymeric materials, preferably polymers molded into films or sheets. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylates, most preferably polyvinyl alcohol copolymers, and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. Preferred average molecular weights are typically from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under trade reference number M8630, as marketed by Chris Craft In. Prod., Gary, Indiana, US), plus plasticizers, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may contain a solid laundry detergent composition or portion thereof separated by a water-soluble membrane and / or a liquid cleaning composition or portion thereof. The compartment for the liquid component may differ in composition from the compartment containing the solid component. See, for example, US 2009 / 0011970.

[0509] Detergent components can be physically separated from each other by compartments in a water-soluble pouch or by different layers of tablets. This avoids undesirable storage interactions between components. Furthermore, the different dissolution profiles of each compartment in the washing solution can cause delayed dissolution of the selected components.

[0510] Non-unit-volume liquid or gel detergents may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70%, up to about 65%, up to about 55%, up to about 45%, or up to about 35%. Other types of liquids, including but not limited to alkanols, amines, diols, ethers, and polyols, may be included in aqueous liquids or gels. Aqueous liquid or gel detergents may contain 0-30% organic solvents. Liquid or gel detergents may be non-aqueous.

[0511] Laundry soap bars

[0512] The enzymes of this invention can be added to laundry soap bars and used for hand washing clothes, fabrics, and / or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, synthetic detergent bars, and detergent bars. The types of bars are generally distinguished by the type of surfactant they contain, and the term laundry soap bar includes those containing soaps derived from fatty acids and / or synthetic soaps. Laundry soap bars have a physical form that is solid at room temperature and therefore not liquid, gel, or powder.

[0513] Laundry soap bars may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or sulfide adducts or hemiacetal adducts), boric acid, borates, borax and / or phenylboronic acid derivatives such as 4-carboxyphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerol, pH-controlling compounds such as fatty acids, citric acid, acetic acid and / or formic acid, and / or salts of monovalent cations and organic anions, wherein the monovalent cation may be, for example, Na+. + K + or NH4 + Furthermore, the organic anion can be, for example, a formate, acetate, citrate, or lactate, such that the salt of the monovalent cation and the organic anion can be, for example, sodium formate.

[0514] Laundry soap bars may also contain complexing agents such as EDTA and HEDP, fragrances and / or different types of fillers, surfactants such as anionic synthetic surfactants, builders, polymeric dirt releasers, detergent chelators, stabilizers, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, defoamers, structural agents, binders, leachants, bleach activators, clay detergents, anti-redeposition agents, polymeric dispersants, brighteners, fabric softeners, fragrances and / or other compounds known in the art.

[0515] Laundry soap bars can be processed in conventional laundry soap bar manufacturing equipment, such as, but not limited to, mixers, pressing machines (e.g., two-stage vacuum pressing machines), extruders, cutters, logo-stamper machines, cooling tunnels, and packaging machines. A premix containing soap, the enzymes of the present invention, optionally one or more other enzymes, protease inhibitors, and salts of monovalent cations and organic anions can be prepared, and then the mixture can be pressed into bars. The enzymes and optionally other enzymes can be added simultaneously, for example, in liquid form, as protease inhibitors. In addition to the mixing and pressing steps, the process may further include grinding, extrusion, cutting, molding, cooling, and / or packaging steps.

[0516] Granular Detergent Formulation

[0517] Enzymes in particulate (powder) detergents are typically used in particulate form (e.g., variants of the present invention), which comprise an enzyme-containing core and optionally one or more coatings. Various methods for preparing the core are well known in the art and include, for example, a) spray drying of a solution containing a liquid enzyme, b) producing a stratified product in which the enzyme is coated as a layer surrounding a pre-formed inert core particle, for example using a fluidized bed apparatus, c) adsorbing the enzyme onto and / or into the surface of a pre-formed core, d) extruding an enzyme-containing paste, e) suspending an enzyme-containing powder in molten wax and atomizing it to produce a granular product, f) granulation by mixing a liquid enzyme-containing component into a dry powder composition of the granulation component, g) reducing the particle size of the enzyme-containing core by grinding or crushing larger particles, pellets, etc., and h) fluidized bed granulation. The enzyme-containing core can be dried (e.g., using a fluidized bed dryer or other known methods for drying particles in the feed or enzyme industry), resulting in a moisture content typically of 0.1%–10% w / w water.

[0518] The enzyme-containing core is optionally coated to improve storage stability and / or reduce dust formation. One type of coating commonly used for enzyme particles in detergents is a salt coating, typically an inorganic salt coating, which can be applied, for example, with a fluid bed of salt solution. Other coating materials that can be used are, for example, polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). The particles may contain more than one coating, such as a salt coating, followed by additional coatings of materials such as PEG, MHPC, or PVA.

[0519] Therefore, the present invention also relates to enzyme particles / granules comprising variations of the present invention. In embodiments, the particle comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0520] The diameter of the core (measured as equivalent sphere diameter (volume-average particle size)) can be 20-2000 µm, particularly 50-1500 µm, 100-1500 µm or 250-1200 µm.

[0521] In the embodiments, the core comprises one or more polypeptides having the protease activity of the present invention.

[0522] The core may include other materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0523] The core may include binders, such as synthetic polymers, waxes, fats, or carbohydrates.

[0524] The core, typically as a homogeneous blend, may include salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components.

[0525] The core may include inert particles into which the enzyme is adsorbed or applied (e.g., by fluidized bed coating) to the surface of the inert particles.

[0526] The diameter of the core can be 20-2000 µm, especially 50-1500 µm, 100-1500 µm or 250-1200 µm.

[0527] The core may be surrounded by at least one coating, for example, to improve storage stability, reduce dust formation during handling, or to color the particles. Optional coatings may include salt coatings or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0528] The coating may be applied at a rate of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%) of the core weight. This amount may be at most 100%, 70%, 50%, 40%, or 30%.

[0529] The coating is preferably at least 0.1 µm thick, particularly at least 0.5 µm, at least 1 µm, or at least 5 µm thick. In some embodiments, the coating thickness is less than 100 µm, such as less than 60 µm or less than 40 µm.

[0530] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating with very few or no pores, such that the sealed / enclosed core unit has very few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0531] The coating may further contain other materials as known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers and / or adhesives, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0532] Salt coatings may contain at least 60% salt by weight, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% salt by weight.

[0533] To provide acceptable protection, the salt coating is preferably at least 0.1 µm thick, for example at least 0.5 µm, at least 1 µm, at least 2 µm, at least 4 µm, at least 5 µm, or at least 8 µm. In particular embodiments, the thickness of the salt coating is less than 100 µm, such as less than 60 µm or less than 40 µm.

[0534] Salt can be added from a salt solution (where the salt is completely dissolved) or from a salt suspension (where the fine particles are less than 50 µm, for example less than 10 µm or less than 5 μm).

[0535] Salt coatings may contain a single salt or a mixture of two or more salts. The salts may be water-soluble, particularly having a solubility of at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g / 100 g of water, for example at least 1 g / 100 g of water, for example at least 5 g / 100 g of water.

[0536] Salts can be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides, or carbonates, or salts of simple organic acids (less than 10 carbon atoms, such as 6 or fewer carbon atoms), such as citrates, malonates, or acetates. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or first transition metal ions, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonic acid, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate, or salts of simple organic acids such as citrate, malonate or acetate can be used.

[0537] The salt in the coating may have a constant humidity of 60% or more, particularly 70%, 80% or more or 85% or more at 20°C, or it may be another hydrated form of such salt (e.g., anhydrous form). Salt coatings may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0538] A specific example of a suitable salt is NaCl (CH4). 20°C = 76%), Na2CO3 (CH 20°C = 92%), NaNO3 (CH 20°C =73%), Na2HPO4 (CH 20°C = 95%), Na3PO4 (CH 25°C = 92%), NH4Cl (CH 20°C = 79.5%), (NH4)2HPO4 (CH 20°C = 93.0%), NH4H2PO4 (CH 20°C = 93.1%), (NH4)2SO4 (CH 20°C = 81.1%), KCl (CH 20°C =85%), K2HPO4 (CH 20°C = 92%), KH2PO4 (CH 20°C = 96.5%), KNO3 (CH 20°C = 93.5%), Na2SO4 (CH 20°C =93%), K2SO4 (CH 20°C = 98%), KHSO4 (CH20°C = 86%), MgSO4 (CH 20°C = 90%), ZnSO4 (CH 20°C = 90%) and sodium citrate (CH 25°C = 86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0539] Salts can be in anhydrous form, or they can be hydrated salts, i.e., crystalline salt hydrates with bound water for crystallization, such as those described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na₂SO₄), anhydrous magnesium sulfate (MgSO₄), magnesium sulfate heptahydrate (MgSO₄·7H₂O), zinc sulfate heptahydrate (ZnSO₄·7H₂O), disodium hydrogen phosphate heptahydrate (Na₂HPO₄·7H₂O), magnesium nitrate hexahydrate (Mg(NO₃)₂(6H₂O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0540] Preferably, the salt is used as a salt solution, for example, in a fluidized bed.

[0541] Coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols containing 12 to 20 carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591.

[0542] The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0543] The core can be prepared by blends of granulated components, for example by methods including granulation techniques such as crystallization, precipitation, pan coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, granulation, spheroidization, particle size reduction, drum granulation and / or high shear granulation.

[0544] Methods for preparing the core can be found in *Handbook of Powder Technology*; CE Capes, *Particle Size Enlargement*; Volume 1; 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, such as:

[0545] (a) Spray-dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets, which are dried as they descend along the drying tower to form an enzyme-containing particulate material. This method can produce very small particles (Michael S. Showell (ed.); Powdered detergents; SurfactantScience Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0546] (b) Layered products in which an enzyme is coated in layers around a pre-formed inert core particle, wherein the enzyme-containing solution is typically atomized in a fluidized bed apparatus, in which the pre-formed core particle is fluidized and the enzyme-containing solution adheres to the core particle and is dried until a dry enzyme layer remains on the surface of the core particle. If useful core particles of the desired size can be found, particles of the desired size can be obtained in this manner. This type of product is described, for example, in WO 97 / 23606.

[0547] (c) Absorbed core particles, wherein the enzyme is absorbed not by coating the core in layers, but by absorbing the enzyme on and / or within the surface of the core. Such a method is described in WO 97 / 39116.

[0548] (d) Extruded or pelletized products, wherein an enzyme-containing paste is pressed into pellets or extruded under pressure through small openings and cut into particles, which are then dried. Such particles are typically of considerable size because the material with the extrusion openings (usually a flat plate with perforations) limits the pressure drop allowed through the extrusion openings. Furthermore, when using small openings, the very high extrusion pressure increases the heat generated in the enzyme paste, which is detrimental to the enzyme (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0549] (e) Spray-granulated products, wherein an enzyme-containing powder is suspended in molten wax, and the suspension is sprayed (e.g., via a rotary sprayer) into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one in which the enzyme is uniformly distributed throughout the inert material rather than concentrated on its surface. This technique is described in U.S. Patent Nos. 4,016,040 and 4,713,245.

[0550] (f) A mixer-granulated product in which an enzyme-containing liquid is added to a dry powder composition of commonly used granulation components. The liquid and powder are mixed in a suitable ratio, and because the water content of the liquid is absorbed by the dry powder, the dry powder components begin to adhere and aggregate, and the particles accumulate to form enzyme-containing granules. Such a method is described in U.S. Patent No. 4,106,991 and related documents EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO 90 / 09428. In specific products of this method, various high-shear mixers can be used as granulators. Granules consisting of enzymes, fillers, and binders are mixed with cellulose fibers to reinforce the granules, resulting in so-called T-granules. The reinforced granules are more robust and release less enzyme dust.

[0551] (g) Particle size reduction, in which core particles are generated by grinding or crushing larger enzyme-containing particles, pellets, flat sheets, and lumps. The desired core particle fraction is obtained by sieving the ground or crushed product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0552] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending microparticles in an airflow and spraying liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets become wetted and sticky. The sticky particles collide with and adhere to other particles to form granules.

[0553] (i) These cores can be dried, for example, in a fluidized bed dryer. Those skilled in the art can use other known methods for drying granules in the feed or enzyme industry. Drying is preferably carried out at a product temperature of 25°C to 90°C. For some enzymes, it is important that the enzyme-containing core contains a small amount of water before salt coating. If water-sensitive enzymes are salt-coated before excess water removal, moisture will be trapped in the core and may negatively affect enzyme activity. After drying, these cores preferably contain 0.1%–10% w / w water.

[0554] Dust-free particles can be produced, such as those disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452, and these particles can optionally be coated by methods known in the art.

[0555] The particles can then contain one or more additional enzymes. Each enzyme will then be present in more particles, ensuring a more uniform distribution of the enzymes and also reducing the physical separation of different enzymes due to the varying particle sizes. Methods for generating multi-enzyme co-particles are disclosed in ip.com disclosure IPCOM000200739D.

[0556] Another example of enzyme formulation using co-particles is disclosed in WO 2013 / 188331.

[0557] The enzyme can also be a protected enzyme prepared according to the method disclosed in EP 238,216.

[0558] In embodiments, the particles further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0559] For further information on enzyme particles and their production, see WO 2013 / 007594, and for example WO 2009 / 092699, EP 1705241, EP 1382668, WO 2007 / 001262, US 6,472,364, WO 2004 / 074419 and WO2009 / 102854.

[0560] use

[0561] The present invention also relates to methods for using variations of the invention or compositions comprising said variations in the washing of textiles and fabrics (e.g., household and industrial garment washing).

[0562] The present invention also relates to methods for using variations or compositions thereof according to the invention in cleaning hard surfaces such as floors, tables, walls, roofs, etc., together with surfaces of hard objects such as automobiles (car wash) and tableware (dishwashing).

[0563] Variations of the invention can be added to detergent compositions, thus making the variation a component of the detergent composition. Therefore, one aspect of the invention relates to the use of variations of the invention in cleaning processes such as laundry and / or hard surface cleaning.

[0564] The detergent compositions of the present invention can be formulated as, for example, hand or machine washing detergent compositions, including a laundry additive composition suitable for pretreating stained fabrics and a fabric softener composition added during rinsing, or formulated as detergent compositions for general household hard surface cleaning operations, or formulated for hand or machine washing of dishes.

[0565] The cleaning or textile care process can be, for example, a laundry process, a dishwashing process, or the cleaning of hard surfaces (such as bathroom tiles, floors, countertops, drains, sinks, and basins). The laundry process can be, for example, household laundry, but can also be industrial laundry. Furthermore, the present invention relates to a method for washing fabrics and / or garments, wherein the method comprises treating the fabric with a washing solution containing a detergent composition and at least one protease variant of the present invention. For example, the cleaning or textile care process can be carried out in machine washing or in manual washing. The washing solution can be, for example, an aqueous washing solution containing a detergent composition.

[0566] In one aspect, variations of the invention are used in cleaning processes, such as laundry processes, which include short wash cycles, typically not exceeding about 30 minutes, for example, not exceeding about 20 minutes, for example, not exceeding about 15 minutes, or not exceeding about 10 minutes. Surprisingly, the Bacillus subtilis enzyme variants of the invention have been found to be remarkably effective in short wash cycles lasting, for example, only about 10-20 minutes. This could be useful, for example, in top-loading washing machines that typically have short wash cycles or for hand washing of clothes.

[0567] On the other hand, variations of the invention can be used in cleaning processes, such as laundry processes, where wash water is used for more than one portion of the laundry. In this case, wash water containing a detergent along with a variation of the invention can be used for a first portion of the laundry in a first wash cycle, and then repeated once or more for subsequent wash cycles with new portions of laundry. It has been found that, even after three or more wash cycles, detergents containing variations of the invention can substantially maintain their cleaning performance for protease-sensitive stains. This may be useful, for example, for hand-washed clothing and / or in dry areas.

[0568] In recent years, there has been a growing interest in alternative detergent components, stemming from the use of renewable biological components such as enzymes and peptides to replace petrochemicals without compromising washing performance. When the composition of a detergent composition changes, there is a need for new enzyme activities or new enzymes with alternative and / or improved properties compared to previously used detergent enzymes (such as proteases, lipases, and amylases) to achieve similar or improved washing performance compared to conventional detergent compositions.

[0569] The present invention further relates to the use of variations of the invention in the process of removing protein stains. Protein stains can be, for example, food stains (e.g., baby food, cocoa, egg, or milk), or other stains (e.g., sebum, blood, ink, or grass), or combinations thereof.

[0570] Washing method

[0571] The present invention provides a method for cleaning fabrics, tableware or hard surfaces using a detergent composition comprising a variant of the present invention.

[0572] The cleaning method involves contacting an object with a detergent composition comprising a protease variant of the present invention under conditions suitable for cleaning the object. In a preferred embodiment, the detergent composition is used in the washing of clothing or tableware.

[0573] Another embodiment relates to a method for removing stains from fabrics or tableware, the method comprising contacting the fabric or tableware with a composition including the protease of the present invention under conditions suitable for cleaning the object. In the cleaning method of the present invention, the object being cleaned can be any suitable object, such as textiles or hard surfaces, such as tableware or floors, tables, walls, etc.

[0574] Compositions and methods for treating fabrics (e.g., desizing textiles) using protease variants of the present invention are also contemplated. The protease variants can be used in any fabric treatment method well known in the art (see, for example, US 6,077,316). For example, in one aspect, the feel and appearance of a fabric are improved by including contacting the fabric with a protease variant in a solution. In another aspect, the fabric is treated with the solution under pressure.

[0575] The detergent compositions of the present invention are suitable for use on clothing and hard surfaces, including dishwashing. Accordingly, the present invention includes a method for washing fabrics or dishwashing, the method comprising contacting the fabric / dishwashing object to be cleaned with a solution comprising the detergent composition according to the present invention. Fabrics may include any fabric that can be washed under normal consumer use conditions. Dishwashing objects may include any tableware, such as earthenware, tableware, ceramics, plastics (e.g., melamine), metals, porcelain, glass, and acrylates. The solution preferably has a pH of about 5.5 to about 11.5. The composition can be used in the solution at concentrations from about 100 ppm (preferably 500 ppm) to about 15,000 ppm. The water temperature range is typically from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, and about 90°C. The water-to-fabric ratio is typically from about 1:1 to about 30:1.

[0576] One or more enzymes in the detergent compositions of the present invention can be stabilized using conventional stabilizers and protease inhibitors, such as polyols like propylene glycol or glycerol, sugars or sugar alcohols, various salts like NaCl; KCl; lactic acid, formic acid, boric acid, or boric acid derivatives (e.g., aromatic borate esters), or phenyl boric acid derivatives (e.g., 4-formylphenylboronic acid), or peptide aldehydes (e.g., dipeptide aldehydes, tripeptide aldehydes, or tetrapeptide aldehydes or aldehyde analogs) (or having the form B1-B0-R, where R is H, CH3, CX3, CHX2, or CH2X (X = halogen), B0 is a single amino acid residue (preferably having optionally substituted aliphatic or aromatic side chains); and B1 consists of one or more amino acid residues (preferably one, two, or three), optionally containing an N-terminal protecting group, or as in WO 2009 / 118375, WO The composition may be a protein-type protease inhibitor, such as RASI, BASI, WASI (a bifunctional α-amylase / subtilisin inhibitor for rice, barley, and wheat), or CI2 or SSI, as described in WO 98 / 13459. The composition may be formulated as described, for example, in WO 92 / 19709, WO 92 / 19708, and US 6,472,364. In some embodiments, the enzymes used herein are stabilized by water-soluble sources of zinc (II), calcium (II), and / or magnesium (II) ions present in the finished composition providing such ions for these enzymes, 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 oxide (IV)).

[0577] The detergent compositions provided herein are typically formulated such that, when used in aqueous cleaning operations, the wash water has a pH of about 5.0 to about 12.5, for example, from about 5.0 to about 11.5, or from about 6.0 to about 10.5. In some embodiments, particulate or liquid laundry detergent products are formulated to have a pH of about 6 to about 8. Techniques for controlling the pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0578] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0579] Example

[0580] This invention relates to novel protease variants that exhibit increased stability and / or improved washing performance in liquid detergent and soap bar compositions compared to parental proteases (e.g., improved removal of protein stains).

[0581] Material

[0582] textile

[0583] Standard textile or small fabric samples can be obtained from the Center for Testmaterials BV (P.E.T. 120, 3133 KT, Vlaardingen, Netherlands), Warwick Equest Ltd (Consett, DH8 6BN, UK) and the Swiss Federal Institute for Materials Science and Technology (EMPA) (Ueberlandstrasse 129, 8600, Dübendorf, Switzerland).

[0584] Detergent form

[0585] The following detergents can be formulated as described herein. One or more proteases for testing small fabric samples can then be added to a standard detergent matrix for testing.

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592] method

[0593] Preparation and purification of peptides

[0594] Mutagenesis was performed using standard methods known in the art, and the expression cassette was introduced into Bacillus subtilis. All DNA manipulation was performed by PCR (e.g., as described in Sambrook et al., 2001) using standard methods known to those skilled in the art.

[0595] The recombinant Bacillus subtilis construct encoding the protease peptide was inoculated into a multi-medium medium (TBgly) and cultured at 37°C under antibiotic selection for 24 h. The overnight culture was then inoculated at a ratio of 1:100 into shake flasks containing a rich medium (PS-1:100 g / L sucrose (Danisco catalog 109-0429), 40 g / L soybean hulls (soybean meal), 10 g / L Na2HPO4·12H2O (Merck catalog 106579), and 0.1 ml / L Dowfax 63N10 (Dow)). The flasks were then cultured at 30°C with shaking at 270 rpm for 4 days.

[0596] The culture supernatant was purified as follows: The culture medium was centrifuged at 26,000 xg for 20 min, and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 µm filter to remove any remaining host cells. The pH of the 0.2 µm filtrate was adjusted to pH 8 with 3 M Tris base, and the pH-adjusted filtrate was applied to a MEP Hypercel column (Pall Corporation) equilibrated in 20 mM Tris / HCl, 1 mM CaCl2 (pH 8.0). After washing the column with equilibration buffer, the column was gradually eluted with 20 mM CH3COOH / NaOH, 1 mM CaCl2 (pH 4.5). The protease activity of the fractions from the column was analyzed using a Suc-AAPF-pNA assay at pH 9, and the peak fractions were combined. The pH of the pooled fractions from the MEP Hypercel column was adjusted to pH 6 with 20% (v / v) CH3COOH or 3 M Tris base, and the pH-adjusted pooled fractions were diluted with deionized water to the same conductivity as 20 mM MES / NaOH, 2 mM CaCl2 (pH 6.0). The diluted pooled fractions were applied to an SP-Sepharose® fast flow column (GE Healthcare) equilibrated in 20 mM MES / NaOH, 2 mM CaCl2 (pH 6.0). After washing the column with equilibration buffer, the protease variants were eluted with a linear NaCl gradient (0 → 0.5 M) in the same buffer for five column volumes. The protease activity of the fractions from the column was analyzed using a Suc-AAPF-pNA assay at pH 9, and the activity fractions were analyzed by SDS-PAGE. Fractions showing only one band on a Coomassie-stained SDS-PAGE gel were pooled as purified formulations for further experiments.

[0597] Protease activity assay I

[0598] The proteolytic activity of the variants of this invention can be determined using the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline, and it is a blocked peptide that can be cleaved by an endopeptide. Following proteolytic cleavage, free pNA molecules, exhibiting a yellow color, are released, and this can be measured spectrophotometrically at a wavelength of 405 nm. The Suc-AAPF-pNA substrate is available from Bachem.

[0599] The sample containing the analyte variant was diluted in residual activity buffer (100 mM Tris, pH 8.6). The assay was performed by transferring 30 µl of the diluted enzyme sample to a 96-well microtiter plate and adding 70 µl of substrate working solution (0.72 mg / mL in 100 mM Tris (pH 8.6)). The solutions were mixed at room temperature, and the absorbance at 405 nm was measured over time (e.g., every 20 seconds for 5 minutes). The slope of the time-dependent absorbance curve (absorbance per minute) was proportional to the proteolytic activity.

[0600] Protease Activity Assay II

[0601] The proteolytic activity of detergent compositions containing variants of the present invention can be determined by using N,N-dimethylcasein (DMC) as a substrate. Peptide bond hydrolysis yields a carboxylic acid and a primary amine. The resulting amine is then reacted with 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma) under alkaline conditions to form a colored complex measurable at 405 nm.

[0602] Dissolve a detergent sample containing a variant of the present invention in 0.08 M sodium sulfite buffer and stir for 10 minutes, then filter the sample (using Waterman 54 filter paper or similar). Use a buffer solution (0.05 M boric acid + 0.16 M sodium sulfite + 0.15 M potassium chloride + 0.0225% (w / v) Brij). ® Sample dilution was performed at L23 (pH 9.00). Analysis was performed using a Konelab 30 analyzer (Thermo Fisher Scientific) with the following reagents according to the assay parameters outlined in Table 19: 1) 3.2 g / L DMC substrate + 0.1 M sodium dihydrogen phosphate monohydrate + 0.07 M Borax + 0.02% (w / v) Brij. ®L23, pH 8.00, 2) 0.1% TNBS, and 3) 0.1% TNBS + 0.4% DSAA. The activity value can then be calculated based on the standard curve.

[0603] Automated Mechanical Stress Measurement (AMSA)

[0604] To evaluate washing performance in laundry, washing experiments were conducted using an automated mechanical stress assay (AMSA). AMSA allows for the examination of the washing performance of large quantities of small-volume enzyme detergent solutions. An AMSA plate consists of several tanks for the test solution and a lid that firmly presses the laundry sample (the textile to be washed) (or, in the case of dishwashing detergent, a melamine brick) across all the tank openings. During washing, the plate, test solution, textile (or the dishwashing brick), and lid are vigorously shaken to bring the test solution into contact with the soiled test sample, applying mechanical stress in a regular, periodic oscillation pattern. For further description, see WO 2002 / 42740, particularly the “Specific Method Examples” paragraph on pages 23-24.

[0605] Washing performance is measured as the brightness of the color of the washed textile. Brightness can be expressed as the intensity of light reflected from the textile sample when illuminated with white light. When the textile is stained, the intensity of the reflected light is lower than that of the clean textile. Therefore, the intensity of the reflected light can be used to measure the washing performance of the target protease.

[0606] Color measurements were performed using a professional flatbed scanner (Epson Expression 10000XL, Atea A / S, Lautrupvang 6, 2750 Ballerup, Denmark) to capture images of the washed textile samples. To extract light intensity values ​​from the scanned images, a specially designed software application (Novozymes Color Vector Analyzer) was used. This program retrieves values ​​from the images and converts them into red, green, and blue (RGB) values. The intensity value (Int) can be calculated by summing the RGB values ​​as vectors and then taking the length of the resulting vector.

[0607]

[0608] Terg-o-tometer (TOM) washing measurement

[0609] The Terg-o-tometer (TOM) is a medium-scale washing assay that can be used to test up to 16 different conditions simultaneously. In short, it consists of 16 x 2 L metal beakers, each equipped with a stirrer that rotates back and forth at a controlled speed to simulate the agitation that occurs in a commercial top-loading washing machine. These beakers are partially immersed in a temperature-controlled water bath. Each beaker is filled with 1 L of detergent solution, and a small piece of fabric sample, ballast, and enzyme are added to the desired level. After a timed wash period, the fabric sample is immediately removed from the beaker and thoroughly rinsed with tap water.

[0610] Next, lay the small fabric sample flat on a rack covered with filter paper, cover it, and allow it to air dry overnight at room temperature. Evaluate all washes the day after washing. Evaluate the light reflectance of the small fabric samples using a Macbeth Color Eye 7000 reflectance spectrophotometer with a large aperture. Measurements are taken under UV-free incident light, and the reflectance (REM) at 460 nm is extracted. Measurements are performed on both unwashed and washed small fabric samples. Place the test fabric sample on top of another small fabric sample of the same type and color.

[0611] The effect of the protease on each fabric sample is calculated by subtracting the reflectance value of the unwashed fabric sample (blank) from the reflectance value of the fabric sample washed with the enzyme. The performance of the new protease (e.g., a protease variant) can be compared to that of a reference protease by calculating the relative performance (RP).

[0612]

[0613] Full Scale Wash (FSW) Test

[0614] The performance of the protease was evaluated on a large scale using a full-scale washing machine (Panasonic XQB65-Q680U, top-loading). Detergent, small fabric samples, ballast, water, and enzyme were added together and washed at a defined temperature for 15 minutes, followed by rinsing. The amounts of detergent, water, enzyme, and ballast used could be adjusted based on various factors, such as regional customs. The small fabric samples were then removed from the wash solution and laid flat to dry overnight at room temperature. The evaluation of the FSW small fabric samples was performed in a similar manner to that of the TOM small fabric samples.

[0615] Mini Terg-o-tometer (mini-TOM) washing test and determination of relative stain removal rate

[0616] The Mini Terg-o-tometer (mini-TOM) is a medium-scale detergent assay that can be used to test up to 16 different conditions simultaneously. In short, it consists of 16 x 0.2 L metal beakers, each equipped with a stirrer that rotates back and forth at a controlled speed to simulate the agitation that occurs in a commercial top-loading washing machine. These beakers are partially immersed in a temperature-controlled water bath. Each beaker is filled with 0.15 L of detergent solution, and a small sample of fabric and enzyme are added to the desired level. At the desired time point, the fabric sample is immediately removed from the beaker and thoroughly rinsed with ice water to prevent hydrolysis of the dirt.

[0617] Next, lay the small fabric sample flat on a rack covered with filter paper, cover it, and allow it to air dry overnight at room temperature. Evaluate all washes the day after washing. Evaluate the light reflectance of the small fabric samples using a Macbeth Color Eye 7000 reflectance spectrophotometer with a large aperture. Measurements are taken under UV-free incident light, and the reflectance (REM) at 460 nm is extracted. Measurements are performed on both unwashed and washed small fabric samples. Place the test fabric sample on top of another small fabric sample of the same type and color.

[0618] The effect of the protease on each small fabric sample was calculated by subtracting the reflectance of the unwashed small fabric sample (blank) from the reflectance of the small fabric sample washed with the protease.

[0619] The relative stain removal rate (R) at a given time point can be calculated. SRR The stain removal rate (SRR) of the novel protease (e.g., a protease variant) is compared to that of a reference protease.

[0620]

[0621] Example 1: FSW testing in detergents in the Asia-Pacific region at low temperature (15°C)

[0622] The washing performance of SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 7):

[0623]

[0624] The protease was included in the washing tests conducted in top-loading washing machines in the Asia-Pacific region using standard detergents and a range of stain types. At low washing temperatures (15°C; see Table 8), SEQ ID NO:9 showed improved washing performance compared to SEQ ID NO:1 and SEQ ID NO:8 on at least six different small fabric samples (representing at least five different stain categories, such as blood, blood milk ink, chocolate, grass, and eggs).

[0625]

[0626] Example 2: FSW test in detergents in the Asia-Pacific region at room temperature (25°C)

[0627] The washing performance of SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 19):

[0628]

[0629] The protease was included in the washing tests conducted in top-loading washing machines in the Asia-Pacific region using standard detergents and a range of stain types. At normal washing temperatures (25°C; see Table 10), SEQ ID NO:9 showed improved washing performance compared to SEQ ID NO:1 and SEQ ID NO:8 on at least eight different small fabric samples (representing at least four different stain categories, e.g., blood, milk ink, chocolate, grass, and eggs)).

[0630]

[0631] Example 3: TOM washing performance evaluation after storage in laundry soap bar matrix

[0632] The washing performance of SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 11):

[0633]

[0634] The protease formulation was incorporated into the base laundry soap bar matrix and incubated at 37°C. After 4 weeks, the performance of the soap bar was evaluated in a terg-o-tometer washing assay. In this assay, the soap sample was ground and washed with seven different stain monitors representing at least four different stain categories (e.g., blood, milk, ink, chocolate, grass, and egg). Compared with SEQ ID NO:1 and SEQ ID NO:8, SEQ ID NO:9 showed improved washing performance for six of the seven stains tested (see Table 12).

[0635]

[0636] Example 4: Evaluation of TOM's washing performance after storage in low-pH European detergents

[0637] The washing performance of SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 13):

[0638]

[0639] After being stored at 25°C in a low-pH European detergent for 24 hours, SEQ ID NO:8 and SEQ ID NO:9 were evaluated in a terg-o-tometer washing assay. A total of seven small fabric samples were used to evaluate washing performance, representing at least four different stain categories (e.g., blood, milk ink, chocolate, grass, and egg). Compared to SEQ ID NO:8, SEQ ID NO:9 showed improved washing performance for all stains tested (see Table 14).

[0640]

[0641] Example 5: Performance Evaluation of TOM Detergent in High-pH European Liquid Detergents

[0642] The washing performance of SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 15):

[0643]

[0644] The protease was evaluated in a terg-o-tometer washing assay using a high-pH European detergent. The results showed that SEQ ID NO:9 had improved washing performance on the selected stains compared to SEQ ID NO:8 (see Table 16).

[0645]

[0646] Example 6: AMSA Assessment in Standard European Liquid Detergents

[0647] The washing performance of SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 17):

[0648]

[0649] SEQ ID NO:1, SEQ ID NO:8, and SEQ ID NO:9 were tested using the AMSA washing assay, and the results are expressed as relative performance compared to SEQ ID NO:1. The results show that SEQ ID NO:9 has improved washing performance in both types of stains tested (see Table 18).

[0650]

[0651] Example 7: Storage stability of liquid detergents with high water content

[0652] The storage stability of SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 19):

[0653]

[0654] SEQ ID NO:8 and SEQ ID NO:9 were incubated in high-water-content liquid detergents at 30°C or 37°C for 4–8 weeks, and then the protease activity was analyzed. Residual protease activity (in %) was determined relative to the protease activity present in the original unincubated sample stored at -18°C. Results showed that SEQ ID NO:9 exhibited improved storage stability in high-water-content liquid detergents compared to SEQ ID NO:8 (see Table 20).

[0655]

[0656] Example 8: Storage stability and mildness to accompanying enzymes in liquid detergents

[0657] The storage stability and mildness of SEQ ID NO:8 and SEQ ID NO:9 were evaluated in commercially available liquid detergents (Bluemoon, China) under the following conditions (Table 21):

[0658]

[0659] Commercially available α-amylase (Amplify Prime 100 L, Novozymes) was incubated with SEQ ID NO:8 or SEQ ID NO:9 at 37°C for 2–4 weeks, followed by protease and α-amylase activity analysis of the samples.

[0660] Residual protease activity (in %) was determined relative to the protease activity present in the original unincubated sample stored at -18°C. Results showed that SEQ ID NO:9 exhibited improved storage stability in liquid detergents compared to SEQ ID NO:8 (see Table 22).

[0661]

[0662] Residual α-amylase activity (expressed as a percentage) was determined relative to the amylase activity present in the original unincubated sample stored at -18°C. The results showed that α-amylase exhibited improved residual activity in the presence of SEQ ID NO:9 compared to SEQ ID NO:8 (see Table 23). Therefore, SEQ ID NO:9 demonstrates improved mildness for concomitant enzymes, particularly α-amylase, compared to SEQ ID NO:8.

[0663]

[0664] Example 9: Stain removal rate in liquid detergents

[0665] The differences in stain removal rates among SEQ ID NO:1, SEQ ID NO:8, and SEQ ID NO:9 were evaluated under the following conditions (Table 24):

[0666]

[0667] Compared to SEQ ID NO:8 and SEQ ID NO:1, SEQ ID NO:9 showed improved stain removal efficiency (see Table 25). SEQ ID NO:9 was also relatively faster in removing dirt compared to SEQ ID NO:8 and SEQ ID NO:1.

[0668]

[0669] Example 10: AMSA Assessment in Liquid Detergents

[0670] The washing performance of the variants of SEQ ID NO:3 and SEQ ID NO:5 was evaluated under the conditions described in Table 26. The results are expressed as relative performance compared to the parent protease.

[0671] As can be seen from Table 27, SEQ ID NO:10 exhibits better washing performance than SEQ ID NO:3.

[0672] As can be seen from Table 28, SEQ ID NO:14 exhibits better washing performance than SEQ ID NO:13 and SEQ ID NO:5.

[0673]

[0674]

[0675]

[0676] Example 11: Storage stability in liquid detergents

[0677] The purified protease sample was diluted with 0.01% Triton X-100 to a suitable concentration based on the absorbance at 280 nm (0.1–0.4 mg / ml for variants of SEQ ID NO:1 and SEQ ID NO:3, and 1.3–5 mg / ml for variant of SEQ ID NO:5). Using a magnetic rod, 30 µl of the protease dilution was mixed with 270 µl of concentrated standard O wash (pH 8 or pH 10) in the wells of a microtiter plate. After mixing and sealing with the plate sealer, the wash plate was incubated at 45°C or 55°C in a Biosan PST-100HL thermal mixer. Each sample was tested at two or three concentrations.

[0678] Residual protease activity was measured after different incubation times (e.g., 0, 1, 4, 24, 48, 96, 192, and 264 hours). 20 µl of sample from a detergent plate was mixed with 150 µl of 0.1 M Tris buffer (pH 8.6). 30 µl of this dilution was transferred to a microtiter plate, followed by the addition of 70 µl of substrate solution (0.72 mg / ml Suc-Ala-Ala-Pro-Phe-pNA in 0.1 M Tris (pH 8.6)) and mixing. The absorbance was then read every 10 seconds at 405 nm for 5 min on a BioTek Synergy H1 plate reader. Activity was determined by linear regression of the slope of the increase in initial absorbance.

[0679] The decrease in activity during incubation with detergent is assumed to be exponential. Half-life (T½) is derived by linear regression of the logarithm of activity relative to incubation time. The half-life improvement factor (T½IF) is calculated as the ratio of the half-life of the protease variant to the half-life of the parent protease.

[0680] As shown in Table 29, SEQ ID NO:9 has improved storage stability compared to SEQ ID NO:8 and SEQ ID NO:1.

[0681] As shown in Table 30, SEQ ID NO:12 exhibits improved storage stability compared to SEQ ID NO:11 and SEQ ID NO:3. Additionally, SEQ ID NO:10 exhibits improved storage stability compared to SEQ ID NO:3.

[0682] As shown in Table 31, SEQ ID NO:14 has improved storage stability compared to SEQ ID NO:13 and SEQ ID NO:5.

[0683]

[0684]

[0685]

[0686] The invention described and claimed herein is not limited to the specific aspects disclosed herein, as these aspects are intended to illustrate several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. In fact, various modifications to the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the disclosure including the definition shall prevail.

[0687] The invention is further defined by the following numbered paragraphs:

[0688] 1. A variant of a parent protease, wherein the variant comprises substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprises substitutions at at least three, such as at least four, at least five, at least six, at least seven, at least eight, or nine positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein the position numbering is based on the numbering of SEQ ID NO:1; wherein the variant has at least 60% sequence identity with the parent protease, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein the variant has protease activity.

[0689] 2. According to the variant described in paragraph 1, the variant comprises that the amino acid residue at position 95 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Asp.

[0690] 3. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 209 corresponding to SEQ ID NO:1 is replaced by Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Tyr, preferably Lys.

[0691] 4. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 9 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Trp, Tyr or Val, preferably Glu.

[0692] 5. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 42 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Cys, Gln, Glu, His, Ile, Leu, Met, Phe, Pro, Ser, Trp, Tyr or Val, preferably Arg.

[0693] 6. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 74 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, preferably Asp.

[0694] 7. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 199 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Tyr, preferably Ile.

[0695] 8. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 200 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asn, Asp, Cys, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp or Val, preferably Leu.

[0696] 9. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 203 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val, preferably Trp.

[0697] 10. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 253 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Trp, Tyr or Val, preferably Asp.

[0698] 11. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 255 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Thr, Trp, Tyr, or Val, preferably Trp.

[0699] 12. A variant according to any one of the preceding paragraphs, wherein the amino acid residue at position 256 corresponding to SEQ ID NO:1 is replaced by Ala, Arg, Asn, Cys, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val, preferably Glu.

[0700] 13. A variant according to any one of the preceding paragraphs, the variant comprising substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprising at least three substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to any one of SEQ ID NO:1.

[0701] 14. A variant according to any one of the preceding paragraphs, the variant comprising substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprising at least four substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to any one of SEQ ID NO:1.

[0702] 15. A variant according to any one of the preceding paragraphs, the variant comprising substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprising at least five substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to any one of SEQ ID NO:1.

[0703] 16. A variant according to any one of the preceding paragraphs, the variant comprising substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprising at least six substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to any one of SEQ ID NO:1.

[0704] 17. A variant according to any one of the preceding paragraphs, the variant comprising substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprising at least seven substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to any one of SEQ ID NO:1.

[0705] 18. A variant according to any one of the preceding paragraphs, the variant comprising substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprising at least eight substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to any one of SEQ ID NO:1.

[0706] 19. A variant according to any one of the preceding paragraphs, the variant comprising substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprising nine substitutions at positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to each of SEQ ID NO:1.

[0707] 20. A variant according to any one of the preceding paragraphs, wherein the total number of substitutions compared to the parent is 5-20, for example 5-15 or 5-10, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions.

[0708] 21. A variant according to any one of the preceding paragraphs, wherein the total number of substitutions compared to the parent is 5 to 11, such as 5, 6, 7, 8, 9, 10 or 11 substitutions.

[0709] 21a. A variant according to any one of paragraphs 1-21, wherein the parent protease is the same as the protease except that it has the following: substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and substitutions at at least three positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 corresponding to SEQ ID NO:1.

[0710] 22. The variant according to any one of paragraphs 1-21, wherein the parent protease has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0711] 23. The variant according to any one of paragraphs 1-21, wherein the parent protease has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0712] 24. The variant according to any one of paragraphs 1-21, wherein the parent protease has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:3.

[0713] 25. The variant according to any one of paragraphs 1-21, wherein the parent protease has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:4.

[0714] 26. The variant according to any one of paragraphs 1-21, wherein the parent protease has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:5.

[0715] 27. The variant according to any one of paragraphs 1-21, wherein the parent protease has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:6.

[0716] 28. The variant according to any one of paragraphs 1-21, wherein the parent protease has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:7.

[0717] 29. A variant according to any one of the preceding paragraphs, wherein the variant comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises at least three, for example at least four, at least five, at least six, at least seven, at least eight or nine substitutions selected from the group consisting of: substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W and L256E of SEQ ID NO:1.

[0718] 30. A variant according to any of the preceding paragraphs, the variant comprising substitutions for G95D and A209K corresponding to SEQ ID NO:1, and further comprising substitutions for S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W and L256E corresponding to SEQ ID NO:1.

[0719] 30. A variant according to any of the preceding paragraphs, the variant comprising additional substitutions at one or more other positions corresponding to positions 60 (e.g., N60D), 97 (e.g., S97D), 99 (e.g., S99E), 116 (e.g., G116N), and 246 (e.g., N246L) of SEQ ID NO:1.

[0720] 31. The variant according to any one of the preceding paragraphs, the variant further comprising one or more substitutions selected from the group consisting of: substitutions corresponding to N60D, S97D, S97E, S99D, S99E, S99H, G116N and N246L of SEQ ID NO:1.

[0721] 32. The variant according to any one of the preceding paragraphs has improved storage stability and / or improved washing performance compared to the parent protease.

[0722] 32a. The variant according to any one of the preceding paragraphs has improved mildness compared to the parent protease.

[0723] 32b. The variant according to any one of the preceding paragraphs has an improved stain removal rate compared to the parent protease.

[0724] 33. A fusion polypeptide comprising a variant and a second polypeptide according to any one of the preceding paragraphs.

[0725] 34. The variant or fusion polypeptide according to any one of the preceding paragraphs, wherein the variant or fusion polypeptide is isolated.

[0726] 35. The variant or fusion polypeptide according to any one of the preceding paragraphs, wherein the variant or fusion polypeptide is purified.

[0727] 36. A particle comprising (a) a core comprising a variant or fusion polypeptide according to any one of paragraphs 1-35; and optionally, (b) a coating consisting of one or more layers surrounding the core.

[0728] 37. A particle comprising: (a) a core; and (b) a coating consisting of one or more layers surrounding the core, wherein the coating comprises a variant or fusion polypeptide according to any one of paragraphs 1-35.

[0729] 38. A liquid composition comprising a variant or fusion polypeptide according to any one of paragraphs 1-35 and an enzyme stabilizer, such as a polyol like propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative such as an aromatic borate ester, or a phenyl boric acid derivative such as 4-formylphenylboronic acid.

[0730] 39. The liquid composition according to paragraph 38, wherein the liquid composition further comprises a filler or carrier material.

[0731] 40. The liquid composition according to paragraph 38 or 39, wherein the liquid composition further comprises a preservative.

[0732] 41. A composition comprising a variant or fusion polypeptide according to any one of paragraphs 1-35, particles according to paragraph 36 or 37, or a liquid composition according to any one of paragraphs 38-40.

[0733] 42. A polynucleotide encoding a variant or fusion polypeptide according to any one of paragraphs 1-35.

[0734] 43. The polynucleotide described in paragraph 42 is isolated.

[0735] 44. The polynucleotide as described in paragraph 42 or 43 is purified.

[0736] 45. A nucleic acid construct or expression vector comprising a polynucleotide according to any one of paragraphs 42-44.

[0737] 46. ​​A recombinant host cell, which is converted with a polynucleotide according to any one of paragraphs 42-44.

[0738] 47. The recombinant host cell according to paragraph 46, wherein the recombinant host cell comprises at least two copies, such as three, four, or five or more copies of the polynucleotide according to any one of paragraphs 42-44.

[0739] 48. The recombinant host cell described in paragraph 46 or 47 is a yeast recombinant host cell, such as cells of the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Saccharomyces*, or *Yersinia*, such as *Kluyveromyces lactis*, *Kalvae*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces davidiana*, *Douglas*, *Kluyveromyces kluyvernsis*, *Nordiya*, *Ovoyces*, or *Yersinia lipolytica*.

[0740] 49. The recombinant host cell described in paragraph 46 or 47 is a filamentous fungal recombinant host cell, such as *Cladosporium*, *Aspergillus*, *Briefomus*, *Cirsium*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Cladosporium*, *Cryptococcus*, *Ustilago*, *Fusarium*, *Pyrophyllus*, *Mucor*, *Hydrophyllus*, *Nematophytes*, *Penicillium*, *Penicillium*. Cells of the genera *Pleurotus*, *Gastromycium*, *Ruminocytium*, *Pleurotus*, *Schizophyllum*, *Basilaria*, *Thermophilic Ascomycetes*, *Fusporium*, *Cyclophorus*, *Valva*, or *Trichoderma*, especially *Aspergillus buergerianus*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus niger*, *Aspergillus oryzae*, *Pseudomonas nigricans*, *Pseudomonas carnegieae*, *Pseudomonas palustris*, *Pseudomonas pannohita*, *Pseudomonas circinata*, and *Pseudomonas rubrum*. , Insectivora, Narrow-sided Auricularia, Keratopteric Auricularia, Lukenowens Auricularia, Coprophytic Auricularia, Rent-spore Auricularia, Queen Auricularia, Tropical Auricularia, Brown Auricularia, Grey-capped Coprinus, Trichoderma, Fusarium solani, Cereal Fusarium, Cuvier's Fusarium, Fusarium macrocarpa, Fusarium graminearum ... Fusarium oxysporum, Fusarium scabiosaefolium, Fusarium pseudobranchii, Fusarium sulfideum, Fusarium rotundum, Fusarium pseudofilariae, Fusarium moniliforme, *Hymenopterus xanthipes*, *Heatophilic hymenopterus*, *Neurospora crassa*, *Penicillium purpureum*, *Pleurotus ostreatus*, *Pleurotus eryngii*, *Pleurotus emarginatus*, *Clostridium emarginatus*, *Clostridium perfringens*, *Trichoderma harzianum*, *Trichoderma cornigrin*, *Trichoderma reesei*, or *Trichoderma viride* cells.

[0741] 50. The recombinant host cell as described in paragraph 46 or 47, wherein the recombinant host cell is a prokaryotic recombinant host cell, for example, Gram-positive cells selected from the group consisting of: Bacillus, Clostridium, Enterococcus, Bacillus terrestris, Lactobacillus, Lactococcus, Bacillus macrocephala, Staphylococcus, Streptococcus, or Streptomyces cells, or Gram-negative bacteria selected from the group consisting of: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Staphylococcus, Neisseria, Pseudomonas, Salmonella. Genera, and Ureaplasma genus cells, such as alkalophilic Bacillus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausti, Bacillus coagulans, Bacillus sclerotiorum, Bacillus splenium, Bacillus tarda, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equi, Streptococcus pyogenes, Streptococcus lactis and Streptococcus equi subsp. vesicant, Streptococcus non-chromogenic, Streptococcus pyogenes, Streptococcus aureus, Streptococcus gray, and Streptococcus purpureus cells.

[0742] 51. The recombinant host cell according to any one of paragraphs 46-50, wherein the recombinant host cell is isolated.

[0743] 52. The recombinant host cell according to any one of paragraphs 46-50, wherein the recombinant host cell is purified.

[0744] 53. A method for producing a protease variant or fusion polypeptide, the method comprising (a) culturing a host cell according to any one of paragraphs 46-52 under conditions suitable for expression of the variant; and (b) recovering the variant.

[0745] 54. A whole culture medium formulation or cell culture composition comprising a variant or fusion polypeptide according to any one of paragraphs 1-35.

[0746] 55. A detergent composition comprising a variant or fusion polypeptide according to any one of paragraphs 1-35.

[0747] 56. The detergent composition according to paragraph 55, wherein the detergent composition is in the form of: strips, uniform tablets, tablets having two or more layers, bags having one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or concentrated liquids.

[0748] 57. The detergent composition according to paragraph 55, wherein the detergent composition is in the form of a liquid detergent, a powder detergent, or a laundry soap bar; preferably wherein the detergent composition is a liquid detergent or a laundry soap bar.

[0749] 58. A method of cleaning an object, the method comprising contacting the object with a detergent composition according to any one of paragraphs 55-57 under conditions suitable for cleaning the object; preferably wherein the object is a fabric, tableware or a hard surface; most preferably wherein the object is a fabric.

[0750] 59. Use of the variants according to any one of paragraphs 1-35 or the detergent composition according to any one of paragraphs 55-57 in cleaning processes, preferably laundry or hard surface cleaning such as automatic dishwashing (ADW).

[0751] The invention is further defined by the following numbered paragraphs:

[0752] 1. A variant of a parent protease, wherein the variant comprises substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprises substitutions at at least three, such as at least four, at least five, at least six, at least seven, at least eight, or nine positions corresponding to any one of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein the position numbering is based on SEQ ID NO:1. The variant is designated as NO:1; wherein, compared to the three-dimensional structure of the parent protease, the variant has a TM score of at least 0.80, such as at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, wherein the three-dimensional structure is calculated using AlphaFold; and wherein the variant has protease activity.

[0753] 2. A variant of a parent protease, wherein the variant comprises substitutions at positions 95 and 209 corresponding to SEQ ID NO:1, and further comprises substitutions at at least three, such as at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any one of SEQ ID NO:1; wherein the position numbering is based on the numbering of SEQ ID NO:1; wherein the variant has at least 60% sequence identity with the parent protease, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity; and wherein the variant has protease activity.

[0754] 3. The variant according to any one of paragraphs 1-2, wherein the parent protease is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; preferably wherein the parent protease is SEQ ID NO:1.

[0755] 4. A variant according to any one of the preceding paragraphs, wherein the variant comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises at least three, for example at least four, at least five, at least six, at least seven, at least eight or nine substitutions selected from the group consisting of: substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W and L256E of SEQ ID NO:1.

[0756] 5. A variant according to any one of the preceding paragraphs, the variant comprising substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprising substitutions corresponding to S9E, N43R, N74D, V199I, Q200L, Y203W, S253D, N255W and L256E of SEQ ID NO:1.

[0757] 6. A variant according to any one of the preceding paragraphs, the variant further comprising substitutions at one or more positions selected from the group consisting of: positions 60, 97, 99, 116 and 246 of SEQ ID NO:1.

[0758] 7. The variant described in paragraph 5, comprising one or more additional substitutions selected from the group consisting of: N60D, S97D, S97E, S99D, S99E, S99H, G116N and N246L of SEQ ID NO:1.

[0759] 8. A variant according to any one of the preceding paragraphs, wherein the variant exhibits improved washing performance and / or improved storage stability compared to the parent protease.

[0760] 9. A polynucleotide encoding a protease variant according to any one of paragraphs 1-8.

[0761] 10. A nucleic acid construct or expression vector comprising the polynucleotides described in paragraph 9.

[0762] 11. A recombinant host cell containing in its genome a nucleic acid construct or expression vector as described in paragraph 10.

[0763] 12. A method for obtaining a variant according to any one of paragraphs 1-8, the method comprising (a) introducing a parent protease substitution at positions 95 and 209 corresponding to SEQ ID NO:1, and further introducing the substitution at at least three, for example at least four, at least five, at least six, at least seven, at least eight or nine, positions corresponding to any one of SEQ ID NO:1, 42, 74, 199, 200, 203, 253, 255 and 256; wherein the position numbering is based on the numbering of SEQ ID NO:1; and (b) recovering the variant.

[0764] 13. A method for producing a variant according to any one of paragraphs 1-8, the method comprising (a) culturing a recombinant host cell according to paragraph 11 under conditions suitable for expressing the variant; and (b) recovering the variant.

[0765] 14. A detergent composition comprising a variant according to any one of paragraphs 1-8; preferably wherein the detergent composition is in the form of: strips, uniform tablets, tablets having two or more layers, bags having one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or concentrated liquids.

[0766] 15. A method of cleaning an object, the method comprising contacting the object with a detergent composition according to paragraph 14 under conditions suitable for cleaning the object; preferably wherein the object is a fabric, tableware or a hard surface; most preferably wherein the object is a fabric.

[0767] 16. Use of the variants according to any one of paragraphs 1-8 or the detergent composition according to paragraph 14 in cleaning processes, preferably laundry or hard surface cleaning such as automatic dishwashing (ADW).

Claims

1. A variant of a parent protease, wherein the variant comprises substitutions for G95D and A209K corresponding to SEQ ID NO:1, and wherein the variant is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:

14.

2. A polynucleotide encoding a protease variant according to claim 1.

3. A nucleic acid construct or expression vector comprising the polynucleotide of claim 2.

4. A recombinant host cell, wherein the recombinant host cell contains the nucleic acid construct or expression vector according to claim 3 in its genome.

5. A method for producing a variant according to claim 1, the method comprising (a) culturing the recombinant host cell of claim 4 under conditions suitable for expressing the variant; and (b) recovering the variant.

6. A detergent composition comprising the variant of claim 1; preferably, wherein the detergent composition is in the form of: strips, uniform tablets, tablets having two or more layers, bags having one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or liquid forms.

7. A method of cleaning an object, the method comprising contacting the object with a detergent composition according to claim 6 under conditions suitable for cleaning the object; preferably wherein the object is a fabric, tableware or a hard surface; most preferably wherein the object is a fabric.

8. Use of the variant of claim 1 or the detergent composition of claim 6 in a cleaning process, preferably in laundry or hard surface cleaning such as automatic dishwashing (ADW).

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