Protease variants with improved solubility

Abstract

The present invention relates to protease variants, polynucleotides encoding said variants, nucleic acid constructs and expression vectors comprising said polynucleotides, host cells expressing said variants, methods for obtaining said variants, detergent compositions comprising said variants, and uses of said variants or said detergent compositions.

Description

[Technical Field]

[0001] Sequence listing reference This application includes a computer-readable sequence listing incorporated herein by reference.

[0002] The present invention relates to a protease variant, a polynucleotide encoding the variant, a nucleic acid construct, an expression vector comprising the polynucleotide, a host cell expressing the variant, a method for obtaining the variant, a detergent composition comprising the variant, and the use of the variant or the detergent composition. [Background technology]

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

[0004] For example, in commercially used proteases for laundry and dishwashing detergents, there is an increasing number of protein-modified variants of naturally occurring wild-type proteases. Furthermore, other protease variants having modifications to the parent protease that result in improvements such as better washing performance, thermal stability, storage stability, or catalytic activity have been described in the art.

[0005] However, further improvements in proteases are facilitated by various factors. For example, washing conditions such as temperature and pH tend to change over time and differ across countries and regions around the world, and many contaminants remain difficult to completely remove under conventional washing conditions.

[0006] Another challenge with proteases is their solubility. Since proteases with low solubility are more likely to crystallize during fermentation and downstream processing, the solubility of proteases is a crucial factor when producing these enzymes. Proteases with high solubility, Processing at higher concentrations makes the protease purification process cheaper, faster, and more sustainable.

[0007] This invention addresses this problem by providing a protease with improved solubility. [Overview of the project] [Means for solving the problem]

[0008] The present invention provides a protease variant having improved solubility. The protease variant of the present invention comprises a positively charged amino acid or polar amino acid at the position corresponding to position 215 of SEQ ID NO: 1.

[0009] Therefore, in the first embodiment, the present invention relates to a protease variant of a parent protease, wherein the variant has at least 80% but less than 100% sequence identity with SEQ ID NO: 1. The variant includes a first substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; The variant comprises at least 3 further modifications, preferably substitutions, selected from the group consisting of X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104I (e.g., V104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., L262E); The variant has protease activity; and The position numbers are based on the numbering of SEQ ID NO: 2.

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

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

[0012] In a fourth aspect, the present invention relates to a host cell expressing the protease variant according to the first aspect.

[0013] In a fifth aspect, the present invention is a method for obtaining a protease variant according to any of claims 1 to 15, comprising (a) Introducing a first substitution to the parent protease selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; and X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104 Introducing at least three further modifications, preferably substitutions (the variants having protease activity), selected from the group consisting of I (e.g., V104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., L262E); and (b) Recover the variant Regarding methods including

[0014] In a sixth embodiment, the present invention relates to a detergent composition comprising a protease variant according to the first embodiment.

[0015] In a seventh embodiment, the present invention relates to the use of a protease variant according to the first embodiment or a detergent composition according to the sixth embodiment in a cleaning process, preferably hard surface cleaning such as laundry or automatic dishwashing (ADW). [Brief explanation of the drawing]

[0016] [Figure 1] The alignment between sequence number 1 and sequence number 2 is shown based on Table 1 of the International Publication No. 1989 / 06279, and the position number corresponding to the position of sequence number 2 can be easily determined. [Modes for carrying out the invention]

[0017] definition Protease: The term "protease" refers to an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC3.4 enzyme group (including each of its 13 subclasses (http: / / en.wikipedia.org / wiki / Category:EC_3.4)). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, and includes Supplements 1-5 published in Eur.J.Biochem.1994,223,1-5; Eur.J.Biochem.1995,232,1-6; Eur.J.Biochem.1996,237,1-5; Eur.J.Biochem.1997,250,1-6; and Eur.J.Biochem.1999,264,610-650. The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Science 6 (1997) 501-523. Serine proteases, or serine peptidases, are a subgroup of proteases characterized by having serine in the active site where they form a covalent adduct with a substrate. Furthermore, subtilases (and serine proteases) are characterized by having two active site amino acid residues other than serine, namely histidine and aspartic acid residues. Subtilases can be classified into six subdivisions: the subtilisin family, the thermitase family, the proteinase K family, the lanthibiotic peptidase family, the kexin family, and the pyrrolicin family. The term "protease activity" means proteolytic activity (EC 3.4). The protease variant of the present invention is endopeptidase (EC3.4.21). For the purposes of the present invention, the protease activity is determined according to the protease activity assay described in the examples below.

[0018] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks the intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that has been processed through a series of steps, including splicing, before appearing as a mature, spliced ​​mRNA.

[0019] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame that begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. A coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0020] Regulatory Sequence: The term "regulatory sequence" means a nucleic acid sequence required for the expression of the polynucleotide encoding the variant of the present invention. Each regulatory sequence may be native (i.e., derived from the same gene) or exogenous (i.e., derived from a different gene) to the polynucleotide encoding the variant, or may be native or exogenous to each other. Examples of such regulatory sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcriptional terminators. At a minimum, regulatory sequences may include promoters and transcriptional stop signals and translational stop signals. The regulatory sequences may be provided with linkers for introducing specific restriction sites that facilitate linking between the coding region of the polynucleotide encoding the variant and the regulatory sequence.

[0021] Expression: The term "expression" includes all processes involved in the production of a variant, such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0022] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule containing polynucleotides encoding a variant, which is operably ligated to a regulatory sequence that results in its expression.

[0023] Fragment: The term "fragment" refers to a polypeptide having one or more (e.g., several) amino acids that are not present at the amino and / or carboxyl termini of a mature polypeptide; this fragment possesses protease activity.

[0024] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which one polypeptide is fused at the N-terminus or C-terminus of a variant of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and involve ligating coding sequences encoding polypeptides so that they are in-frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein techniques in which the fusion polypeptide is produced post-translationally (Cooper et al., 1993, EMBO J.12:2575-2583; Dawson et al., 1994, Science 266:776-779). A fusion polypeptide may further include a cleavage site between the two polypeptides. When the fusion protein is secreted, this site is cleaved, releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed below: 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., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0025] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc., by the nucleic acid construct or expression vector containing the polynucleotide of the present invention. The term "host cell" encompasses all offspring of a parent cell that are not identical to the parent cell due to mutations that occur during replication.

[0026] Hybrid polypeptide: The term "hybrid polypeptide" refers to a polypeptide that contains domains derived from two or more polypeptides, for example, a binding module from one polypeptide and a catalytic domain from another polypeptide. The domains may be fused at the N-terminus or C-terminus.

[0027] Improved Properties: The term "improved properties" refers to characteristics associated with a variant that are improved compared to the parent. Such improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, oxidation stability, pH activity, pH stability, polyester degradation activity, polyester specificity, protein degradation stability, solubility, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermal stability.

[0028] In one embodiment, variants of the present invention have improved solubility. In particular, variants of the present invention exhibit, for example, reduced protease crystal formation during fermentation and / or increased protease crystal solubility (or in other words, improved protease crystal resolubilization). Protease crystal formation and protease crystal solubility can be determined by the procedure described in Example 1 below. Protease crystal solubility can also be determined as the rate of protease crystal dissolution. Using this method, proteases are crystallized by increasing their concentration in an aqueous buffer (e.g., via a rotary concentrator) and increasing the salt concentration and adjusting the pH value until conditions suitable for crystallization are reached. After crystallization, protease crystal solubility can be determined by measuring the rate of crystal dissolution.

[0029] In one embodiment, the variant of the present invention has equivalent or improved solubility. Protease activity is determined according to the protease activity assay described in the examples below.

[0030] Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other designated material or component that has been separated from at least one other material or component that naturally associates as it would in nature, including, but not limited to, other proteins, nucleic acids, or cells. Examples of isolated polypeptides include, but are not limited to, culture broths containing secreted polypeptides.

[0031] Mature polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide after N-terminal processing (e.g., removal of the signal peptide).

[0032] Mutant: The term "mutant" refers to a polynucleotide that codes for a variant.

[0033] Nucleic acid construct: The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, modified to contain a nucleic acid segment in a manner not normally found in nature, or a synthetic compound containing one or more regulatory sequences.

[0034] Operablely linked: The term "operably linked" refers to a configuration in which a control sequence is positioned appropriately relative to the coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence.

[0035] Parent or Parent Protease: The terms "parent" or "parent protease" mean a protease that has been modified to produce the enzyme variant of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

[0036] Polymer: The term "polymer" means a chemical compound or mixture of compounds whose structure consists of multiple monomers (repeating units) linked by covalent chemical bonds. In the context of the present invention, the term polymer includes natural or synthetic polymers composed of a single type of repeating unit (i.e., homopolymer) or a mixture of different repeating units (i.e., copolymer or heteropolymer). According to the present invention, the term "oligomer," when used in reference to polymer, means a molecule containing 2 to about 20 monomers.

[0037] Purified: The term "purified" means a nucleic acid or polypeptide that is substantially free of other components when measured by analytical techniques well known in the art (for example, a purified polypeptide or nucleic acid may form separated bands in an electrophoretic gel, in a chromatographic eluate, and / or in a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide 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%, or about 99.8% or more pure (e.g., by weight percentage on a molar basis). In a related sense, a composition is concentrated if the concentration of molecules increases substantially after the application of a purification or concentration technique. The term "concentrated" refers to compounds, polypeptides, cells, nucleic acids, amino acids, or other specific materials or components present in the composition at a higher relative or absolute concentration than that of the starting composition.

[0038] Recombinant: When used in relation to cells, nucleic acids, proteins, or vectors, the term "recombinant" means that they have been modified from their natural state. For example, a recombinant cell expresses a gene not found in the cell's natural form (non-recombinant), or expresses a natural gene at a different level or under different conditions than those found in nature. A recombinant nucleic acid differs from its natural sequence by one or more nucleotides and / or is operably ligated to a heterologous sequence, such as a heterologous promoter in an expression vector. A recombinant protein may differ from its natural sequence by one or more amino acids and / or is fused to a heterologous sequence. A vector containing nucleic acids encoding a polypeptide is a recombinant vector. The term "recombinant" is synonymous with "genetically modified" and "gene transfer."

[0039] Sequence identity: The relationship between two amino acid sequences or two nucleotide sequences is described by a parameter called "sequence identity."

[0040] For the purposes of this invention, sequence homology 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-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The nobrief option must be specified on the command line for the Needle program to report the longest identity. The Needle output, labeled “longest identity”, is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment)

[0041] For the purposes of this invention, sequence identity between two polynucleotide sequences is determined as the “longest identity” output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, cited above), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, cited above) version 6.6.0 or later. The parameters used are a gap start penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The nobrief option must be specified on the command line for the Needle program to report the longest identity. The Needle output labeled “longest identity” is calculated as follows: (100 identical deoxyribonucleotides) / (length of alignment - total number of gaps in the alignment).

[0042] Variants and Protease Variants: The terms “variant” and “protease variant” refer to polypeptides having protease activity that includes substitutions, insertions, and / or deletions at one or more (e.g., several) positions compared to the parent. Substitution means replacing an amino acid at a certain position with a different amino acid; deletion means removing an amino acid at a certain position; and insertion means adding one amino acid adjacent to and immediately following an amino acid at a certain position. For the purposes of the present invention, protease activity is determined by the procedure described in the examples below.

[0043] Wild-type: The term "wild-type" in relation to amino acid sequences or nucleic acid sequences means that the amino acid sequence or nucleic acid sequence is a natural sequence or a sequence that exists in nature. As used herein, the term "naturally occurring" refers to something found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "not naturally occurring" refers to something not found in nature (e.g., recombinant nucleic acids and modifications of protein sequences or wild-type sequences produced in the laboratory).

[0044] Conventional methods for naming protease variants For the purposes of the present invention, the polypeptide of SEQ ID NO: 2 is used to determine the corresponding amino acid residue numbers in the variant of the present invention. The amino acid sequence of the variant of the present invention is aligned with SEQ ID NO: 2, and based on this alignment, the amino acid position numbers correspond to any amino acid residue in the variant of the present invention.

[0045] The numbering used herein with respect to Sequence IDs 1, 3, 4, 5, and 6 is based on the numbering of Sequence ID 2. Therefore, with respect to Sequence IDs 1, 3, 4, 5, and 6, the amino acid residues are numbered based on the corresponding amino acid residues in Sequence ID 2. Specifically, the numbering shows the alignment of five proteases, including the mature polypeptide of the subtilise BPN' (BASBPN) sequence (sequence c in the table) and the mature polypeptide of subtilisin 309 from Bacillus clausii, also known as Savinase® (BLSAVI) (sequence a in the table), based on the alignment in Table 1 of International Publication No. 1989 / 06279. Those skilled in the art will know that the positional numbers used for subtilisin 309 and other proteases in the patent literature are often based on the corresponding positional numbers of BPN' in this alignment.

[0046] Attached Figure 1 is provided for reference purposes and shows the alignment between Sequence ID 1 and Sequence ID 2 based on Table 1 of International Publication No. 1989 / 06279, where the position number corresponding to the position of Sequence ID 2 can be easily determined.

[0047] The identification of corresponding amino acid residues in other proteases can be determined by alignment of multiple polypeptide sequences using several computer programs with their respective default parameters. These programs include, but are not limited to, the following: MUSCLE (multiple sequence comparison using logarithmic prediction; version 3.5 and later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 and later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and EMBOSS EMMA (version 1.83 and later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680) employing ClustalW.

[0048] In describing the variants of the present invention, the nomenclature described below is applied for ease of reference. Accepted IUPAC one- or three-letter amino acid abbreviations are used.

[0049] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of threonine to alanine at position 226 is designated as "Thr226Ala" or "T226A". Multiple substitutions are separated by an addition sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" indicates the substitution of glycine (G) at position 205 with arginine (R) and serine (S) at position 411 with phenylalanine (F). Alternatively, multiple substitutions may be separated by a comma (","), for example, "Gly205Arg,Ser411Phe" or "G205R,S411F".

[0050] Deletions: In the case of amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, a deletion of glycine at position 195 is designated as "Gly195*" or "G195*". Multiple deletions are separated by an addition sign ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*". Alternatively, multiple deletions may be separated by a comma (","), for example, "Gly195*,Ser411*" or "G195*,S411*".

[0051] Insertion: In the case of amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, the insertion of lysine after glycine at position 195 is designated as "Gly195GlyLys" or "G195GK". 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 lysine and alanine after glycine at position 195 is indicated as "Gly195GlyLysAla" or "G195GKA".

[0052] In such cases, the inserted amino acid residue is numbered by adding a lowercase letter to the position number of the amino acid residue preceding it. In the example above, the sequence would be as follows:

[0053] [Table 1]

[0054] Multiple Modifications: Variants containing multiple modifications are separated by an additional marker ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+Gly195E" represent the substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple modifications may be separated by commas (","), for example, "Arg170Tyr,Gly195Glu" or "R170Y,G195E".

[0055] Different Modifications: If different modifications can be introduced at a certain position, the different modifications are separated by commas. For example, "Arg170Tyr,Glu" represents the substitution of arginine with tyrosine or glutamate at position 170. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" specifies the following variant: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0056] Array Overview Sequence ID 1 is the amino acid sequence of Savinase® protease. Sequence ID 2 is the amino acid sequence of BPN' protease. Sequence ID 3 is the amino acid sequence of a variant of Sequence ID 1. Sequence ID 4 is the amino acid sequence of a variant of Sequence ID 1. Sequence ID 5 is the amino acid sequence of a variant of Sequence ID 1. Sequence ID 6 is the amino acid sequence of a variant of Sequence ID 1.

[0057] Detailed description of the present invention The present invention provides a novel protease with improved solubility. The protease variant of the present invention contains a positively charged amino acid or polar amino acid at the position corresponding to position 215 of SEQ ID NO: 1 (i.e., position A215 of SEQ ID NO: 1). The introduction of a positively charged amino acid or polar amino acid at this position results in improved solubility, particularly a reduction in protease crystal formation and an increase in protease crystal solubility, as described in the examples below.

[0058] Protease variant The present invention relates to a protease variant of a parent protease, wherein the variant has 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%, or at least 99% sequence identity with SEQ ID NO: 1, but less than 100%, and the variant is X215K The variant includes a first substitution selected from the group consisting of X215R, X215Q, X125N, X215S, and X215T, wherein the variant is X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., For example, S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104I (e.g., V104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., At least three, for example, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more further modifications, preferably including substitutions, selected from the group consisting of S259D), X261W (e.g., N261W), and X262E (e.g., L262E), wherein the variant has protease activity and its positional number is based on the numbering of Sequence ID No. 2.

[0059] In one embodiment, the first substitution is selected from the group consisting of X215K, X215Q, X125N, X215S, and X215T; preferably, the first substitution is selected from the group consisting of X215K, X215Q, X125N, and X215T.

[0060] In one embodiment, the first substitution is selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; preferably, the first substitution is selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T; most preferably, the first substitution is selected from the group consisting of A215K, A215Q, A215N, and A215T.

[0061] In one embodiment, the variants are X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S10 3T), X104I (e.g., V104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., In a preferred embodiment, the variant includes at least three, for example, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more further modifications, preferably substitutions, selected from the group consisting of L262E.

[0062] In a preferred embodiment, the protease variant is a) S3T, V4I, S99D, S101E, S103A, G160S, and V205I; b)I35ID, N76D, H120D, G195E, K235L; c) S9E, N43R, N76D, S99F, S101L, S103T, V104I, V205I, Q206L, Y209W, S259D, N261W, and L262E; and d) S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E The modifier includes at least three, for example, at least four, or five further modifications, preferably substitutions, selected from the group of substitutions selected from the group consisting of the above.

[0063] In a preferred embodiment, the protease variant includes at least three, for example, at least four, at least five, at least six, or seven further substitutions selected from the group consisting of S3T, V4I, S99D, S101E, S103A, G160S, and V205I.

[0064] In a preferred embodiment, the protease variant includes at least three, for example, at least four or five further modifications, preferably substitutions, selected from the group consisting of I35ID, N76D, H120D, G195E, and K235L.

[0065] In a preferred embodiment, the protease variant includes at least three, for example, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or thirteen further substitutions selected from the group consisting of S9E, N43R, N76D, S99F, S101L, S103T, V104I, V205I, Q206L, Y209W, S259D, N261W, and L262E.

[0066] In a preferred embodiment, the protease variant includes at least three, for example, at least four, at least five, at least six, at least seven, at least eight, or nine further substitutions selected from the group consisting of S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E.

[0067] In a preferred embodiment, the protease variant is a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; preferably a substitution selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; most preferably, contains, is essentially, or consists of SEQ ID NO: 1 having a substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T.

[0068] In a preferred embodiment, the protease variant is a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; preferably a substitution selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; most preferably, includes, is essentially, or consists of SEQ ID NO: 3 having a substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T.

[0069] In a preferred embodiment, the protease variant is a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; preferably a substitution selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; most preferably, contains, is essentially, or consists of SEQ ID NO: 4 having a substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T.

[0070] In a preferred embodiment, the protease variant is a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; preferably a substitution selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; most preferably, it includes, is essentially, or consists of SEQ ID NO: 5 having a substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T.

[0071] In a preferred embodiment, the protease variant is a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; preferably a substitution selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; most preferably, contains, is essentially, or consists of SEQ ID NO: 6 having a substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T.

[0072] In addition to the substitutions described above, the variant may also include substitutions at one or more other positions.

[0073] Amino acid changes can be minor, such as 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-terminal or carboxyl-terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to 20–25 residues; or minor extensions, such as polyhistidine sequences, antigen epitopes, or binding domains, that facilitate purification by altering the net charge or another function.

[0074] Examples of conservative substitutions include basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, in H. Neurath and RLHill, 1979, *The Proteins*, Academic Press, New York. Common substitutions include 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.

[0075] Alternatively, amino acid changes can alter the physicochemical properties of polypeptides. For example, amino acid changes may improve the thermal stability of polypeptides, alter substrate specificity, or change the optimal pH.

[0076] Essential amino acids in polypeptides can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced into any residue within the molecule to identify amino acid residues critical to the molecule's activity, and the resulting mutant molecules are tested for activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interactions can also be determined by physical analysis of the structure, measured by techniques such as nuclear magnetic conjugate, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of the putative contact site amino acids. For example, see de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J.Mol.Biol.224:899-904; Wlodaver et al., 1992, FEBS Lett.309:59-64. The identity of essential amino acids can also be inferred from alignment with related polypeptides.

[0077] The variant of the present invention has improved solubility. In particular, the variant of the present invention exhibits, for example, reduced protease crystal formation during fermentation of host cells expressing the variant, and increased solubility of such protease crystals, as described in Example 1 below. Improved solubility can be determined by using various methods known to those skilled in the art. Preferably, improved solubility is determined as reduced protease crystal formation or increased protease crystal solubility according to Example 1 below.

[0078] In one embodiment, the protease variant has improved solubility compared to a protease that is otherwise identical but does not have a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T. In a preferred embodiment, the protease variant does not have a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T, and does not have a substitution selected from the group consisting of X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D, X99F (For example, S99F), X101E (for example, S101E), X101L (for example, S101L), X103A (for example, S103A), X103T (for example, S103T), X104I (for example, V104I), X120D (for example, H120D), X160S (for example, G160S), X195E (for example, G195E), X205I (for example, V205I), X206L (for example, Q Compared to the same protease without substitutions, the modified protease has at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 40%, at least 50%, or more improved solubility compared to at least 300%, at least 40%, at least 5

[0079] In preferred embodiments, the protease variant has improved solubility at 10-30°C, preferably 15-25°C, more preferably about 20°C, and most preferably 20°C.

[0080] In preferred embodiments, the protease variant has improved solubility at pH 3 to 9, preferably at pH 4 to 8, more preferably at pH 4 to 6, even more preferably at pH 4 to 5, and most preferably at pH 4.5.

[0081] In preferred embodiments, the protease variant has improved solubility at 15-25°C and pH 4-6. Preferably, the protease variant has improved solubility at 20°C and pH 4-5.

[0082] In one embodiment, the protease variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 1.

[0083] In one embodiment, the protease variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 3.

[0084] In one embodiment, the protease variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 4.

[0085] In one embodiment, the protease variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 5.

[0086] In one embodiment, the protease variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 6.

[0087] In addition to improved solubility, variants of the present invention may have one or more improved properties compared to the parent. One or more improved properties may be selected from the group consisting of catalytic efficiency, catalytic rate, chemical stability, oxidation stability, pH activity, pH stability, proteolytic stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermal stability.

[0088] The variants of the present invention have protease activity, preferably equivalent or improved protease activity. In one embodiment, the protease variant has improved solubility compared to a protease that is otherwise identical but does not have a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T. In a preferred embodiment, the protease variant does not have a first substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T, and does not have a first substitution selected from the group consisting of X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104I (e.g., V104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L ( For example, the protease activity is equivalent to or improved by, for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% compared to the same protease without substitutions, which has at least three further modifications selected from the group consisting of Q206L, X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., L262E).

[0089] In one embodiment, the variant has protease activity equivalent to, or for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more than, that of SEQ ID NO: 1.

[0090] In one embodiment, the variant has protease activity equivalent to, or for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more than, that of SEQ ID NO: 3.

[0091] In one embodiment, the variant has protease activity equivalent to, or for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more than, that of SEQ ID NO: 4.

[0092] In one embodiment, the variant has protease activity equivalent to, or for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more than, that of SEQ ID NO: 5.

[0093] In one embodiment, the variant has protease activity equivalent to, or for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more than, that of SEQ ID NO: 6.

[0094] In one embodiment, the present invention relates to a polypeptide having at least 80%, for example, 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%, and at least 99%, but less than 100% sequence identity with SEQ ID NO: 1, preferably an isolated or purified polypeptide, wherein the variant includes a substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T, the variant has protease activity, and the position number is based on the numbering of SEQ ID NO: 2. In a preferred embodiment, the variant includes a substitution selected from the group consisting of A215K, A215R, A215Q, A125N, A215S, and A215T. In a preferred embodiment, the variant includes a substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T. In one embodiment, the variant has protease activity equivalent to or improved by, for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more compared to SEQ ID NO: 1. In one embodiment, the variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 1.In preferred embodiments, the variant has improved solubility at 10 to 30°C, preferably 15 to 25°C, more preferably about 20°C, and most preferably 20°C. In preferred embodiments, the variant has improved stability at pH 3 to 9, preferably pH 4 to 8, more preferably pH 4 to 6, even more preferably pH 4 to 5, and most preferably pH 4.5. In preferred embodiments, the variant contains, is essentially, or consists of SEQ ID NO: 1 having substituted A215K. In preferred embodiments, the variant contains, is essentially, or consists of SEQ ID NO: 1 having substituted A215R. In preferred embodiments, the variant contains, is essentially, or consists of SEQ ID NO: 1 having substituted A215Q. In preferred embodiments, the variant contains, is essentially, or consists of SEQ ID NO: 1 having substituted A215N. In preferred embodiments, the variant contains, is essentially, or consists of SEQ ID NO: 1 having substituted A215S. In a preferred embodiment, the variant includes, is essentially derived from, or consists of SEQ ID NO: 1 having substitution A215T.

[0095] In one embodiment, the present invention relates to a polypeptide having at least 80%, for example, 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%, and at least 99%, but less than 100%, sequence identity with respect to a polypeptide, preferably an isolated or purified polypeptide, wherein the variant comprises a first substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T, and the variant comprises at least three, for example, at least four, at least five, at least six, or seven further substitutions selected from the group consisting of S3T, V4I, S99D, S101E, S103A, G160S, and V205I, wherein the variant has protease activity, and the positional number is based on the numbering of Sequence ID No. 2. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215R, A215Q, A125N, A215S, and A215T. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215Q, A215N, and A215T. In one embodiment, the variant has protease activity equivalent to or improved by, for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 3.In one embodiment, the variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 3. In a preferred embodiment, the variant has improved solubility at 10 to 30°C, preferably at 15 to 25°C, more preferably at about 20°C, and most preferably at 20°C. In a preferred embodiment, the variant has improved stability at pH 3 to 9, preferably at pH 4 to 8, more preferably at pH 4 to 6, even more preferably at pH 4 to 5, and most preferably at pH 4.5. In a preferred embodiment, the variant includes, is essentially, or consists of, SEQ ID NO: 3 having substitution A215K. In a preferred embodiment, the variant includes, is essentially, or consists of, SEQ ID NO: 3 having substitution A215R. In a preferred embodiment, the variant includes, is essentially, or consists of, SEQ ID NO: 3 having substitution A215Q. In a preferred embodiment, the variant includes, is essentially, or consists of, SEQ ID NO: 3 having substitution A215N. In a preferred embodiment, the variant includes, is essentially, or consists of, SEQ ID NO: 3 having substitution A215S. In a preferred embodiment, the variant includes, is essentially, or consists of, SEQ ID NO: 3 having substitution A215T.

[0096] In one embodiment, the present invention relates to a polypeptide having at least 80%, for example, 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%, and at least 99%, but less than 100%, sequence identity with respect to a variant having at least 80%, for example, 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%, and at least 99%, but less than 100%, sequence identity with respect to an isolated or purified polypeptide, wherein the variant comprises a first substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T, and the variant comprises at least three, for example, at least four, or five further substitutions selected from the group consisting of I35ID, N76D, H120D, G195E, and K235L, wherein the variant has protease activity, and the positional number is based on the numbering of sequence number 2. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215R, A215Q, A125N, A215S, and A215T. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215Q, A215N, and A215T. In one embodiment, the variant has protease activity equivalent to or improved by, for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 4.In one embodiment, the variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 4. In a preferred embodiment, the variant has improved solubility at 10 to 30°C, preferably at 15 to 25°C, more preferably at about 20°C, and most preferably at 20°C. In a preferred embodiment, the variant has improved stability at pH 3 to 9, preferably at pH 4 to 8, more preferably at pH 4 to 6, even more preferably at pH 4 to 5, and most preferably at pH 4.5. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 4 having substitution A215K. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 4 having substitution A215R. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 4 having substitution A215Q. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 4 having substitution A215N. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 4 having substitution A215S. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 4 having substitution A215T.

[0097] In one embodiment, the present invention relates to a polypeptide having at least 80%, for example, 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%, and at least 99% but less than 100% sequence identity with SEQ ID NO: 5, preferably an isolated or purified polypeptide, wherein the variant is a first selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T. The variant includes substitutions, and further includes at least three, for example, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or thirteen further substitutions selected from the group consisting of S9E, N43R, N76D, S99F, S101L, S103T, V104I, V205I, Q206L, Y209W, S259D, N261W, and L262E, wherein the variant has protease activity and its positional number is based on the numbering of Sequence ID No. 2. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215R, A215Q, A125N, A215S, and A215T. In a preferred embodiment, the variant comprises a first substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T. In one embodiment, the variant has protease activity equivalent to, or for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more improved compared to SEQ ID NO: 5.In one embodiment, the variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 5. In a preferred embodiment, the variant has improved solubility at 10 to 30°C, preferably at 15 to 25°C, more preferably at about 20°C, and most preferably at 20°C. In a preferred embodiment, the variant has improved stability at pH 3 to 9, preferably at pH 4 to 8, more preferably at pH 4 to 6, even more preferably at pH 4 to 5, and most preferably at pH 4.5. In a preferred embodiment, the variant includes, is essentially, or consists of, sequence number 5 having substitution A215K. In a preferred embodiment, the variant includes, is essentially, or consists of, sequence number 5 having substitution A215R. In a preferred embodiment, the variant includes, is essentially, or consists of, sequence number 5 having substitution A215Q. In a preferred embodiment, the variant includes, is essentially, or consists of, sequence number 5 having substitution A215N. In a preferred embodiment, the variant includes, is essentially, or consists of, sequence number 5 having substitution A215S. In a preferred embodiment, the variant includes, is essentially, or consists of, sequence number 5 having substitution A215T.

[0098] In one embodiment, the present invention relates to polypeptides having at least 80%, for example, 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%, and at least 99%, but less than 100% sequence identity with respect to Sequence ID No. 6, preferably isolated or purified polypeptides, wherein variants are X215K, X215R, X215Q, and X125. The variant includes a first substitution selected from the group consisting of N, X215S, and X215T, and includes at least three, for example, at least four, at least five, at least six, at least seven, at least eight, or nine further substitutions selected from the group consisting of S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E, and the variant has protease activity and the position number is based on the numbering of Sequence ID No. 2. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215R, A215Q, A125N, A215S, and A215T. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T. In a preferred embodiment, the variant includes a first substitution selected from the group consisting of A215K, A215Q, A215N, and A215T. In one embodiment, the variant has protease activity equivalent to or improved by, for example, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more compared to SEQ ID NO: 6.In one embodiment, the variant has improved solubility by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to SEQ ID NO: 6. In a preferred embodiment, the variant has improved solubility at 10 to 30°C, preferably at 15 to 25°C, more preferably at about 20°C, and most preferably at 20°C. In a preferred embodiment, the variant has improved stability at pH 3 to 9, preferably at pH 4 to 8, more preferably at pH 4 to 6, even more preferably at pH 4 to 5, and most preferably at pH 4.5. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 6 having substitution A215K. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 6 having substitution A215R. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 6 having substitution A215Q. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 6 having substitution A215N. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 6 having substitution A215S. In a preferred embodiment, the variant includes, is essentially, or consists of SEQ ID NO: 6 having substitution A215T.

[0099] Parent protease The protease variants of the present invention may be based on any parent protease. The parent may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

[0100] In one embodiment, the parent protease has sequence identity to the polypeptide of SEQ ID NO: 1 of at least 80%, for example, 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 only up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In one embodiment, the parent contains, is essentially, or consists of the amino acid sequence of SEQ ID NO: 1.

[0101] In one embodiment, the parent protease has sequence identity to the polypeptide of SEQ ID NO: 3 of at least 80%, for example, 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, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In one embodiment, the parent contains, is essentially, or consists of the amino acid sequence of SEQ ID NO: 3.

[0102] In one embodiment, the parent protease has sequence identity to the polypeptide of SEQ ID NO: 4 by at least 80%, for example, 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, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In one embodiment, the parent contains, is essentially, or consists of the amino acid sequence of SEQ ID NO: 4.

[0103] In one embodiment, the parent protease has sequence identity to the polypeptide of SEQ ID NO: 5 of at least 80%, for example, 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, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In one embodiment, the parent contains, is essentially, or consists of the amino acid sequence of SEQ ID NO: 5.

[0104] In one embodiment, the parent protease has sequence identity to the polypeptide of SEQ ID NO: 6 by at least 80%, for example, 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 only up to 20 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In one embodiment, the parent contains, is essentially, or consists of the amino acid sequence of SEQ ID NO: 6.

[0105] The parent polypeptide may be a hybrid polypeptide in which the region of one polypeptide is fused to the N-terminus or C-terminus of the region of the other polypeptide.

[0106] The parent may be a fusion polypeptide or a cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the present invention. The fusion polypeptide is 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 polypeptides such that they are in-frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein techniques in which the fusion polypeptide is produced post-translationally (Cooper et al., 1993, EMBO J.12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0107] The fusion polypeptide may also contain a cleavage site between the two polypeptides. When the fusion protein is secreted, this site is cleaved, releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed below: 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., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0108] The parent may be obtained from a microorganism of any genus. For the purposes of this invention, the term “obtained from,” when used herein in relation to a given source, means that the parent encoded by polynucleotides is produced by the source or by a strain into which polynucleotides from the source have been inserted. In one embodiment, the parent is secreted extracellularly.

[0109] The parent can be a bacterial protease. For example, the parent may be a Gram-positive protease from the genera Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. It may be a gram-negative bacterial polypeptide, or a protease of a gram-negative bacterial species such as Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma.

[0110] In one manifestation, the parents are Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, and Bacillus stearothermophilus. It is a protease of Bacillus subtilis or Bacillus thuringiensis (stearothermophilus).

[0111] In another embodiment, the parent is a protease from Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus.

[0112] In another embodiment, the parent is a protease of Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans.

[0113] With respect to the aforementioned species, it will be understood that the present invention encompasses other taxonomic equivalents, such as anamorphs, regardless of whether they are in complete or incomplete states, and regardless of the names of the species in which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0114] Strains of these species are publicly and readily available in several culture collections, including the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection and Northern Regional Research Center (NRRL).

[0115] The parent may be identified and obtained using the above-mentioned probes from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural substances (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. The polynucleotide encoding the parent may then be obtained by similarly screening a genomic DNA or cDNA library or mixed DNA sample of another microorganism. After detecting the polynucleotide encoding the parent using one or more probes, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989).

[0116] Protease variant preparation The present invention also relates to a method for obtaining a protease variant, (a) Introducing a first substitution to the parent protease selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; and X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104 Introducing at least three further modifications, preferably substitutions (the variants having protease activity), selected from the group consisting of I (e.g., V104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., L262E); and (b) Recover the variant Regarding methods including

[0117] In one embodiment, the first substitution is selected from the group consisting of X215K, X215Q, X125N, X215S, and X215T; preferably, the first substitution is selected from the group consisting of X215K, X215Q, X125N, and X215T.

[0118] In one embodiment, the first substitution is selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; preferably, the first substitution is selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T; most preferably, the first substitution is selected from the group consisting of A215K, A215Q, A215N, and A215T.

[0119] In one embodiment, at least three further modifications, preferably substitutions, are selected from the group consisting of S3T, V4I, S9E, I35ID, N43R, N76D, S99D, S99F, S101E, S101L, S103A, S103T, V104I, H120D, G160S, G195E, V205I, Q206L, Y209W, K235L, S259D, N261W, and L262E.

[0120] In one embodiment, at least three further modifications, preferably substitutions, are a) S3T, V4I, S99D, S101E, S103A, G160S, and V205I; b)I35ID, N76D, H120D, G195E, K235L; c) S9E, N43R, N76D, S99F, S101L, S103T, V104I, V205I, Q206L, Y209W, S259D, N261W, and L262E; and d) S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E It is selected from one of the groups consisting of [the specified group].

[0121] Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, and shuffling.

[0122] Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in the polynucleotide encoding the parent.

[0123] Site-directed mutagenesis can be achieved in vitro by PCR, which involves the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, which involves restriction enzyme cleavage at a site in a plasmid containing the parent-coding polynucleotide, followed by ligation of an oligonucleotide containing the mutation in this polynucleotide. Typically, the restriction enzymes used to digest the plasmid and oligonucleotide are the same, and the attached ends of the plasmid and insert can be ligated to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0124] Site-directed mutagenesis can also be achieved in vivo by methods known in the art. See, for example, 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.

[0125] Any site-directed mutagenesis procedure can be used in the present invention. Numerous commercially available kits can be used to prepare variants.

[0126] Synthetic gene construction methods require the in vitro synthesis of polynucleotide molecules designed to encode the target polypeptide. Gene synthesis can be carried out using many techniques, including the multi-microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), and similar techniques in which oligonucleotides are synthesized and assembled on photoprogrammable microfluidic chips.

[0127] Substitutions, deletions, and / or insertions of single or multiple amino acids may be made and tested using known mutagenesis, recombination, and / or shuffling methods, followed by relevant 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; International Publication No. 95 / 17413; or International Publication No. 95 / 22625. Other methods that may be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30;10832-10837; U.S. Patent No. 5,223,409; International Publication No. 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46;145; Ner et al., 1988, DNA 7;127).

[0128] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods for detecting the activity of cloned mutagenesis polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutation-induced DNA molecules encoding active polypeptides can be recovered from host cells and readily sequenced using standard methods in the art. This method allows for rapid determination of the importance of individual amino acid residues within the polypeptide.

[0129] Semi-synthetic gene construction is achieved by combining synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or shuffling. Semi-synthetic construction is exemplified by a process utilizing a synthesized polynucleotide fragment in combination with PCR technology. Thus, a defined region of the gene may be newly synthesized, other regions may be amplified using site-directed mutagenesis primers, and yet other regions may be subjected to error-prone PCR or non-error-prone PCR amplification. The polynucleotide subsequence may then be shuffled.

[0130] Polynucleotides The present invention also relates to isolated polynucleotides encoding variants of the present invention.

[0131] Techniques used to isolate or clone polynucleotides are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. Cloning of polynucleotides from genomic DNA can be achieved, for example, by polymerase chain reaction (PCR) or by using antibody screening of an expression library to detect cloned DNA fragments having shared structural features. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation-activated transcription (LAT), and polynucleotide-based amplification (NASBA) may also be used.

[0132] nucleic acid construct The present invention also relates to nucleic acid constructs comprising polynucleotides encoding variants of the present invention, wherein the polynucleotides are operably linked to one or more regulatory sequences that induce the expression of the coding sequence in a suitable host cell under conditions compatible with the regulatory sequences.

[0133] Polynucleotides can be manipulated in various ways to induce variant expression. It may be desirable or necessary to manipulate polynucleotides depending on the expression vector before insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA are well known in the art.

[0134] The regulatory sequence may be a promoter (a polynucleotide recognized by the host cell for the expression of the polynucleotide encoding the variant of the present invention). The promoter contains a transcriptional regulatory sequence that mediates the expression of the variant. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including variants, truncated forms, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide of either the host or a heterogeneous species.

[0135] Examples of suitable promoters for inducing transcription of the nucleic acid constructs of the present invention in bacterial host cells include the α-amylase gene (amyQ) of Bacillus amyloliquefaciens, the α-amylase gene (amyL) of Bacillus licheniformis, the penicillinase gene (penP) of Bacillus licheniformis, the maltose-producing amylase gene (amyM) of Bacillus stearothermophilus, the levansculas gene (sacB) of Bacillus subtilis, the xylA and xylB genes of Bacillus subtilis, and Bacillus thuringiensis. These include the cryIIIA gene of *E. coli* (Agaisse and Lereclus, 1994, Molecular Microbiology 13;97-107), the Lac operon of *E. coli*, the trc promoter of *E. coli* (Egon et al., 1988, Gene 69;301-315), the agarase gene (dagA) of *Streptomyces coelicolor*, and promoters derived from the β-lactamase gene of prokaryotes (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in Gilbert et al., 1980, Scientific American 242:74-94, “Useful proteins from recombinant bacteria”; and Sambrook et al., 1989. An example of a tandem promoter is disclosed in International Publication No. 99 / 43835.

[0136] Examples of suitable promoters for inducing transcription of the nucleic acid constructs of the present invention in filamentous fungal host cells include acetamidase of Aspergillus nidulans, neutral alpha-amylase of Aspergillus niger, acid-stable alpha-amylase of Aspergillus niger, glucoamylase (glaA) of Aspergillus niger or Aspergillus awamori, TAKA amylase of Aspergillus oryzae, alkaline protease of Aspergillus oryzae, triose phosphate isomerase of Aspergillus oryzae, and Fusarium oxysporum. Trypsin-like protease of Fusarium oxysporum (International Publication No. 96 / 00787), amyloglucosidase of Fusarium venenatum (International Publication No. 00 / 56900), Daria of Fusarium venenatum (International Publication No. 00 / 56900), Quinn of Fusarium venenatum (International Publication No. 00 / 56900), lipase of Rhizomucor miehei, aspartate proteinase of Rhizomucor miehei, beta-glucosidase of Trichoderma reesei, Trichoderma reesei Cellobiohydrolase I of Trichoderma reesei, cellobiohydrolase II of Trichoderma reesei, endoglucanase I of Trichoderma reesei, endoglucanase II of Trichoderma reesei, Trichoderma reeseiEndoglucanase III of Trichoderma reesei, endoglucanase V of Trichoderma reesei, xylanase I of Trichoderma reesei, xylanase II of Trichoderma reesei, xylanase III of Trichoderma reesei, beta-xylosidase of Trichoderma reesei, and Trichoderma reesei Promoters derived from the translation elongation factor of reesei, and the NA2-tpi promoter (a modified promoter derived from the neutral alpha-amylase gene of the genus Aspergillus in which the untranslated leader is replaced by an untranslated leader derived from the triose phosphate isomerase gene of the genus Aspergillus; a non-limiting example is a modified promoter derived from the neutral alpha-amylase gene of Aspergillus niger in which the untranslated leader is replaced by an untranslated leader derived from the triose phosphate isomerase gene of Aspergillus nidulans or Aspergillus oryzae); as well as their variants, truncated and hybrid promoters. Other promoters are described in U.S. Patent No. 6,011,147.

[0137] In the yeast host, useful promoters can be obtained from genes related to Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0138] The control sequence may also be a transcriptional terminator recognized by the host cell to terminate transcription. The terminator is operably ligated to the 3' end of the polynucleotide encoding the variant. Any functional terminator in the host cell may be used in this invention.

[0139] Preferred terminators for bacterial host cells are derived from the alkaline protease (aprH) of Bacillus clausii, the α-amylase (amyL) of Bacillus licheniformis, and the ribosomal RNA (rrnB) of Escherichia coli.

[0140] Preferred terminators for filamentous fungal host cells are acetamidase from Aspergillus nidulans, anthranilate synthase from Aspergillus nidulans, glucoamylase from Aspergillus niger, alpha-glucosidase from Aspergillus niger, TAKA amylase from Aspergillus oryzae, trypsin-like protease from Fusarium oxysporum, beta-glucosidase from Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, and Trichoderma reesei These genes are derived from the genes related to cellobiohydrolase II, endoglucanase I, endoglucanase II, endoglucanase III, endoglucanase V, xylanase I, xylanase II, xylanase III, beta-xylosidase, and translation elongation factor of Trichoderma reesei.

[0141] Preferred terminators for yeast hosts are obtained from the genes for enolase, cytochrome C (CYC1), and glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Other useful terminators for yeast host cells are described by Romanos et al., 1992, cited above.

[0142] Regulatory sequences can also be mRNA stabilization regions downstream of promoters and upstream of gene coding sequences that increase gene expression.

[0143] Suitable mRNA stabilization regions can be found in the cryIIIA gene of Bacillus thuringiensis (International Publication No. 94 / 25612) and the SP82 gene of Bacillus subtilis (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0144] The regulatory sequence may also be the untranslated region of the mRNA leader, which is crucial for translation by the host cell. The leader is operably ligated to the 5' end of the polynucleotide encoding the variant. Any functional leader within the host cell may be used.

[0145] Preferred leaders for filamentous fungal host cells are derived from the genes related to TAKA amylase from Aspergillus oryzae and triose phosphate isomerase from Aspergillus nidulans.

[0146] Suitable leaders for yeast host cells are derived from genes related to Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0147] The regulatory sequence may also be a polyadenylation sequence that is operably ligated to the 3' end of a polynucleotide and, when transcribed, is recognized as a signal by the host cell, adding a polyadenosine residue to the transcribed mRNA. Any polyadenylation sequence that functions within the host cell may be used.

[0148] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for anthranilate synthase from Aspergillus nidulans, glucoamylase from Aspergillus niger, α-glucosidase from Aspergillus niger, TAKA amylase from Aspergillus oryzae, and trypsin-like protease from Fusarium oxysporum.

[0149] Useful polyadenylated sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0150] The regulatory sequence may also be a signal peptide coding region that codes for a signal peptide ligated to the N-terminus of the variant and directs the variant into the cell's secretory pathway. The 5' end of the polynucleotide coding sequence may essentially contain the signal peptide coding sequence that is originally ligated in the translational reading frame, along with the segment of the coding sequence that codes for the variant. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required if 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 the secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the host cell's secretory pathway may be used.

[0151] Effective signal peptide coding sequences for bacterial host cells are those derived from genes related to Bacillus NCIB 11837 maltose-producing amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0152] Effective signal peptide coding sequences for filamentous fungal host cells are those derived from genes related to neutral amylase of Aspergillus niger, glucoamylase of Aspergillus niger, TAKA amylase of Aspergillus oryzae, cellulase of Humicola insolens, endoglucanase V of Humicola insolens, lipase of Humicola lanuginosa, and aspartate proteinase of Rhizomucor miehei.

[0153] Useful signal peptides for yeast host cells are obtained from the α-factor and invertase genes of Saccharomyces cerevisiae. Other useful signal peptide coding sequences are described by Romanos et al., 1992, cited above.

[0154] The regulatory sequence may also be a polypeptide coding sequence that encodes a propeptide located at the N-terminus of the variant. The resulting polypeptide is known as a proenzyme or propeptide (or possibly an enzyme precursor). Propeptides are generally inactive and can be converted to active variants by catalytic or autocatalytic cleavage of the propeptide from this propeptide. Propeptide coding sequences may be obtained from the genes for alkaline protease (aprE) of Bacillus subtilis, neutral protease (nprT) of Bacillus subtilis, laccase of Myceliophthora thermophila (International Publication No. 95 / 33836), aspartate proteinase of Rhizomucor miehei, and α-factor of Saccharomyces cerevisiae.

[0155] When both a signal peptide 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.

[0156] In some cases, it is desirable to add regulatory sequences that control the expression of a variant in relation to the proliferation of host cells. Examples of regulatory sequences are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. Examples of regulatory sequences in prokaryotes include the lac operator system, the tac operator system, and the trp operator system. In yeast, the ADH2 system or the GAL1 system may be used. In filamentous fungi, the glucoamylase promoter of Aspergillus niger, the TAKA alpha-amylase promoter and the glucoamylase promoter of Aspergillus oryzae, the cellobiohydrolase I promoter and the cellobiohydrolase II promoter of Trichoderma reesei may be used. Another example of a regulatory sequence is one that enables gene amplification. In eukaryotes, examples of these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein gene, which is amplified by heavy metals. In these cases, the polynucleotide encoding the variant is likely to be operablely ligated to the regulatory sequence.

[0157] Expression vector The present invention also relates to a recombinant expression vector comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcription and translation termination signals. Various nucleotides and regulatory sequences may be ligated together to construct a recombinant expression vector that includes one or more convenient restriction sites, allowing for the insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a vector suitable for expression. When constructing the expression vector, the coding sequence is positioned within the vector so that it is operably ligated to a regulatory sequence suitable for expression.

[0158] Recombinant expression vectors can be any vector (e.g., plasmid or virus) that allows for convenient recombinant DNA procedures and enables the expression of polynucleotides. The selection of this vector typically depends on the compatibility between the vector and the host cell into which it is introduced. The vector may be a linear or closed-circular plasmid.

[0159] The vector may be a self-replicating vector, i.e., a vector whose replication exists as an extrachromosomal element independent of chromosomal replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. This vector may include any means necessary for reliable self-replication. Alternatively, the vector may be integrated into the genome upon introduction into a host cell and replicated together with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, may be used, containing the entire DNA or transposons to be introduced into the host cell's genome.

[0160] The vector preferably contains one or more selection markers that enable easy selection of transformed, transfected, transduced, or like cells. The selection markers are genes whose products result in biocide or viral resistance, heavy metal resistance, prototrophicity to trophic requirements, and the like.

[0161] Examples of bacterial selection markers include the dal gene of Bacillus licheniformis or Bacillus subtilis, or markers conferring antibiotic resistance such as ampicillin resistance, chloramphenicol resistance, kanamycin resistance, neomycin resistance, spectinomycin resistance, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothrysine acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenylyl sulfate transferase), and trpC (anthranilate synthase), as well as their equivalents. Preferred genes for use in Aspergillus cells are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus. Preferred genes for use in Trichoderma cells are adeA, adeB, amdS, hph, and pyrG.

[0162] The selection marker may be a dual selection marker system as described in International Publication No. 2010 / 039889. In one embodiment, the dual selection marker is an hph-tk dual selection marker system.

[0163] The vector preferably contains elements that enable the integration of the vector into the host cell's genome or the self-replication of the vector within the cell independently of the genome.

[0164] Regarding integration into the host cell genome, the vector may rely on a sequence of polynucleotides encoding a variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration into the host cell genome by homologous recombination at a precise location within the chromosome. To increase the likelihood of integration at a precise location, the integration element should contain a sufficient number of nucleic acids (e.g., 100–10,000 base pairs, 400–10,000 base pairs, and 800–10,000 base pairs), and this nucleic acid should have high sequence identity to the corresponding target sequence to increase the likelihood of homologous recombination. The integration element can be any sequence homologous to the target sequence in the host cell genome. Furthermore, the integration element may or may not encode polynucleotides. On the other hand, the vector may be integrated into the host cell genome by non-homologous recombination.

[0165] In the case of autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously within the host cell in question. The origin of replication can be any plasmid replication origin that mediates autonomous replication that functions within the cell. The terms “origin of replication” or “plasmid replication origin” mean a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0166] Examples of bacterial origins of replication include the pBR322, pUC19, pACYC177, and pACYC184 plasmids that can replicate in Escherichia coli (E. coli), and the pUB110, pE194, pTA1060, and pAMβ1 plasmids that can replicate in Bacillus species.

[0167] Examples of origins of replication used in yeast host cells include the 2-micron origins of replication ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0168] Examples of useful origins of replication in filamentous fungal cells include AMA1 and ANS1 (Gems et al., 1991, Gene 98; 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; International Publication No. 00 / 24883). Isolation of the AMA1 gene and construction of plasmids or vectors containing this gene can be achieved according to the methods disclosed in International Publication No. 00 / 24883.

[0169] The generation of variants can be increased by inserting two or more copies of the polynucleotide of the present invention into host cells. Increasing the copy number of the polynucleotide can be achieved by incorporating at least one further copy of the sequence into the host cell genome, or by including an amplified selectable marker gene in the polynucleotide, and by culturing the cells in the presence of a suitable selector, cells containing the amplified copy of the selectable marker gene, and therefore cells containing the further copies of the polynucleotide, can be selected.

[0170] The procedure used to construct the recombinant expression vector of the present invention by linking the above elements is well known to those skilled in the art (e.g., Sambrook et al., 1989).

[0171] host cell The present invention also relates to recombinant host cells comprising polynucleotides encoding the variants of the present invention, which are operably linked to one or more regulatory sequences that induce the generation of the variants of the present invention. The construct or vector comprising the polynucleotides is introduced into the host cell so that the construct or vector is maintained as a chromosomal construct or as a self-replicating extrachromosomal vector as described above. The term “host cell” includes any offspring of a parent cell that are not identical to the parent cell due to mutations that occurred during replication. The selection of the host cell will depend largely on the gene encoding the variant and its source.

[0172] The host cell may be any cell useful in the recombinant production of the variant, such as a prokaryote or a eukaryote.

[0173] The host cells of prokaryotes can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, the genera Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, the genera Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0174] Bacterial host cells are 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, and Bacillus pmyrus. The host cells may be any Bacillus species, including Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Preferably, the bacterial host cells are Bacillus licheniformis cells.

[0175] The bacterial host cells may be any Streptococcus cell, including, but not limited to, cells of Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus.

[0176] The bacterial host cells may be any Streptomyces cell, including, but not limited to, cells of Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans.

[0177] DNA introduction into Bacillus cells can be achieved by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168:111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81:823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56:209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). DNA introduction into Escherichia coli (E. coli) can be achieved by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or by electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). DNA introduction into Streptomyces cells can be achieved by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294).DNA introduction into Pseudomonas cells can be achieved by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57). The introduction of DNA into Streptococcus cells can be achieved by innate transformation (see, e.g., Perry and Kuramitsu, 1981, Infect.Immun.32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, e.g., Buckley et al., 1999, Appl.Environ.Microbiol.65:3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol.Rev.45:409-436). However, any method known in the art for introducing DNA into host cells may be used.

[0178] The host cell can also be a eukaryote, such as a mammal, insect, plant, or fungal cell.

[0179] The host cell may be a fungal cell. As used herein, “fungus” includes the Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined in Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0180] The fungal host cell may be a yeast cell. As used herein, "yeast" refers to ascomycetes (Endomycetales), basidiomycetes, and imperfect fungi (Blastomycetes). Because the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0181] The yeast host cells are those of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, and Saccharomyces kuruiberi. These may be cells of *Saccharomyces kluyveri*, *Saccharomyces norbensis*, *Saccharomyces oviformis*, or *Yarrowia lipolytica*.

[0182] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subphylum Fungi and Oomycetes (as defined in Hawksworth et al., 1995, see above). 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 obligate aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by unicellular thallus budding, and carbon catabolism may be fermentative.

[0183] The host cells of filamentous fungi include the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, and Ne. These may be cells from the genera Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma.

[0184] For example, filamentous fungal host cells include Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, and Ceriporiopsis gilvense. Chrysosporium gilvescens), Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslangum queenslandicum), Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactrioidesFusarium bactridioides), Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochrome Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purprogenum purpurogenum), Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor (TrametesThese may be cells of Trichoderma versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0185] Fungal cells can be transformed by processes including protoplast formation, protoplast transformation, and cell wall regeneration, in methods known to the present day. Preferred procedures for the transformation of Aspergillus and Trichoderma host cells are described in European Patent No. 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio / Technology 6:1419-1422. Preferred methods for the transformation of Fusarium species are described in Malardier et al., 1989, Gene 78:147-156, and in International Publication No. 96 / 00787. Yeast can be transformed using the procedures described by Becker and Guarente, In Abelson, J.Nand Simon, MI, editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0186] Generation method The present invention also relates to a method for generating a variant, comprising (a) culturing recombinant host cells of the present invention under conditions that facilitate the generation of the variant; and optionally (b) recovering the variant.

[0187] Recombinant host cells are cultured in a nutrient medium suitable for variant generation using methods known in the art. For example, cells may be cultured in a suitable medium and under conditions that allow for variant expression and / or isolation, such as flask shaking culture in experimental or industrial fermenters, or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation). This culture is carried out in a suitable nutrient medium containing a carbon source, a nitrogen source, and inorganic salts, using procedures known in the art. The suitable medium is available from commercial suppliers or can be prepared according to publicly available compositions (e.g., compositions published in the American Type Culture Collection catalog). 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 the cell solubil.

[0188] The variant can be detected using methods known in the art that are specific to the variant. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, the activity of the variant may be determined using an enzyme assay.

[0189] The variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium by conventional procedures including, but not limited to, sampling, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one embodiment, the total fermentation broth is recovered.

[0190] The variants may be purified by various procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing), solubility differentiation (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989).

[0191] In an alternative embodiment, the variant is not recovered, and instead, the host cells of the present invention expressing the variant are used as the source of the variant.

[0192] Fermented broth formulation or cell composition The present invention also relates to fermentation broth formulations or cell compositions containing the variant of the present invention. The fermentation broth product further includes additional components used in the fermentation process, such as cells (including host cells containing genes encoding the variant of the present invention, and used to produce the desired variant), cell fragments, biomass, fermentation medium and / or fermentation product. In some embodiments, the composition is a whole broth of dead cells containing organic acids, dead cells and / or cell fragments, and culture medium.

[0193] 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, fermentation broth is produced when a microbial culture is grown to saturation and incubated under carbon-restricted conditions that allow for protein synthesis (e.g., enzyme expression by host cells) and secretion into the cell culture medium. Fermentation broth may contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, fermentation broth is not fractionated and contains used culture medium and cell fragments present after microbial cells (e.g., filamentous fungal cells) have been removed, for example, by centrifugation. In some embodiments, fermentation broth contains used cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0194] In some embodiments, the fermentation broth formulation and cell composition include a first organic acid component comprising at least one 1-5 carbon organic acid and / or a salt thereof, and a second organic acid component comprising at least one 6 or more carbon organic acid and / or a salt thereof. In specific 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 above, 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 above.

[0195] In one embodiment, the composition contains one or more organic acids and optionally further contains dead cells and / or cell fragments. In one embodiment, the dead cells and / or cell fragments are removed from the total broth of dead cells to provide a composition that does not contain these components.

[0196] The fermented broth formulation or cell composition may further include, but is not limited to, preservatives and / or antimicrobial agents (e.g., bacteriostatic agents) including sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0197] The whole broth or composition of dead cells may contain the unfractionated contents of the fermentation material obtained at the end of fermentation. Typically, the whole broth or composition of dead cells contains used culture medium and cell fragments present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-restricted conditions that allow for protein synthesis. In some embodiments, the whole broth or composition of dead cells contains used cell culture medium, extracellular enzymes, and dead filamentous fungal cells. In some embodiments, microbial cells present in the whole broth or composition of dead cells may be permeabilized and / or lysed using methods known in the art.

[0198] The total broth or cell composition described herein is typically a liquid, but may contain insoluble components such as dead cells, cell fragments, culture medium components, and / or insoluble enzymes. In some embodiments, the insoluble components may be removed to provide a clarified liquid composition.

[0199] The whole broth formulations and cell compositions of the present invention may be prepared by the methods described in International Publication No. 90 / 15861 or International Publication No. 2010 / 096673.

[0200] Detergent composition The present invention also relates to compositions comprising the protease variant of the present invention, for example, detergents or cleaning compositions.

[0201] The present invention also relates to a composition comprising the protease variant of the present invention and further comprising one or more detergent components 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 detergent components that do not exist in nature.

[0202] The present invention also relates to a composition comprising the protease variant of the present invention and further comprising one or more additional enzymes selected from the group consisting of amylase, catalase, cellulase (e.g., endoglucanase), cutinase, haloperoxygenase, lipase, mannanase, pectinase, pectin lyase, peroxidase, protease, xanthanase, lichenase, and xyloglucanase, or any mixture thereof.

[0203] The detergent composition may be in the form of, for example, a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a structured or dense powder, granules, paste, gel, or a structured, dense or concentrated liquid. In a preferred embodiment, the detergent composition is in the form of a liquid or gel, particularly a liquid laundry detergent.

[0204] The present invention also relates to the use of the composition of the present invention in cleaning processes such as cleaning hard surfaces, including laundry or dishwashing.

[0205] The selection of additional components for the detergent composition is within the scope of the art of those skilled in the art and includes conventional components, including the following exemplary, non-limiting components. The selection of components may involve considering the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning is performed, and the formulation of the detergent product for fabric care.

[0206] In certain embodiments, the detergent composition includes the protease variant of the present invention as well as one or more non-natural detergent components such as surfactants, hydrotropes, builders, cobuilders, chelators or chelating agents, bleaching agents or bleaching components, polymers, fabric colorants, fabric softeners, foaming accelerators, foaming inhibitors, dispersants, color transfer inhibitors, fluorescent whitening agents, fragrances, optical glossing agents, bactericides, fungicides, dirt suspenders, dirt-releasing polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0207] In one embodiment, the protease variant of the present invention may be added to the detergent composition in an amount corresponding to 0.01 to 200 mg of enzyme protein per liter of the detergent solution, preferably 0.05 to 50 mg of enzyme protein per liter of the detergent solution, and particularly 0.1 to 10 mg of enzyme protein per liter of the detergent solution.

[0208] Automatic dishwashing (ADW) compositions may contain, for example, 0.001% to 30% of enzyme proteins by weight of the composition, such as 0.5% to 10%, 0.1% to 15%, 0.01% to 20%, or 0.001% to 30%.

[0209] The granular composition for laundry may contain, for example, 0.001% to 20% of enzyme protein by weight of the composition, such as 0.05% to 5%, or 0.01% to 10%.

[0210] The liquid composition for laundry may contain, for example, 0.0001% to 10% of enzyme protein by weight of the composition, such as 0.1% to 5%, or 0.001% to 7%.

[0211] Enzymes such as the protease variants of the present invention may be stabilized using conventional stabilizers, such as polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid, or boric acid derivatives, such as aromatic boric acid esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid. The compositions may be formulated, for example, as described in International Publication No. 1992 / 19709 and International Publication No. 1992 / 19708, or the variants of the present invention may be stabilized using peptaldehydes or ketones, such as those described in International Publication No. 2005 / 105826 and International Publication No. 2009 / 118375.

[0212] The protease variant of the present invention is a) At least 0.01 mg of active protease variant per liter of detergent, b) At least one surfactant in an amount of 2 wt% to 60 wt% c) At least one type of builder with a concentration of 5 wt% to 50 wt% It can be incorporated into liquid laundry compositions, such as liquid laundry compositions containing [the specified ingredient].

[0213] Detergent compositions may be incorporated into granular detergents for laundry. Such detergents a) At least 0.01 mg of an active protease variant per gram of the composition b) Anionic surfactant, preferably 5 wt% to 50 wt% c) Nonionic surfactant, preferably 1 wt% to 8 wt% d) Builders such as carbonates, zeolites, phosphate builders, calcium scavenging builders, or complexing agents, preferably 5 wt% to 40 wt% It may include.

[0214] The components listed below are classified by general headings according to their specific functionalities, but this should not be interpreted as limiting, as those skilled in the art may understand that the components may include additional functionalities.

[0215] surfactant The detergent composition may contain one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semipolar 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. Surfactants are typically present at levels of about 0.1% to 60% by weight, such as about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactants are selected based on the desired cleaning application and include any conventional surfactants known in the art. Any surfactant known in the art may be used for use in detergents. The surfactants reduce the surface tension in the detergent, lifting, dispersing, and subsequently rinsing away the dirt being cleaned.

[0216] If included, detergents typically contain approximately 1% to 40% by weight of anionic surfactants, for example, approximately 5% to 15%, or approximately 20% to 25%, including approximately 5% to 30% anionic surfactants. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diyrbis(sulfate), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), aliphatic alcohol sulfates (FAS), primary alcohol sulfates (PAS), and alcohol ethers. Examples include sulfates (AES, AEOS, or FES, also known as alcohol ethoxysulfates or aliphatic alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, α-sulfo fatty acid methyl esters (α-SFMe or SES) including methyl ester sulfonates (MES), alkyl- or alkenyl succinates, dodecenyl / tetradecenyl succinates (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinates or soaps, and combinations thereof.

[0217] If present, detergents typically contain 0% to about 10% by weight of cationic surfactants. Non-exclusive examples of cationic surfactants include alkyldimethylethanol quaternary amines (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0218] If present, detergents typically contain approximately 0.2% to 40% by weight of nonionic surfactants, for example, approximately 0.5% to 30%, particularly approximately 1% to 20%, approximately 3% to 10%, for example, approximately 3% to 5%, or approximately 8% to 12%. Non-exclusive examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated aliphatic alcohols (PFA), alkoxylated fatty acid alkyl esters, e.g., ethoxylated fatty acid alkyl esters and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide, GA, or fatty acid glucamide, FAGA), as well as products available under the trademark names SPAN and TWEEN, and combinations thereof.

[0219] If present, detergents typically contain approximately 0% to 10% by weight of semipolar surfactants. Non-exclusive examples of semipolar surfactants include amine oxides (AOs) such as alkyldimethylamine oxide, N-(cocoalkyl)-N,N-dimethylamine oxide and N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0220] If included, detergents typically contain approximately 0% to 10% by weight of zwitterionic surfactants. Non-exclusive examples of zwitterionic surfactants include betaine, alkyldimethyl betaine, sulfobetaine, and combinations thereof.

[0221] Builders and co-builders Detergent compositions may contain about 0-65% by weight, for example, about 5-45%, of detergent builders or co-builders, or mixtures thereof. In dishwashing detergents, the builder level is typically 40-65%, particularly 50-65%. Builders and chelators soften the wash water, for example, by removing metal ions from the liquid. Builders and / or co-builders may be chelating agents that form 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, e.g., sodium triphosphate (STP or STPP), carbonates, e.g., sodium carbonate, soluble silicates, e.g., sodium metasilicate, layered silicates (e.g., Hoechst's SKS-6), ethanolamines, e.g., 2-aminoethane-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2''-nitrilotriethanol), and carboxymethyl inulin (CMI), as well as combinations thereof.

[0222] The detergent composition may also contain 0 to 20% by weight, for example, about 5% to about 10%, of detergent cobuilders, or mixtures thereof. The detergent composition may contain cobuilders alone or in combination with a builder, such as a zeolite builder. Non-limiting examples of cobuilders include homopolymers or copolymers thereof of polyacrylates, for example, poly(acrylic acid) (PAA) or copoli(acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrates, chelating agents, for example, aminocarboxylates, aminopolycarboxylates, and phosphonates, as well as 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), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), and ethylenediaminetetra(methylenephosphonic acid). Aspartic acid (EDTMPA), diethylenetriaminepentakis(methylenephosphonic acid) (DTPMPA or DTMPA), 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), sulfanyl Examples include 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 thereof and salts. Further exemplary builders and / or co-builders are described, for example, in International Publication No. 2009 / 102854 and U.S. Patent No. 5,977,053.

[0223] The protease variant of the present invention also, a) At least 0.01 mg of an active protease variant according to the present invention, and b) Preferably, 10 to 50 wt% of a builder 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 A dishwashing composition containing this may be incorporated into an automatic dishwashing composition (ADW).

[0224] Bleaching type Detergents may contain 0 to 50% by weight of a bleaching agent, for example, about 0.1% to about 25%. Bleaching agents often remove discoloration by oxidation, and many bleaches also have strong bactericidal properties and are used for disinfection and sterilization. Any bleaching agent known in the art for use in laundry detergents may be used. Suitable bleaching agent components include bleaching catalysts, photodecolorizing agents, bleaching activators, sources of hydrogen peroxide such as sodium percarbonate, and sodium perborate, pre-formed peracids and mixtures thereof. Suitable pre-formed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonates and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxon(R), and mixtures thereof. A non-limiting example of a bleaching system is a peroxide-based bleaching system, which, in combination with a peracid-forming bleaching activator, may include, for example, an inorganic salt containing alkali metal salts such as sodium perboric acid (usually monohydrate or tetrahydrate), percarbonate, persulfate, superphosphate, or persilicate.

[0225] The term "bleach activator" as used herein means a compound that reacts with a peroxygen bleaching agent, such as hydrogen peroxide, to form a peracid. The peracid thus formed constitutes activated bleaching. Suitable bleach activators for use 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), diperoxide dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and / or those disclosed in International Publication No. 98 / 17767. A specific family of bleach activators of interest is disclosed in European Patent No. 624154, of which acetyltriethyl citrate (ATC) is particularly preferred. ATC or short-chain triglyceride-like triacetin has the advantage of being environmentally friendly because it ultimately decomposes into citric acid and alcohol. Furthermore, acetyltriethyl citrate and triacetin have good hydrolysis stability of the product during storage, making them effective bleach activators. Finally, ATC provides good builder ability for laundry additives. Alternatively, the bleaching system may include, for example, amide, imide, or sulfone-type peroxy acids. The bleaching system may also include peracids such as 6-(phthalimide)peroxyhexanoic acid (PAP). The bleaching system may also include a bleaching catalyst or a bleaching accelerator.

[0226] 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 complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2 and [2,2',2''-nitrilotris(ethane-1,2-diirazanylylidene-κN-metanylylidene)triphenolate-κ3O]manganese(III). The bleaching catalyst may also be other metal compounds such as iron or cobalt complexes.

[0227] In some embodiments, the bleaching component may be an organic catalyst selected from the group consisting of organic catalysts having the following formula: [ka] (iii) and mixtures thereof (wherein each R1 is independently a branched alkyl group containing 9 to 24 carbon atoms or a linear alkyl group containing 11 to 24 carbon atoms, preferably each R1 is independently a branched alkyl group containing 9 to 18 carbon atoms or a linear alkyl group containing 11 to 18 carbon atoms, 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 International Publication Nos. 2007 / 087258, 2007 / 087244, 2007 / 087259, and 2007 / 087242. A suitable photobleaching agent may be, for example, zinc phthalocyanine sulfonate.

[0228] Hydrotrope Hydrotropes are compounds that solubilize hydrophobic compounds in aqueous solutions (or substances of opposite polarity in a nonpolar environment). While hydrotropes typically possess both hydrophilic and hydrophobic properties (so-called amphiphilic properties, as seen in surfactants), the molecular structure of hydrotropes generally does not readily undergo spontaneous self-aggregation; see, for example, the overview 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 found with respect to surfactants and lipids forming micelle phases, lamellar phases, or other distinct mesophases. In fact, many hydrotropes exhibit a persistent aggregation process where aggregate size increases with increasing concentration. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing polar and nonpolar substances, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries ranging from pharmaceuticals, personal care, and food to technological applications. The use of hydrotropes in detergent compositions allows for more concentrated formulations of surfactants (for example, in the process of concentrating liquid detergents by removing water) without inducing undesirable phenomena such as phase separation or high viscosity.

[0229] Detergents may contain 0 to 5% by weight of hydrotropes, for example, about 0.5 to about 5%, or about 3 to about 5%. Any hydrotrope known in the art for use in detergents may be used. 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.

[0230] polymer The detergent may contain 0 to 10% by weight of a polymer, for example, 0.5 to 5%, 2 to 5%, 0.5 to 2%, or 0.2 to 1%. Any polymer known in the art for use in detergents may be used. The polymer may function as a cobuilder as described above, or it may impart anti-re-adhesion, fiber protection, dirt release, color transfer prevention, grease cleaning, and / or defoaming properties. Some polymers may have two or more of the above properties and / or two or more of the following motifs. Examples of 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 copolymer, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycol, 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 oxides and polypropylene oxides (PEO-PPO), and diquaternium ethoxysulfate. Other exemplary polymers are disclosed, for example, in International Publication No. 2006 / 130575. Salts of the above polymers are also being considered.

[0231] Fabric color adjusters The detergent composition of the present invention may also contain a fabric colorant such as a dye or pigment, which, when incorporated into the detergent composition, can deposit on the fabric when the cleaning solution containing the detergent composition comes into contact with the fabric, thereby altering the fabric's hue through the absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric colorants absorb at least some of the visible light spectrum, thus altering the surface hue. Suitable fabric colorants include dyes and dye-clay conjugates, which may also include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include, for example, small molecule dyes selected from the group consisting of dyes classified under the color index (CI) classification of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, as described in International Publication No. 2005 / 003274, International Publication No. 2005 / 003275, International Publication No. 2005 / 003276 and European Patent No. 1876226 (incorporated herein by reference), or mixtures thereof. The detergent composition preferably contains about 0.00003 wt% to about 0.2 wt%, about 0.00008 wt% to about 0.05 wt%, or about 0.0001 wt% to about 0.04 wt% of a fabric colorant. The composition may also contain 0.0001 wt% to about 0.2 wt% of a fabric colorant, which is particularly preferred when the composition is in the form of unit dose pouches. Suitable colorants are disclosed, for example, in International Publication No. 2007 / 087257 and International Publication No. 2007 / 087243.

[0232] additional enzymes The detergent composition may contain one or more additional enzymes such as amylase, arabinase, carbohydrase, cellulase (e.g., endoglucanase), cutinase, deoxyribonuclease, galactanase, haloperoxygenase, lipase, mannanase, oxidase (e.g., lactase and / or peroxidase), pectinase, pectin lyase, further proteases, xylanase, xanthanase, xyloglucanase, or oxidoreductase.

[0233] When the composition contains one or more additional enzymes, the additional enzymes are preferably amylase and / or lipase, particularly amylase.

[0234] The properties of the selected enzyme should be compatible with the selected detergent (e.g., optimal pH, compatibility with other enzymatic and non-enzymatic components).

[0235] Protease: The composition may contain, in addition to the protease variant of the present invention, one or more additional proteases, including those of bacterial, fungal, plant, viral, or animal origin. Bacterial proteases are preferred. The protease 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 another metalloprotease such as those from the M5, M7, or M8 family.

[0236] Examples of metalloproteases include those derived from Bacillus amyloliquefaciens, as described in International Publication No. 2007 / 044993 (Genencor Int.), which are neutral metalloproteases.

[0237] Suitable commercially available protease enzymes include Alcalase(registered trademark), Duralase(registered trademark), Durazym(registered trademark), Relase(registered trademark), Relase(registered trademark) Ultra, Savinase(registered trademark), Savinase(registered trademark) Ultra, Primase(registered trademark), Polarzyme(registered trademark), Kannase(registered trademark), Liquanase(registered trademark), Liquanase(registered trademark) Ultra, Ovozyme(registered trademark), Coronase(registered trademark), Coronase(registered trademark) Ultra, Blaze(registered trademark), Blaze Evity(registered trademark) 100T, Blaze Evity(registered trademark) 125T, Blaze Evity(registered trademark) 150T, Neutrase(registered trademark), Everlase(registered trademark), Esperase(registered trademark), Progress(registered trademark) Uno, and Progress(registered trademark) Excel(Novozyme). Products sold under A / S include the following trademarks: Maxatase (registered trademark), Maxacal (registered trademark), Maxapem (registered trademark), Purafect (registered trademark), Purafect (registered trademark) Ox, Purafect (registered trademark) OxP, Purafect Prime (registered trademark), Puramax (registered trademark), FN2 (registered trademark), FN3 (registered trademark), FN4 (registered trademark), Excellase (registered trademark), Excellenz P1000 (trademark), Excellenz P1250 (trademark), Eraser (registered trademark), Preferenz (registered trademark) P100, Preferenz (registered trademark) P110, Effectenz P1000 (trademark), Effectenz P1050 (trademark), Effectenz P2000 (trademark), Purafast (registered trademark), Properase (registered trademark), Opticlean (registered trademark), and Optimase (registered trademark) (Danisco / DuPont), Axapem (trademark) (Gist-Brocases NV)Examples of products sold under this designation include BLAP (the sequence shown in Figure 29 of U.S. Patent No. 5,352,604) and its variant (Henkel AG), as well as KAP (Bacillus alkalophilus subtilisin) from Kao.

[0238] Lipase and cutinase Suitable lipases and cutinases include those of bacterial or fungal origin. These include chemically modified mutant enzymes or protein-manipulated mutant enzymes. Examples include those from the genus Thermomyces, such as T. lanuginosus (formerly Humicola lanuginosa), as described in European Patent No. 258068 and European Patent No. 305216. Lipase derived from (formerly named lanuginosa), for example, cutinase from Humicola species such as H. insolens (International Publication No. 96 / 13580), for example, P. alcaligenes or P. pseudoalcaligenes (European Patent No. 218272), P. cepacia (European Patent No. 331376), P. sp. strain SD705 (International Publication No. 95 / 06720 and International Publication No. 96 / 27002), P. wisconsinensis (P.Lipases derived from strains of the genus Pseudomonas (some of which are now renamed Burkholderia), such as Pseudomonas wisconsinensis (International Publication No. 96 / 12012), GDSL-type Streptomyces lipase (International Publication No. 2010 / 065455), cutinase derived from the rice blast fungus (Magnaporthe grisea) (International Publication No. 2010 / 107560), cutinase derived from Pseudomonas mendocina (U.S. Patent No. 5,389,536), lipase derived from Thermobifida fusca (International Publication No. 2011 / 084412), and Geobacillus stearothermophilus. Examples include lipase derived from *Stearothermophilus* (International Publication No. 2011 / 084417), lipase derived from *Bacillus subtilis* (International Publication No. 2011 / 084599), and lipase derived from *Streptomyces griseus* (International Publication No. 2011 / 150157) and *S. pristinaespiralis* (International Publication No. 2012 / 137147).

[0239] Other examples include lipase variants described in European Patent No. 407225, International Publication No. 92 / 05249, International Publication No. 94 / 01541, International Publication No. 94 / 25578, International Publication No. 95 / 14783, International Publication No. 95 / 30744, International Publication No. 95 / 35381, International Publication No. 95 / 22615, International Publication No. 96 / 00292, International Publication No. 97 / 04079, International Publication No. 97 / 07202, International Publication No. 00 / 34450, International Publication No. 00 / 60063, International Publication No. 01 / 92502, International Publication No. 2007 / 87508, and International Publication No. 2009 / 109500.

[0240] Preferred commercially available lipase products include Lipolase®, Lipex®; Lipolex® and Lipoclean® (Novozymes A / S), Lumafast (originally from Genencor), and Lipomax (originally from Gist-Brocades).

[0241] Further examples include lipases, sometimes called acyltransferases or perhydrolases, such as an acyltransferase homologous to Candida antarctica lipase A (International Publication No. 2010 / 111143), an acyltransferase derived from Mycobacterium smegmatis (International Publication No. 2005 / 056782), a perhydrolase derived from the CE 7 family (International Publication No. 2009 / 067279), and variants of M. smegmatis perhydrolase, particularly the S54V variant used in the Gentle Power Bleach product manufactured by Huntsman Textile Effects Pte Ltd (International Publication No. 2010 / 100028).

[0242] amylase Suitable amylases that can be used with the protease variant of the present invention may be alpha-amylases or glucoamylases, and may be of bacterial or fungal origin. These include chemically modified mutants or protein-manipulated mutants. Examples of amylases include alpha-amylases obtained from specific strains of the genus Bacillus, such as Bacillus licheniformis, which is described in more detail in British Patent No. 1,296,839.

[0243] Preferred amylases include amylases having SEQ ID NO: 2 in International Publication No. 95 / 10603 or variants having 90% sequence identity with SEQ ID NO: 3. Preferred variants are those described in SEQ ID NO: 4 in International Publication Nos. 94 / 02597, 94 / 18314, 97 / 43424 and 99 / 19467, and are variants having substitutions at one or more of the following positions, for example: 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.

[0244] Other preferred amylases include the amylase having SEQ ID NO: 6 in International Publication No. 2002 / 10355 or a variant thereof having 90% sequence identity with SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 have deletions at positions 181 and 182 and a substitution at position 193.

[0245] Other preferred amylases are hybrid alpha-amylases or variants having 90% sequence identity thereto, comprising residues 1-33 of alpha-amylase derived from B. amyloliquefaciens, as shown in SEQ ID NO. 6 of International Publication No. 2006 / 066594, and residues 36-483 of alpha-amylase from B. licheniformis, as shown in SEQ ID NO. 4 of International Publication No. 2006 / 066594. Preferred variants of this hybrid alpha-amylase have substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid alpha-amylase containing residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO. 6 of International Publication No. 2006 / 066594 and residues 36-483 of SEQ ID NO. 4 is the substitution: M197T; H156Y+A181T+N190F+A209V+Q264S; or G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S It possesses the following characteristics.

[0246] Other preferred amylases are those having the sequence of SEQ ID NO: 6 in International Publication No. 99 / 19467, or variants thereof having 90% sequence identity with SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 have 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 have deletions at positions R181 and G182, or at positions H183 and G184.

[0247] 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 in International Publication No. 96 / 23873, or variants thereof having 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7 have substitutions, deletions, or insertions at one or more of the following positions, using SEQ ID NO: 2 in International Publication No. 96 / 23873 for numbering: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476. More preferred variants have deletions at two positions selected from 181, 182, 183, and 184, for example, 181 and 182, 182 and 183, or positions 183 and 184. The most preferred amylase variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 has deletions at positions 183 and 184 and substitutions at one or more positions 140, 195, 206, 243, 260, 304, and 476.

[0248] Other amylases that may be used are amylases having SEQ ID NO: 2 in International Publication No. 2008 / 153815, SEQ ID NO: 10 in International Publication No. 01 / 66712, or variants thereof having 90% sequence identity with SEQ ID NO: 2 in International Publication No. 2008 / 153815 or 90% sequence identity with SEQ ID NO: 10 in International Publication No. 01 / 66712. Preferred variants of SEQ ID NO: 10 in International Publication No. 01 / 66712 have substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0249] Further preferred amylases are those having SEQ ID NO: 2 in International Publication No. 2009 / 061380 or variants having 90% sequence identity with SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 have C-terminal shortening and / or substitution, deletion or insertion at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. A more preferred variant of SEQ ID NO: 2 has 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 R180 and / or S181 or T182 and / or G183. The most preferred amylase variant of SEQ ID NO: 2 has 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 It has the following characteristics: The variant has a shortened C-terminus and optionally further includes a substitution at position 243 and / or deletions at positions 180 and / or 181.

[0250] A further suitable amylase is an amylase having SEQ ID NO: 1 in WO 2013 / 184577 pamphlet or a variant having 90% sequence identity with SEQ ID NO: 1 thereof. 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, I203, S241, R458, T459, D460, G476 and G477. The most preferred variant of SEQ ID NO: 1 is a substitution at one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K, and / or a deletion at position R178 and / or S179 or T180 and / or G181. The most preferred amylase variant of SEQ ID NO: 1 is the substitution: E187P + I203Y + G476K including E187P + I203Y + R458N + T459S + D460T + G476K, and optionally further including a substitution at position 241 and / or a deletion at position 178 and / or 179.

[0251] A further suitable amylase is an amylase having SEQ ID NO: 1 in WO 2010 / 104675 pamphlet or a variant having 90% sequence identity with SEQ ID NO: 1 thereof. 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.

[0252] The most preferred variant of SEQ ID NO: 1 has 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 includes the substitution N21D+D97N+V128I and optionally further includes a substitution at position 200 and / or deletions at positions 180 and / or 181.

[0253] Other preferred amylases are alpha-amylases having SEQ ID NO: 12 in International Publication No. 01 / 66712, or variants having at least 90% sequence identity with SEQ ID NO: 12. Preferred amylase variants have substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO: 12 in International Publication No. 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. Specific preferred amylases include variants having deletions of D183 and G184 and substitutions R118K, N195F, R320K and R458K, as well as variants further having substitutions at one or more positions selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, and the most preferred variant further having substitutions at all of these positions.

[0254] Other examples include amylase variants, such as those described in International Publication No. 2011 / 098531, International Publication No. 2013 / 001078, and International Publication No. 2013 / 001087. Commercially available amylases include Duramyl™, Teramyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, BAN™, Amplify™ and Amplify™ Prime (manufactured by Novozymes A / S), as well as Rapidase™, Purastar™ / Effectenz™, Powerase, Preferenz S1000, Preferenz S110, and Preferenz S110 (manufactured by Genencor International Inc. / DuPont).

[0255] One preferred amylase is a variant of the amylase having Sequence ID No. 13 in International Publication No. 2016 / 180748, having Modified H1*+N54S+V56T+K72R+G109A+F113Q+R116Q+W167F+Q172G+A174S+G182*+D183*+G184T+N195F+V206L+K391A+P473R+G476K.

[0256] Another preferred amylase is a variant of the amylase having Sequence ID No. 1 in International Publication No. 2013 / 001078, having Modified D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K.

[0257] Another preferred amylase is a variant of the amylase having Sequence ID No. 1 in International Publication No. 2018 / 141707, having Modified H1*+G7A+G109A+W140Y+G182*+D183*+N195F+V206Y+Y243F+E260G+N280S+G304R+E391A+G476K.

[0258] A further preferred amylase is the variant of amylase having Sequence ID No. 1 in International Publication No. 2017 / 191160, which has Modified L202M + T246V.

[0259] Deoxyribonuclease (DNase) A suitable deoxyribonuclease (DNase) is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thereby degrading DNA. DNases obtainable from bacteria are preferred, particularly those obtainable from species of the genus Bacillus; in particular, DNases obtainable from Bacillus subtilis or Bacillus licheniformis are preferred. Examples of such DNases are described in International Publication No. 2011 / 098579 and International Publication No. 2014 / 087011.

[0260] Oxidoreductase In one embodiment, the composition may contain an oxidoreductase, which is an enzyme that catalyzes a reduction-oxidation reaction. A preferred oxidoreductase is superoxide dismutase.

[0261] Peroxidase / Oxidase Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. These include chemically modified mutants or protein-manipulated mutants. Examples of useful peroxidases include those from the genus Coprinus, such as the peroxidase from C. cinereus, and its variants, as described in International Publication No. 93 / 24618, International Publication No. 95 / 10602, and International Publication No. 98 / 15257.

[0262] A commercially available peroxidase is Guardzyme® (Novozymes A / S).

[0263] Auxiliary materials Any detergent components known in the art for use in laundry detergents may also be used. Other optional detergent components include, alone or in combination, anticorrosive agents, anti-shrinkage agents, anti-redeposition agents, anti-wrinkle agents, disinfectants, binders, corrosion inhibitors, disintegrants / disintegrating agents, dyes, enzyme stabilizers (polyols such as boric acid, borates, CMC, and / or propylene glycol), fabric softeners containing clay, fillers / processing aids, fluorescent whitening agents / optical gloss agents, foaming accelerators, foam (soap foam) regulators, fragrances, stain suspenders, softeners, foaming inhibitors, yellowing inhibitors, and wicking agents. Any component known in the art for use in laundry detergents may be used. The selection of such components is well within the scope of the art of those skilled in the art.

[0264] 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 copolymer acids or salts thereof, where the polycarboxylic acid contains at least two carboxyl groups separated from each other by two or fewer carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant Science Series volume 71, Marcel Dekker, Inc., 1997.

[0265] Anti-color transfer agents: The detergent composition of the present invention may also contain one or more anti-color transfer agents. Suitable polymer anti-color transfer agents 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 of the subject, the anti-color transfer agent may be present at a level of about 0.0001% to about 10% by weight, about 0.01% to about 5% by weight, or about 0.1% to about 3% by weight of the composition.

[0266] Fluorescent whitening agents: The detergent compositions of the present invention may also preferably contain additional components that can impart color to the articles being cleaned, such as fluorescent whitening agents or optical glossing agents. If present, the glossing agent is preferably at a level of about 0.01% to about 0.5%. Any fluorescent whitening agent suitable for use in laundry detergent compositions may be used in the compositions of the present invention. The most commonly used fluorescent whitening agents belong to the classes of diaminostilbene sulfonic acid derivatives, diarylpyrazoline derivatives, and bisphenyl distyryl derivatives. Examples of diaminostilbene-sulfonic acid derivative type fluorescent whitening agents include 4,4'-bis-(2-diethanolamino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2,4-dianilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; and 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazine-6-ylamino)stilbene-2,2'-disulfonate. Examples include the sodium salts of ruben-2,2'-disulfonate, 4,4'-bis-(4-phenyl-2,1,3-triazole-2-yl)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, and 2-(stilbil-4''-naphtho-1.,2':4,5)-1,2,3-triazole-2''-sulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, commercially available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl)disulfonate. A preferred fluorescent whitening agent is the commercially available Parawhite KX from Paramount Minerals and Chemicals, Mumbai, India.Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazoline and 7-alkylaminocoumarin. Suitable levels of fluorescent gloss include lower limits of about 0.01, 0.05, about 0.1, or about 0.2 wt%, and upper limits of 0.5 or even 0.75 wt%.

[0267] Dirt-Removing Polymers: The detergent compositions of the present invention may also contain one or more dirt-removing polymers that assist in the removal of dirt from fabrics such as cotton and polyester fabrics, particularly hydrophobic dirt from polyester fabrics. The dirt-removing polymers may be, for example, nonionic or anionic terephthalate polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, or polyester polyamides; see, for example, Chapter 7, Powdered Detergents, Surfactant Science Series Volume 71, Marcel Dekker, Inc. Another type of dirt-removing polymer is an amphiphilic alkoxylated oil-cleaning polymer comprising a core structure and a plurality of alkoxylate groups bonded to this core structure. The core structure may include a polyalkylene imine structure or a polyalkanolamine structure, as detailed in International Publication No. 2009 / 087523 (incorporated herein by reference). Furthermore, random graft copolymers are preferred dirt-removing polymers. Suitable graft copolymers are described in more detail in International Publication No. 2007 / 138054, International Publication No. 2006 / 108856, and International Publication No. 2006 / 113314 (incorporated herein by reference). Other fouling-free polymers are substituted polysaccharide structures, particularly substituted cellulose structures, such as modified cellulose derivatives, as described in European Patent No. 1867808 or International Publication No. 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 anionic modified cellulose, nonionic modified cellulose, cationic modified cellulose, zwitterionic modified cellulose, and mixtures thereof.Suitable cellulose polymers include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxymethyl cellulose, and mixtures thereof.

[0268] Redeposition inhibitor: The detergent composition of the present invention may also contain one or more redeposition inhibitors such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), a homopolymer of acrylic acid, a copolymer of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. Under the aforementioned soil-release polymer, the above cellulose polymer can also function as a redeposition inhibitor.

[0269] Other suitable auxiliary materials include anti-shrinkage agents, anti-wrinkle agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foaming regulators, fragrances, pigments, foaming agents, solvents, and structuring agents and / or structure elasticizing agents for liquid detergents.

[0270] Formulation of detergent products The detergent enzyme, that is, the protease variant 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 these 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, particularly non-dusting granules, liquids, particularly stabilized liquids, or slurries.

[0271] The detergent composition of the present invention may be in any convenient form, such as bars, homogeneous tablets, tablets having two or more layers, pouches having one or more compartments, structured or dense powders, granules, pastes, gels, or structured, dense or concentrated liquids. There are several forms of detergent formulations, such as layers (same or different phases), pouches, and forms for mechanical dispensing units.

[0272] The pouch can be configured as one or more compartments. This may be any form, shape, and material suitable for holding the composition, for example, to prevent the composition from being released from the pouch before contact with water. The pouch is made from a water-soluble film that encloses the contents. The internal volume can be divided into compartments in the pouch. Preferred films are polymer materials, preferably polymers 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, most preferably polyvinyl alcohol copolymers, and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer in the film, for example PVA, is at least about 60%. The preferred average molecular weight is usually about 20,000 to about 150,000. The film may also be hydrolyzable and may be a blend composition comprising a water-soluble polymer blend, such as polylactide and polyvinyl alcohol (known by trade name M8630, sold by Chris Craft In. Prod. of Gary, Indiana, US), and plasticizers such as glycerol, ethyleneglycerol, propylene glycol, sorbitol, and mixtures thereof. The pouch may contain a solid laundry detergent composition or a portion of its components separated by the water-soluble film and / or a liquid cleaning composition or a portion of its components. The compartment for the liquid components may have a different composition from the compartment containing the solids. (See, for example, U.S. Patent Application Publication No. 2009 / 0011970).

[0273] Detergent components can be physically separated from each other by compartments within a water-soluble pouch or by various layers of a tablet. This avoids undesirable storage interactions between components. The varying dissolution profiles of each compartment also allow for delayed dissolution of selected components in the cleaning solution.

[0274] Liquid or gel detergents that are not in unit doses may be water-soluble and typically contain at least 20% by weight and up to 95% water, for example, up to about 70%, up to about 65%, up to about 55%, up to about 45%, or up to about 35% water. Concentrated liquid detergents may have a lower water content, for example, up to about 30% or less or up to about 20%, in the range of about 1% to about 20%, such as about 2% to about 15%. Other types of liquids, such as alkanols, amines, diols, ethers, and polyols, may be included in water-soluble liquids or gels, but are not limited to these. Water-soluble liquid or gel detergents may contain 0 to 30% organic solvents. Liquid or gel detergents may be non-water-soluble.

[0275] Liquid detergent compositions may be formulated to have a moderate pH of about 6 to about 10, such as about pH 7, about pH 8, or about pH 9, or they may be formulated to have a higher pH of about 10 to about 12, such as about pH 10, about pH 11, or about pH 12.

[0276] Unless otherwise indicated, the term “liquid” as used herein should be understood to include all types of liquid detergent compositions, such as concentrated liquids, gels, or, for example, the liquid or gel portion of a pouch having one or more compartments.

[0277] Laundry soap bar The enzymes of the present invention may be added to laundry soap bars and used for hand-washing laundry, fabrics and / or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, synthetic detergent bars and detergent bars. The types of bars usually differ by the type of surfactant they contain, and the term laundry soap bar includes those containing fatty acid-derived soaps and / or synthetic soaps. Laundry soap bars are solid and therefore have a physical form that is not liquid, gel or powder at room temperature.

[0278] The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptaldehyde (or hydrosulfite adducts or hemiacetal adducts), boric acid, borates, borax and / or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerin, pH-controlling compounds such as fatty acids, citric acid, acetic acid and / or formic acid, and / or salts of monovalent cations and organic anions, where the monovalent cation is, for example, Na + , K + or NH4 + The organic anion may be, for example, a formate, acetate, citrate, or lactate, and as a result, the salt of the monovalent cation and organic anion may be, for example, sodium formate.

[0279] Laundry soap bars may also contain complexing agents such as EDTA and HEDP, fragrances and / or different types of fillers, surfactants, such as anionic synthetic surfactants, builders, polymeric stain removers, detergent chelators, stabilizers, fillers, dyes, colorants, stain transfer inhibitors, alkoxylated polycarbonates, foam inhibitors, structuring agents, binders, leaching agents, bleach activators, clay stain removers, redeposition inhibitors, polymeric dispersants, glossing agents, fabric softeners, fragrances and / or other compounds known in the art.

[0280] Laundry soap bars can be processed in conventional laundry soap bar manufacturing apparatus, such as agitators, compressors, two-stage vacuum compressors, extruders, cutters, logo stampers, cooling tunnels, and packaging machines, but not limited to these. A pre-mixed mixture 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 the mixture is subsequently prodded. The enzyme and optionally additional enzymes may be added simultaneously with the protease inhibitor, for example, in liquid form. In addition to the mixing and prodding steps, the process may further include steps of grinding, extrusion, cutting, stamping, cooling, and / or packaging.

[0281] Granular detergent formulation Enzymes in the form of enzyme-containing cores and granules containing optionally one or more coatings are commonly used in granular (powder) detergents. Various methods for preparing cores are well known in the art and include, for example, a) spray drying of a liquid enzyme-containing solution, b) production of a layered product having enzyme coated as a layer around pre-formed inert core particles using, for example, a fluidized bed apparatus, c) absorption of enzyme onto and / or into the surface of a pre-formed core, d) extrusion of an enzyme-containing paste, e) spraying of enzyme-containing powder in molten wax resulting in suspension and metal particle products, f) agitated granulation by adding an enzyme-containing liquid to a dry powder composition of granulation components, g) reduction in size of enzyme-containing cores by grinding or crushing larger particles, pellets, etc., and h) fluidized bed granulation. Enzyme-containing cores can be dried, for example, using a fluidized bed dryer or other known method for drying granules in the feed or enzyme industry, typically resulting in a water content of 0.1–10% w / w water.

[0282] The enzyme-containing core is optionally provided with a coating that improves storage stability and / or reduces 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, for example, as a solution of salt using a fluidized bed. Other coating materials that can be used are, for example, polyethylene glycol (PEG), methylhydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). The granules may contain two or more coatings, for example, a salt coating followed by an additional coating of a material such as PEG, MHPC, or PVA.

[0283] For further information on enzyme granules and their production, please refer to International Publication No. 2013 / 007594, as well as, for example, International Publication No. 2009 / 092699, European Patent No. 1705241, European Patent No. 1382668, International Publication No. 2007 / 001262, U.S. Patent No. 6,472,364, International Publication No. 2004 / 074419, and International Publication No. 2009 / 102854.

[0284] Instructions for use and cleaning The present invention also relates to a method for using a protease variant or composition thereof according to the present invention in the washing of fabrics and textiles, such as in household laundry and industrial laundry.

[0285] The present invention also relates to a method for using variants or compositions thereof in cleaning hard surfaces such as floors, tables, walls, and roofs, as well as the surfaces of hard objects such as cars (car washing) and tableware (dishwashing).

[0286] The protease variant of the present invention can be added and thereby become a component of a detergent composition. Accordingly, one aspect of the present invention relates to the use of the protease variant in cleaning processes such as laundry and / or hard surface cleaning.

[0287] The detergent compositions of the present invention can be formulated, for example, as hand or machine laundry detergent compositions, for example, as laundry additive compositions suitable for pre-treating soiled fabrics and as rinsing additive fabric softener compositions, or as detergent compositions for use in general household hard surface cleaning operations, or for use in hand or machine dishwashing operations.

[0288] The cleaning process or textile care process may be, for example, a laundry process, a dishwashing process, or cleaning of hard surfaces such as bathroom tiles, floors, tabletops, drainpipes, skin, and washbasins. The laundry process may be, for example, home laundry, but may also be industrial laundry. Furthermore, the present invention relates to a process for washing fabrics and / or garments, the process comprising treating the fabric with a cleaning solution containing a detergent composition and at least one protease variant of the present invention. The cleaning process or textile care process may be carried out, for example, by machine washing or by hand. The cleaning solution may be, for example, an aqueous cleaning solution containing a detergent composition.

[0289] In recent years, there has been growing interest in replacing components in detergents derived from petrochemicals with renewable biological components such as enzymes and polypeptides without compromising cleaning performance. When the components of a detergent composition change, novel enzymes with novel enzymatic activity or alternative and / or improved properties may be required compared to conventional detergent enzymes such as proteases, lipases, and amylases, in order to achieve similar or improved cleaning performance compared to conventional detergent compositions.

[0290] The present invention further relates to the use of the protease variant of the present invention in a process of removing protein-based stains. Protein-based stains may include food stains, such as baby food, cocoa, eggs or milk, or other stains such as sebum, blood, ink or grass, or combinations thereof.

[0291] Cleaning method The present invention relates to a method for cleaning fabrics, tableware, or hard surfaces with a detergent composition containing the protease variant of the present invention.

[0292] The cleaning method involves contacting an object with a detergent composition containing the 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.

[0293] Another embodiment relates to a method for removing dirt from fabrics or tableware, comprising contacting the fabrics or tableware with a composition comprising the protease of the present invention under conditions suitable for cleaning the object. In the cleaning method of the present invention, the object being cleaned may be any suitable object such as textiles or tableware or hard surfaces such as floors, tables, or walls.

[0294] Compositions and methods for treating fabrics (for example, for desizing textiles) using one or more protease variants of the present invention are also intended. The proteases can be used in any fabric treatment method well known in the art (see, for example, U.S. Patent No. 6,077,316). For example, in one embodiment, the feel and appearance of a fabric are improved by a method comprising contacting the fabric with a protease in a solution. In one embodiment, the fabric is treated with a solution under pressure.

[0295] The detergent composition of the present invention is suitable for use in hard surface applications, including laundry and dishwashing. Accordingly, the present invention includes a method for washing fabrics or dishes, comprising contacting the fabric / dish to be cleaned with a solution containing the detergent composition according to the present invention. Fabrics may include any fabric that can be washed under normal consumer use conditions. Dishes may include any tableware such as pottery, porcelain, ceramics, plastics such as melamine, metal, porcelain, glass, and acrylic resin. The solution preferably has a pH of about 5.5 to about 11.5. The composition may be used in solution at a concentration of about 100 ppm, preferably 500 ppm to about 15,000 ppm. Water temperatures typically range from approximately 5°C to 95°C, including around 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, and 90°C. The water-to-fabric ratio is typically around 1:1 to 30:1.

[0296] The enzyme in the detergent composition of the present invention is a conventional stabilizer and protease inhibitor, for example, polyols such as propylene glycol or glycerol, sugars or sugar alcohols, different salts such as NaCl or KCl; lactic acid, formic acid, boric acid, or boric acid derivatives, for example, aromatic boric acid esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid, or peptaldehydes such as di-, tri-, or tetrapeptide aldehydes or aldehyde analogs (forms B1-B0-R, where R is H, CH3, CX3, CHX2, or CH2X (X = halogen), and B0 is , is a single amino acid residue (preferably having an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (preferably 1, 2, or 3), optionally including an N-terminal protecting group (as described in International Publication No. 2009 / 118375, International Publication No. 98 / 13459) or can be stabilized using protein-type protease inhibitors such as RASI, BASI, WASI (bifunctional alpha-amylase / subtilisin inhibitors for rice, barley, and wheat) or CI2 or SSI. The composition may be formulated as described, for example, in International Publication No. 92 / 19709, International Publication No. 92 / 19708 and U.S. Patent No. 6,472,364. In some embodiments, the enzymes used 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 yield 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)).

[0297] The detergent compositions provided herein are typically formulated so that the wash water has a pH of about 5.0 to about 12.5, such as about 5.0 to about 11.5, or about 6.0 to about 10.5, during use in aqueous cleaning operations. In some embodiments, the granular or liquid laundry products are formulated to have a pH of about 6 to about 8. Techniques for controlling the pH at the recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0298] The present invention can be further described by the following embodiments, which should not be construed as limiting the scope of the invention. [Examples]

[0299] Preparation and purification of polypeptides The introduction of mutations and expression cassettes into Bacillus subtilis was carried out by standard methods known in the art. All DNA manipulation was performed by PCR using standard methods known to those skilled in the art (e.g., as described by Sambrook et al, 2001). Recombinant B. subtilis constructs encoding protease polypeptides were seeded in compound medium (TBgly) under antibiotic selectivity and cultured at 37°C for 24 hours. The culture was seeded overnight in a nutrient-rich medium (PS-1) containing 100 g / L sucrose (Danisco cat. no. 109-0429), 40 g / L hulled soybeans (soybean flour), 10 g / L Na2HPO412H2O (Merck cat. no. 106579), and 0.1 ml / L Dowfax63N10 (Dow) in a ratio of 1:100 in a shaking flask. Shaking flask culture was carried out at 30°C for 4 days while shaking at 270 rpm.

[0300] Purification of the culture supernatant was performed as follows: The culture broth was centrifuged at 26,000xg for 20 minutes, and the supernatant was carefully discarded from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filtration unit to remove any remaining host cells. The pH of the 0.2 μm filtrate was adjusted to pH 8 with 3 M Tris base, and the pH-adjusted filtrate was applied to a MEP Hypercel column (Pall Corporation) equilibrated in 20 mM Tris / HCl, 1 mM CaCl2, pH 8.0. After washing the column with equilibration buffer, the column was eluted stepwise in 20 mM CH3COOH / NaOH, 1 mM CaCl2, pH 4.5. The fraction from the column was analyzed for protease activity using a Suc-AAPF-pNA assay at pH 9, and the 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 3M Tris base. The pH-adjusted pool is then 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, the protease variant is eluted over 5 column volumes with a linear NaCl gradient (0→0.5M) in the same buffer. The fraction from the column is analyzed for protease activity using a Suc-AAPF-pNA assay at pH 9, and the active fraction is analyzed by SDS-PAGE. Fractions in which only one band is observed on the Coomassi-stained SDS-PAGE gel are pooled as purified preparations and used for further experiments.

[0301] Protease activity assay Proteolytic activity can be determined by a method using the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide, which is a blocked peptide that can be cleaved by endoprotease. After proteolytic cleavage, yellow free pNA molecules are released and can be measured by visible spectrophotometry at a wavelength of 405 nm. The Suc-AAPF-PNA substrate is manufactured by Bachem (catalog number L1400, dissolved in DMSO).

[0302] The protease sample to be analyzed is diluted in the remaining active buffer (100 mM Tris, pH 8.6). The assay is performed by transferring 30 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 70 μl of the substrate standard solution (0.72 mg / ml in 100 mM Tris, pH 8.6). The solution is mixed at room temperature, and the absorption is measured at OD 405 nm every 20 seconds for 5 minutes.

[0303] The slope of the time-dependent absorption curve (absorbance per minute) is directly proportional to the activity of the protease of interest under a given set of conditions. The protease sample should be diluted to a level where the slope is linear.

[0304] Example 1. Improved solubility of protease variants Definition: Fermentation broth: ● A CB = Total protease activity in the fermentation broth (including crystallized protease). ● A CB SUP = Dissolved protease activity in the fermentation broth. ● A CB PEL = Pellet protease activity in the fermentation broth (including crystallized protease). ● A INIT = Percentage of dissolved protease activity in the fermentation broth.

[0305] Protease crystal dissolution: ● A FULL =Complete protease activity (including crystallized protease) in diluted fermentation broth. ● A EXP =Expected total protease activity of diluted fermented broth based on the protease activity of fermented broth and dilution factors:

number

number

number

[0306] Materials and methods: Initial dissolved protease activity in fermented broth Fermentation broths were collected from host cells expressing protease variants with and without substitution at the position corresponding to A215 of Sequence ID No. 1, and the protease crystals were analyzed. The presence of protease crystals was confirmed by optical microscopy (Olympus BX51) and powder X-ray diffraction (XRPD, PANalytical Empyrean) as described in Acta Cryst. (Frankaer, CG, et.al. (2014). Acta Cryst. D70, 1115-1123).

[0307] Crystallization / formation is related to the initial dissolved protease activity (A) in fermentation broth. INITThe activity of the dissolved protease (non-crystallized protease fraction) was evaluated by investigating the activity of the dissolved protease. The following samples were collected: ● A CB Complete fermented broth sample (containing crystallized protease) ● A CB SUP :Supernatant sample (dissolved protease) ● A CB PEL : Pellet sample from culture broth containing crystallized protease.

[0308] A CB SUP and A CB PEL The sample was sampled and fractionated by high-speed centrifugation (5 minutes, 10,000x RCF, 20°C). The sample was then analyzed by the protease activity assay described above. A CB and A CB SUP Using the protease activity in,

number

number

[0309] Protease crystal dissolution: To evaluate the crystalline dissolution of protease variants with and without substitution at the position corresponding to A215 of Sequence ID No. 1, fermentation broth was diluted five-fold with H2O, the pH level was adjusted to 4.5 with acetic acid (20%), and the conductivity was adjusted to 9 mS / cm with CaCl2 (34%). Dissolution was carried out at a constant temperature of 20°C and with thorough mixing.

[0310] The experiment began immediately after dissolution. After a total of 15 minutes and 60 minutes, the fully protease-active sample (A) was obtained. FULL ) and supernatant protease activity sample (A SUP ) was recovered. A SUP Samples were collected by high-speed centrifugation (5 minutes, 10,000x RCF, 20°C), and the supernatant was discarded. Subsequently, all samples were analyzed by the protease activity assay described above.

[0311] To evaluate crystal dissolution in fermentation broth, the expected complete protease activity (A EXP ) was calculated based on the protease activity of the fermentation broth and dilution factors:

number

[0312] A recovered during the dissolution experiment FULL Using the sample, A CB and A FULL By calculating the difference between the measured activity in the fermentation broth and the expected total protease activity in the fermentation broth, A EXP We verified:

number

[0313] Crystal dissolution occurred after 60 minutes when the dissolved protease (A DISS It was evaluated by calculating the fraction of:

number

[0314] Ultimately, protease +A215X A regarding DISS A is the same protease that does not have the A215X substitution. DISS This was standardized, resulting in a difference in protease crystal dissolution in the fermentation broth, which was given as a multiplier increase:

number

[0315] result: Table 1 shows the initial lysed protease activity of A215X variants in cultured broth. Thus, A215K substitution resulted in a 2.9–15.6-fold increase in initial lysed protease activity measured in fermented broth across the five tested proteases. In addition, substitutions A215Q and A215N resulted in 5.1-fold and 2.4-fold increases, respectively, in initial lysed activity. A215T substitution had a smaller effect, resulting in a 1.1-fold increase, and no effect on initial lysed activity was observed with respect to the A215S variant. These data indicate that the degree of protease crystal formation is reduced by the introduction of A215X substitution.

[0316] [Table 2]

[0317] Table 2 shows the dissolved protease activity of the A215X variants after 60 minutes. Thus, the A215K substitution resulted in a 1.1–6.0-fold increase in dissolved protease activity for all five proteases tested. Substitutions A215Q, A215N, A215T, and A215S resulted in a 1.6–4.3-fold increase in crystal solubility. The A215S substitution did not affect the degree of protease crystal formation (see Table 1), but this substitution resulted in a 1.7-fold increase in dissolved activity after 60 minutes. Therefore, these data indicate that the solubility of protease crystals is increased by the introduction of the A215X substitution.

[0318] [Table 3]

[0319] The invention described and claimed herein should not be limited to the scope of the specific embodiments disclosed herein, for these embodiments are intended to be illustrative of several embodiments of the invention. Embodiments of any equivalent 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 be apparent to those skilled in the art from the foregoing. Such modifications are also intended to be within the scope of the appended claims. In case of any conflict, the present disclosure, including definitions, shall prevail. Some aspects of the present invention are described below. 1. A protease variant of a parent protease, wherein the variant has at least 80% but less than 100% sequence identity with SEQ ID NO: 1. The variant includes a first substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; The aforementioned variants are X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104I (e.g., V104I), X120D ( For example, this includes at least three further modifications, preferably substitutions, selected from the group consisting of H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., L262E); The variant has protease activity; and The position number is based on the numbering of Sequence ID No. 2, and it is a protease variant. 2. The protease variant according to item 1, wherein the first substitution is selected from the group consisting of X215K, X215Q, X125N, X215S, and X215T; preferably, the first substitution is selected from the group consisting of X215K, X215Q, X125N, and X215T. 3. The protease variant according to item 1, wherein the first substitution is selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; preferably, the first substitution is selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T; most preferably, the first substitution is selected from the group consisting of A215K, A215Q, A215N, and A215T. 4. The protease variant according to item 1, wherein the at least three further modifications, preferably substitutions, are selected from the group consisting of S3T, V4I, S9E, I35ID, N43R, N76D, S99D, S99F, S101E, S101L, S103A, S103T, V104I, H120D, G160S, G195E, V205I, Q206L, Y209W, K235L, S259D, N261W, and L262E. 5. The above at least three further modifications, preferably substitutions, a) S3T, V4I, S99D, S101E, S103A, G160S, and V205I; b)I35ID, N76D, H120D, G195E, K235L; c) S9E, N43R, N76D, S99F, S101L, S103T, V104I, V205I, Q206L, Y209W, S259D, N261W, and L262E; and d) S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E A protease variant selected from one of the groups consisting of the following, as described in item 4. 6. A protease variant according to any one of items 1 to 5, having improved solubility compared to the parent protease; preferably, the solubility is improved by at least 4%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to the parent protease. 7. The parent protease does not have the first substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T, and does not have X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104I (e.g., V A protease variant according to item 6, which is a protease that has no substitutions but is otherwise identical to the above, comprising at least three further modifications selected from the group consisting of 104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., L262E). 8. The protease variant according to item 6, wherein the variant has improved solubility compared to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 6. 9. The protease variant according to any one of items 6 to 8, wherein the protease variant has improved solubility at 10 to 30°C, preferably 15 to 25°C, and most preferably 20°C. 10. The protease variant according to any one of items 6 to 9, wherein the protease variant has improved solubility at pH 3 to 9, preferably at pH 4 to 8, more preferably at pH 4 to 6, even more preferably at pH 4 to 5, and most preferably at pH 4.5. 11. The protease variant according to any one of items 9 to 10, wherein the protease variant has improved solubility at 15 to 25°C and pH 4 to 6, preferably at 20°C and pH 4 to 5. 12. A protease variant according to any one of items 6 to 11, wherein the improved solubility is determined as a reduction in protease crystal formation and / or an increase in protease crystal solubility according to Example 1. 13. A protease variant according to any one of items 1 to 12, having equivalent or improved protease activity; preferably, the protease activity is at least 100%, for example, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% compared to the parent protease. 14. The parent protease does not have the first substitution selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T, and does not have X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104I (e.g., V A protease variant according to item 13, which is a protease that has no substitutions but is otherwise identical to the above, comprising at least three further modifications selected from the group consisting of 104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., L262E). 15. A protease variant according to any one of items 13 to 14, wherein the variant has protease activity equivalent to or improved compared to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 6. 16. A polynucleotide encoding a protease variant as described in any of items 1-15. 17. Nucleic acid constructs or expression vectors containing polynucleotides as described in item 16. 18. Host cells expressing any of the protease variants described in items 1-15. 19. A method for obtaining a protease variant described in any of items 1 to 15, (a) Introducing a first substitution to the parent protease selected from the group consisting of X215K, X215R, X215Q, X125N, X215S, and X215T; and X3T (e.g., S3T), X4I (e.g., V4I), X9E (e.g., S9E), I35ID, X43R (e.g., N43R), X76D (e.g., N76D), X99D (e.g., S99D), X99F (e.g., S99F), X101E (e.g., S101E), X101L (e.g., S101L), X103A (e.g., S103A), X103T (e.g., S103T), X104I (e.g., The introduction of at least three further modifications, preferably substitutions, selected from the group consisting of V104I), X120D (e.g., H120D), X160S (e.g., G160S), X195E (e.g., G195E), X205I (e.g., V205I), X206L (e.g., Q206L), X209W (e.g., Y209W), X235L (e.g., K235L), X259D (e.g., S259D), X261W (e.g., N261W), and X262E (e.g., L262E), wherein the variants have protease activity; and (b) Recovering the variant A method that includes this. 20. A detergent composition comprising a protease variant described in any of items 1 to 15 and one or more detergent components. 21. The detergent composition according to item 20, wherein the composition is in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a typical or dense powder, granules, paste, gel, or a typical, dense or concentrated liquid. 22. The detergent composition according to item 20, wherein the composition is in liquid form. 23. Use of a protease variant according to any of items 1 to 15 or a detergent composition according to any of items 20 to 22 in a cleaning process, preferably laundry or dishwashing, for example, hard surface cleaning such as an automatic dishwasher.

Claims

1. A protease variant of a parent protease, wherein the variant has at least 90% but less than 100% sequence identity with SEQ ID NO:

1. The variant includes a first substitution selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; The variant includes at least three further modifications selected from the group consisting of S3T, V4I, S9E, I35ID, N43R, N76D, S99D, S99F, S101E, S101L, S103A, S103T, V104I, H120D, G160S, G195E, V205I, Q206L, Y209W, K235L, S259D, N261W, and L262E; The variant has protease activity; The variant has improved solubility compared to the parent protease, and the parent protease is a protease that is otherwise identical but does not have the first substitution at position 215; and The position number is based on the numbering of sequence number 2, and it is a protease variant.

2. The protease variant according to claim 1, wherein the first substitution is selected from the group consisting of A215K, A215Q, A215N, A215S, and A215T.

3. The protease variant according to claim 1, wherein the first substitution is selected from the group consisting of A215K, A215Q, A215N, and A215T.

4. The above-mentioned at least three further modifications are a) S3T, V4I, S99D, S101E, S103A, G160S, and V205I; b) I35ID, N76D, H120D, G195E, K235L; c) S9E, N43R, N76D, S99F, S101L, S103T, V104I, V205I, Q206L, Y209W, S259D, N261W, and L262E; and d) S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E A protease variant according to claim 1, selected from one of the group consisting of the following.

5. The protease variant according to any one of claims 1 to 4, wherein the solubility is improved by at least 4%, 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 compared to the parent protease.

6. The protease variant according to any one of claims 1 to 5, wherein the variant has improved solubility compared to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO:

6.

7. The protease variant according to any one of claims 1 to 6, wherein the protease variant has improved solubility at 10 to 30°C and / or the protease variant has improved solubility at pH 3 to 9.

8. The protease variant according to any one of claims 1 to 7, wherein the protease variant has improved solubility at 15 to 25°C and pH 4 to 6.

9. The protease variant according to any one of claims 1 to 8, wherein the protease variant has improved solubility at 20°C and pH 4 to 5.

10. The protease variant according to any one of claims 1 to 9, wherein the improved solubility is determined as a reduction in protease crystal formation and / or an increase in protease crystal solubility according to Example 1.

11. A protease variant according to any one of claims 1 to 10, having equivalent or improved protease activity; the protease activity being at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% compared to the parent protease.

12. The protease variant according to any one of claims 1 to 11, wherein the variant has protease activity equivalent to or improved compared to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO:

6.

13. A polynucleotide encoding a protease variant according to any one of claims 1 to 12.

14. A nucleic acid construct or expression vector comprising a polynucleotide as described in claim 13.

15. A host cell expressing the protease variant described in any one of claims 1 to 12.

16. A method for obtaining a protease variant according to any one of claims 1 to 12, (a) Introducing a first substitution to the parent protease selected from the group consisting of A215K, A215R, A215Q, A215N, A215S, and A215T; and introducing at least three further modifications selected from the group consisting of S3T, V4I, S9E, I35ID, N43R, N76D, S99D, S99F, S101E, S101L, S103A, S103T, V104I, H120D, G160S, G195E, V205I, Q206L, Y209W, K235L, S259D, N261W, and L262E, wherein the variants have protease activity; and (b) Recovering the variant A method that includes this.

17. A detergent composition comprising a protease variant according to any one of claims 1 to 12 and one or more detergent components.

18. The detergent composition according to claim 17, wherein the composition is in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a typical or dense powder, granules, paste, gel, or a typical, dense or concentrated liquid.

19. The detergent composition according to claim 17, wherein the composition is in liquid form.

20. Use of a protease variant according to any one of claims 1 to 12 or a detergent composition according to any one of claims 17 to 19 in laundry or hard surface cleaning.

21. Use of a protease variant according to any one of claims 1 to 12 or a detergent composition according to any one of claims 17 to 19 in dishwashing.

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