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

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] 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. [Background technology]

[0003] In the detergent industry, enzymes have been incorporated into cleaning compositions for decades. Enzymes used in such compositions include proteases, lipases, amylases, cellulases, mannanases, and other enzymes, or mixtures thereof. Commercially, the most important enzymes are proteases.

[0004] For example, an increasing number of proteases used commercially in laundry and dishwashing detergents are engineered variants of naturally occurring wild-type proteases, which have been described in the art as possessing modifications that confer improvements, such as better cleaning performance, thermal stability, storage stability, or catalytic activity, compared to the parent protease.

[0005] However, further improvements in proteases are favored by various factors. For example, washing conditions, such as temperature and pH, tend to change over time and also differ between different countries or regions of the world. In addition, many stains remain difficult to completely remove under conventional washing conditions. Therefore, novel protease variants with improved cleaning performance under various conditions remain commercially relevant. In addition, novel protease variants with improved storage stability are guaranteed to maintain sufficient proteolytic activity after a certain period of storage in a detergent composition, either during the manufacture and distribution of the detergent composition and / or after storage of the detergent composition by the end-user before use, thereby ensuring the maintenance of the protease variant's cleaning performance. Furthermore, novel protease variants that exhibit improved mildness toward companion enzymes (i.e., other enzymes present in the detergent composition) are guaranteed to avoid unnecessary proteolysis and to preserve the enzymatic activity of such enzymes. Finally, new protease variants with improved stain removal rates are desirable because they act faster and are therefore particularly suitable for cleaning methods involving shorter wash cycles (e.g., laundry and dishwashing methods). Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to protease variants with improved properties. The variants of the present invention exhibit improved cleaning performance and improved storage stability under several different temperature and pH conditions. The variants of the present invention also have improved mildness to companion enzymes, thereby broadening the compatibility of the variants of the present invention with other enzymes and reducing the need for co-formulation with protease inhibitors. The variants of the present invention also exhibit improved stain removal speed, making them particularly suitable for cleaning methods with short wash cycles.

[0007] The variants of the present invention are particularly suitable for high pH liquid detergents, having a pH of 10 or greater, and laundry bars. In high pH liquid detergents, the variants of the present invention exhibit improved cleaning performance and improved storage stability. When included in laundry bars, the variants of the present invention improve proteinaceous stain removal and reduce the need for stabilizers, thereby further reducing the manufacturing costs associated with producing laundry bars.

[0008] In a first aspect, the present invention relates to a variant of a parent protease, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, such as at least 4, at least 5, at least 6, at least 7, at least 8, or 9 positions, wherein the numbering of the positions is based on the numbering of SEQ ID NO:1, wherein the variant has 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 was calculated using AlphaFold, and wherein the variant has protease activity.

[0009] In a second aspect, the present invention relates to a variant of a parent protease, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, wherein the numbering of the positions is based on the numbering of SEQ ID NO:1, wherein the variant has at least 60% but less than 100% sequence identity to the parent protease, and wherein the variant has protease activity.

[0010] In a third aspect, the present invention relates to a polynucleotide encoding a variant of the first or second aspect.

[0011] In a fourth aspect, the present invention relates to a nucleic acid construct or expression vector comprising a polynucleotide of the third aspect.

[0012] In a fifth aspect, the present invention relates to a recombinant host cell comprising in its genome a nucleic acid construct or an expression vector according to the fourth aspect.

[0013] In a sixth aspect, the present invention relates to a method of obtaining a variant according to the first or second aspect, comprising: (a) introducing substitutions into a parent protease at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further introducing substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255 and 256 of SEQ ID NO:1, such as introducing further substitutions at at least 4, at least 5, at least 6, at least 7, at least 8 or 9 positions, wherein the numbering of the positions is based on the numbering of SEQ ID NO:1, and wherein the variant has protease activity; and (b) recovering the variant.

[0014] In a seventh aspect, the present invention relates to a method of producing a variant of the first or second aspect, the method comprising: (a) culturing a recombinant host cell of the fifth aspect under conditions suitable for expression of the variant; and (b) recovering the variant.

[0015] In an eighth aspect, the present invention relates to a detergent composition comprising a variant of the first aspect or the second aspect.

[0016] In a ninth aspect, the present invention relates to a method of cleaning an object, the method comprising contacting the object with the detergent composition of the eighth aspect under conditions suitable for cleaning the object.

[0017] In a tenth aspect, the present invention relates to the use of the detergent composition of the first aspect or a variant of the second aspect or the eighth aspect in a washing process. [Brief explanation of the drawings]

[0018] [Figure 1] 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:2.

[0019] [Figure 2] 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:3.

[0020] [Figure 3] 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:4.

[0021] [Figure 4] 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:5.

[0022] [Figure 5] 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:6.

[0023] [Figure 6] 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:7.

[0024] [Figure 7] Summary of corresponding substitutions in SEQ ID NOs: 1-7. DETAILED DESCRIPTION OF THE INVENTION

[0025] Array Overview SEQ ID NO: 1 is Subtilisin Savinase (Savinase®) from Bacillus lentus.

[0026] SEQ ID NO: 2 is subtilisin BPN' from Bacillus amyloliquefaciens.

[0027] SEQ ID NO: 3 is Subtilisin Carlsberg (Alcalase®) from Bacillus licheniformis.

[0028] SEQ ID NO: 4 is a protease derived from Bacillus gibsonii.

[0029] SEQ ID NO: 5 is a protease derived from Bacillus gibsonii.

[0030] SEQ ID NO: 6 is a protease from Bacillus sp. TY-145.

[0031] SEQ ID NO: 7 is a protease from Actinomadura keratinilytica.

[0032] SEQ ID NO:8 is a stabilized variant of SEQ ID NO:1 with the substitutions S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E.

[0033] SEQ ID NO:9 is SEQ ID NO:1 with the substitutions S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.

[0034] SEQ ID NO: 10 is SEQ ID NO: 3 with the substitutions A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S, and S258P.

[0035] SEQ ID NO: 11 is SEQ ID NO: 3 with the substitutions P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261E.

[0036] SEQ ID NO: 12 is SEQ ID NO: 3 with the substitutions P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E.

[0037] SEQ ID NO: 13 is SEQ ID NO: 5 with the substitutions T9E, N42R, N74D, V199I, Q200L, Y203W, N253D, S255W, and Q256E.

[0038] SEQ ID NO: 14 is SEQ ID NO: 5 with the substitutions T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D, and S255W.

[0039] definition In accordance with this Detailed Description, the following definitions apply: Please note that the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0040] Unless otherwise defined or clearly indicated by 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 belongs.

[0041] Protease: The term "protease" refers to an enzyme having peptidase activity (EC 3.4; also known as proteolytic activity or protease activity) that catalyzes the hydrolysis of peptide bonds. The EC 3.4 group includes several subgroups, such as EC 3.4.21 (serine endopeptidases), which in turn includes several subgroups, such as EC 3.4.21.62 (subtilisins). For purposes of the present invention, protease activity may be determined according to Protease Activity Assay I or Protease Activity Assay II, as described in the Examples herein.

[0042] AlphaFold structure prediction: AlphaFold is a computational method for predicting the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., Highly accurate protein structure prediction with AlphaFold. Nature, 2021). Predicted structures for millions of polypeptides deposited in the UniProt database have been deposited 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 polypeptide can be obtained by searching its UniProt accession number.

[0043] In addition to the many three-dimensional structures already published, code for reconstructing and predicting the structure of novel polypeptides is available in source code repositories such as Github.com under deepmind / alphafold / (using notebooks / AlphaFold.ipynb) using AlphaFold v2.3.1 or later. Additionally, this code can be found on Github.com under sokrypton / ColabFold using v1.5.2 or later using AlphaFold2.ipynb. For technical details, see Jumper et al. (see above).

[0044] AlphaFold generates a per-residue estimate of its confidence on a scale of 0 to 100. This confidence measure is called the pLDDT and corresponds to the model's predicted score on the lDDT-Cα metric. It is stored in the B-factor field of the downloadable mmCIF and PDB files (unlike the B-factor, the higher the pLDDT, the better). Regions with a pLDDT score above 90 are expected to be modeled with high accuracy. They should be suitable for any application that benefits from high accuracy (e.g., binding site characterization). Regions with a pLDDT score between 70 and 90 are expected to be well modeled, corresponding to generally good backbone predictions.

[0045] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a spliced ​​mature mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed by a series of steps, including splicing, before appearing as a spliced ​​mature mRNA.

[0046] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0047] Control sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence may be native (i.e., derived from the same gene) or heterologous (i.e., derived from a different gene) to the polynucleotide encoding the variant, and may be native or heterologous to each other. Such control sequences include, but are not limited to, leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, control sequences include a promoter, and transcription and translation stop signals. Control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding the variant.

[0048] Expression: The term "expression" includes all steps involved in the production of a variant (for example, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion).

[0049] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a variant, the coding sequence operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0050] Extended: The term "extended" refers to the addition of one or more amino acids to the amino and / or carboxyl termini of a variant, such that the "extended" variant has protease activity.

[0051] Fragment: The term "fragment" refers to a variant in which one or more amino acids are missing from the amino and / or carboxyl terminus of the variant, wherein the fragment retains protease activity.

[0052] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which a polypeptide is fused at the N-terminus and / or C-terminus of a variant of the present invention. Fusion polypeptides can be produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention to each other. Techniques for producing fusion polypeptides are known in the art and include ligating coding sequences encoding the polypeptides in frame and under the control of the same promoter and terminator so that expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. When the fusion protein is secreted, this site is cleaved to release the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0053] Heterologous: The term "heterologous," with reference to a host cell, means that the polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous," with reference to a polypeptide or nucleic acid, means that the regulatory sequences (e.g., promoter) of the polypeptide or nucleic acid are not naturally associated with the polypeptide or nucleic acid, i.e., the regulatory sequences are derived from a gene other than the gene encoding the mature polypeptide.

[0054] Host strain or host cell: A "host strain" or "host cell" refers to an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a variant has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts made from cells.

[0055] Improved property: The term "improved property" refers to a characteristic associated with a variant that is improved compared to the parent. 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 cleaning performance.

[0056] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection," "transformation," or "transduction," as known in the art.

[0057] Isolated: The term "isolated" refers to a variant, nucleic acid, cell, or other specified substance or component that is separated from at least one other substance or component, including, but not limited to, other proteins, nucleic acids, cells, etc. Thus, an isolated polypeptide, nucleic acid, cell, or other substance is in a form that does not occur in nature. Isolated polypeptides include, but are not limited to, culture broths containing secreted variants expressed in host cells.

[0058] Mature polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide after N-terminal and / or C-terminal processing (eg, removal of a signal peptide).

[0059] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having protease activity.

[0060] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0061] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0062] Nucleic Acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids may be single- or double-stranded and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, multiple codons may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0063] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain a segment of nucleic acid in a manner not normally occurring in nature, or that is synthetic, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0064] Operably linked: The term "operably linked" means that the components specified are in a relationship, including, but not limited to, juxtaposition, permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that expression of the coding sequence is under the control of the regulatory sequence.

[0065] Parent or Parent Protease: The term "parent" or "parent protease" refers to a protease that is modified to produce a protease variant of the present invention.

[0066] Purified: The term "purified" refers to a nucleic acid, variant, or cell that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified variant or nucleic acid may form a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or variant is 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., percent by weight on a molar basis). In a related sense, a composition is enriched with respect to a molecule if there is a substantial increase in the concentration of that molecule after application of a purification or concentration technique. The term "enriched" refers to a compound, variant, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a higher relative or absolute concentration compared to the starting composition.

[0067] In one aspect, the term "purified," as used herein, refers to a variant or cell that is essentially free of components, particularly insoluble components, from the producing organism. In another aspect, the term "purified" refers to a variant that is essentially free of insoluble components, particularly insoluble components, from the natural organism from which it is obtained. In one aspect, the variant has been separated from a portion of the soluble components of the organism and culture medium from which it is recovered. The variant may be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0068] The variant may thus be purified so that only trace amounts of other proteins (particularly other polypeptides) are present. The term "purified," as used herein, may refer to the removal of other components (particularly other proteins, most particularly other enzymes) present in the cell of origin of the polypeptide. The variant may also be "substantially pure," i.e., free of other components from the organism in which the variant is produced (e.g., the host organism in the case of recombinantly produced variants). In one aspect, the polypeptide is at least 40% pure by weight of total polypeptide material present in a preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of total polypeptide material present in a preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation that contains up to 10%, preferably up to 8%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, even more preferably up to 2%, most preferably up to 1%, and most preferably up to 0.5% by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated.

[0069] Thus, a substantially pure variant is preferably 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, by weight of total polypeptide material present in the preparation. The variants of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is naturally or recombinantly associated). This can be achieved, for example, by preparing the variant by well-known recombinant methods, or by preparing the variant by classical purification methods.

[0070] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a constellation that differs from that found in nature. The term recombinant refers to a cell, nucleic acid, variant, or vector that has been modified from its natural state. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes at levels or under different conditions than those found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0071] Recover: The term "recover" or "recovery" refers to the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified materials, e.g., recovery of a polypeptide from whole fermentation broth or from cell-free fermentation broth, by collecting polypeptide crystals, by filtration (e.g., depth filtration (using filter aid or packed filter material, fabric filtration in chamber filters, rotary drum filtration, drum filtration, rotary vacuum drum filters, candle filters, horizontal leaf filters, or similar, with seed or pad filtration in a framework or modular setup) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration, in either cross-flow, dynamic cross-flow, or dead-end operation)), or by centrifugation (using a decanter centrifuge, disc centrifuge, hydrocyclone, or similar), or by precipitating the polypeptide and collecting it from the broth medium by particle size classification using an appropriate solid-liquid separation method. Recovering includes isolating and / or purifying the polypeptide.

[0072] Sequence homology: The relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence homology."

[0073] For purposes of the present invention, sequence identity between two amino acid sequences is determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 6.6.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified on the command line. The Needle output labeled "longest identity" is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0074] For purposes of the present invention, sequence identity between two polynucleotide sequences is preferably determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 6.6.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified on the command line. The Needle output labeled "longest identity" is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment−total number of gaps in the alignment).

[0075] Signal peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside of the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0076] Structural similarity: The relatedness between two amino acid sequences is conventionally described by the parameter "sequence identity." However, because the biological function of a polypeptide is defined by its three-dimensional structure, not its amino acid sequence, a better way to assess the functional relationship between polypeptides is by comparing their three-dimensional structures. Therefore, for purposes of the present invention, the relatedness between the three-dimensional structures of two polypeptides is described by the parameter "structural similarity."

[0077] The three-dimensional structure of any polypeptide can be obtained experimentally, for example, via X-ray crystallography, or can be obtained using in silico methods such as AlphaFold (see above). The structural similarity between the three-dimensional structures can then be calculated using the following general formula (Zhang & Skolnick, Proteins 57:702-710, 2004):

number

[0078] For the purposes of this invention, L Nis always the length of the reference protein, and indicates the use of a fixed reference length L to prevent artificially large TM-scores from substructure alignments:

number

[0079] Before a TM-score can be calculated, structural alignment of the three-dimensional structures of the two polypeptides is required. This is achieved by an algorithm that optimizes structural overlap, and several methods are available, such as CEAlign (Shindyalov and Bourne, Protein Eng., 11, 739-747, 1998), DALI (Holm and Sander, Trends Biochem. Sci., 20, 478-480, 1995), or TM-Align (Nucleic Acids Res. 33:2302-2309, 2005).

[0080] For the purpose of this invention, TM-Align is applied. For convenience, TM-Score is integrated into TM-Align software available from the author's website. The version of TM-Align is preferably updated to 2019-08-22 or later, and the TM-Score between the reference protein and the query protein can be calculated by this command: TM Align<query.pdb><reference.pdb> -L<reference length> (where,<query.pdb> is the name of the PDB file containing the coordinates of the query polypeptide,<reference.pdb> is the name of the PDB file containing the coordinates of the reference polypeptide) The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

[0081] The maximum TM-score is 1, for example 1.0, which corresponds to identical three-dimensional structures.

[0082] Subsequence: The term "subsequence" refers to a polynucleotide lacking one or more nucleotides from the 5' and / or 3' end of a mature polypeptide coding sequence, which subsequence encodes a fragment that has protease activity.

[0083] Variant: The term "variant" refers to a polypeptide having protease activity and which contains a substitution, insertion (including extension), and / or deletion (e.g., truncation) at one or more positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0084] Wild-type: The term "wild-type" with respect to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of a wild-type sequence).

[0085] Rules for naming variants For purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid position in another protease. The amino acid sequence of the other protease is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program in the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0086] An alignment of SEQ ID NO: 1 with SEQ ID NOs: 2, 3, 4, 5, 6, and 7 is provided as Figures 1 to 6. A summary of the corresponding substitutions in SEQ ID NOs: 1 to 7 is provided in Figure 7.

[0087] In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference: accepted IUPAC single-letter or three-letter amino acid abbreviations are employed.

[0088] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of Ala for Thr at position 226 would be designated "T226A." Multiple mutations are separated by an additional symbol ("+") or a comma, e.g., "G205R+S411F" or "G205R,S411F" represent a substitution of Arg (R) for Gly (G) and Phe (F) for Ser (S) at positions 205 and 411, respectively. Because the amino acid residue at a given position varies between parents, the substituted amino acid may be indicated by an X, e.g., "X226A."

[0089] Deletions: For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, a deletion of the amino acid Gly at position 195 is designated as "Gly195*". Multiple deletions are separated by an additional symbol ("+") or a comma, for example, "G195*+S411*" or "G195*+S411*". Because the amino acid residue at a given position varies between parents, the deleted amino acid may be indicated by an X, for example, "X195*".

[0090] Insertions: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of Lys after the amino acid Gly at position 195 would be designated "G195GK." Because the amino acid residue at a given position varies between parents, an insertion of lysine after the amino acid at position 195 could be designated "X195*."

[0091] Insertions of multiple amino acids are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, the insertion of Lys and Ala after amino acid Gly at position 195 is designated as "G195GKA." In such cases, the inserted amino acid residues are numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue. Thus, in the previous example, the sequence would be:

[0092] [Table 1] This becomes:

[0093] Alternatively, the insertion of an amino acid residue such as lysine after amino acid position 195 can be indicated as "195aK", and the insertion of two or more additional amino acid residues such as Lys and Ala after amino acid position 195 can be indicated as "195aK, 195bA".

[0094] Multiple changes: Variants containing multiple changes are separated by an additional symbol ("+"), for example, "R170Y+G195E" represents the substitution of Arg and Gly by Tyr and Gly at positions 170 and 195, respectively.

[0095] Variant: Where variants can be introduced at a position, the variants are separated by commas, for example, "R170Y,E" represents the substitution of Arg with Tyr or Glu at position 170. Thus, "Y167G,A+R170G,A" represents the following variant: Specify "Y167G+R170G", "Y167G+R170A", "Y167A+R170G", and "Y167A+R170A".

[0096] Detailed Description of the Invention The present invention relates to protease variants with improved properties. The variants of the present invention exhibit improved cleaning performance and improved storage stability under several different temperature and pH conditions. The variants of the present invention also have improved mildness to companion enzymes, thereby broadening the compatibility of the variants of the present invention with other enzymes and reducing the need for co-formulation with protease inhibitors. The variants of the present invention also exhibit improved stain removal speed, making them particularly suitable for cleaning methods with short wash cycles.

[0097] The variants of the present invention are particularly suitable for high pH liquid detergents, having a pH of 10 or greater, and laundry bars. In high pH liquid detergents, the variants of the present invention exhibit improved cleaning performance and improved storage stability. When included in laundry bars, the variants of the present invention improve proteinaceous stain removal and reduce the need for stabilizers, thereby further reducing the manufacturing costs associated with producing laundry bars.

[0098] The present invention also relates to polynucleotides encoding the variants of the invention, nucleic acid constructs and expression vectors comprising such polynucleotides, recombinant host cells expressing the variants of the invention, methods of obtaining the variants of the invention, methods of producing the variants of the invention, detergent compositions comprising the variants of the invention, and uses of the variants of the invention.

[0099] variant In a first aspect, the present invention provides a variant of a parent protease, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, such as at least 4, at least 5, at least 6, at least 7, at least 8, or 9 positions, wherein the numbering of the positions is based on the numbering of SEQ ID NO:1, and wherein the variant has a TM-score of at least 0.80 compared to the three-dimensional structure of the parent protease, 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 has been calculated using AlphaFold and the variant has protease activity.

[0100] In one embodiment, the variant has a TM-score of at least 0.90, e.g., 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 the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0101] In one embodiment, the variant has a TM-score of at least 0.95 compared to the three-dimensional structure of the parent protease, e.g., 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.

[0102] In one embodiment, the variant has a TM-score of at least 0.980, e.g., 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.

[0103] In one embodiment, the variant has a TM-score of at least 0.990 compared to the three-dimensional structure of the parent protease, e.g., 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, wherein the three-dimensional structure is calculated using AlphaFold.

[0104] In certain 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.

[0105] 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.

[0106] 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.

[0107] In one embodiment, the parent protease is an otherwise identical protease that does not have substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and does not have substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0108] In another 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 SEQ ID NO: 1, e.g., 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 has been 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 has been 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 has been calculated using AlphaFold.

[0109] In another 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 SEQ ID NO:2, e.g., 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 has been 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 has been 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 has been calculated using AlphaFold.

[0110] In another 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 SEQ ID NO: 3, e.g., 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 has been calculated using AlphaFold. Preferably, the parent is SEQ ID NO: 3 and the variant has a TM-score of at least 0.980, e.g., 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 has been calculated using AlphaFold. Most preferably, the parent is SEQ ID NO: 3 and the variant has a TM-score of at least 0.990, e.g., 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 has been calculated using AlphaFold.

[0111] In another embodiment, the parent is SEQ ID NO: 4 and the variant has a TM-score of at least 0.80 compared to the three-dimensional structure of SEQ ID NO: 4, e.g., 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 has been 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 has been 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 has been calculated using AlphaFold.

[0112] In another embodiment, the parent is SEQ ID NO: 5 and the variant has a TM-score of at least 0.80 compared to the three-dimensional structure of SEQ ID NO: 5, e.g., 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 has been 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 has been 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 has been calculated using AlphaFold.

[0113] In another embodiment, the parent is SEQ ID NO: 6 and the variant has a TM-score of at least 0.80 compared to the three-dimensional structure of SEQ ID NO: 6, e.g., 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. 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 has been 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 has been calculated using AlphaFold.

[0114] In another embodiment, the parent is SEQ ID NO: 7 and the variant has a TM-score of at least 0.80 compared to the three-dimensional structure of SEQ ID NO: 7, e.g., 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. Preferably, the parent is SEQ ID NO: 7 and the variant has a TM-score of at least 0.980, e.g., 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 has been calculated using AlphaFold. Most preferably, the parent is SEQ ID NO: 7 and the variant has a TM-score of at least 0.990, e.g., 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 has been calculated using AlphaFold.

[0115] For some parent proteases, three-dimensional structures are publicly available. The three-dimensional structure of SEQ ID NO: 1 (Savinase®) is available under UniProt accession number P29600 or 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.

[0116] 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 No. 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 No. P29600.

[0117] 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 No. 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 No. P00782.

[0118] 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 No. 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 No. P00780.

[0119] In a second aspect, the present invention relates to a variant of a parent protease, which variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprises substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, such as at least 4, at least 5, at least 6, at least 7, at least 8, or 9 positions, wherein the numbering of the positions is based on the numbering of SEQ ID NO:1, and which variant has at least 60% sequence identity to 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%, and which variant retains protease activity.

[0120] In certain 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.

[0121] 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.

[0122] 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.

[0123] In one embodiment, the parent protease is an otherwise identical protease that does not have substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and does not have substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0124] In another embodiment, the parent is SEQ ID NO: 1 and the variant has at least 60% sequence identity to the polypeptide of SEQ ID NO: 1, 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%.

[0125] In another embodiment, the parent is SEQ ID NO: 2 and the variant has at least 60% sequence identity to the polypeptide of SEQ ID NO: 2, 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%.

[0126] In another embodiment, the parent is SEQ ID NO: 3 and the variant has at least 60% sequence identity to the polypeptide of 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 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%.

[0127] In another embodiment, the parent is SEQ ID NO: 4 and the variant has at least 60% sequence identity to the polypeptide of SEQ ID NO: 4, 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%.

[0128] In another embodiment, the parent is SEQ ID NO: 5 and the variant has at least 60% sequence identity to the polypeptide of SEQ ID NO: 5, 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%.

[0129] In another embodiment, the parent is SEQ ID NO: 6 and the variant has at least 60% sequence identity to the polypeptide of SEQ ID NO: 6, 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%.

[0130] In another embodiment, the parent is SEQ ID NO: 7 and the variant has at least 60% sequence identity to the polypeptide of SEQ ID NO: 7, 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%.

[0131] In one aspect, the number of substitutions in a variant of the invention is 5 to 30 substitutions, such as 5 to 25, 5 to 20, 5 to 15, and 5 to 10 substitutions, 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 a variant of the invention is 5 to 11 substitutions, such as 5, 6, 7, 8, 9, 10, or 11 substitutions.

[0132] In another embodiment, the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprises at least three substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0133] In another embodiment, the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprises at least four substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0134] In another embodiment, the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprises at least five substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0135] In another embodiment, the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprises six substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0136] In another embodiment, the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprises at least seven substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0137] In another embodiment, the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprises at least eight substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0138] In another embodiment, the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprises nine substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0139] Variants of the invention comprise a substitution at a position corresponding to position 95 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 95 of 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 with Asp.

[0140] A variant of the invention comprises a substitution at a position corresponding to position 209 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 209 of 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 with Lys.

[0141] Variants of the invention may comprise a substitution at a position corresponding to position 9 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 9 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Trp, Tyr, or Val, preferably with Glu.

[0142] Variants of the invention may comprise a substitution at a position corresponding to position 42 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 42 of SEQ ID NO: 1 is substituted with Ala, Arg, Cys, Gln, Glu, His, Ile, Leu, Met, Phe, Pro, Ser, Trp, Tyr, or Val, preferably with Arg.

[0143] Variants of the invention may comprise a substitution at a position corresponding to position 74 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 74 of 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 with Asp.

[0144] Variants of the invention may comprise a substitution at a position corresponding to position 199 of SEQ ID NO: 1. In one aspect, the amino acid at a position corresponding to position 199 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with He.

[0145] Variants of the invention may comprise a substitution at a position corresponding to position 200 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 200 of 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 with Leu.

[0146] Variants of the invention may comprise a substitution at a position corresponding to position 203 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 203 of 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 with Trp.

[0147] Variants of the invention may comprise a substitution at a position corresponding to position 253 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 253 of 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 with Asp.

[0148] Variants of the invention may comprise a substitution at a position corresponding to position 255 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 255 of 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 with Trp.

[0149] Variants of the invention may comprise a substitution at a position corresponding to position 256 of SEQ ID NO: 1. In one aspect, the amino acid at the position corresponding to position 256 of 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 with Glu.

[0150] In one aspect, a variant of the invention comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises at least three 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, such as at least four, at least five, at least six, at least seven, at least eight, or nine substitutions.

[0151] In one aspect, a variant of the invention comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises at least four 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.

[0152] In one aspect, a variant of the invention comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises at least five 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.

[0153] In one aspect, a variant of the invention comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises at least six 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.

[0154] In one aspect, a variant of the invention comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises at least seven 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.

[0155] In one aspect, a variant of the invention comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises at least eight 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.

[0156] In one aspect, a variant of the invention comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1, and further comprises 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.

[0157] In one aspect, the invention provides a variant of SEQ ID NO: 1 comprising the substitutions X95D (e.g. G95D) and X209K (e.g. A209K), and further comprising at least three 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), such as at least four, at least five, at least six, at least eight ... and further comprising at least 6, at least 7, at least 8, or 9 substitutions, where the numbering of positions is based on the numbering of SEQ ID NO: 1, and the variant has at least 60% sequence identity to SEQ ID NO: 1, e.g., 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%, and the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 1 with the substitutions S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.

[0158] In one aspect, the invention provides a variant of SEQ ID NO: 2 comprising the substitutions X97D (e.g., G97D) and X215K (e.g., G215K), and further comprising at least three 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), such as at least 4, at least 5, at least 6, or at least 7 substitutions. wherein the numbering of positions is based on the numbering of SEQ ID NO: 2, and the variant has at least 60% sequence identity to SEQ ID NO: 2, e.g., 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%, and the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 2 with the substitutions S9E, K43R, N76D, G97D, Q206L, L209W, G215K, F261W, and Y262E.

[0159] In one aspect, the invention provides a variant of SEQ ID NO: 3 comprising the substitutions X96D (e.g. N96D) and X214K (e.g. A214K), and further comprising at least three 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), such as at least 4, at least 5, at least and further comprising 6, at least 7, or 8 substitutions, where the numbering of positions is based on the numbering of SEQ ID NO: 3, and the variant has at least 60% sequence identity to SEQ ID NO: 3, e.g., 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%, and the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the substitutions P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E.

[0160] In one aspect, the invention provides a variant of SEQ ID NO: 4 comprising the substitutions X95D (e.g. G95D) and X209K (e.g. A209K), and further comprising at least three 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), such as at least 4, at least 5, at least 6 , at least 7, or 8 substitutions, where the numbering of positions is based on the numbering of SEQ ID NO: 4, and the variant has at least 60% sequence identity to SEQ ID NO: 4, e.g., 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%, and the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 4 with the substitutions T9E, T42R, N74D, G95D, Q200L, Y203W, A209K, N253D, S255W, and Q256E.

[0161] In one aspect, the invention provides a variant of SEQ ID NO: 5 comprising the substitutions X95D (e.g. G95D) and X209K (e.g. V209K), and further comprising at least three 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), such as at least four, at least five, at least six, at least eight ... and further comprising at least 6, at least 7, at least 8, or 9 substitutions, where the numbering of positions is based on the numbering of SEQ ID NO: 5, and the variant has at least 60% sequence identity to SEQ ID NO: 5, e.g., 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%, and the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 5 with the substitutions T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D, S255W, and Q256E.

[0162] In one aspect, the invention provides a variant of SEQ ID NO: 6 comprising the substitutions X107D (e.g. G107D) and X245K (e.g. N245K), and further comprising at least three 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), such as at least 4, at least 5, at least and further comprising at least 6, at least 7, or at least 8 substitutions, where the numbering of positions is based on the numbering of SEQ ID NO: 6, and the variant has at least 60% sequence identity to SEQ ID NO: 6, e.g., 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%, and the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 6 with the substitutions K12E, D44R, S86D, G107D, V235I, E236L, N245K, T297D, D299W, and D300E.

[0163] In one aspect, the invention provides a variant of SEQ ID NO: 7 comprising the substitutions X99D (e.g., N99D) and X215K (e.g., N215K), and further comprising at least three 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), such as further comprising at least four, at least five, or six substitutions; wherein the numbering of positions is based on the numbering of SEQ ID NO: 7, and the variant has at least 60% sequence identity to SEQ ID NO: 7, e.g., 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%, and the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 7 with the substitutions D12E, G51R, N99D, T206L, N215K, T266D, N268W, and L269E.

[0164] In one aspect, the invention relates to a variant of SEQ ID NO: 7, comprising the substitutions X99D (e.g., N99D) and X215K (e.g., N215K), and further comprising the substitutions X12E (e.g., D12E), X51R (e.g., G51R), and X206L (e.g., T206L), wherein the numbering of positions is based on the numbering of SEQ ID NO: 7, and wherein the variant has at least 60% sequence identity to SEQ ID NO: 7, 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%, and wherein the variant has protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 7 with the substitutions D12E, G51R, N99D, T206L, and N215K.

[0165] In an alternative aspect, the present invention provides a variant of SEQ ID NO: 3 comprising the substitutions X96D (e.g. N96D) or X214K (e.g. A214K), preferably X96D (e.g. N96D) and X214K (e.g. A214K), and also including X68S (e.g. A68S), X77N (e.g. T77N), X78I (e.g. X78I (e.g. T78I), X127S (e.g. X127S), 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), for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 substitutions, where the numbering of positions is based on the numbering of SEQ ID NO: 3, and the variant has at least 60% sequence identity to 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%, and which variant has protease activity. In one embodiment, the variant comprises the substitutions N96D and A214K, and is selected from the group consisting of A68S, T77N, T78I, G127S, A128P, G165Q, N184Q, A20 and further comprising at least one substitution selected from the group consisting of: T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S, and S258P, e.g., further comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 substitutions. In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the 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 substitution 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), such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8 substitutions. In an even more preferred embodiment, the variant further comprises at least one substitution selected from the group consisting of P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261E, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8 substitutions. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the substitutions P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258P, F260W, and Y261E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the substitutions P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258D, F260W, and Y261E.

[0166] In an alternative aspect, the invention provides a variant of SEQ ID NO: 3 comprising the substitution X214K (e.g., A214K) and any of the following substitutions: X9T (e.g., P9T), X17H (e.g., Q17H), X77N (e.g., T77N), X78I (e.g., T78I), X96D (e.g., N96D), X103F (e.g., Y103F), X127T (e.g., G127T), X136T (e.g., G136T), X146T (e.g., G146T), X156T (e.g., G156T), X166T (e.g., G166T), X17H (e.g., Q17H), X77N (e.g., T77N), X78I at least one substitution selected from the group consisting of 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), e.g., at least two, and further comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or 14 substitutions, where the numbering of the positions is based on the numbering of SEQ ID NO: 3, and the variant has at least 60% sequence identity to SEQ ID NO: 3, e.g., 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%, and the variant has protease activity. In one embodiment, the variant comprises the substitution A214K and further comprises at least one substitution selected from the group consisting of P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, and S258P, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 substitutions.In a preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the 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 substitution 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), such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8 substitutions. In an even more preferred embodiment, the variant further comprises at least one substitution selected from the group consisting of P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261E, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8 substitutions. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the substitutions P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the substitutions P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E.In a most preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the substitutions P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO: 3 with the substitutions P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E.

[0167] Variants 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 variant 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.

[0168] Variants of the invention may further comprise an extension of one or more amino acids at the N-terminus and / or C-terminus.

[0169] Alternatively, the variants of the invention may further comprise a truncation of one or more amino acids at the N-terminus and / or C-terminus.

[0170] The amino acid changes introduced into the parent protease to obtain the variants of the present invention can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of 20-25 residues or less; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine tracts, antigenic epitopes, or binding domains.

[0171] Examples of conservative substitutions are within the group of 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 low molecular weight amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, in H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions 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.

[0172] Alternatively, the amino acid changes may be of such a nature that the physicochemical properties of the polypeptide are altered, for example, the amino acid changes may improve the thermostability of the polypeptide, alter its substrate specificity, change its pH optimum, etc.

[0173] Essential amino acids in a polypeptide 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, single alanine mutations are introduced at every residue in a molecule, and the resulting molecules are tested for protease activity to identify amino acid residues essential for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., 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. Identification of essential amino acids can also be inferred from alignment with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides, or with polypeptides / proteins (typically with similar three-dimensional structure, function, and significant sequence similarity) derived from a common ancestor within a polypeptide or protein family. Additionally, or alternatively, protein structure prediction tools can be used for protein structure modeling to identify important amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, "Highly accurate protein structure prediction with AlphaFold," Nature 596:583-589.

[0174] In one aspect, the variants of the invention have improved stability under storage conditions (i.e., improved storage stability) compared to a reference protease. In one embodiment, the storage stability is improved by at least 5%, e.g., 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.

[0175] In some embodiments, the reference protease is a parent protease. In one embodiment, the parent protease is an identical protease except that it does not have substitutions at positions corresponding to 95 and 209 of SEQ ID NO:1 and does not have substitutions at at least three positions corresponding to any 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.

[0176] In some embodiments, the variant has improved storage stability compared to an otherwise identical protease that does not have the substitutions at positions 95 and 209 of SEQ ID NO: 1. In one embodiment, the storage stability is improved by at least 5%, e.g., 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.

[0177] In one embodiment, the variant has improved storage stability in liquid detergents having a pH of 8-14, preferably a pH of 9-13, most preferably a pH of 10-12.

[0178] In one embodiment, the variant has improved storage stability in liquid detergents having a pH of 7-11, preferably a pH of 7-10, most preferably a pH of 8-10.

[0179] In one embodiment, the variant has improved storage stability at temperatures between 10 and 40°C, preferably between 10 and 30°C, most preferably between 15 and 25°C.

[0180] In one embodiment, the variant has improved storage stability at temperatures between 10 and 60°C, more preferably between 30 and 55°C, most preferably between 45 and 55°C.

[0181] In a preferred embodiment, the variant has improved storage stability in liquid detergent as determined according to Example 7 herein.

[0182] In a preferred embodiment, the variant has improved storage stability in liquid detergents, preferably in Model O detergents having a pH of 8-10 or in Model B detergents having a pH of 7-8, as determined according to Example 11 herein.

[0183] In one aspect, the variants of the invention have improved wash performance compared to a reference parent protease, hi one embodiment, the wash performance is improved by at least 5%, e.g., 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.

[0184] In some embodiments, the reference protease is a parent protease. In one embodiment, the parent protease is an identical protease except that it does not have substitutions at positions corresponding to 95 and 209 of SEQ ID NO:1 and does not have substitutions at at least three positions corresponding to any 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 wash performance compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved cleaning performance compared to SEQ ID NO: 5. In a preferred embodiment, the variant has improved cleaning performance compared to SEQ ID NO: 6. In a preferred embodiment, the variant has improved cleaning performance compared to SEQ ID NO: 7.

[0185] In some embodiments, the variant has improved wash performance compared to an otherwise identical protease that does not have the substitutions at positions 95 and 209 of SEQ ID NO: 1. In one embodiment, wash performance is improved by at least 5%, e.g., 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 wash performance compared to SEQ ID NO: 8. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO: 11. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO: 13.

[0186] In one embodiment, the variant has improved cleaning performance in liquid detergents having a pH of 8-14, preferably a pH of 9-13, most preferably a pH of 10-12.

[0187] In one embodiment, the variant has improved cleaning performance in liquid detergents having a pH of 7-11, preferably a pH of 7-10, most preferably a pH of 8-10.

[0188] In one embodiment, the variant has improved washing performance at washing temperatures of 10-60°C, preferably 10-40°C, more preferably 10-30°C, most preferably 15-25°C.

[0189] In one embodiment, the variant has improved cleaning performance as determined in Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, or Example 10 herein. In a preferred embodiment, the variant has improved cleaning performance in a liquid detergent as determined according to Example 1 herein. In a preferred embodiment, the variant has improved cleaning performance in a liquid detergent as determined according to Example 2 herein. In a preferred embodiment, the variant has improved cleaning performance in a liquid detergent as determined according to Example 3 herein. In a preferred embodiment, the variant has improved cleaning performance in a liquid detergent as determined according to Example 4 herein. In a preferred embodiment, the variant has improved cleaning performance in a liquid detergent as determined according to Example 5 herein. In a preferred embodiment, the variant has improved cleaning performance in a liquid detergent as determined according to Example 6 herein.

[0190] In a preferred embodiment, the variant has improved cleaning performance in a liquid detergent, preferably in a Model O detergent having a pH of 8-10 or in a Model B detergent having a pH of 7-8, as determined according to Example 10 herein.

[0191] In one aspect, the variants of the invention have improved mildness compared to a reference protease. Improved mildness, in the context of the present invention, means that the protease variant is less aggressive towards other enzymes (also called companion enzymes or co-enzymes) in the detergent matrix, thereby resulting in improved residual activity of the companion enzymes after storage with the protease variant of the invention in a detergent composition. In one embodiment, the mildness is improved by at least 5%, e.g., 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.

[0192] In some embodiments, the reference protease is a parent protease. In one embodiment, the parent protease is an identical protease except that it does not have substitutions at positions corresponding to 95 and 209 of SEQ ID NO:1 and does not have substitutions at at least three positions corresponding to any 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.

[0193] In some embodiments, the variant has improved mildness compared to an otherwise identical protease that does not have the substitutions at positions 95 and 209 of SEQ ID NO: 1. In one embodiment, the mildness is improved by at least 5%, e.g., 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.

[0194] In some embodiments, the variants result in improved residual activity of the companion enzyme. In a preferred embodiment, the residual activity of the companion enzyme is improved by at least 5%, e.g., 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. In one embodiment, the companion enzyme is selected from the group consisting of amylase (e.g., α-amylase), arabinase, carbohydrase, cellulase (e.g., endoglucanase), cutinase, DNase, galactanase, haloperoxygenase, lipase, mannanase, oxidase (e.g., laccase or peroxidase), pectinase, pectin lyase, protease, xylanase, xanthanase, or xyloglucanase. In a preferred embodiment, the companion enzyme is α-amylase. In a preferred embodiment, the residual activity is determined according to Example 8 herein.

[0195] In one embodiment, the variant provides improved residual activity of the companion enzyme (preferably α-amylase) after storage in a liquid detergent having a pH of 8-14, preferably a pH of 9-13, and most preferably a pH of 10-12.

[0196] In one embodiment, the variants provide improved residual activity of a companion enzyme (preferably α-amylase) after storage with a protease variant of the invention when stored in a liquid detergent at temperatures between 10 and 60°C, more preferably between 15 and 50°C, and most preferably between 20 and 40°C.

[0197] In a preferred embodiment, the variant has improved mildness in liquid detergents as determined according to Example 8 herein.

[0198] In a preferred embodiment, the variant provides improved residual activity of α-amylase in liquid detergent as determined according to Example 8 herein.

[0199] In a preferred embodiment, the variant has improved mildness in Model O liquid detergent.

[0200] In a preferred embodiment, the variant provides improved residual activity of α-amylase in Model O liquid detergent.

[0201] In one aspect, the variants of the invention have improved stain removal rates compared to a reference protease. Improved stain removal rates, in the context of the present invention, mean that the variants remove proteinaceous soils relatively faster within a given time frame compared to the parent protease. In one embodiment, the stain removal rate is improved by at least 5%, e.g., 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.

[0202] In some embodiments, the reference protease is a parent protease. In one embodiment, the parent protease is an identical protease except that it does not have substitutions at positions corresponding to 95 and 209 of SEQ ID NO:1 and does not have substitutions at at least three positions corresponding to any 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 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.

[0203] In some embodiments, the variant has an improved stain removal rate compared to an otherwise identical protease that does not have the substitutions at positions 95 and 209 of SEQ ID NO: 1. In one embodiment, the stain removal rate is improved by at least 5%, e.g., 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 an improved stain removal rate compared to SEQ ID NO: 8. In a preferred embodiment, the variant has an improved stain removal rate compared to SEQ ID NO: 11. In a preferred embodiment, the variant has an improved stain removal rate compared to SEQ ID NO: 13.

[0204] In one embodiment, the variant has improved stain removal rate at wash temperatures of 10-60°C, more preferably 15-50°C, most preferably 20-40°C.

[0205] In a preferred embodiment, the variant has improved stain removal rate in liquid detergent as determined according to Example 9 herein.

[0206] A variant of the invention may be a fusion polypeptide comprising a variant of the invention.

[0207] In some aspects, the variants of the present invention are isolated.

[0208] In another embodiment, the variants of the present invention are purified.

[0209] Parent protease The parent protease can be a polypeptide that has at least 60% sequence identity to 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, e.g., 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%.

[0210] In one aspect, the parent protease is a polypeptide that has at least 60% sequence identity to 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, e.g., 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%.

[0211] In one aspect, the parent protease is a polypeptide that has at least 60% sequence identity to the polypeptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, e.g., 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%.

[0212] In one embodiment, the parent has at least 60% sequence identity to the polypeptide of SEQ ID NO: 1, e.g., 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%, and has protease activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 1 by up to 20 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 1.

[0213] In one embodiment, the parent has at least 60% sequence identity to the polypeptide of SEQ ID NO: 2, e.g., 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%, and has protease activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 2 by up to 20 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 2.

[0214] In one embodiment, the parent has at least 60% sequence identity to the polypeptide of SEQ ID NO: 3, e.g., 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%, and has protease activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 3 by up to 20 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 3.

[0215] In one embodiment, the parent has at least 60% sequence identity to the polypeptide of SEQ ID NO: 4, e.g., 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%, and has protease activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 4 by up to 20 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 4.

[0216] In one embodiment, the parent has at least 60% sequence identity to the polypeptide of SEQ ID NO: 5, e.g., 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%, and has protease activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 5 by up to 20 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 5.

[0217] In one embodiment, the parent has at least 60% sequence identity to the polypeptide of SEQ ID NO: 5, e.g., 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%, and has protease activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 5 by up to 20 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 5.

[0218] In one embodiment, the parent has at least 60% sequence identity to the polypeptide of SEQ ID NO: 6, e.g., 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%, and has protease activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 6 by up to 20 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 6.

[0219] In one embodiment, the parent has at least 60% sequence identity to the polypeptide of SEQ ID NO: 7, e.g., 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%, and has protease activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 7 by up to 20 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 7.

[0220] The parent may be a fusion polypeptide or a cleavable fusion polypeptide. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to the polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and involve ligating coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0221] The fusion polypeptide may further comprise a cleavage site between the two polypeptides that is cleaved to release the two polypeptides when the fusion protein is secreted. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0222] The parent can be obtained from a microorganism of any genus. For purposes of the present invention, the term "obtained from," when used herein in reference to a given source, shall mean that the parent encoded by the polynucleotide is produced by that source or is produced by a strain into which a polynucleotide derived from that source has been inserted. In one aspect, the parent is secreted extracellularly.

[0223] In one embodiment, the parent is a Bacillus lentus protease, such as the protease of SEQ ID NO: 1. In one embodiment, the parent is a Bacillus amyloliquefaciens protease, such as the protease of SEQ ID NO: 2. In one embodiment, the parent is a Bacillus licheniformis protease, such as the protease of SEQ ID NO: 3. In one embodiment, the parent is a Bacillus gibsonii protease, such as the protease of SEQ ID NO: 4. In one embodiment, the parent is a Bacillus gibsonii protease, such as the protease of SEQ ID NO: 5. In one aspect, the parent is a Bacillus sp. TY-145 protease, such as the protease of SEQ ID NO: 6. In one aspect, the parent is an Actinomadura keratinilytica protease, such as the protease of SEQ ID NO: 7.

[0224] Preparation of variants The present invention also relates to a method for obtaining a variant having protease activity, comprising: (a) introducing substitutions into a parent protease at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further introducing substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, e.g., introducing substitutions at at least four, at least five, at least six, at least seven, at least eight, or at least nine positions, wherein the variant has protease activity; and (b) recovering the variant.

[0225] The variants may 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, and the like.

[0226] Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent gene.

[0227] Site-directed mutagenesis can be achieved in vitro by PCR, involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, involving restriction enzyme cleavage at a site in a plasmid containing a parent encoding polynucleotide, followed by ligation of an oligonucleotide containing the mutation into this polynucleotide. Usually, the restriction enzymes that digest the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to be ligated together. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0228] Site-directed mutagenesis can also be achieved in vivo by methods known in the art (see, e.g., U.S. Patent Application Publication No. 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, Fungal Genet. Newslett. 43:15-16).

[0229] Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

[0230] Synthetic gene construction involves the in vitro synthesis of polynucleotide molecules designed to encode a polypeptide of interest. Gene synthesis can be performed using several techniques, such as the multiplexed microchip-based technique described in Tian et al., 2004, Nature 432:1050-1054, and similar techniques in which oligonucleotides are synthesized and assembled on a photoprogrammable microfluidic chip.

[0231] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed in 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; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0232] The mutagenesis / shuffling method can be combined with a high-throughput automated screening method to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and easily sequenced using standard methods in the art. This method allows for the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0233] Semisynthetic gene construction is achieved by combining aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semisynthetic construction is typically by a process utilizing synthesized polynucleotide fragments in combination with PCR techniques. Thus, defined regions of a gene may be synthesized de novo, while other regions may be amplified using site-directed mutagenesis primers, and still other regions may be subjected to error-prone or non-error-prone PCR amplification. The polynucleotide subsequences may then be shuffled.

[0234] Polynucleotides The present invention also relates to polynucleotides encoding the variants of the invention.

[0235] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof.

[0236] In one aspect, the polynucleotide is isolated.

[0237] In another embodiment, the polynucleotide is purified.

[0238] nucleic acid construct The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0239] The polynucleotide may be manipulated in a variety of ways to result in expression of the variant. It may be desirable or necessary to manipulate the polynucleotide depending on the expression vector prior to insertion into the vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0240] promoter The control sequence may be a promoter, which is a polynucleotide recognized by a host cell for expression of a polynucleotide encoding the variant of the invention. The promoter contains transcriptional control sequences that mediate expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0241] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY; Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier; and Song et al., 2016, PLOS One 11(7):e0158447.

[0242] Examples of suitable promoters for directing transcription of a polynucleotide of the invention in a filamentous fungal host cell are promoters obtained from cells of the genera Aspergillus, Fusarium, Rhizomucor, and Trichoderma, such as those described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0243] For expression in yeast hosts, examples of useful promoters 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 Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications", Fungal Biology.

[0244] Terminator The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. This terminator is operably linked to the 3' end of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.

[0245] Preferred terminators for bacterial host cells can be obtained from the genes for alkaline protease (aprH) of Bacillus clausii, α-amylase (amyL) of Bacillus licheniformis, and ribosomal RNA (rrnB) of Escherichia coli.

[0246] Preferred terminators for filamentous fungal host cells can be obtained from species of the genus Aspergillus or Trichoderma, such as those derived from the genes for Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as those described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0247] Preferred terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other terminators useful for yeast host cells are described in Romanos et al., 1992, Yeast 8:423-488.

[0248] mRNA stabilizers The regulatory sequence can also be an mRNA stabilizing region downstream of the promoter and upstream of the coding sequence of a gene that increases expression of the gene.

[0249] Examples of suitable mRNA stabilization regions are obtained from the cryIIIA gene of Bacillus thuringiensis (WO 94 / 25612) and the SP82 gene of Bacillus subtilis (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0250] Examples of mRNA stabilization regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824, and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0251] Leader sequence The control sequence may also be a leader, untranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5' end of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

[0252] Suitable leaders for bacterial host cells are described in Hambraeus et al., 2000, Microbiology 146(12):3051-3059, and Kaberdin and Blaesi, 2006, FEMS Microbiol. Rev. 30(6):967-979.

[0253] Preferred leaders for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0254] Suitable leaders for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0255] Polyadenylation sequence The control sequence may also be a polyadenylation sequence, which is operably linked to the 3' end of the polynucleotide and, upon transcription, is recognized by a host cell as a signal for adding polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0256] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0257] Useful polyadenylation sequences for yeast host cells are described in Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0258] signal peptide The regulatory sequence may also be a signal peptide coding region linked to the N-terminus of the variant, encoding a signal peptide that directs the variant into the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the variant. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence foreign to the coding sequence. A foreign signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of the host cell may be used.

[0259] Effective signal peptide coding sequences for filamentous fungal host cells include those obtained from the genes encoding Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as those described in Xu et al., 2018, Biotechnology Letters 40:949-955.

[0260] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described in Romanos et al., 1992, supra.

[0261] Propeptide The regulatory sequence may also be a propeptide coding sequence encoding a propeptide located at the N-terminus of the variant. The resulting polypeptide is known as a proenzyme or propolypeptide (or sometimes a zymogen). Propolypeptides are generally inactive and can be converted to active variants by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences can be obtained from the genes for alkaline protease (aprE) of Bacillus subtilis, neutral protease (nprT) of Bacillus subtilis, laccase of Myceliophthora thermophila (WO 95 / 33836), aspartic proteinase of Rhizomucor miehei, and α-factor of Saccharomyces cerevisiae.

[0262] When both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the variant, and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence.

[0263] Regulatory sequences It may be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are those that turn gene expression on or off in response to chemical or physical stimuli, such as the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA α-amylase promoter, the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are sequences that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified by heavy metals.

[0264] transcription factors The regulatory sequence can also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. This transcription factor can function alone and / or 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 often attaches to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. This transcription factor can regulate the expression of a protein of interest directly (i.e., by activating the transcription of a gene encoding the protein of interest by binding to the promoter) or indirectly (i.e., by activating the transcription of an additional transcription factor that regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the additional transcription factor). 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.

[0265] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcriptional and translational stop signals. Various nucleotide and control sequences can be ligated together to generate a recombinant expression vector that may contain one or more convenient restriction sites, allowing for insertion or substitution of a polynucleotide encoding a variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating an expression vector, a coding sequence is placed into the vector so that it is operably linked to appropriate control sequences for expression.

[0266] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can bring about expression of a polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0267] The vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids may be used that together contain the total DNA or transposon to be introduced into the genome of the host cell.

[0268] The vectors preferably contain one or more selectable markers which permit easy selection of transformed, transfected, transduced or like cells. A selectable marker is a gene the product of which confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0269] The vector preferably contains at least one element that allows the vector to be integrated into the genome of the host cell or to replicate autonomously within the cell independently of the genome.

[0270] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0271] For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in a host cell of interest. The origin of replication may be any plasmid origin of replication that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid origin of replication" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0272] Multiple copies of the polynucleotides of the present invention can be inserted into a host cell to increase polypeptide production. For example, two, three, four, five, or more copies can be inserted into the host cell. Increasing the copy number of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene in the polynucleotide; cells containing an amplified copy of the selectable marker gene can be selected by culturing the cells in the presence of an appropriate selection agent, thereby selecting cells containing additional copies of the polynucleotide.

[0273] host cell The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.

[0274] Once the construct or vector containing the polynucleotide is introduced into a host cell, the construct or vector is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector, as described above. The choice of host cell will largely depend on the gene encoding the variant and its source. The recombinant host cell may contain a single copy of the polynucleotide of the invention, or may contain at least two copies, e.g., three, four, five, or more copies.

[0275] The host cell may be any cell useful for the recombinant production of the variants of the invention, for example a prokaryotic or fungal cell.

[0276] The host cell can be any microbial cell useful for the recombinant production of the polypeptides of the invention, for example, a prokaryotic or fungal cell.

[0277] Prokaryotic host cells can be any gram-positive or gram-negative bacterium, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0278] The bacterial host cell can be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus In some embodiments, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, or Bacillus subtilis cell.

[0279] For purposes of the present invention, the class / genus / species of Bacillus shall be defined as set forth in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0280] 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 cells.

[0281] A 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.

[0282] Methods for introducing DNA into prokaryotic host cells are well known in the art, and any suitable method may be used, including, but not limited to, protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, where the DNA is introduced as a linear or circular polynucleotide. A person skilled in the art will be able to readily identify a suitable method for introducing DNA into a given prokaryotic cell, for example, depending 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.

[0283] The host cell may be a fungal cell. As used herein, "fungi" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0284] Fungal cells can be transformed by processes including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistics, and shock wave-mediated transformation, which are reviewed in Li et al., 2017, Microbial Cell Factories 16:168, and 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 a linear or circular polynucleotide.

[0285] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiomycete yeasts, and yeasts belonging to the fungi imperfecti (Blastomycetes). Because the classification of yeasts may change in the future, for purposes of the present invention, yeasts will be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0286] The yeast host cell can be a cell of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, The yeast host cell may be a cell of Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica. In a preferred embodiment, the yeast host cell is a cell of the genus Pichia or Komagataella, such as a cell of Pichia pastoris (Komagataella phaffii).

[0287] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0288] The filamentous fungal host cells may be selected from the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, The cell may be a cell of the genus Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In preferred embodiments, the filamentous fungal host cell is an Aspergillus, Trichoderma, or Fusarium cell, and in even more preferred embodiments, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0289] For example, the filamentous fungal host cell may be selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, and Ceriporiopsis girvescens. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenstrandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutushirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambusinum sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersoniiemersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0290] In one embodiment, the host cell is isolated.

[0291] In another embodiment, the host cell is purified.

[0292] Manufacturing method The present invention also relates to a method of producing a variant of the invention, comprising (a) culturing a recombinant host cell of the invention under conditions conducive to the production of the variant; and, optionally, (b) recovering the variant.

[0293] The host cells are cultured in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cells can be cultured in shake flask cultures or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors in a suitable medium and under conditions that allow for expression and / or isolation of the variant. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., compositions published in catalogs of the American 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.

[0294] The variants may be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or enzyme assays to determine the relative or specific activity of the variant.

[0295] The variant may be recovered from the culture medium using methods known in the art, including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one embodiment, the whole fermentation broth is recovered. In another embodiment, the cell-free fermentation broth containing the polypeptide is recovered.

[0296] The variants can be purified by various procedures known in the art to obtain substantially pure variants and / or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0297] In an alternative embodiment, the variant is not recovered.

[0298] Protease Granules The present invention also relates to enzyme granules / particles comprising the variants of the present invention. In one embodiment, the granules comprise a core and, optionally, one or more coatings (outer layers) surrounding the core.

[0299] The core may have a diameter, measured as the equivalent spherical diameter (average particle size by volume), of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm, or 250 to 1200 μm. The core diameter, measured as the equivalent spherical diameter, may be determined using laser diffraction, for example, using a Malvern Mastersizer and / or the method described in ISO 13320 (2020).

[0300] In some embodiments, the core comprises a variant of the present invention.

[0301] The core may contain additional substances such as fillers, fibrous materials (cellulose or synthetic), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0302] The core may include a binder such as a synthetic polymer, a wax, a fat, or a carbohydrate.

[0303] The core may include salts of multivalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components, typically as a homogeneous blend.

[0304] The core may comprise inert particles, with the variant being absorbed therein or applied onto the surface, for example by fluidized bed coating.

[0305] The core may have a diameter of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm, or 250 to 1200 μm.

[0306] The core may be surrounded by at least one coating, for example, to improve storage stability, to reduce dust formation during handling, or to color the granules. Optional coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA).

[0307] The coating may be applied in an amount of at least 0.1% by weight of the core, such as at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%, which may be up to 100%, 70%, 50%, 40%, or 30%.

[0308] 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, hi some embodiments, the coating thickness is less than 100 μm, e.g., less than 60 μm, or less than 40 μm.

[0309] The coating must encapsulate the core unit by forming a substantially continuous layer, which is to be understood as a coating with few or no holes, so that there are few or no uncoated areas of the core unit. This layer or coating must in particular be uniform in thickness.

[0310] The coating may further include other materials known in the art, such as fillers, anti-adherents, pigments, dyes, plasticizers, and / or binders, such as titanium dioxide, kaolin, calcium carbonate, or talc.

[0311] The salt coating may comprise at least 60% by weight salt, e.g., 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% by weight salt.

[0312] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, e.g., 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 thick. In certain embodiments, the salt coating is less than 100 μm thick, e.g., less than 60 μm or less than 40 μm thick.

[0313] The salt may be added from a salt solution in which the salt is completely dissolved, or from a salt suspension in which the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm.

[0314] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility of at least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 g per 100 g of water, such as at least 1 g per 100 g of water, for example at least 5 g per 100 g of water.

[0315] The salt may be an inorganic salt, such as a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride, or carbonate, or a salt of a simple organic acid (having less than 10 carbon atoms, e.g., 6 or fewer carbon atoms), such as a citrate, malonate, or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, ammonium ions, or first transition series metal ions such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or earth alkali metal salts of sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides or carbonates, or salts of simple organic acids such as citrates, malonates or acetates may be used.

[0316] The salt in the coating may have a humidity of more than 60%, in particular more than 70%, more than 80%, or more than 85% at 20° C., or may be another hydrated form of such salt (e.g., anhydrous). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0317] A specific example of a suitable salt is NaCl(CH 20℃ = 76%), Na2CO3(CH 20℃ = 92%), NaNO3(CH 20℃ = 73%), Na2HPO4(CH 20℃ = 95%), Na3PO4(CH 25℃ = 92%), NH4Cl(CH 20℃ =79.5%), (NH4)2HPO4(CH 20℃ =93.0%), NH4H2PO4(CH 20℃ = 93.1%), (NH4)2SO4(CH20℃ = 81.1%), KCl(CH 20℃ = 85%), K2HPO4(CH 20℃ = 92%), KH2PO4(CH 20℃ =96.5%), KNO3(CH 20℃ = 93.5%), Na2SO4(CH 20℃ = 93%), K2SO4(CH 20℃ = 98%), KHSO4(CH 20℃ = 86%), MgSO4(CH 20℃ = 90%), ZnSO4(CH 20℃ = 90%), and sodium citrate (CH 25℃ =86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0318] The salts may be in anhydrous form or may be hydrated salts (i.e., crystalline salt hydrates containing bound water of crystallization, as described, for example, in WO 99 / 32595). Specific examples include anhydrous sodium sulfate (NaSO), anhydrous magnesium sulfate (MgSO), magnesium sulfate heptahydrate (MgSO · 7H2O), zinc sulfate heptahydrate (ZnSO4 · 7H2O), dibasic sodium phosphate heptahydrate (Na2HPO4 · 7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0319] Preferably, the salt may be applied as a solution of the salt, for example using a fluidized bed.

[0320] The coating material may be a wax coating material or a film-forming coating material. Examples of wax coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1,483,591.

[0321] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA). Examples of multi-coated enzyme granules are described in WO 93 / 07263 and WO 97 / 23606.

[0322] The cores may be prepared by granulating a blend of ingredients, and may be prepared by methods including granulation techniques such as crystallization, precipitation, pan coating, fluidized bed coating, fluidized bed agglomeration, rotary micronization, extrusion, prilling, spheronization, size reduction, drum granulation, and / or high shear granulation.

[0323] Methods for preparing the cores can be found in Handbook of Powder Technology; Particle size enlargement by CECapes; Vol. 1; 1980; Elsevier. Preparation methods include known feed and granule formulation techniques, such as: (a) Spray-dried products, in which a liquid enzyme-containing solution is atomized in a spray-drying tower to form droplets that are dried as they move down the drying tower to form enzyme-containing particulate material, thus producing microparticles (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pages 140-142; Marcel Dekker).

[0324] (b) Layered products, in which the enzyme is coated as a layer surrounding a preformed inert core particle; the enzyme-containing solution is typically atomized in a fluidized bed apparatus; the preformed core particle is fluidized; the enzyme-containing solution adheres to the core particle and is completely dried, leaving a layer of dried enzyme on the surface of the core particle. If a useful core particle of a desired size can be found, particles of the desired size can be obtained in this manner. Products of this type are described, for example, in WO 97 / 23606.

[0325] (c) Absorbed core particles, where the enzyme is absorbed onto and / or into the surface of the core rather than coating the variant as a layer around the core. Such a process is described in WO 97 / 39116.

[0326] (d) Extruded or pelletized products, in which the variant-containing paste is pressed into pellets or extruded under pressure through small openings and cut into particles, which are then dried. Such particles usually have a significant size because the material from which the extrusion openings are made (usually a perforated plate) limits the allowable pressure drop across the extrusion openings. When small openings are used, very high extrusion pressures also increase the heat generation in the enzyme paste, which is detrimental to the enzymes (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71; pages 140-142; Marcel Dekker).

[0327] (e) Granulated products, in which a variant-containing powder is suspended in molten wax and the suspension is sprayed, for example, with a rotating disk atomizer, into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (editor); Powdered Detergents; Surfactant Science Series; 1998; Vol. 71; pages 140-142; Marcel Dekker). The resulting product is one in which the variant is uniformly distributed throughout the inert material instead of being concentrated on its surface. U.S. Pat. Nos. 4,016,040 and 4,713,245 describe this technology.

[0328] (f) Mixer granulation, in which a variant-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and powder are mixed in the appropriate ratio, and as the moisture from the liquid is absorbed into the dry powder, the dry powder components begin to adhere and aggregate, resulting in particle deposition and the formation of enzyme-containing granules. Such processes are described in U.S. Pat. No. 4,106,991, European Patent Nos. 170360, 304332, 304331, and International Publication Nos. 90 / 09440 and 90 / 09428. In certain embodiments 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 particles, producing so-called T-granules. The reinforced particles are more robust and less likely to release enzyme dust.

[0329] (g) Size reduction, in which larger particles, pellets, tablets, briquettes, etc. containing the enzyme are crushed or crushed to produce cores. The crushed or crushed product is sieved to obtain the desired core particle fraction. The large and small particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0330] (h) Fluidized Bed Granulation. Fluidized bed granulation involves suspending particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. Particles hit by the spray droplets become wet and sticky. The sticky particles collide with other particles, causing them to adhere together, forming granules.

[0331] (i) The cores may be subjected to drying, such as in a fluidized bed dryer. Other known methods for drying granules in the feed or enzyme industry may be used by those skilled in the art. Drying is preferably carried out at a product temperature of 25-90°C. For some enzymes, it is important that the cores containing the variants contain a small amount of water before coating with salt. If a water-sensitive enzyme is coated with salt before removing the excess water, the excess water may become trapped within the core, which may adversely affect the activity of the enzyme. After drying, the cores preferably contain 0.1-10% w / w water.

[0332] Non-shattering granules may be prepared, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art.

[0333] The granules may further comprise one or more additional enzymes, with each enzyme being present in more granules to ensure more uniform enzyme distribution and less physical separation of the various enzymes due to different particle sizes. A method for making multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0334] Another example of formulating yeast using co-granules is disclosed in WO 2013 / 188331.

[0335] The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238216.

[0336] In some embodiments, the granules 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 acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl 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.

[0337] Liquid formulations The present invention also relates to liquid compositions comprising the variants of the invention, which may contain an enzyme stabilizer (e.g., a polyol such as 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 phenylboronic acid derivative, such as 4-formylphenylboronic acid).

[0338] In some embodiments, a filler or carrier material is included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate, and silicate, as well as talc, clay, and the like. Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols, such as polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the composition comprises from about 5% to about 90% of such materials.

[0339] In one aspect, the liquid formulation comprises 20-80% w / w polyol, hi one embodiment, the liquid formulation comprises 0.001-2% w / w preservative.

[0340] In another embodiment, the present invention provides (A) 0.001 to 25% w / w of the variant of the invention; (B) 20 to 80% w / w of polyol; (C) optionally 0.001 to 2% w / w of a preservative; and (D)Water The present invention relates to a liquid formulation comprising:

[0341] In another embodiment, the present invention provides (A) 0.001 to 25% w / w of the variant of the invention; (B) 0.001 to 2% w / w of a preservative; (C) optionally 20 to 80% w / w of a polyol; and (D)Water The present invention relates to a liquid formulation comprising:

[0342] In another embodiment, the liquid formulation comprises one or more formulating agents, for example, 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, acetates, and phosphates, preferably 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-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight less than about 600, and polypropylene glycol (PPG) having an average molecular weight less than about 600, more preferably selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0343] In another embodiment, the liquid formulation comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol. In one embodiment, the liquid formulation comprises 20% to 80% polyol, e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0344] 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 comprises 0.02-1.5% w / w of the preservative, e.g., 0.05-1% w / w of the preservative, or 0.1-0.5% w / w of the preservative. In one embodiment, the liquid formulation comprises 0.001-2% w / w of the preservative (i.e., total amount of preservative), e.g., 0.02-1.5% w / w of the preservative, 0.05-1% w / w of the preservative, or 0.1-0.5% w / w of the preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0345] In another embodiment, the liquid formulation further comprises one or more additional enzymes, for example, hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl 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.

[0346] Fermentation broth preparation or cell composition The present invention also relates to fermentation broth preparations or cell compositions comprising the variants of the invention. The fermentation broth preparations or cell compositions further include additional components used in the fermentation process, such as cells (e.g., host cells containing a gene encoding the variant of the invention used to produce the variant of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a killed whole broth containing organic acid, killed cells and / or cell debris, and culture medium.

[0347] The term "fermentation broth," as used herein, refers to a preparation produced by cell fermentation with no or minimal recovery and / or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation and incubated under carbon-limited conditions that allow protein synthesis (e.g., expression of enzymes by the host cells) and secretion into the cell culture medium. A fermentation broth can include unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, a fermentation broth is unfractionated and includes spent culture medium and cellular debris present after the microbial cells (e.g., filamentous fungal cells) have been removed, for example, by centrifugation. In some embodiments, the fermentation broth includes spent cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0348] In some embodiments, the fermentation broth preparation or cell composition comprises a first organic acid component comprising at least one 1-5 carbon organic acid and / or salt thereof, and a second organic acid component comprising at least one 6 or more carbon organic acid and / or salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, 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, a salt thereof, or a mixture of two or more of the foregoing.

[0349] In one aspect, the composition comprises an organic acid and, optionally, further comprises dead cells and / or cell debris, hi some embodiments, the dead cells and / or cell debris are removed from the cell-killed whole broth to obtain a composition free of these components.

[0350] The fermentation broth preparation or cell composition may further comprise preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0351] The cell-killed whole broth or cell composition may comprise the unfractionated contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killed whole broth or cell composition contains spent 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 that allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition comprises spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or cell composition may be permeabilized and / or lysed using methods known in the art.

[0352] The whole broth or cell compositions described herein are typically liquid but may contain insoluble components such as dead cells, cell debris, culture medium components, and / or insoluble enzymes. In some embodiments, the insoluble components can be removed to obtain a clarified liquid composition.

[0353] The whole broth preparations and cell compositions of the present invention may be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0354] Detergent Composition The present invention also relates to compositions comprising the variants of the invention, for example detergent or cleaning compositions.

[0355] The present invention also relates to compositions comprising the variants of the present invention and further comprising one or more detergent ingredients; and / or one or more additional enzymes. In a preferred embodiment, the composition is a detergent composition comprising one or more detergent components, in particular one or more non-naturally occurring detergent components.

[0356] The present invention also relates to compositions, including variants of the present invention, further comprising one or more additional enzymes selected from the group consisting of amylases (e.g., α-amylases), catalases, cellulases (e.g., endoglucanases), cutinases, DNases, haloperoxygenases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, xanthanases, lichenases, and xyloglucanases, or any mixture thereof.

[0357] The detergent composition may be in the form of, for example, a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compacted powder, granules, a paste, a gel, or a regular, compacted, or concentrated liquid.

[0358] In one preferred embodiment, the detergent composition is a liquid composition.

[0359] In one preferred embodiment, the detergent composition is a powder composition.

[0360] In one preferred embodiment, the detergent composition is a laundry bar soap.

[0361] The present invention also relates to the use of the compositions of the present invention in cleaning processes (eg laundry or hard surface cleaning such as dishwashing).

[0362] The selection of additional components for detergent compositions is within the skill of one in the art and includes conventional ingredients, such as the non-limiting component examples set forth below. Component selection may include consideration of the type of fabric to be washed, the type and / or degree of soiling, the temperature at which the wash will be performed, and the detergent product formulation for fabric care.

[0363] In certain embodiments, detergent compositions comprise a variant of the present invention and one or more non-naturally occurring detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching systems or components, polymers, fabric hueing agents, fabric softeners, suds boosters, suds suppressors, dispersants, dye transfer inhibitors, optical brighteners, fragrances, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0364] In one embodiment, the variant of the invention may be added to a detergent composition in an amount corresponding to 0.01 to 200 mg of enzyme protein per litre of wash liquor, preferably corresponding to 0.05 to 50 mg of enzyme protein per litre of wash liquor, in particular corresponding to 0.1 to 10 mg of enzyme protein per litre of wash liquor.

[0365] An automatic dishwashing (ADW) composition may, for example, comprise 0.001% to 30% (such as 0.01 to 20%, such as 0.1 to 15%, such as 0.5 to 10%) of enzyme protein by weight of the composition.

[0366] A laundry granular composition may, for example, contain from 0.001% to 20% (eg, from 0.01% to 10%, such as from 0.05% to 5%) enzyme protein by weight of the composition.

[0367] A liquid laundry composition may, for example, contain from 0.0001% to 10% (eg, from 0.001% to 7%, such as from 0.1% to 5%) of enzyme protein by weight of the composition.

[0368] Enzymes such as the variants of the invention may be stabilized using conventional stabilisers, for example polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, for example aromatic boric acid esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid, and the compositions may be formulated as described, for example, in WO 92 / 19709 and WO 92 / 19708, or variants according to the invention may be stabilized using peptide aldehydes or ketones, such as described in WO 2005 / 105826 and WO 2009 / 118375.

[0369] The variants of the present invention may be a) at least 0.01 mg of active variant per liter of detergent; b) 2% to 60% by weight of at least one surfactant c) 5% to 50% by weight of at least one builder The composition may be formulated into a liquid detergent composition, such as a liquid laundry composition comprising:

[0370] The detergent composition may be formulated into a granular laundry detergent. a) at least 0.01 mg of active protease variant per gram of composition b) anionic surfactants, preferably 5% to 50% by weight c) a nonionic surfactant, preferably 1% to 8% by weight d) builders such as carbonates, zeolites, phosphate builders, calcium scavenger builders, or complexing agents, preferably 5% to 40% by weight may include:

[0371] The components below are categorized under general headings according to specific functionality, but this should not be construed as limiting, as components may include additional functionality as will be understood by those skilled in the art.

[0372] surfactants The detergent composition 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 certain embodiments, the detergent composition contains a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant is typically present at a level of about 0.1% to 60% by weight, e.g., about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant is selected based on the desired cleaning application and includes any conventional surfactant known in the art. Any surfactant known in the art for use in detergents may be utilized. The surfactant reduces surface tension in the detergent, lifting, dispersing, and subsequently rinsing away stains being cleaned.

[0373] When included, the detergent will typically contain from about 1% to about 40% by weight of anionic surfactant, e.g., from about 5% to about 30%, such as from about 5% to about 15%, or from about 20% to about 25%. Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), LAS isomers, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, alkenesulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethers, and the like. alcohol ethoxy sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, α-sulfofatty acid methyl esters (α-SFMe or SES) such as methyl ester sulfonate (MES), alkyl- or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, di- and monoesters of sulfosuccinic acid, or soaps, and combinations thereof.

[0374] When included, the detergent will typically contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0375] When included, the detergent will typically contain from about 0.2% to about 40% by weight of nonionic surfactant, for example 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%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFAs), alkoxylated fatty acid alkyl esters such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APEs), nonylphenol ethoxylates (NPEs), alkyl polyglycosides (APGs), alkoxylated amines, fatty acid monoethanolamides (FAMs), fatty acid diethanolamides (FADAs), ethoxylated fatty acid monoethanolamides (EFAMs), propoxylated fatty acid monoethanolamides (PFAMs), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides; GAs, or fatty acid glucamides; FAGAs), and products available under the tradenames SPAN and TWEEN®, and combinations thereof.

[0376] When included, the detergent will typically contain from about 0% to about 10% by weight of semi-polar surfactants. Non-limiting examples of semi-polar surfactants include amine oxides (AOs) such as alkyl dimethyl amine oxide, N-(coco alkyl)-N,N-dimethyl amine oxide, and N-(tallow alkyl)-N,N-bis(2-hydroxyethyl) amine oxide, fatty acid alkanolamides, and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0377] When included, the detergent will typically contain from about 0% to about 10% by weight of zwitterionic surfactants. Non-limiting examples of zwitterionic surfactants include betaines, alkyldimethylbetaines, sulfobetaines, and combinations thereof.

[0378] Builders and co-builders The detergent compositions may contain about 0 to 65% by weight (e.g., about 5% to about 45%) of detergent builder or co-builder or mixtures thereof. In dishwashing detergents, builder levels are typically about 40 to 65%, particularly about 50 to 65%. Builders and chelators soften wash water, for example, by removing metal ions from the liquid. The builder and / or co-builder may be, in particular, a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples of 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-aminoethan-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2''-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.

[0379] The detergent compositions may also contain 0 to 20% by weight (e.g., about 5% to about 10%) of a detergent co-builder or mixtures thereof. The detergent compositions may contain the co-builder alone or in combination with a builder (e.g., a zeolite builder). Non-limiting examples of co-builders include polyacrylate homopolymers or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrates, chelators such as aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl or alkenyl succinic acids.Additional specific examples include 2,2',2''-nitrilotriacetic 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- (methylene phosphonic acid) (EDTMPA), diethylenetriaminepentakis(methylene phosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N- (2-Sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (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)-ethylidenediamine-N,N',N''-triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or co-builders are described, for example, in WO 2009 / 102854 and U.S. Pat. No. 5,977,053.

[0380] The variants of the present invention may also be formulated into dishwashing compositions, preferably containing: a) at least 0.01 mg of an active protease variant according to the invention, and b) 10 to 50% by weight of a 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 c) at least one bleaching component The composition may also be formulated into an automatic dishwashing composition (ADW) comprising:

[0381] bleaching type The detergent may contain 0 to 50% by weight (e.g., about 0.1% to about 25%) of a bleaching system. Bleaching systems often remove discoloration by oxidation, and many bleaches also have strong germicidal properties and are used for disinfecting and sterilizing. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleach catalysts, photobleaches, bleach activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, preformed peracids, and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonates and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, such as Oxone®, and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may include inorganic salts including, for example, alkali metal salts of sodium perborates (usually the mono- or tetrahydrate), percarbonates, persulfates, perphosphates, persilicates, and the like, in combination with a peracid-forming bleach activator.

[0382] The term bleach activator, as used herein, refers to a compound that reacts with a peroxygen bleach, such as hydrogen peroxide, to form a peracid. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the classes of esters, amides, imides, or anhydrides. Suitable examples include tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzenesulfonate (ISONOBS), diperoxydodecanoic 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 specific family of bleach activators of interest is disclosed in EP 624154, and within this family, acetyl triethyl citrate (ATC) is particularly preferred. ATC, or the short-chain triglyceride-like triacetin, has the advantage of being environmentally friendly because it ultimately decomposes into citric acid and alcohol. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability of the product upon storage, making them effective bleach activators. Finally, ATC provides good builder capacity for laundry additives. Alternatively, the bleaching system can include peroxyacids, for example, of the amide, imide, or sulfone type. The bleaching system can also include peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system can also include a bleach catalyst or bleach accelerator.

[0383] 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 dinuclear manganese complexes [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2 and [2,2',2''-nitrilotris(ethane-1,2-diylazanylylidene-κN-methanylylidene)triphenolato-κ3O]manganese(III). The bleach catalyst may also be other metal compounds, such as iron or cobalt complexes.

[0384] In some embodiments, the bleach component has the formula: [ka] wherein each R1 is independently a branched alkyl group containing 9 to 24 carbons or a linear alkyl group containing 11 to 24 carbons; preferably, each R1 is independently a branched alkyl group containing 9 to 18 carbons or a linear 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, iso-nonyl, iso-decyl, iso-tridecyl, and iso-pentadecyl. Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259, and WO 2007 / 087242. A suitable photobleach can be, for example, a sulfonated zinc phthalocyanine.

[0385] Hydrotrope Hydrotropes are compounds that solubilize hydrophobic compounds in aqueous solutions (or in non-polar environments, substances of the opposite polarity). Typically, hydrotropes have both hydrophilic and hydrophobic characteristics (so-called amphiphilic properties, as known from surfactants), but the molecular structure of hydrotropes usually 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. Hydrotropes do not exhibit a critical concentration at which self-aggregation occurs, as is found for surfactants and lipids that form micellar, lamellar, or other distinct mesophases. In fact, many hydrotropes exhibit a sustained aggregation process in which the size of the aggregates increases with increasing concentration. However, many hydrotropes change the phase behavior, stability, and colloidal properties of systems containing polar and non-polar substances, such as mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries ranging from pharmaceutical, personal care, and food to technical applications. The use of hydrotropes in detergent compositions allows, for example, more concentrated formulations of surfactants (as in the process of concentrating liquid detergents by removing water) without inducing undesirable phenomena such as phase separation or high viscosity.

[0386] The detergent may contain 0 to 5% by weight (e.g., about 0.5 to about 5%, or about 3% to about 5%) of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0387] polymer The detergent may contain 0-10% by weight (e.g., 0.5-5%, 2-5%, 0.5-2%, or 0.2-1%) of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as described above or may provide anti-redeposition, fabric protection, soil release, dye transfer prevention, grease cleaning, and / or defoaming properties. Some polymers may have more than one of the above properties and / or more than one of the following motifs: 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 polycarboxylates such as PAA, PAA / PMA, polyaspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethylene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the aforementioned polymers are also contemplated.

[0388] Fabric color toning agent The detergent compositions of the present invention may also contain fabric hueing agents, such as dyes or pigments, which, when incorporated into the detergent composition, can be deposited on fabrics when the fabrics come into contact with a washing liquor containing the detergent composition, changing the color of the fabric by absorbing / reflecting visible light. Fluorescent brighteners emit at least some visible light. In contrast, fabric hueing agents absorb at least a portion of the visible light spectrum, thereby changing the color of the surface. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include, for example, small molecule dyes selected from the group consisting of dyes classified under the Color Index (CI) classifications Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, or mixtures thereof, as described in WO 2005 / 003274, WO 2005 / 003275, WO 2005 / 003276, and EP 1 876 226 (incorporated herein by reference). The detergent compositions preferably comprise from about 0.00003% to about 0.2%, from about 0.00008% to about 0.05%, or even from about 0.0001% to about 0.04% by weight of a fabric hueing agent. The composition may include 0.0001% to about 0.2% by weight of a fabric hueing agent, which may be particularly preferred when the composition is in the form of a unit dose pouch. Suitable fabric hueing agents are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0389] Additional enzymes Detergent additives or detergent compositions comprising the variants of the present invention may comprise one or more enzymes such as amylases (e.g., α-amylases), arabinases, carbohydrases, cellulases (e.g., endoglucanases), cutinases, DNases, galactanases, haloperoxygenases, lipases, mannanases, oxidases such as laccases and / or peroxidases, pectinases, pectin lyases, proteases, xylanases, xanthanases, or xyloglucanases.

[0390] The properties of the selected enzyme should be compatible with the selected detergent (eg, pH optimum, compatibility with other enzymatic and non-enzymatic components, etc.).

[0391] cellulase The term "cellulase" refers to one or more (e.g., several) enzymes that hydrolyze cellulose materials. The terms "cellulase" and "polypeptide having cellulase activity" are used interchangeably. The cellulase may 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 endoglucanases (e.g., EC 3.2.1.4), cellobiohydrolases, β-glucosidases, or combinations thereof.

[0392] Suitable cellulases include single-component and mixtures of bacterial or fungal enzymes. Chemically modified or protein-engineered variants are also contemplated. Cellulases can be, for example, single-component or mixtures of single-component endo-1,4-β-glucanases, also referred to as endoglucanases.

[0393] Suitable cellulases include those derived from the genera Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include fungal cellulases derived from Humicola insolens (U.S. Pat. No. 4,435,307) or from Trichoderma, such as T. reesei or T. viride. Other suitable cellulases are those from the genus Thielavia, such as Thielavia terrestris, as described in WO 96 / 29397, or the fungal cellulases produced by Myceliophthora thermophila and Fusarium oxysporum, as disclosed in U.S. Patent Nos. 5,648,263, 5,691,178, 5,776,757, WO 89 / 09259, and WO 91 / 17244. Also of interest are cellulases from the genus Bacillus, as described in WO 02 / 099091 and JP 2000210081 A1. Suitable cellulases are alkaline or neutral cellulases with care benefits. Examples of cellulases are described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940.Other examples are cellulase variants such as those described in WO 94 / 07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593, 5,763,254, WO 95 / 24471, WO 98 / 12307.

[0394] The other cellulase is an endo-β-1,4-glucanase enzyme having a sequence that is at least 97% identical to the amino acid sequence of positions 1 to 773 of SEQ ID NO:2 of WO 2002 / 099091, or a Family 44 xyloglucanase enzyme having a sequence that is at least 60% identical to the amino acid sequence of positions 40 to 559 of SEQ ID NO:2 of WO 2001 / 062903.

[0395] Yet another group of suitable cellulases comprises a stabilized linker between the core and the CBM. Particularly useful are those cellulases having at least 80% identity to SEQ ID NO: 397, SEQ ID NO: 398, or SEQ ID NO: 399 of WO 2023 / 061928.

[0396] 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, Revitalenz 1000, Revitalenz 200, and Revitalenz 2000 (Dupont Industrial Biosciences); KAC-500(B)™ (Kao Corporation); and Biotouch DC and Biotouch FLX1 (AB Enzymes).

[0397] Two basic approaches for measuring cellulolytic enzyme activity are (1) measuring total cellulolytic enzyme activity and (2) measuring individual cellulolytic enzyme activities (endoglucanase, cellobiohydrolase, and β-glucosidase), as reviewed in Zhang et al., 2006, Biotechnology Advances 24:452-481. Total cellulolytic enzyme activity can be measured using insoluble substrates such as Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, and pretreated lignocellulose. The most common total cellulolytic activity assay is the filter paper assay, which uses 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).

[0398] Proteases The composition may contain one or more additional proteases, including those of bacterial, fungal, plant, viral, or animal origin, such as proteases of plant or microbial origin. Microbial origin is preferred. Chemically modified or engineered variants are included. This may be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease may be, for example, from the S1 family, such as trypsin, or from the S8 family, such as subtilisin. The metalloprotease may be, for example, thermolysin from the M4 family, or other metalloproteases, such as those from the M5, M7, or M8 families.

[0399] An example of a metalloprotease is the neutral metalloprotease described in WO 2007 / 044993 (Genencor Int.), such as that from Bacillus amyloliquefaciens.

[0400] Suitable commercially available protease enzymes include those sold under the 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 A / S), Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Those sold under the trade names Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Opticlean®, Optimase®, Preferenz® P200, Preferenz® P300, and Preferenz® P400 (DuPont / IFF), Axapem™ (Gist-Brocades NV), BLAP (sequence shown in Figure 29 of U.S. Pat. No. 5,352,604) and variants thereof (Henkel AG), and KAP (Bacillus alkalophilus) by Kao.

[0401] Lipase and cutinase Suitable lipases and cutinases include those of bacterial or fungal origin, including chemically modified or protein engineered mutant enzymes. Examples include lipases from Thermomyces, such as those from T. lanuginosus (formerly Humicola lanuginosa), as described in EP 258068 and EP 305216. lanuginosa), cutinases from Humicola, for example H. insolens cutinases (WO 96 / 13580), lipases from strains of Pseudomonas (some of which have now been renamed Burkholderia), for example P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (P.Lipases from strains of Pseudomonas wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 2010 / 065455), cutinases from Magnaporthe grisea (WO 2010 / 107560), cutinases from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipases from Thermobifida fusca (WO 2011 / 084412), and Geobacillus stearothermophilus (Geobacillus stearothermophilus lipase (WO 2011 / 084417), lipase from Bacillus subtilis (WO 2011 / 084599), and lipases from Streptomyces griseus (WO 2011 / 150157) and S. pristinaespiralis (WO 2012 / 137147).

[0402] 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.

[0403] Preferred commercially available lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A / S), Lumafast (originally from Genencor), and Lipomax (originally from Gist-Brocades).

[0404] Further examples are lipases, sometimes called acyltransferases or perhydrolases, such as acyltransferases with homology 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 M. smegmatis perhydrolase, in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 2010 / 100028).

[0405] amylase Suitable amylases for use with the variants of the present invention may be α-amylases or glucoamylases and may be of bacterial or fungal origin, including chemically modified or engineered variants. Examples of amylases include α-amylases obtained from Bacillus species, such as the α-amylase obtained from a specialized strain of Bacillus licheniformis, which is described in more detail in British Patent No. 1,296,839.

[0406] Suitable amylases include the amylase having SEQ ID NO: 2 in WO 95 / 10603, or a variant thereof having 90% sequence identity to SEQ ID NO: 3. Preferred variants are set forth in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424, and WO 99 / 19467, such as variants having substitutions at one or more of the following positions: 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.

[0407] A different suitable amylase is the amylase having SEQ ID NO: 6 in WO 02 / 10355, or a variant thereof having 90% sequence identity to SEQ ID NO: 6. A preferred variant of SEQ ID NO: 6 is one having deletions at positions 181 and 182 and a substitution at position 193.

[0408] Another suitable amylase is a hybrid α-amylase comprising residues 1-33 of the α-amylase from B. amyloliquefaciens as set forth in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of the α-amylase from B. licheniformis as set forth in SEQ ID NO: 4 of WO 2006 / 066594, or a variant with 90% sequence identity thereof. 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, 1201, A209, and Q264. The most preferred variant of the hybrid α-amylase comprising residues 1-33 of the α-amylase from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 contains the following substitutions: M197T; H156Y+A181T+N190F+A209V+Q264S; or G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S It has the following characteristics.

[0409] Another preferred amylase is an amylase having the sequence of SEQ ID NO: 6 in WO 99 / 19467, or a variant thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having 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 having deletions at positions R181 and G182, or H183 and G184.

[0410] Additional amylases that may be used are those having 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 to 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 that have 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, using SEQ ID NO: 2 of WO 96 / 023873 for numbering purposes. More preferred variants have deletions at two positions selected from 181, 182, 183, and 184, for example, deletions at positions 181 and 182, 182 and 183, or 183 and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 have deletions at positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304, and 476.

[0411] Other amylases that may be used are amylases having SEQ ID NO: 2 of WO 2008 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 2008 / 153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 are those that have substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0412] Further suitable amylases are those having SEQ ID NO: 2 of WO 2009 / 061380, or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having C-terminal truncations and / or substitutions, deletions or insertions 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 with 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 deletions at positions R180 and / or S181, or T182 and / or G183. Most preferred amylase variants of SEQ ID NO:2 are those with the following substitutions: N128C+K178L+T182G+Y305R+G475K; N128C+K178L+T182G+F202Y+Y305R+D319T+G475K; S125A+N128C+K178L+T182G+Y305R+G475K; or S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K which variant is C-terminally truncated and optionally further comprises a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0413] A further suitable amylase is the amylase having SEQ ID NO: 1 of WO 2013 / 184577, or a variant thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 are those having substitutions, deletions, or insertions at one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, 1203, S241, R458, T459, D460, G476, and G477. More preferred variants of SEQ ID NO: 1 are those with substitutions at one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K, and G477K, and / or deletions at positions R178 and / or S179, or T180 and / or G181. Most preferred amylase variants of SEQ ID NO: 1 are those with the following substitutions: E187P+I203Y+G476K E187P+I203Y+R458N+T459S+D460T+G476K Including, Optionally, it further comprises a substitution at position 241 and / or a deletion at positions 178 and / or 179.

[0414] A further suitable amylase is the amylase having SEQ ID NO: 1 of WO 2010 / 104675, or a variant thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 are those having 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.

[0415] 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 deletions at positions R179 and / or S180, or I181 and / or G182. The most preferred amylase variant of SEQ ID NO: 1 comprises the substitutions N21D+D97N+V128I, and optionally further comprises a substitution at position 200, and / or a deletion at positions 180 and / or 181.

[0416] Another suitable amylase is the α-amylase having SEQ ID NO: 12 in WO 01 / 66712, or a variant having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those that have substitutions, deletions or insertions at one or more of the following positions of 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 a deletion of D183 and G184 and the substitutions R118K, N195F, R320K, and R458K, as well as variants further having substitutions at one or more positions selected from the following group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with variants further having substitutions at all of these positions being most preferred.

[0417] Other examples are amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087. Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, and BAN™ (from Novozymes A / S), and Rapidase™, Purastar™ / Effectenz™, Powerase, Preferenz S1000, Preferenz S100, and Preferenz S110 (from Genencor International Inc. / DuPont).

[0418] Peroxidase / Oxidase Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin, and include chemically modified or protein-engineered variants. Examples of useful peroxidases include peroxidases from the genus Coprinus, such as those from C. cinereus, as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257, and variants thereof.

[0419] Commercially available peroxidases include Guardzyme™ (Novozymes A / S).

[0420] Auxiliary materials Any detergent component known in the art for use in laundry detergents can also be utilized.Other optional detergent components include, alone or in combination, anti-corrosion agents, anti-shrinkage agents, soil redeposition inhibitors, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegrating agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and / or polyols such as propylene glycol), fabric softeners including clay, fillers / processing aids, optical brighteners / optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil suspending agents, softeners, foam suppressors, tan inhibitors, and wicking agents.Any component known in the art for use in laundry detergents can also be utilized.The selection of such components is well within the skill of those skilled in the art.

[0421] Dispersants: The detergent compositions of the present invention may also contain dispersants. In particular, powder detergents may contain dispersants. Suitable water-soluble organic materials include homo- or copolymeric acids or salts thereof, where 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, Inc., 1997.

[0422] Dye Transfer Inhibitors: The detergent compositions of the present invention may also 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, polyvinyloxazolidone, and polyvinylimidazole, or mixtures thereof. When present in the composition, the dye transfer inhibitors may be present at levels of from about 0.0001% to about 10%, from about 0.01% to about 5%, or from about 0.1% to about 3%, by weight of the composition.

[0423] Fluorescent Brighteners: The detergent compositions of the present invention will also preferably contain additional components that can alter the color tone of the washed items, such as fluorescent or optical brighteners. When present, the brightener is preferably present at a level of about 0.01% to about 0.5%. Any fluorescent brightener suitable for use in laundry detergent compositions can be used in the compositions of the present invention. The most commonly used fluorescent brighteners belong to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and bisphenyl-distyryl derivatives. Examples of fluorescent whitening agents of the diaminostilbene-sulfonic acid derivative type include: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)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-triazin-6-ylamino)stilbene-2,2'-disulfonate, and the sodium salt of 2-(stilbyl-4"-naphtho-1,2':4,5)-1,2,3-triazole-2"-sulfonate. Preferred optical brighteners 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-triazin-6-ylamino)stilbene disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl)disulfonate. Also a preferred optical brightener is Parawhite KX, commercially available from Paramount Minerals and Chemicals, Mumbai, India.Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazolines and 7-alkylaminocoumarins. Suitable fluorescent whitening agent levels include lower levels of about 0.01, 0.05, about 0.1, or about 0.2% by weight, to upper levels of 0.5 or 0.75% by weight.

[0424] Soil-releasing polymer: The detergent composition of the present invention may also contain one or more soil-releasing polymers that aid in the removal of soils from fabrics such as cotton and polyester-based fabrics, particularly hydrophobic soils from polyester-based fabrics. The soil-releasing polymer may be, for example, a nonionic or anionic terephthalate-based polymer, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, or polyester polyamides, as described, for example, in Chapter 7 of Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker, Inc. Another type of soil-releasing polymer is an amphiphilic alkoxylated grease-cleaning polymer that includes a core structure and multiple alkoxylate groups attached to the core structure. The core structure may include a polyalkyleneimine structure or a polyalkanolamine structure, as described in detail in WO 2009 / 087523 (incorporated herein by reference). Additionally, random graft copolymers are suitable soil-releasing polymers. Suitable graft copolymers are described in more detail in International Publication Nos. 2007 / 138054, 2006 / 108856, and 2006 / 113314 (incorporated herein by reference). Other soil-release polymers are substituted polysaccharide structures, particularly substituted cellulosic structures, such as modified cellulose derivatives such as those described in EP 1867808 or WO 03 / 040279 (both incorporated herein by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, ester carboxymethyl cellulose, and mixtures thereof.

[0425] Anti-redeposition agents: The detergent compositions of the present invention may also 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 cellulosic polymers described above under soil release polymers may also function as anti-redeposition agents.

[0426] Other suitable adjuvants include, but are not limited to, shrinkage inhibitors, anti-wrinkle agents, disinfectants, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, fragrances, pigments, suds suppressors, solvents, and structurants and / or structural elasticizers for liquid detergents.

[0427] Detergent product formulation The detergent enzymes (i.e., the protease variants of the present invention and, optionally, one or more additional enzymes) can be included in the detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive containing all of the enzymes. The detergent additive containing one or more enzymes can be formulated, for example, as granules, liquids, slurries, etc. Preferred detergent additive formulations include granules, especially non-shattering granules, liquids, especially stabilized liquids, or slurries.

[0428] The detergent compositions of the present invention may be in any convenient form, such as a bar, a homogeneous tablet, a tablet with two or more layers, a pouch with one or more compartments, a regular or compacted powder, granules, a paste, a gel, or a regular, compacted, or concentrated liquid. There are several detergent formulation forms, such as layers (same or different phases), pouches, and forms for mechanical dosing units.

[0429] The pouch may be configured as a single or multiple compartments. The pouch may be of any form, shape, and material suitable for retaining the composition without, for example, releasing the composition from the pouch prior to contact with water. The pouch is fabricated from a water-soluble film that encloses an internal volume. This internal volume may be divided into pouch compartments. Preferred films are polymeric materials, preferably polymers that can be formed 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, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer (e.g., PVA) in the film is at least about 60%. Preferred average molecular weights are typically from about 20,000 to about 150,000. The film can also be a blend composition comprising a hydrolytically degradable and water-soluble polymer blend (e.g., polylactide and polyvinyl alcohol) (known under trade reference M8630 as sold by Chris Craft In, Prod. of Gary, Indiana, US) and a plasticizer such as glycerol, ethylene glycerol, propylene glycol, sorbitol, and mixtures thereof. The pouch can contain a solid laundry cleaning composition or component and / or a liquid cleaning composition or component separated by a water-soluble film. The compartment for the liquid component can be different in composition from the compartment containing the solid; see, for example, US Patent Application Publication No. 2009 / 0011970.

[0430] The detergent components may be physically separated from one another by compartments in various layers of the water-soluble pouch or tablet, which may avoid undesirable storage interactions between the components. The different dissolution profiles of each of the compartments may also result in delayed dissolution of selected components in the wash solution.

[0431] Non-unit-dose liquid or gel detergents may be aqueous, typically containing at least 20% by weight and up to 95% water, e.g., up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, or up to about 35% water. Other types of liquids, including but not limited to alkanols, amines, diols, ethers, and polyols, may also be included in aqueous liquids or gels. Aqueous liquid or gel detergents may contain 0-30% organic solvents. Liquid or gel detergents may also be non-aqueous.

[0432] Laundry bar soap The enzymes of the present invention can be added to laundry bars and used on hand-washed laundry, fabrics, and / or textiles. The term laundry bar includes laundry bars, soap bars, combo bars, synthetic detergent bars, and detergent bars. Bar types typically differ in the type of surfactant they contain, and the term laundry bar includes those containing fatty acid-derived soaps and / or synthetic soaps. Laundry bars are solids, and therefore have a physical form that is not a liquid, gel, or powder at room temperature.

[0433] The laundry bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adducts or hemiacetal adducts), boric acid, borate salts, borax, and / or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerin, fatty acids, pH adjusting compounds such as citric acid, acetic acid, and / or formic acid, and / or salts of monovalent cations and organic anions, where the monovalent cations are, for example, Na + , K. + , or NH 4 + and the organic anion can be, for example, formate, acetate, citrate, or lactate, so that the salt of the monovalent cation and organic anion can be, for example, sodium formate.

[0434] The laundry bar may also contain complexing agents such as EDTA and HEDP, perfumes and / or different types of fillers, surfactants such as anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizers, fillers, pigments, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressors, structurants, binders, leaching agents, bleach activators, clay soil removers, anti-redeposition agents, polymeric dispersants, brighteners, fabric softeners, perfumes, and / or other compounds known in the art.

[0435] The laundry bar may be processed in conventional laundry bar manufacturing equipment, including, but not limited to, mixers, presses (e.g., two-stage vacuum presses), extruders, cutters, logo imprinters, cooling tunnels, and packaging machines. A premix containing soap, the enzyme of the present invention, optionally one or more additional enzymes, a protease inhibitor, and salts of monovalent cations and organic anions may be prepared, and then the mixture is plodded. The enzyme and optional additional enzymes may be added simultaneously with the protease inhibitor, e.g., in liquid form. In addition to the mixing and plodding steps, the process may further include steps of grinding, extruding, cutting, stamping, cooling, and / or packaging.

[0436] Granular detergent formulations Enzymes, such as the variants of the present invention, in the form of granules comprising an enzyme-containing core and, optionally, one or more coatings, are commonly used in granular (powdered) detergents. Various methods for preparing the cores are known in the art, including: a) spray drying of a liquid enzyme-containing solution; b) producing a layered product having the enzyme coated as a layer around a preformed inert core particle, e.g., using fluidized bed equipment; c) absorbing the enzyme onto and / or into the surface of a preformed core; d) extrusion of an enzyme-containing paste; e) suspending an enzyme-containing powder in molten wax and spraying to produce a small-particle product; f) agitator granulation by adding an enzyme-containing liquid to a dry powder composition of the granulated components; g) size reduction of the enzyme-containing core by grinding or crushing large particles, pellets, etc.; and h) fluidized bed granulation. The enzyme-containing cores may be dried, e.g., using a fluidized bed dryer or other known methods for drying granules in the feed or enzyme industry, to typically produce a moisture content of 0.1 to 10% w / w water.

[0437] The enzyme-containing core is optionally provided with a coating to improve storage stability and / or reduce dust formation. One type of coating often used for enzyme granules for detergents is a salt coating, typically an inorganic salt coating, which can be applied as a salt solution, for example, using a fluidized bed. Other coating materials that can be used include, for example, polyethylene glycol (PEG), methylhydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). The granules can include multiple coatings, for example, a salt coating followed by an additional coating of a material such as PEG, MHPC, or PVA.

[0438] Therefore, the present invention also relates to enzyme granules / particles comprising the variants of the present invention. In one embodiment, the granules comprise a core and, optionally, one or more coatings (outer layers) surrounding the core.

[0439] The core may have a diameter, measured as the equivalent spherical diameter (average particle size by volume), of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm, or 250 to 1200 μm.

[0440] In one embodiment, the core comprises one or more polypeptides having protease activity of the present invention.

[0441] The core may contain additional substances such as fillers, fibrous materials (cellulose or synthetic), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0442] The core may include a binder such as a synthetic polymer, a wax, a fat, or a carbohydrate.

[0443] The core may include salts of multivalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components, typically as a homogeneous blend.

[0444] The core may contain inert particles with enzymes absorbed therein or applied onto the surface, for example by fluidized bed coating.

[0445] The core may have a diameter of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm, or 250 to 1200 μm.

[0446] 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 granules. Optional coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methylhydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0447] The coating may be applied in an amount of at least 0.1% by weight of the core, such as at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%, which may be up to 100%, 70%, 50%, 40%, or 30%.

[0448] 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, hi some embodiments, the coating thickness is less than 100 μm, e.g., less than 60 μm, or less than 40 μm.

[0449] The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating that has few or no pores, so that the core unit it encapsulates / encloses has few or no uncovered spots. The layer or coating should in particular be uniform in thickness.

[0450] The coating may further include other materials known in the art, such as fillers, anti-adherents, pigments, dyes, plasticizers, and / or binders, such as titanium dioxide, kaolin, calcium carbonate, or talc.

[0451] The salt coating may comprise at least 60% by weight salt, e.g., 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% by weight salt.

[0452] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, e.g., 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 thick. In certain embodiments, the salt coating is less than 100 μm thick, e.g., less than 60 μm or less than 40 μm thick.

[0453] The salt may be added from a salt solution in which the salt is completely dissolved, or from a salt suspension in which the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm.

[0454] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility of at least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 g per 100 g of water, such as at least 1 g per 100 g of water, for example at least 5 g per 100 g of water.

[0455] The salt may be an inorganic salt, such as a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride, or carbonate, or a salt of a simple organic acid (fewer than 10 carbon atoms, e.g., 6 or fewer carbon atoms), such as a citrate, malonate, or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, ammonium ions, or first transition series metal ions such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or earth alkali metal salts of sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides or carbonates, or salts of simple organic acids such as citrates, malonates or acetates may be used.

[0456] The salt in the coating may have a humidity of more than 60%, in particular more than 70%, more than 80%, or more than 85% at 20° C., or may be another hydrated form of such salt (e.g., anhydrous). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0457] A specific example of a suitable salt is NaCl(CH 20℃ = 76%), Na2CO3(CH 20℃ = 92%), NaNO3(CH 20℃ = 73%), Na2HPO4(CH 20℃ = 95%), Na3PO4(CH 25℃ = 92%), NH4Cl(CH 20℃ =79.5%), (NH4)2HPO4(CH 20℃ =93.0%), NH4H2PO4(CH 20℃ = 93.1%), (NH4)2SO4(CH20℃ = 81.1%), KCl(CH 20℃ = 85%), K2HPO4(CH 20℃ = 92%), KH2PO4(CH 20℃ =96.5%), KNO3(CH 20℃ = 93.5%), Na2SO4(CH 20℃ = 93%), K2SO4(CH 20℃ = 98%), KHSO4(CH 20℃ = 86%), MgSO4(CH 20℃ = 90%), ZnSO4(CH 20℃ = 90%), and sodium citrate (CH 25℃ =86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0458] The salts may be in anhydrous form or may be hydrated salts (i.e., crystalline salt hydrates containing bound water of crystallization, as described, for example, in WO 99 / 32595). Specific examples include anhydrous sodium sulfate (NaSO), anhydrous magnesium sulfate (MgSO), magnesium sulfate heptahydrate (MgSO·7H0), zinc sulfate heptahydrate (ZnSO·7H0), dibasic sodium phosphate heptahydrate (NaHPO·7H0), magnesium nitrate hexahydrate (Mg(NO)(6H0)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0459] Preferably, the salt may be applied as a solution of the salt, for example using a fluidized bed.

[0460] The coating material may be a wax coating material or a film-forming coating material. Examples of wax coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1,483,591.

[0461] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA). Examples of multi-coated enzyme granules are described in WO 93 / 07263 and WO 97 / 23606.

[0462] The cores may be prepared by granulating a blend of ingredients, and may be prepared by methods including granulation techniques such as crystallization, precipitation, pan coating, fluidized bed coating, fluidized bed agglomeration, rotary micronization, extrusion, prilling, spheronization, size reduction, drum granulation, and / or high shear granulation.

[0463] Methods for preparing the cores can be found in Handbook of Powder Technology; Particle size enlargement by CECapes; Vol. 1; 1980; Elsevier. Preparation methods include known feed and granule formulation techniques, such as: (a) Spray-dried products, in which a liquid enzyme-containing solution is atomized in a spray-drying tower to form droplets that are dried as they move down the drying tower to form enzyme-containing particulate material, thus producing microparticles (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pages 140-142; Marcel Dekker).

[0464] (b) Layered products, in which the enzyme is coated as a layer surrounding a preformed inert core particle; the enzyme-containing solution is typically atomized in a fluidized bed apparatus; the preformed core particle is fluidized; the enzyme-containing solution adheres to the core particle and is completely dried, leaving a layer of dried enzyme on the surface of the core particle. If a useful core particle of a desired size can be found, particles of the desired size can be obtained in this manner. Products of this type are described, for example, in WO 97 / 23606.

[0465] (c) Absorbed core particles, where the enzyme is absorbed onto and / or into the surface of the core rather than being coated as a layer around the core. Such a process is described in WO 97 / 39116.

[0466] (d) Extruded or pelletized products, in which the enzyme-containing paste is pressed into pellets or extruded under pressure through small orifices and cut into particles, which are then dried. Such particles usually have a significant size because the material from which the extrusion orifices are made (usually a perforated plate) limits the allowable pressure drop across the orifice. When small orifices are used, very high extrusion pressures also increase the heat generation in the enzyme paste, which is detrimental to the enzymes (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71; pages 140-142; Marcel Dekker).

[0467] (e) Granulated products, in which an enzyme-containing powder is suspended in molten wax and the suspension is sprayed, for example, with a rotating disk atomizer, into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pages 140-142; Marcel Dekker). The resulting product is one in which the enzyme is uniformly distributed throughout the inert material instead of being concentrated on its surface. U.S. Pat. Nos. 4,016,040 and 4,713,245 describe this technology.

[0468] (f) Mixer-granulated products, in which an enzyme-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and powder are mixed in the appropriate ratio, and as the moisture from the liquid is absorbed into the dry powder, the dry powder components begin to adhere and aggregate, resulting in particle deposition and the formation of enzyme-containing granules. Such a process is described in U.S. Pat. No. 4,106,991 and related documents EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO 90 / 09428. In certain products of this process, various high-shear mixers can be used as granulators. Granules consisting of enzymes, fillers, and binders are mixed with cellulose fibers to reinforce the particles, producing so-called T-granules. The reinforced particles are more robust and less likely to release enzyme dust.

[0469] (g) Size reduction, in which larger particles, pellets, tablets, briquettes, etc. containing the enzyme are crushed or crushed to produce cores. The crushed or crushed product is sieved to obtain the desired core particle fraction. The large and small particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0470] (h) Fluidized Bed Granulation. Fluidized bed granulation involves suspending particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. Particles hit by the spray droplets become wet and sticky. The sticky particles collide with other particles, causing them to adhere together, forming granules.

[0471] (i) The cores may be subjected to drying, such as in a fluidized bed dryer. Other known methods for drying granules in the feed or enzyme industry may be used by those skilled in the art. Drying is preferably carried out at a product temperature of 25-90°C. For some enzymes, it is important that the enzyme-containing cores contain a small amount of water before coating with salt. If a water-sensitive enzyme is coated with salt before removing the excess water, the excess water may become trapped within the core, which may adversely affect the activity of the enzyme. After drying, the cores preferably contain 0.1-10% w / w of water.

[0472] Non-shattering granules may be prepared, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art.

[0473] The granules may further comprise one or more additional enzymes, with each enzyme being present in more granules to ensure more uniform enzyme distribution and less physical separation of the various enzymes due to different particle sizes. A method for making multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0474] Another example of formulating enzymes through the use of co-granules is disclosed in WO 2013 / 188331.

[0475] The enzyme may also be a protected enzyme prepared according to the method disclosed in EP 238,216.

[0476] In some embodiments, the granules 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 acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl 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.

[0477] For further information on enzyme granules and their production, see WO 2013 / 007594 and, for example, WO 2009 / 092699, EP 1705241, EP 1382668, WO 2007 / 001262, U.S. Pat. No. 6,472,364, WO 2004 / 074419, and WO 2009 / 102854.

[0478] use The present invention is also directed to methods for using the variants of the present invention or compositions comprising said variants in the laundering of textiles and fabrics, including domestic laundry washing and industrial laundry washing.

[0479] The present invention is also directed to methods for using the variants according to the invention or compositions thereof when cleaning hard surfaces such as floors, tables, walls, roofs, and hard object surfaces such as cars (car washing) and dishes (dish washing).

[0480] The variants of the present invention may be added to, and thereby become a component of, detergent compositions, and therefore one aspect of the present invention relates to the use of the variants of the present invention in cleaning processes such as laundry and / or hard surface cleaning.

[0481] The detergent compositions of the present invention may be formulated, for example, as hand or machine laundry detergent compositions comprising a laundry additive composition suitable for pre-treating soiled fabrics and a fabric softener composition with added rinse aid, as detergent compositions for use in general household hard surface cleaning operations, or for hand or machine dishwashing operations.

[0482] The cleaning process 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, tabletops, drains, sinks, and washbasins. The laundry process can be, for example, a domestic laundry, but can also be an industrial laundry. Furthermore, the present invention relates to a process for washing fabrics and / or clothes, which process comprises treating fabrics with a washing solution comprising a detergent composition and at least one protease variant of the present invention. The cleaning process or textile care process can be, for example, carried out by machine washing or by hand. The washing solution can be, for example, an aqueous washing solution comprising the detergent composition.

[0483] In one aspect, the variants of the invention are used in cleaning processes involving short wash cycles (typically wash cycles of about 20 minutes or less, e.g., about 30 minutes or less, such as about 15 minutes or less or about 10 minutes or less), e.g., in laundry processes. Surprisingly, it has been found that the subtilase variants of the invention are highly effective in short wash cycles, e.g., lasting only about 10-20 minutes. This may be useful, for example, in top-loading washing machines, where wash cycles are often short, or for hand-washing.

[0484] In another aspect, the variants of the present invention are used in a cleaning process, for example, a laundry process, and the wash water is used for two or more wash loads. In this case, the wash water containing the detergent with the variant of the present invention can be used in the first wash cycle of the first wash load and then reused for one or more additional wash cycles in the new wash load. It has been found that detergents containing the variants of the present invention can substantially maintain their cleaning performance against protease-sensitive stains even after three or more wash cycles. This can be useful, for example, for laundry washed by hand and / or in water-scarce areas.

[0485] In recent years, there has been growing interest in replacing petrochemical-derived components in detergents with renewable biological components such as enzymes and polypeptides without compromising cleaning performance. As the components of detergent compositions change, new enzymes with novel enzymatic activities or alternative and / or improved properties compared to previously used detergent enzymes such as proteases, lipases, and amylases may be required to achieve similar or improved cleaning performance compared to conventional detergent compositions.

[0486] The invention further relates to the use of the variants of the invention in a process for removing proteinaceous stains, which may be food stains, such as baby food, cocoa, egg or milk stains, or other stains such as sebum, blood, ink or grass, or combinations thereof.

[0487] Cleaning method The present invention provides a method of cleaning fabrics, dishware, or hard surfaces with a detergent composition comprising a variant of the present invention.

[0488] The method of cleaning comprises 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 a laundry or dishwashing process.

[0489] Another embodiment relates to a method for removing stains from fabrics or dishware, comprising contacting the fabric or dishware with a composition comprising a protease of the present invention under conditions suitable for cleaning the object. In the cleaning methods of the present invention, the object being cleaned can be any suitable object, such as a textile, or dishware, or a hard surface such as a floor, table, wall, etc.

[0490] Also contemplated are compositions and methods for treating fabrics (e.g., for textile desizing) using the protease variants of the present invention. The protease variants may be used in any fabric treatment method known in the art (see, e.g., U.S. Pat. No. 6,077,316). For example, in one aspect, the feel and appearance of a fabric are improved by a method comprising contacting the fabric with a protease variant in a solution. In one aspect, the fabric is treated with a solution under pressure.

[0491] The detergent compositions of the present invention are suitable for use in laundry and hard surface applications, including dishwashing. Accordingly, the present invention includes a method of laundering fabrics or cleaning dishware, comprising contacting the fabrics / dishware to be washed with a solution containing a detergent composition of the present invention. The fabrics may include any fabric washable under normal consumer use conditions. The dishware may include any dishware made of ceramic, cutlery, china, plastics such as melamine, metal, porcelain, glass, and acrylic. The solution preferably has a pH of about 5.5 to about 11.5. The compositions may be utilized in concentrations of about 100 ppm (preferably 500 ppm) to about 15,000 ppm in solution. The water temperature typically ranges from about 5° C. to about 95° C., for example, 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 about 1:1 to about 30:1.

[0492] The enzymes of the detergent compositions of the present invention may be stabilized with conventional stabilizers and protease inhibitors, e.g., polyols such as propylene glycol or glycerol, sugars or sugar alcohols, different salts such as NaCl; KCl; lactic acid, formic acid, boric acid, or boric acid derivatives, e.g., aromatic borate esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid, or peptide aldehydes such as di-, tri-, or tetrapeptide aldehydes or aldehyde analogs (of the form B1-B0-R, where R is H, CH3, CX3, CHX2, or CH2X (X = halogen), and B0 is a single amino acid residue (preferably with an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (preferably 1, 2, or 3), optionally containing an N-terminal protecting group or stabilized using a protein-type protease inhibitor such as RASI, BASI, WASI (rice, barley, and wheat bifunctional α-amylase / subtilisin inhibitor), or CI2 or SSI, as described in WO 92 / 19709, WO 92 / 19708, and U.S. Pat. No. 6,472,364. This composition may be formulated, for example, as described in WO 92 / 19709, WO 92 / 19708, and U.S. Pat. No. 6,472,364. In some embodiments, the enzymes utilized herein are stabilized by the presence of a water-soluble source of zinc(II), calcium(II), and / or magnesium(II) ions in the final composition that provides such ions to the enzyme, as well as other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and oxovanadium(IV)).

[0493] The detergent compositions provided herein are typically formulated to provide a wash water pH of about 5.0 to about 12.5, e.g., about 5.0 to about 11.5, or about 6.0 to about 10.5, during use in aqueous cleaning operations. In some embodiments, granular or liquid laundry products are formulated to provide a pH of about 6 to about 8. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, and the like, and are well known to those skilled in the art.

[0494] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0495] The present invention relates to novel protease variants that exhibit increased stability and / or improved cleaning performance (exemplified by improved removal of proteinaceous stains) in liquid detergent and bar soap compositions compared to the parent proteases.

[0496] material textile products Standard textiles or swatches were obtained from the Center for Test materials BV (PO Box 120, 3133 KT Vlaardingen, The Netherlands), Warwick Equest Ltd (Consett, DH8 6BN, United Kingdom), and EMPA (Ueberlandstrasse 129, 8600 Duebendorf, Switzerland).

[0497] Detergent Format The following detergents used may be made up as described herein: The protease to be tested on the swatches may then be added to the model detergent base for testing.

[0498] [Table 2]

[0499] [Table 3]

[0500] [Table 4]

[0501] [Table 5]

[0502] [Table 6]

[0503] [Table 7]

[0504] method Polypeptide preparation and purification Mutation and introduction of the expression cassette into Bacillus subtilis were performed by standard methods known in the art. All DNA manipulations were performed by PCR using standard methods known to those skilled in the art (e.g., as described in Sambrook et al., 2001).

[0505] Recombinant B. subtilis constructs encoding protease polypeptides were inoculated into complex medium (TBgly) and grown at 37°C for 24 hours under antibiotic selection. Shake flasks containing rich medium (PS-1: 100 g / L sucrose (Danisco catalog no. 109:0429), 40 g / L crust soy (soybean flour), 10 g / L NaHPO 12H0 (Merck catalog no. 106579), and 0.1 ml / L Dowfax 63N10 (Dow)) were inoculated with overnight cultures at a 1:100 ratio. Shake flask cultivation was carried out at 30°C for 4 days with shaking at 270 rpm.

[0506] Purification of the culture supernatant was performed as follows: the culture broth was centrifuged at 26,000 × g for 20 minutes, and the supernatant was carefully decanted from the sediment. The supernatant was filtered through a Nalgene 0.2 μm filtration unit to remove residual 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 with 20 mM Tris / HCl, 1 mM CaCl2, pH 8.0. After washing the column with the equilibration buffer, the column was step-eluted with 20 mM CH3COOH / NaOH, 1 mM CaCl2, pH 4.5. Fractions from the column were analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9, and peak fractions were pooled. The pH of the pool from the MEP Hypercel column is adjusted to pH 6 with 20% (v / v) CH3COOH or 3 M Tris base, and the pH-adjusted pool is diluted with deionized water to the same conductivity as 20 mM MES / NaOH, 2 mM CaCl2, pH 6.0. The diluted pool is applied to an SP-Sepharose® Fast Flow column (GE Healthcare) equilibrated with 20 mM MES / NaOH, 2 mM CaCl2, pH 6.0. After washing the column with equilibration buffer, protease variants are eluted with a linear NaCl gradient (0 to 0.5 M) in the same buffer over 5 column volumes. Fractions from the column are analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9, and active fractions are analyzed by SDS-PAGE. Fractions in which only one band is observed on a Coomassie-stained SDS-PAGE gel are pooled as a purified preparation and used for further experiments.

[0507] Protease Activity Assay I The proteolytic activity of the variants of the present invention can be determined by a method utilizing the Suc-AAPF-PNA substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide, a blocked peptide that can be cleaved by endoproteases. After proteolytic cleavage, yellow free pNA molecules are liberated and can be measured by visible spectrometry at a wavelength of 405 nm. The Suc-AAPF-PNA substrate can be purchased from Bachem.

[0508] The sample containing the variant to be analyzed is diluted with the remaining activity buffer (100 mM Tris, pH 8.6). The assay is performed by transferring 30 μl of 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 solution is mixed at room temperature, and the absorbance at 405 nm is measured over time, for example, every 20 seconds for 5 minutes. The slope of the time-dependent absorbance curve (absorbance / min) is directly proportional to the proteolytic activity.

[0509] Protease Activity Assay II The proteolytic activity of detergent compositions containing the variants of the present invention can be determined by a method using N,N-dimethylcasein (DMC) as a substrate. Hydrolysis of peptide bonds produces carboxylic acids and primary amines. The amines then react with 2,4,6-trinitrobenzene-sulfonic acid (TNBS, Sigma) under alkaline conditions to form colored complexes, which can be measured at 405 nm.

[0510] A detergent sample containing the variant of the invention is dissolved in 0.08 M sodium sulfite buffer and stirred for 10 minutes, after which the sample is filtered (Whatman filter number 54 or similar). Sample dilution is performed using buffer (0.05 M boric acid + 0.16 M sodium sulfite + 0.15 M potassium chloride + 0.0225% (w / v) Brij® L23, pH 9.00). Reagents such as 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 are employed in performing the assay using a Konelab 30 Analyzer (ThermoFisher Scientific) according to the assay parameters outlined in Table 19. Activity values ​​can then be calculated based on the standard curve.

[0511] Automated Mechanical Stress Assay (AMSA) To evaluate washing performance in laundry, washing experiments are performed using an automated mechanical stress assay (AMSA). The AMSA allows for testing the washing performance of large amounts of small-volume enzyme-detergent solutions. The AMSA plate has several slots for test solutions and a lid that firmly presses the laundry sample (or melamine tile in the case of dishwashing detergent) into all slot openings. During the wash time, the plate, test solution, textile (or tile in the case of dishwashing), and lid are vigorously shaken to bring the test solution into contact with the soiled test sample and apply mechanical stress in a regular, periodic vibrational manner. For further explanation, see WO 2002 / 42740 (especially the paragraph "Special Method Embodiments" on pages 23-24).

[0512] The cleaning performance is measured as the whiteness of the color of the washed textile. Whiteness can be expressed as the intensity of light reflected from a textile sample when illuminated with white light. If the textile is soiled, the intensity of the reflected light is lower compared to the light reflected from a clean textile. Therefore, the intensity of the reflected light can be used to measure the cleaning performance of a protease of interest.

[0513] Color measurements are performed using a professional flatbed scanner (EPSON EXPRESSION 10000XL, Atea A / S, Lautrupvang 6, 2750 Ballerup, Denmark) used to capture images of the washed textile samples. A specially designed software application (Novozymes Color Vector Analyzer) is used to extract light intensity values ​​from the scanned images. This program takes values ​​from the image and converts them to values ​​for red, green, and blue (RGB). Intensity values ​​(Int) can be calculated by adding the RGB values ​​together with a vector and then subtracting the length of the resulting vector.

number

[0514] Terg-O-tometer (TOM) washing assay The Terg-o-tometer (TOM) is a mid-scale cleaning assay that can be adapted to test up to 16 different conditions simultaneously. Briefly, it consists of 16 x 2 L metal beakers, each fitted with an agitator that rotates back and forth at a controlled speed to simulate the agitation experienced in commercial top-loading washers. The beakers are partially submerged in a thermostatic water bath, which allows for temperature control. Each beaker is filled with 1 L of detergent solution and contains test swatches, ballast, and enzymes to the required levels. After the timed cleaning period, the swatches are quickly removed from the beakers and rinsed thoroughly with tap water.

[0515] The swatches are then spread flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after washing. Evaluation of the light reflectance of the swatches is carried out using a Macbeth Color Eye 7000 reflectance spectrophotometer with a large aperture. Measurements are carried out without UV in the incident light, extracting the reflectance (REM) at 460 nm. Measurements are carried out on unwashed swatches and on washed swatches. The test swatch to be measured is placed on top of another swatch of the same type and color.

[0516] The effect of the protease on each swatch is calculated by subtracting the reflectance value of the swatch washed without enzyme (blank) from the swatch washed with enzyme. The performance of the novel protease (e.g., protease variant) is calculated as Relative Performance (RP):

number

[0517] Full-scale washing (FSW) test A full-scale washing machine (Panasonic XQB65-Q680U, top loader) is used to evaluate protease performance on a large scale. Detergent, swatches, ballast, water, and enzymes are combined and washed at a specified temperature, in this case for 15 minutes, followed by rinsing. The levels of detergent, water, enzyme, and ballast used may be adjusted based on various factors, such as local practices. The swatches are then removed from the wash solution and allowed to dry flat at room temperature overnight. Evaluation of the FSW swatches is performed in a similar manner to the TOM swatches.

[0518] Mini Terg-o-tometer (mini-TOM) cleaning assay and determination of relative stain removal rates The Mini Terg-o-tometer (mini-TOM) is a mid-scale cleaning assay that can be adapted to simultaneously test up to 16 different conditions. Briefly, it consists of 16 x 0.2 L metal beakers, each equipped with an agitator that rotates back and forth at a controlled speed to simulate the agitation experienced in commercial top-loading washers. The beakers are partially submerged in a temperature-controlled water bath. Each beaker is filled with 0.15 L of detergent solution, and test swatches and enzymes are added to the required level. At the desired time point, the swatches are quickly removed from the beakers and rinsed thoroughly with ice water to stop soil hydrolysis.

[0519] The swatches are then spread flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after washing. Evaluation of the light reflectance of the swatches is carried out using a Macbeth Color Eye 7000 reflectance spectrophotometer with a large aperture. Measurements are carried out without UV in the incident light, extracting the reflectance (REM) at 460 nm. Measurements are carried out on unwashed swatches and on washed swatches. The test swatch to be measured is placed on top of another swatch of the same type and color.

[0520] The effect of the protease on each swatch is calculated by subtracting the reflectance value of the swatch washed without enzyme (blank) from the swatch washed with enzyme.

[0521] The stain removal rate (SRR) of a novel protease (e.g., protease variant) was calculated as the relative stain removal rate (R SRR ) can be compared to the stain removal rate of a reference protease.

number

[0522] Example 1: FSW Test in Asia Pacific Detergent at Low Temperature (15°C) The cleaning performance of SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 9 was evaluated under the following conditions (Table 7).

[0523] [Table 8]

[0524] The proteases were included in wash tests run in an Asia Pacific top loader washer using model detergents and various stain types. At least six different swatches representing at least five different stain categories (e.g., blood, blood milk ink, chocolate, grass, and egg) showed improved wash performance with SEQ ID NO:9 compared to SEQ ID NO:1 and SEQ ID NO:8 at low wash temperatures (15°C; see Table 8).

[0525] [Table 9]

[0526] Example 2: FSW Test in Asia Pacific Detergent at Standard Temperature (25°C) The cleaning performance of SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 9 was evaluated under the following conditions (Table 19).

[0527] [Table 10]

[0528] The proteases were included in wash tests run in an Asia Pacific top loader washer using model detergents and various stain types. At least eight different swatches representing at least four different stain categories (e.g., blood, milk ink, chocolate, grass, and egg) showed improved wash performance with SEQ ID NO:9 compared to SEQ ID NO:1 and SEQ ID NO:8 at standard wash temperature (25°C; see Table 10).

[0529] [Table 11]

[0530] Example 3: TOM Cleaning Performance Evaluation After Storage in Laundry Bar Matrices The cleaning performance of SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 9 was evaluated under the following conditions (Table 11).

[0531] [Table 12]

[0532] The protease formulations were incorporated into a basic laundry bar matrix and incubated at 37°C. After four weeks, the bars were evaluated for performance in a terg-o-tometer cleaning assay. In this assay, soap samples were grated and washed with seven different stain monitors representing at least four different stain categories (e.g., blood, milk ink, chocolate, grass, and egg). SEQ ID NO:9 showed improved cleaning performance compared to SEQ ID NO:1 and SEQ ID NO:8 for six of the seven stains tested (see Table 12).

[0533] [Table 13]

[0534] Example 4: TOM cleaning performance evaluation after storage in low pH European detergents The cleaning performance of SEQ ID NO: 8 and SEQ ID NO: 9 was evaluated under the following conditions (Table 13).

[0535] [Table 14]

[0536] SEQ ID NO:8 and SEQ ID NO:9 were evaluated in a terg-o-tometer cleaning assay after storage for 24 hours at 25°C in a low pH European detergent. A total of seven swatches representing at least four different stain categories (e.g., blood, milk ink, chocolate, grass, and egg) were used to evaluate cleaning performance. SEQ ID NO:9 showed improved cleaning performance compared to SEQ ID NO:8 across all stains tested with SEQ ID NO:9 (see Table 14).

[0537] [Table 15]

[0538] Example 5: TOM cleaning performance evaluation in high pH European liquid detergents The cleaning performance of SEQ ID NO: 8 and SEQ ID NO: 9 was evaluated under the following conditions (Table 15).

[0539] [Table 16]

[0540] The proteases were evaluated in a terg-o-tometer cleaning assay using a high pH European detergent. The results showed improved cleaning performance on selected stains with SEQ ID NO: 9 compared to SEQ ID NO: 8 (see Table 16).

[0541] [Table 17]

[0542] Example 6: AMSA Evaluation in Model European Liquid Detergents The cleaning performance of SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 9 was evaluated under the following conditions (Table 17).

[0543] [Table 18]

[0544] SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 9 were tested using the AMSA cleaning assay and the results expressed in terms of relative performance to SEQ ID NO: 1. The results showed improved cleaning performance for SEQ ID NO: 9 across both stains tested (see Table 18).

[0545] [Table 19]

[0546] Example 7: Storage Stability in High Moisture Liquid Detergent The storage stability of SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 19).

[0547] [Table 20]

[0548] SEQ ID NO:8 and SEQ ID NO:9 were incubated in high-moisture liquid model detergents at 30 or 37°C for 4 to 8 weeks and then analyzed for protease activity. Residual protease activity was determined relative to the protease activity present in the original, unincubated sample stored at -18°C and expressed as a %. The results show that SEQ ID NO:9 has improved storage stability in high-moisture liquid detergents compared to SEQ ID NO:8 (see Table 20).

[0549] [Table 21]

[0550] Example 8: Storage Stability and Mildness to Companion Enzymes in Liquid Detergents The storage stability and mildness of SEQ ID NO:8 and SEQ ID NO:9 were evaluated in a commercial liquid detergent (BlueMoon, China) under the following conditions (Table 21).

[0551] [Table 22]

[0552] Commercially available α-amylase (Amplify Prime 100L, Novozymes A / S) was incubated with either SEQ ID NO: 8 or SEQ ID NO: 9 at 37°C for 2-4 weeks, after which samples were analyzed for protease and α-amylase activity.

[0553] Residual protease activity was determined relative to the protease activity present in the original, unincubated sample stored at -18°C and expressed as a %. The results show that SEQ ID NO:9 has improved storage stability in liquid detergent compared to SEQ ID NO:8 (see Table 22).

[0554] [Table 23]

[0555] Residual α-amylase activity was determined relative to the amylase activity present in the original, unincubated sample stored at -18°C and expressed as a %. The results show that α-amylase has improved residual activity in the presence of SEQ ID NO:9 compared to SEQ ID NO:8 (see Table 23). Thus, SEQ ID NO:9 has improved mildness towards companion enzymes (particularly α-amylase) compared to SEQ ID NO:8.

[0556] [Table 24]

[0557] Example 9: Soil removal rate in liquid detergent The difference in stain removal rate between SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 9 was evaluated under the following conditions (Table 24).

[0558] [Table 25]

[0559] The stain removal rate of SEQ ID NO:9 is improved compared to SEQ ID NO:8 and SEQ ID NO:1 (see Table 25). SEQ ID NO:9 is relatively faster in soil removal compared to SEQ ID NO:8 and SEQ ID NO:1.

[0560] [Table 26]

[0561] Example 10: AMSA Evaluation in Liquid Detergents The cleaning performance of variants of SEQ ID NO:3 and SEQ ID NO:5 was evaluated under the conditions described in Table 26. Results are expressed in terms of relative performance compared to the parent protease.

[0562] As can be seen from Table 27, SEQ ID NO: 10 shows superior cleaning performance compared to SEQ ID NO:3.

[0563] As can be seen from Table 28, SEQ ID NO:14 shows superior cleaning performance compared to SEQ ID NO:13 and SEQ ID NO:5.

[0564] [Table 27]

[0565] [Table 28]

[0566] [Table 29]

[0567] Example 11: Storage stability in liquid detergent Purified protease samples were diluted with 0.01% Triton® X-100 to the appropriate concentration based on 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 variants of SEQ ID NO:5). 30 μl of this protease dilution was mixed with 270 μl of concentrated Model O detergent (pH 8 or pH 10) in a microtiter plate well using a magnetic bar. After mixing and sealing with a plate seal, the detergent plate was incubated at 45°C or 55°C in a Biosan PST-100HL thermomixer. All samples were tested at duplicate or triplicate concentrations.

[0568] After various incubation times (e.g., 0, 1, 4, 24, 48, 96, 192, and 264 hours), residual protease activity was measured. 20 μl of sample from the 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), mixing, and then the absorbance at 405 nm was read every 10 seconds for 5 minutes using a BioTek Synergy H1 plate reader. Activity was determined from the slope of the initial absorbance increase by linear regression.

[0569] The decrease in activity during incubation with detergent appears to be exponential. The half-life (T1 / 2) was found from linear regression of the logarithm of activity against incubation time. The half-life improvement (T1 / 2IF) was calculated as the ratio of the half-life of the protease variant to that of the parent protease.

[0570] As can be seen in Table 29, SEQ ID NO:9 has improved storage stability compared to SEQ ID NO:8 and SEQ ID NO:1.

[0571] As can be seen in Table 30, SEQ ID NO: 12 has improved storage stability compared to SEQ ID NO: 11 and SEQ ID NO: 3. In addition, SEQ ID NO: 10 has improved storage stability compared to SEQ ID NO: 3.

[0572] As can be seen in Table 31, SEQ ID NO:14 has improved storage stability compared to SEQ ID NO:13 and SEQ ID NO:5.

[0573] [Table 30]

[0574] [Table 31]

[0575] [Table 32]

[0576] The present invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, since these embodiments are intended to be illustrative of some aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of 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 the case of conflicts, the present disclosure, including definitions, will control.

[0577] The present invention is further defined by the following numbered paragraphs: 1. A variant of a parent protease, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, e.g., at least 4, at least 5, at least 6, at least 7, at least 8, or 9 positions, wherein the numbering of positions is based on the numbering of SEQ ID NO:1, said variant having at least 60% sequence identity to the parent protease, e.g., 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%, and wherein said variant has protease activity.

[0578] 2. A variant according to paragraph 1, comprising a substitution of the amino acid residue at the position corresponding to position 95 of SEQ ID NO: 1 with Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably with Asp.

[0579] 3. A variant according to paragraph 1 or 2, comprising a substitution of the amino acid residue at the position corresponding to position 209 of SEQ ID NO: 1 with Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Lys.

[0580] 4. A variant according to any one of paragraphs 1 to 3, comprising a substitution of the amino acid residue at the position corresponding to position 9 of SEQ ID NO: 1 with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Trp, Tyr, or Val, preferably with Glu.

[0581] 5. A variant according to any one of paragraphs 1 to 4, comprising a substitution of the amino acid residue at the position corresponding to position 42 of SEQ ID NO: 1 with Ala, Arg, Cys, Gln, Glu, His, Ile, Leu, Met, Phe, Pro, Ser, Trp, Tyr, or Val, preferably with Arg.

[0582] 6. A variant according to any one of paragraphs 1 to 5, comprising a substitution of the amino acid residue at the position corresponding to position 74 of SEQ ID NO: 1 with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Asp.

[0583] 7. The variant according to any one of paragraphs 1 to 6, comprising a substitution of the amino acid residue at the position corresponding to position 199 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Ile.

[0584] 8. A variant according to any one of paragraphs 1 to 7, comprising a substitution of the amino acid residue at the position corresponding to position 200 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, or Val, preferably with Leu.

[0585] 9. The variant according to any one of paragraphs 1 to 8, comprising a substitution of the amino acid residue at the position corresponding to position 203 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, preferably with Trp.

[0586] 10. A variant according to any one of paragraphs 1 to 9, comprising a substitution of the amino acid residue at the position corresponding to position 253 of SEQ ID NO: 1 with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Trp, Tyr, or Val, preferably with Asp.

[0587] 11. A variant according to any one of paragraphs 1 to 10, comprising a substitution of the amino acid residue at the position corresponding to position 255 of SEQ ID NO: 1 with Ala, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Thr, Trp, Tyr, or Val, preferably with Trp.

[0588] 12. A variant according to any one of paragraphs 1 to 11, comprising a substitution of the amino acid residue at the position corresponding to position 256 of SEQ ID NO: 1 with Ala, Arg, Asn, Cys, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, preferably with Glu.

[0589] 13. The variant of any one of paragraphs 1 to 12, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising at least three substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0590] 14. The variant of any one of paragraphs 1 to 13, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising at least four substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0591] 15. The variant of any one of paragraphs 1 to 14, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising at least five substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0592] 16. The variant of any one of paragraphs 1 to 15, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising at least six substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0593] 17. The variant of any one of paragraphs 1 to 16, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising at least seven substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0594] 18. The variant of any one of paragraphs 1 to 17, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising at least eight substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0595] 19. The variant of any one of paragraphs 1 to 18, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising nine substitutions at positions corresponding to positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, respectively.

[0596] 20. The variant of any one of paragraphs 1 to 19, wherein the total number of substitutions compared to the parent is 5 to 20 substitutions, such as 5 to 15 or 5 to 10 substitutions, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions.

[0597] 21. The variant of any one of paragraphs 1 to 20, wherein the total number of substitutions compared to the parent is between 5 and 11 substitutions, such as 5, 6, 7, 8, 9, 10, or 11 substitutions.

[0598] 21a. The variant of any one of paragraphs 1 to 21, wherein the parent protease is an identical protease except that it does not have substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and does not have substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0599] 22. The variant of any one of paragraphs 1 to 21, wherein the parent protease has at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1, e.g., 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%.

[0600] 23. The variant of any one of paragraphs 1 to 21, wherein the parent protease has at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 2, e.g., 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%.

[0601] 24. The variant of any one of paragraphs 1 to 21, wherein the parent protease has at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 3, e.g., 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%.

[0602] 25. The variant of any one of paragraphs 1 to 21, wherein the parent protease has at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 4, e.g., 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%.

[0603] 26. The variant of any one of paragraphs 1 to 21, wherein the parent protease has at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 5, e.g., 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%.

[0604] 27. The variant of any one of paragraphs 1 to 21, wherein the parent protease has at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 6, e.g., 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%.

[0605] 28. The variant of any one of paragraphs 1 to 21, wherein the parent protease has at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 7, e.g., 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%.

[0606] 29. The variant of any one of paragraphs 1 to 28, wherein the variant comprises substitutions corresponding to G95D and A209K in SEQ ID NO: 1, and further comprises at least three substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E in SEQ ID NO: 1, such as further comprising at least four, at least five, at least six, at least seven, at least eight, or nine substitutions.

[0607] 30. The variant of any one of paragraphs 1 to 29, comprising substitutions corresponding to G95D and A209K in SEQ ID NO:1, and further comprising substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E in SEQ ID NO:1.

[0608] 30. The variant of any one of paragraphs 1 to 29, 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.

[0609] 31. The variant of any one of paragraphs 1 to 30, 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.

[0610] 32. The variant of any one of paragraphs 1 to 31, which has improved storage stability and / or improved cleaning performance compared to the parent protease.

[0611] 32a. The variant of any one of paragraphs 1 to 32, having improved mildness compared to the parent protease.

[0612] 32b. A variant of any one of paragraphs 1 to 32a, having improved stain removal rate compared to the parent protease.

[0613] 33. A fusion polypeptide comprising a variant according to any one of paragraphs 1 to 32b and a second polypeptide.

[0614] 34. The variant or fusion polypeptide of any one of paragraphs 1 to 33, which is isolated.

[0615] 35. The variant or fusion polypeptide of any one of paragraphs 1 to 34, which is purified.

[0616] 36. A granule comprising: (a) a core comprising a variant or fusion polypeptide according to any one of paragraphs 1 to 35; and optionally (b) a coating consisting of one or more layers surrounding the core.

[0617] 37. A granule 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 described in any one of paragraphs 1 to 35.

[0618] 38. A liquid composition comprising a variant or fusion polypeptide according to any one of paragraphs 1 to 35, and an enzyme stabilizer, for example a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative, for example an aromatic borate ester, or a phenylboronic acid derivative, such as 4-formylphenylboronic acid.

[0619] 39. The liquid composition according to paragraph 38, further comprising a filler or carrier material.

[0620] 40. The liquid composition according to paragraph 38 or 39, further comprising a preservative.

[0621] 41. A composition comprising a variant or fusion polypeptide according to any one of paragraphs 1 to 35, a granule according to paragraph 36 or 37, or a liquid composition according to any one of paragraphs 38 to 40.

[0622] 42. A polynucleotide encoding a variant or fusion polypeptide according to any one of paragraphs 1 to 35.

[0623] 43. The polynucleotide of paragraph 42, which is isolated.

[0624] 44. The polynucleotide of paragraph 42 or 43, which is purified.

[0625] 45. A nucleic acid construct or expression vector comprising a polynucleotide according to any one of paragraphs 42 to 44.

[0626] 46. ​​A recombinant host cell transformed with a polynucleotide according to any one of paragraphs 42 to 44.

[0627] 47. A recombinant host cell according to paragraph 46, comprising at least two copies of the polynucleotide according to any one of paragraphs 42 to 44, such as three, four, or five or more copies.

[0628] 48. Yeast recombinant host cells, for example, cells of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, for example, Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, 48. The recombinant host cell of paragraph 46 or 47, which is a cell of Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.

[0629] 49. Filamentous fungal recombinant host cells, such as Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastiq. The cell of the genus Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma, specifically, Aspergillus awamori. awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirinaaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium meldarium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulantumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum 48. The recombinant host cell of paragraph 46 or 47, which is a Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cell.

[0630] 50. A prokaryotic recombinant host cell, for example, a Gram-positive cell selected from the group consisting of cells of the genera Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces, or a cell of the genera Campylobacter, Escherichia coli (E.and Gram-negative bacteria selected from the group consisting of cells of the genera Bacillus coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis uberis), and Streptococcus equi subsp.48. The recombinant host cell of paragraph 46 or 47, which is a cell of Streptomyces zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans.

[0631] 51. The recombinant host cell of any one of paragraphs 46 to 50, which is isolated.

[0632] 52. The recombinant host cell of any one of paragraphs 46 to 50, which is purified.

[0633] 53. A method for producing a protease variant or fusion polypeptide, comprising: (a) culturing a host cell according to any one of paragraphs 46 to 52 under conditions suitable for expression of said variant; and (b) recovering said variant.

[0634] 54. A whole broth preparation or cell culture composition comprising a variant or fusion polypeptide according to any one of paragraphs 1 to 35.

[0635] 55. A detergent composition comprising a variant of the fusion polypeptide according to any one of paragraphs 1 to 35.

[0636] 56. The detergent composition according to paragraph 55 in the form of a bar, a homogeneous tablet, a tablet with two or more layers, a pouch with one or more compartments, a regular or compacted powder, granules, a paste, a gel, or a regular, compacted or concentrated liquid.

[0637] 57. The detergent composition according to paragraph 55 in the form of a liquid detergent, a powder detergent, or a laundry bar, preferably a liquid detergent or a laundry bar.

[0638] 58. A method of cleaning an object, comprising contacting said object with the detergent composition of any one of paragraphs 55 to 57 under conditions suitable for cleaning said object, preferably said object is a fabric, dishware, or a hard surface; most preferably said object is a fabric.

[0639] 59. Use of a variant according to any one of paragraphs 1 to 35 or a detergent composition according to any one of paragraphs 55 to 57 in a cleaning process, preferably in laundry or hard surface washing, such as automatic dishwashing (ADW).

Claims

1. A variant of a parent protease, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, e.g., at least 4, at least 5, at least 6, at least 7, at least 8, or 9 positions, wherein the numbering of the positions is based on the numbering of SEQ ID NO:1, and wherein the variant has a TM-score of at least 0.80 compared to the three-dimensional structure of the parent protease, e.g., 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 the variant has protease activity.

2. 1. A variant of a parent protease, comprising substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further comprising substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, e.g., at least 4, at least 5, at least 6, at least 7, at least 8, or 9 positions, wherein the numbering of positions is based on the numbering of SEQ ID NO:1, said variant having at least 60% sequence identity to said parent protease, e.g., 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%, and wherein said variant has protease activity.

3. 3. The variant of claim 1 or 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, the parent protease is SEQ ID NO:

1.

4. 4. The variant of any one of claims 1 to 3, wherein the variant comprises substitutions corresponding to G95D and A209K in SEQ ID NO: 1, and further comprises at least three substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E in SEQ ID NO: 1, such as at least four, at least five, at least six, at least seven, at least eight, or nine substitutions.

5. 5. The variant of any one of claims 1 to 4, comprising substitutions corresponding to G95D and A209K in SEQ ID NO:1, and further comprising substitutions corresponding to S9E, N43R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E in SEQ ID NO:

1.

6. The variant of any one of claims 1 to 5, further comprising a substitution at one or more positions selected from the group consisting of positions 60, 97, 99, 116, and 246 of SEQ ID NO:

1.

7. 6. The variant of claim 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.

8. The variant of any one of claims 1 to 7, wherein the variant exhibits improved cleaning performance and / or improved storage stability compared to the parent protease.

9. A polynucleotide encoding the protease variant according to any one of claims 1 to 8.

10. A nucleic acid construct or expression vector comprising the polynucleotide of claim 9.

11. A recombinant host cell comprising in its genome the nucleic acid construct or expression vector of claim 10.

12. 9. A method of obtaining a variant according to any one of claims 1 to 8, comprising: (a) introducing substitutions into a parent protease at positions corresponding to positions 95 and 209 of SEQ ID NO:1, and further introducing substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1, for example, introducing substitutions at at least four, at least five, at least six, at least seven, at least eight, or at least nine positions, wherein the numbering of the positions is based on the numbering of SEQ ID NO:1; and (b) recovering the variant.

13. A method for producing a variant of any one of claims 1 to 8, comprising: (a) culturing a recombinant host cell of claim 11 under conditions suitable for expression of the variant; and (b) recovering the variant.

14. 9. A detergent composition comprising a variant according to any one of claims 1 to 8, preferably in the form of a bar, a homogeneous tablet, a tablet with two or more layers, a pouch with one or more compartments, a regular or compacted powder, granules, a paste, a gel, or a regular, compacted or concentrated liquid.

15. 15. A method of cleaning an object, comprising contacting said object with the detergent composition of claim 14 under conditions suitable for cleaning said object, preferably said object is a fabric, dishware, or hard surface; most preferably said object is a fabric.

16. Use of a variant according to any one of claims 1 to 8 or a detergent composition according to claim 14 in a cleaning process, preferably in laundry or hard surface cleaning such as automatic dishwashing (ADW).

Images