Detergent composition comprising protease and anionic surfactant

Abstract

The present invention relates to compositions comprising a protease and an anionic surfactant. The compositions of the invention are suitable as, e.g., cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions.

Description

[0001] DETERGENT COMPOSITION COMPRISING PROTEASE AND ANIONIC SURFACTANT REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to compositions comprising a protease and an anionic surfactant. The compositions of the invention are suitable as, e.g., cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions.

[0005] BACKGROUND OF THE INVENTION

[0006] Enzymes and surfactants have been widely used in the detergent industry for many years. The addition of enzymes to detergent compositions provides many benefits, including improved stain removal, shorter wash cycles, and reduced energy and water consumption, thereby leading to more environmentally friendly detergents.

[0007] Among the enzymes, proteases play a crucial role due to their ability to break down protein-based stains, enhancing the overall cleaning performance. Surfactants, such as anionic surfactants, play a role by emulsifying oils and suspending dirt particles, facilitating their removal during washing.

[0008] Despite the commercial success of currently available detergent products, further improvements remain of commercial interest. Washing conditions such as temperature and pH tend to change over time and are also different from country to country and across different regions. In addition, some stains are still difficult to remove completely with currently available detergents products. Hence, there is a continuous need for new and improved enzymatic detergent compositions.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides detergent compositions comprising a combination of proteases and an anionic surfactant that provides a synergistic improvement of stain removal. In particular, protease variants having improved protease activity in the presence of anionic surfactants, in particular alkyl ether carboxylate surfactants, compared to the parent protease have been identified, giving rise to improved wash performance of a detergent composition comprising the protease variant and an anionic surfactant.In a first aspect, the present invention relates to a detergent composition comprising: a protease variant of a parent protease, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold; and wherein the protease variant has protease activity; and an anionic surfactant.

[0011] In a second aspect, the present invention relates to a detergent composition comprising: a protease variant of a parent protease, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the protease variant has protease activity; and an anionic surfactant.

[0012] In a third aspect, the present invention relates to a method of cleaning an object, preferably a fabric, dishware, or a hard surface, comprising contacting the object with a detergent composition according to the first aspect or the second aspect under conditions suitable for cleaning the object.

[0013] In a fourth aspect, the present invention relates to the use of a detergent composition according to the first aspect or the second aspect in a cleaning process, preferably laundry or hard surface cleaning.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:2.

[0016] Figure 2 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:3.

[0017] Figure 3 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:4.

[0018] Figure 4 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:5.

[0019] Figure 5 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:6.Figure 6 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:7.

[0020] Figure 7 is an overview of corresponding substitutions in SEQ ID NOs:1-7.

[0021] SEQUENCE OVERVIEW SEQ ID NO:1 is Subtilisin Savinase (Savinase®) from Bacillus lentus.

[0022] SEQ ID NO:2 is Subtilisin BPN’ from Bacillus amyloliquefaciens.

[0023] SEQ ID NO:3 is Subtilisin Carlsberg (Alcalase®) from Bacillus licheniformis.

[0024] SEQ ID NO:4 is a protease from Bacillus gibsonii.

[0025] SEQ ID NO:5 is a protease from Bacillus gibsonii.

[0026] SEQ ID NO:6 is a protease from Bacillus sp. TY-145.

[0027] SEQ ID NO:7 is a protease from Actinomadura keratinilytica.

[0028] SEQ ID NO:8 is a stabilized variant of SEQ ID NO:1 with the substitutions S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E.

[0029] SEQ ID NO:9 is SEQ ID NO:1 with the substitutions S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.

[0030] DEFINITIONS

[0031] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0032] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0033] Protease: The term “protease” means an enzyme having peptidase activity (EC 3.4; also known as proteolytic activity or protease activity) that catalyzes the hydrolysis of peptide bonds. The EC 3.4 group includes several sub-groups, including EC 3.4.21 (serine endopeptidase), which further contains several sub-groups, including EC. 3.4.21.62 (subtilisin). For purpose of the present invention, protease activity may be determined according to Protease Activity Assay I or Protease Activity Assay II described in the Examples herein.

[0034] Protease fragment: The term “protease fragment” (or “fragment” when used in the context of a protease) means a polypeptide having protease activity comprising a deletion at one or more (or one or several) positions at the N- and / or C-terminus as compared to its parent protease which is a protease having the identical amino acid sequence of said fragment but not having the N- and / or C-terminal deletion.

[0035] Protease parent: The term “protease parent” or “parent protease” (or “parent” when used in the context of a protease) means a protease to which an alteration is made to produce a protease variant, including protease fragments. Thus, the parent protease is a protease havingthe identical ammo acid sequence of said protease variant but not having the alterations at one or more of said specified positions. It will be understood, that in the present context the expression “having identical amino acid sequence” relates to 100 % sequence identity. The parent protease may be a naturally occurring (wild-type) polypeptide or a variant thereof.

[0036] Protease variant: The term “protease variant” (or “variant” when used in the context of a protease) means a polypeptide having protease activity comprising an alteration, i.e., a substitution, insertion (including extension), and / or deletion (e.g. truncation), preferably substitution, at one or more (or one or several) positions compared to its parent protease which is a protease having the identical amino acid sequence of said variant but not having the alterations at one or more of said specified positions. Substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding amino acids, e.g., 1 to 10 amino acids, preferably 1-3 amino acids adjacent to an amino acid occupying a position. Amino acid substitutions may exchange a native amino acid for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative. In one embodiment, the protease variant is a deletion variant, for example a fragment of a parent protease. The protease variants have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the protease activity of the mature parent protease from which they have been derived.

[0037] Surfactant: The term “surfactant” as used herein, refers to any conventional understanding of a surfactant within the art, which may be anionic and / or non-ionic and / or semi-polar and / or zwitterionic and / or cationic, or a mixture thereof.

[0038] Anionic surfactant: The term “anionic surfactant” as used herein, refers to the nonlimiting examples of anionic surfactants, including carboxylates, sulfates and sulfonates. In some embodiments, the anionic surfactant is selected from the group of ether carboxylates, such as alkyl ether carboxylates, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.

[0039] AlphaFold structure prediction: AlphaFold is a computational method for predicting the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., Highlyaccurate protein structure prediction with AlphaFold. Nature, 2021). Predicted structures for millions of polypeptides deposited in the UniProt database have been deposited in the AlphaFold Protein Structure Database, using the AlphaFold Monomer v2.0 model (Varadi et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research, 2021). In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.

[0040] In addition to the many three-dimensional structures that are already publicly available, code is available for reproducing and predicting structures of new polypeptides at source code repositories such as Github.com under deepmind / alphafold / , using notebooks / AlphaFold.ipynb, which uses AlphaFold v2.3.1 or newer. Additionally, it can be found in Github.com under sokrypton / ColabFold using v1.5.2 or newer, using AlphaFold2.ipynb. For technical details, please see Jumper et al. (vide supra).

[0041] AlphaFold produces a per-residue estimate of its confidence on a scale from 0 to 100. This confidence measure is called pLDDT and corresponds to the model’s predicted score on the IDDT-Ca metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better). Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction.

[0042] Extension: The term “extension” means an addition of one or more amino acids to the amino and / or carboxyl terminus of a variant, wherein the “extended” variant has protease activity.

[0043] Fragment: The term “fragment” means a variant having one or more amino acids absent from the amino and / or carboxyl terminus of the variant; wherein the fragment has protease activity.

[0044] Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and / or the 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, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed inMartin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol.

[0045] 7Q: 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 etal., 1986, Biochemistry 25: 505-512; Collins-Racie etal., 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.

[0046] Effective amount of enzyme: The term "effective amount of enzyme" refers to the quantity of enzyme necessary to achieve the enzymatic activity required in the specific application, e.g., in a defined detergent composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme used, the cleaning application, the specific composition of the detergent composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like. The term "effective amount" of a enzyme variant refers to the quantity of enzyme variant described hereinbefore that achieves a desired level of enzymatic activity, e.g., in a defined detergent composition.

[0047] Improved property: The term “improved property” means a characteristic associated with a protease variant that is improved compared to the protease parent or with a detergent composition comprising said protease variant. A detergent composition comprising such protease variant may thus also be said to have an improved property over a detergent composition comprising a protease parent of said variant. The improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, mildness, oxidation stability, pH activity, pH stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, thermostability, and wash performance. In a preferred embodiment, the protease variant of the invention had improved properties, such as improved wash performance, compared to a parent protease. Similarly, a detergent composition comprising the protease variant of the invention has improved properties, such as improved wash performance or stain removal efficiency, compared to a detergent composition comprising the parent protease. Furthermore, the protease variant and anionic surfactant may show synergy and thereby provide a detergent composition having an even further improved wash performance when compared to a detergent composition comprising the parent protease and an anionic surfactant.

[0048] Fabric: The term "fabric" encompasses any textile material. Thus, it is intended that the term encompass garments, as well as fabrics, yarns, fibres, non-woven materials, natural materials, synthetic materials, and any other textile material.

[0049] Textile: The term "textile" refers to woven fabrics, as well as staple fibres and filaments suitable for conversion to or use as yarns, woven, knit, and non-woven fabrics. The term encompasses yarns made from natural, as well as synthetic (e.g., manufactured) fibres. The term,textile materials is a general term for fibres, yarn intermediates, yarn, fabrics, and products made from fabrics {e.g., garments and other articles).

[0050] Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0051] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) 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), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0052] (Identical Residues x 100) / (Length of Alignment -Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0053] (Identical Deoxyribonucleotides x 100) / (Length of Alignment- Total Number of Gaps in Alignment) Structural Similarity: The relatedness between two amino acid sequences has conventionally been described by the parameter “sequence identity”. However, since the biological function of a polypeptide is defined by its three-dimensional structure rather than its amino acid sequence, a better way of assessing a functional relationship between polypeptides is by comparing their three-dimensional structures. Thus, for the purposes of the present invention, the relatedness between the three-dimensional structure of two polypeptides is described by the parameter “structural similarity”.

[0054] A three-dimensional structure of any polypeptide may be obtained experimentally via, e.g. , X-ray crystallography or using in silico methods such as AlphaFold (vide supra). The structural similarity between three-dimensional structures may then be determined by the TM-score, which is calculated using the following general formula (Zhang & Skolnick, Proteins 57:702-710, 2004):TM-score = Max

[0055]

[0056] where LN is the length of the native structure, LT is the length of the aligned residues to the template structure, d, is the distance between the / th pair of aligned residues and do is a scale to normalize the match difference. ‘Max’ denotes the maximum value after optimal spatial superposition.

[0057] For the purposes of the present invention, LN is always the length of the reference protein, indicating the use of a fixed reference length L to prevent artificially large TM-scores from alignment of substructures:

[0058] LT

[0059] 1 'V 1

[0060] TM-score = - > - =•

[0061] fd -\2

[0062] i=1l + hr

[0063]

[0064] \do /

[0065] A structural alignment of the three-dimensional structures of two polypeptides is necessary before the TM-score can be calculated. This is achieved via algorithms that optimize the structural overlap, and several methods are available, such as CEalign (Shindyalov and Bourne, Protein Eng., 11, 739-747, 1998), DALI (Holm and Sander, Trends Biochem. Sci., 20, 478-480, 1995), orTM-align (Nucleic Acids Res. 33:2302-2309, 2005).

[0066] For the purposes of the present invention, TM-align is applied. For convenience, TM-score is integrated in the TM-align software, which is available from the author’s website. The version of TM-align is preferably updated 2019-08-22 or later, and the TM-score between a reference and a query protein is determined by running this command:

[0067] TMalign <query. pdb> <ref erence . pdb> -L <length of reference> Where <query.pdb> is the name of the PDB file containing coordinates of the query polypeptide, <reference.pdb> is the name of the PDB file containing coordinates of the reference polypeptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

[0068] The maximal TM-score is 1, e.g., 1.0, corresponding to identical three-dimensional structures.

[0069] Conventions for Designation of Variants

[0070] For purposes of the present invention, the polypeptide disclosed in SEQ ID NO:1 is used to determine the corresponding amino acid positions in another protease. The amino acid sequence of another protease is aligned with the polypeptide disclosed in SEQ ID NO:1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO:1 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) 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), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0071] Alignments of SEQ ID NO:1 and SEQ ID NOs: 2, 3, 4, 5, 6, and 7 are provided as Figures 1-6. An overview of corresponding substitutions in SEQ ID NOs: 1-7 is provided in Figure 7.

[0072] In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted III PAG single letter or three letter amino acid abbreviation is employed.

[0073] Substitutions: For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of Thr at position 226 with Ala is designated as “T226A”. Multiple mutations are separated by addition marks (“+”) or by commas, e.g., “G205R+S411F” or “G205R,S411F”, representing substitutions at positions 205 and 411 of Gly (G) with Arg (R) and Ser (S) with Phe (F), respectively. Because the amino acid residue at a given position varies from parent protease to parent, the amino acid to be substituted may be indicated with X, e.g., “X226A”.

[0074] Deletions: For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of the amino acid Gly at position 195 is designated as “Gly195*”. Multiple deletions are separated by addition marks (“+”) or by commas, e.g., “G195*+S411*” or “G195*,S411*”. Because the amino acid residue at a given position varies from parent protease to parent, the amino acid to be deleted may be indicated with X, e.g., “X195*”.

[0075] Insertions: Foran amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of Lys after the amino acid Gly at position 195 is designated “G195GK”. Because the amino acid residue at a given position varies from parent protease to parent, the insertion of lysine after the amino acid at position 195 may be indicated with “X195*”.

[0076] An insertion of multiple amino acids is designated [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of Lys and Ala after the amino acid Gly at position 195 is indicated as “G195GKA”. In such cases, the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:Parent: Variant:

[0077] 195 195 195a 195b

[0078] G G - K- A

[0079]

[0080] Alternatively, an insertion of an amino acid residue such as lysine after the amino acid at position 195 may be indicated by “195aK”, and the insertion of two or more additional amino acid residues such as Lys and Ala after the amino acid at position 195 may be indicated by “195aK,195bA”.

[0081] Multiple alterations: Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “R170Y+G195E” representing a substitution of Arg and Gly at positions 170 and 195 with Tyr and Glu, respectively.

[0082] Different alterations: Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., “R170Y.E” represents a substitution of Arg at position 170 with Tyr or Glu. Thus, “Y167G,A+R170G,A” designates the following variants: “Y167G+R170G”, Y167G+R170A”, “Y167A+R170G”, and “Y167A+R170A”.

[0083] DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention invention relates to detergent compositions with improved properties. In particular, the detergent compositions of the invention exhibit improved wash performance. As can been seen from the Example disclosed herein, the detergent compositions of the invention comprise a combination of protease variants and anionic surfactant that provide a synergistic improvement in stain removal.

[0085] Thus, in a first aspect, the present invention relates to a detergent composition comprising: a protease variant of a parent protease and an anionic surfactant, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold; and wherein the protease variant has protease activity.

[0086] In a second aspect, the present invention relates to a detergent composition comprising: a protease variant of a parent protease and an anionic surfactant, wherein the protease variantcomprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprise substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the protease variants have protease activity.

[0087] Protease variant

[0088] In the first aspect of the invention, the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold; and wherein the protease variant has protease activity.

[0089] In an embodiment, the protease variant has a TM-score of at least 0.90, e.g., at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0090] In an embodiment, the protease variant has a TM-score of at least 0.95, e.g., at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0091] In an embodiment, the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0092] In an embodiment, the protease variant has a TM-score of at least 0.990, e.g., at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997,at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0093] In an embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In a preferred embodiment, the parent protease is SEQ ID NO:1.

[0094] In another embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0095] In another embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0096] In an embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ I D NO: 1.

[0097] In another embodiment, the parent protease is SEQ ID NO:1 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent protease is SEQ ID NO:1 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent protease is SEQ ID NO:1 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO: 1, wherein the three-dimensional structure is calculated using AlphaFold.

[0098] In another embodiment, the parent protease is SEQ ID NO:2 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent protease is SEQ ID NO:2 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent protease is SEQ ID NO:2 andthe protease variant has a TM-score of at least 0.990, e.g., at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold.

[0099] In another embodiment, the parent protease is SEQ ID NO:3 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent protease is SEQ ID NO:3 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent protease is SEQ ID NO:3 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold.

[0100] In another embodiment, the parent protease is SEQ ID NO:4 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent protease is SEQ ID NO:4 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent protease is SEQ ID NO:4 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold.

[0101] In another embodiment, the parent protease is SEQ ID NO:5 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structureof SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent protease is SEQ ID NO:5 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent protease is SEQ ID NO:5 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold.

[0102] In another embodiment, the parent protease is SEQ ID NO:6 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent protease is SEQ ID NO:6 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent protease is SEQ ID NO:6 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold.

[0103] In another embodiment, the parent protease is SEQ ID NO:7 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent protease is SEQ ID NO:7 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent protease is SEQ ID NO:7 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991, at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold.For some parent proteases, a three-dimensional structure is publicly available. A three-dimensional structure of SEQ ID NO:1 (Savinase®) is available under UniProt accession number P29600 or, alternatively, PDB accession number 1SVN. A three-dimensional structure of SEQ ID NO:2 (BPN’) is available under UniProt accession number P00782. A three-dimensional structure of SEQ ID NO:3 (Alcalase®) is available under UniProt accession number P00780.

[0104] In an embodiment, the parent protease is SEQ ID NO:1 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600. Preferably, the parent protease is SEQ ID NO:1 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600. Most preferably, the parent protease is SEQ ID NO:1 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600.

[0105] In an embodiment, the parent protease is SEQ ID NO:2 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782. Preferably, the parent protease is SEQ ID NO:2 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782. Most preferably, the parent protease is SEQ ID NO:2 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782.

[0106] In an embodiment, the parent protease is SEQ ID NO:3 and the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780. Preferably, the parent protease isSEQ ID NO:3 and the protease variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780. Most preferably, the parent protease is SEQ ID NO:3 and the protease variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780.

[0107] In the second aspect of the invention, the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprise substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the protease variants have protease activity.

[0108] In an embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In a preferred embodiment, the parent protease is SEQ ID NO:1.

[0109] In another embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0110] In another embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0111] In an embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ I D NO: 1.

[0112] In another embodiment, the parent protease is SEQ ID NO:1 and the protease variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1.

[0113] In another embodiment, the parent protease is SEQ ID NO:2 and the protease variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%,at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:2.

[0114] In another embodiment, the parent protease is SEQ ID NO:3 and the protease variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:3.

[0115] In another embodiment, the parent protease is SEQ ID NO:4 and the protease variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:4.

[0116] In another embodiment, the parent protease is SEQ ID NO:5 and the protease variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:5.

[0117] In another embodiment, the parent protease is SEQ ID NO:6 and the protease variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:6.

[0118] In another embodiment, the parent protease is SEQ ID NO:7 and the protease variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:7.

[0119] In an aspect, the number of substitutions in the protease variants of the present invention is 5-30, e.g., 5-25, 5-20, 5-15 and 5-10, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, substitutions. In a preferred embodiment, the number of substitutions in the protease variants of the present invention is 5-11, such as 5, 6, 7, 8, 9, 10, or 11 substitutions.

[0120] In another aspect, a protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least three substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.In another aspect, a protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least four substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0121] In another aspect, a protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least five substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0122] In another aspect, a protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least six substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0123] In another aspect, a protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least seven substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0124] In another aspect, a protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least eight substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0125] In another aspect, a protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises nine substitutions at positions corresponding to each of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0126] The protease variants of the invention comprise a substitution at a position corresponding to position 95 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 95 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Asp.

[0127] The protease variants of the invention comprise a substitution at a position corresponding to position 209 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 209 of SEQ ID NO:1 is substituted with Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Lys.

[0128] The protease variants of the invention may comprise a substitution at a position corresponding to position 9 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 9 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Cys, Gin, Glu, Gly, His, lie, Leu, Met, Phe, Trp, Tyr, or Vai, preferably with Glu.

[0129] The protease variants of the invention may comprise a substitution at a position corresponding to position 42 of SEQ ID NO:1. In one aspect, the amino acid at a positioncorresponding to position 42 of SEQ ID NO:1 is substituted with Ala, Arg, Cys, Gin, Glu, His, lie, Leu, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, preferably with Arg.

[0130] The protease variants of the invention may comprise a substitution at a position corresponding to position 74 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 74 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Asp.

[0131] The protease variants of the invention may comprise a substitution at a position corresponding to position 199 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 199 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with lie.

[0132] The protease variants of the invention may comprise a substitution at a position corresponding to position 200 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 200 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Trp, or Vai, preferably with Leu.

[0133] The protease variants of the invention may comprise a substitution at a position corresponding to position 203 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 203 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Trp.

[0134] The protease variants of the invention may comprise a substitution at a position corresponding to position 253 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 253 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Trp, Tyr, or Vai, preferably with Asp.

[0135] The protease variants of the invention may comprise a substitution at a position corresponding to position 255 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 255 of SEQ ID NO:1 is substituted with Ala, Arg, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Pro, Thr, Trp, Tyr, or Vai, preferably with Trp.

[0136] The protease variants of the invention may comprise a substitution at a position corresponding to position 256 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 256 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Cys, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Glu.

[0137] In one aspect, the protease variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0138] In one aspect, the protease variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least four substitutions selectedfrom the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0139] In one aspect, the protease variants of the invention comprise substitutions corresponding to G95D and A209Kof SEQ ID NO:1 and further comprise at least five substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0140] In one aspect, the protease variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least six substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0141] In one aspect, the protease variants of the invention comprise substitutions corresponding to substitutions G95D and A209K of SEQ ID NO:1 and further comprise at least seven substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0142] In one aspect, the protease variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least eight substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0143] In one aspect, the protease variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprising nine substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0144] In one aspect, the present invention relates to protease variants of SEQ ID NO:1 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., A209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of X9E (e.g., S9E), X42R (e.g., N42R), X74D (e.g., N74D), X199I (e.g., V199I), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., S253D), X255W (e.g., N255W), and X256E (e.g., L256E); wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:1; and wherein the protease variants have protease activity. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:1 with the substitutions S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.

[0145] In one aspect, the present invention relates to protease variants of SEQ ID NO:2 comprising the substitutions X97D (e.g., G97D) and X215K (e.g., G215K) and further comprising at least three, e.g., at least four, at least five, at least six, or seven, substitutions selected fromthe group consisting of X9E (e.g., S9E), X43R (e.g., K43R), X76D (e.g., N76D), X206L (e.g., Q206L), X209W(e.g., L209W), X261W(e.g., F261W), and X262E (e.g., Y262E); wherein position numbering is based on the numbering of SEQ ID NO:2; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:2; and wherein the protease variants have protease activity. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:2 with the substitutions S9E, K43R, N76D, G97D, Q206L, L209W, G215K, F261W, and Y262E.

[0146] In one aspect, the present invention relates to protease variants of SEQ ID NO:3 comprising the substitutions X96D (e.g., N96D) and X214K (e.g., A214K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g.,Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261E (e.g., Y261E); wherein position numbering is based on the numbering of SEQ ID NO:3; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:3; and wherein the protease variants have protease activity. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E.

[0147] In one aspect, the present invention relates to protease variants of SEQ ID NO:4 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., A209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., T9E), X42R (e.g., T42R), X74D (e.g., N74D), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., N253D), X255W (e.g., S255W), and X256E (e.g., Q256E); wherein position numbering is based on the numbering of SEQ ID NO:4; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:4; and wherein the protease variants have protease activity. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:4 with the substitutions T9E, T42R, N74D, G95D, Q200L, Y203W, A209K, N253D, S255W, and Q256E.

[0148] In one aspect, the present invention relates to protease variants of SEQ ID NO:5 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., V209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of X9E (e.g., T9E), X42R (e.g., N42R), X74D(e.g. N74D), X199I (e.g., V199I), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., N253D), X255W (e.g., S255W), and X256E (e.g., Q256E); wherein position numbering is based on the numbering of SEQ ID NO:5; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:5; and wherein the protease variant have protease activity. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:5 with the substitutions T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D, S255W, and Q256E.

[0149] In one aspect, the present invention relates to protease variants of SEQ ID NO:6 comprising the substitutions X107D (e.g., G107D) and X245K (e.g., N245K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X12E (e.g., K12E), X44R (e.g., D44R), X86D (e.g., S86D), X235I (e.g., V235I), X236L (e.g., E236L), X297D (e.g., T297D), X299W (e.g., D299W), and X300E (e.g., D300E); wherein position numbering is based on the numbering of SEQ ID NO:6; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:6; and wherein the protease variants have protease activity. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:6 with the substitutions K12E, D44R, S86D, G107D, V235I, E236L, N245K, T297D, D299W, and D300E.

[0150] In one aspect, the present invention relates to protease variants of SEQ ID NO:7 comprising the substitutions X99D (e.g., N99D) and X215K (e.g., N215K) and further comprising at least three, e.g., at least four, at least five, or six, substitutions selected from the group consisting of X12E (e.g., D12E), X51R (e.g., G51R), X206L (e.g., T206L), X266D (e.g., T266D), X268W (e.g., N268W), and X269E (e.g., L269E); wherein position numbering is based on the numbering of SEQ ID NO:7; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:7; and wherein the protease variants have protease activity. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:7 with the substitutions D12E, G51R, N99D, T206L, N215K, T266D, N268W, and L269E.

[0151] In one aspect, the present invention relates to protease variants of SEQ ID NO:7 comprising the substitutions X99D (e.g., N99D) and X215K (e.g., N215K) and further comprising the substitutions X12E (e.g., D12E), X51R (e.g., G51R), and X206L (e.g., T206L); wherein position numbering is based on the numbering of SEQ ID NO:7; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:7; and wherein the protease variants have protease activity. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:7 with the substitutions D12E, G51 R, N99D, T206L, and N215K.

[0152] In an alternative aspect, the present invention relates to protease variants of SEQ ID NO:3 comprising the substitutions X96D (e.g., N96D) or X214K (e.g., A214K), preferably X96D (e.g., N96D) and X214K (e.g., A214K), and further comprising at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten, substitutions selected from the group consisting of X68S (e.g., A68S), X77N (e.g., T77N), X78I (e.g., T78I), X127S (e.g., G127S), X128P (e.g., A128P), X165Q (e.g., G165Q), X184Q (e.g., N184Q), X202V (e.g., A202V), X217S (e.g., N217S), and X258P (e.g., S258P); wherein position numbering is based on the numbering of SEQ ID NO:3; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:3; and wherein the protease variants have protease activity. In an embodiment, the protease variant comprises the substitutions N96D and A214K and further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten, substitutions selected from the group consisting of A68S, T77N, T78I, G127S, A128P, G165Q, N184Q, A202V, N217S, and S258P. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S, and S258P. In a more preferred embodiment, the protease variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261E (e.g., Y261E). In an even more preferred embodiment, the protease variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261E. In a most preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258P, F260W, and Y261E. In a most preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258D, F260W, and Y261E.

[0153] In an alternative aspect, the present invention relates to protease variants of SEQ ID NO:3 comprising the substitution X214K (e.g., A214K) and further comprising at least one, e.g., at leasttwo, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least elleven, at least twelve, at least thirteen, or fourteen, substitutions selected from the group consisting of X9T, (e.g., P9T), X17H, (e.g., Q17H), X77N (e.g., T77N), X78I (e.g., T78I), X96D (e.g., N96D), X103F (e.g., Y103F), X127T (e.g., G127T), X128K (e.g., A128K), X129Q, (e.g., S129Q), X165Q (e.g., G165Q), X184Q (e.g., N184Q), X202V (e.g., A202V), X203E (e.g., G203E), and X258P (e.g., S258P); wherein position numbering is based on the numbering of SEQ ID NO:3; wherein the protease variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:3; and wherein the protease variants have protease activity. In an embodiment, the protease variant comprises the substitution A214K and further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least elleven, at least twelve, at least thirteen, or fourteen, substitutions selected from the group consisting of P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, and S258P. In a preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, A214K, and S258P. In a more preferred embodiment, the protease variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261E (e.g., Y261E). In an even more preferred embodiment, the protease variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261E. In a most preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261E. In a most preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E. In a most preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261E. In a most preferred embodiment, the protease variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261E.The protease variants of the invention may comprise further substitutions at one or more other positions corresponding to positions 60 (e.g., N60D), 97 (e.g., S97E), 99 (e.g., S99E), 116 (e.g., G116N) , and 246 (e.g., N246L) of SEQ ID NO:1. Preferably, the protease variants comprise one or more further substitutions selected from the group consisting of substitutions corresponding to N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L) of SEQ ID NO:1.

[0154] The protease variants of the invention may further comprise an extension of one or more amino acids at the N-terminal and / or C-terminal ends.

[0155] Alternatively, the protease variants of the invention may further comprise a truncation of one or more amino acids at the N-terminal and / or C-terminal ends.

[0156] The amino acid changes introduced into parent proteases to provide protease variants according to the present invention may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0157] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly.

[0158] Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

[0159] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos eta!., 1992, Science 255: 306-312; Smith etal., 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 an alignment with a related polypeptide, and / or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides / proteins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity. Additionally, or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and / or active sites of polypeptides. See, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

[0160] In preferred embodiments of the invention, the detergent composition comprising the protease variant and an anionic surfactant exhibits improved wash performance compared to a detergent composition comprising the parent protease and an anionic surfactant.

[0161] In an embodiment, the protease variants of the invention have improved wash performance compared to a reference protease. In an embodiment, wash performance is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.

[0162] In some embodiments, the reference protease is a parent protease. In an embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In one embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the protease variant has improved wash performance compared to SEQ ID NO:1. In a preferred embodiment, the protease variant has improved wash performance compared to SEQ ID NO:2. In a preferred embodiment, the protease variant has improved wash performance compared to SEQ ID NO:3. In a preferred embodiment, the protease variant has improved wash performance compared to SEQ ID NO:4. In a preferred embodiment, the protease variant has improved wash performance compared to SEQ ID NO:5. In a preferred embodiment, the protease variant has improved wash performance compared to SEQ ID NO:6. In a preferred embodiment, the protease variant has improved wash performance compared to SEQ ID NO:7.In some embodiments, the protease variant has improved wash performance compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1. In one embodiment, wash performance is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%. In a preferred embodiment, the protease variant has improved wash performance compared to SEQ ID NO:8.

[0163] In an embodiment, the protease variant has improved wash performance in a liquid detergent having pH 8-14, preferably pH 9-13, most preferably pH 10-12.

[0164] In an embodiment, the protease variant has improved wash performance in a liquid detergent having pH 7-11, preferably pH 7-10, most preferably pH 8-10.

[0165] In an embodiment, the protease variant has improved wash performance at a washing temperature of 10-60 °C, preferably 10-40 °C, more preferably 10-30 °C, most preferably 15-25 °C.

[0166] In an embodiment, the protease variant has improved wash performance as determined in Example 1 herein. In a preferred embodiment, the protease variant has improved wash performance in liquid detergent as determined according to Example 1 herein.

[0167] Parent protease

[0168] The parent protease may be a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to the polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0169] In an aspect, the parent protease is a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0170] In an aspect, the parent protease is a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0171] In an aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent protease differs by up to 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:1. In another aspect, the parent protease comprises or consists of the polypeptide of SEQ ID NO:1.

[0172] In an aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent protease differs by up to 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:2. In another aspect, the parent protease comprises or consists of the polypeptide of SEQ ID NO:2.

[0173] In an aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:3 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent protease differs by up to 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:3. In another aspect, the parent protease comprises or consists of the polypeptide of SEQ ID NO:3.

[0174] In an aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:4 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent protease differs by up to 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:4. In another aspect, the parent protease comprises or consists of the polypeptide of SEQ ID NO:4.

[0175] In an aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:5 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent protease differs by up to 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:5. In another aspect, the parent protease comprises or consists of the polypeptide of SEQ ID NO:5.

[0176] In an aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:6 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent protease differs by up to 20 amino acids, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:6. In another aspect, the parent protease comprises or consists of the polypeptide of SEQ ID NO:6.

[0177] In an aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:7 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent protease differs by up to 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:7. In another aspect, the parent protease comprises or consists of the polypeptide of SEQ ID NO:7.

[0178] The parent protease may be a fusion polypeptide or cleavable fusion polypeptide. 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 include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson etal., 1994, Science 266: 776-779).

[0179] A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward etal., 1995, Biotechnology 13: 498-503; and Contreras etal., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter etal., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0180] The parent protease may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent protease encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent protease is secreted extracellularly.

[0181] In one aspect, the parent protease is a Bacillus lentus protease, e.g., the protease of SEQ ID NO:1. In one aspect, the parent protease is a Bacillus amyloliquefaciens protease, e.g., the protease of SEQ ID NO:2. In one aspect, the parent protease is a Bacillus licheniformis protease, e.g., the protease of SEQ ID NO:3. In one aspect, the parent protease is a Bacillus gibsonii protease, e.g., the protease of SEQ ID NO:4. In one aspect, the parent protease is a Bacillus gibsonii protease, e.g., the protease of SEQ ID NO:5. In one aspect, the parent protease is aBacillus sp. TY145 protease, e.g., the protease of SEQ ID NO:6. In one aspect, the parent protease is a Actinomadura keratinilytica protease, e.g., the protease of SEQ ID NO:7.

[0182] Anionic surfactant

[0183] The detergent composition of the invention comprises an anionic surfactant.

[0184] In an embodiment, the anionic surfactant is selected from the group consisting of: ether carboxylates, such as alkyl ether carboxylates, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.

[0185] In an embodiment, the anionic surfactant is an ether carboxylate according to the general Formula (I):

[0186] R-O-(OCH2CH2)n-CH2-COOX Formula (I)

[0187] wherein

[0188] R represents a linear or branched C4-C22alkyl or alkenyl group;

[0189] n represents a number from 1-20; and

[0190] X represents a hydrogen, sodium, potassium, ammonium and / or alkanolammonium. Preferably, the radical R in Formula (I) contains from 8 to 18 carbon atoms, and the R preferably derives from fatty alcohols, Ziegler alcohols, oxo alcohols, Guerbet alcohols and alkylphenols. Preference is given to Cs- to Cis-alkyl ether carboxylates, and octyl-, nonyl- and tributylphenyl ether carboxylic acids. Particularly preferred R radicals are Cs-alkyl , Ci2 / i4-alkyl , iso-Ci3-al kyl , Ci4 / i5-oxoalkyl, Ci6 / is-alkyl , lauryl, oleyl and / or tallow fat alkyl. The index n in Formula (I) is preferably from 1 to 20, preferably from 1 to 15, more preferably from 1 to 12 and most preferably from 2 to 10. In a preferred embodiment, X in Formula (I) is hydrogen.

[0191] In some embodiments, the anionic surfactant is a carboxylate anionic surfactant. In the context of the invention, the term "carboxylate anionic surfactant" means an anionic surfactant comprising one or more carboxylic or carboxylate functions (-COOH or -COO-).

[0192] In an embodiment, the anionic surfactant is an ether carboxylate surfactant. In a preferred embodiment, the anionic surfactant is an alkyl ether carboxylate surfactant.

[0193] In the context of the invention, an alkyl ether carboxylic acid, also referred to as an alkyl ether carboxylate, may be chosen from a variety of specific compounds that exhibit desirableproperties for detergent applications. These compounds can include a range of chain lengths and branching structures, allowing formulators to tailor the detergent's performance characteristics to meet specific cleaning needs. For example, the alkyl ether carboxylate may be chosen based on the desire that a detergent composition is effective on various types of stains and soils.

[0194] In an embodiment, the anionic surfactant is a linear or branched alkyl ether carboxylate. In an embodiment, the anionic surfactant is an alkyl ether carboxylate having an average carbon chain length of about 10 to about 26, about 10 to about 20, or about 16 to about 18.

[0195] In an embodiment, the anionic surfactant is an alkyl ether carboxylate having an average level of ethoxylation of about 2 to about 20, about 7 to about 13, about 8 to about 12, or about 9.5 to about 10.5. The acid form or salt form of the alkyl ether carboxylate may be used. The alkyl chain of the alkyl ether carboxylate may contain one cis or trans double bond. Commercial alkyl ether carboxylates are available, for example, from Kao (sold under the tradename Akypo®), Huntsman (sold under the tradename Empicol®), and Clariant (sold under the tradename Emulsogen®). For example the anionic surfactant may be Emulsogen® COL 020, COL 050, COL 080 and COL 100, which are Oleic alcohol polyethylene glycol ethers carboxylic acid with average ethoxylation of 2EO, 5EO, 8EO, and 10EO respectively, all of which are commercially available from Clariant. Preferably, Emulsogen® COL 100 is used as anionic surfactant for the detergent compositions of the invention.

[0196] In a preferred embodiment, the anionic surfactant is an ether carboxylate selected from the group of oleyl-O-(OCH2CH2)s-CH2-COOH or oleyl-O-(OCH2CH2) -CH2-COOH. In a particular embodiment, the anionic surfactant is oleyl-O-(OCH2CH2) -CH2-COOH.

[0197] In an embodiment, the anionic surfactant is selected from linear alkylbenzene sulfonate (LAS) or alcohol ether sulfate (AEOS).

[0198] In an embodiment, the detergent composition comprises the anionic surfactant alcohol ether sulfate (AEOS).

[0199] In some embodiments, the detergent composition comprises one or more anionic surfactants.

[0200] In some embodiments, the detergent composition comprises a mixture of two or more surfactants. The term “a mixture of two or more surfactants” as used herein, refers to the combination of at least two surfactants which may be of the same group of surfactants, such as a combination of one anionic surfactant and second anionic surfactant, or it may be a combination of surfactants of different groups, such as one anionic surfactant and one non-ionic surfactant.

[0201] In an embodiment, the detergent composition comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. The term “non-ionic surfactant” as used herein, refers to the non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates(APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0202] In another embodiment, the detergent composition comprises one or more nonionic surfactant, such as AEG. The abbreviations AEOS and AES refer to alcohol ether sulfates, which are also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates.

[0203] In an embodiment, the at least one surfactant is a mix of a first surfactant and a second surfactant. In an embodiment, the first surfactant is a first anionic surfactant and the second surfactant is a second anionic surfactant. In another embodiment, the first surfactant is an anionic surfactant and the second surfactant is a non-ionic surfactant.

[0204] It is believed that by combining an anionic surfactant with the protease variant of the invention in a detergent composition, the overall performance of the detergent composition is improved, as seen for example by an improved wash performance. Futher, the concentration of surfactant may even be lowered when combined with a protease variant as described herein, and the same effect, such as wash performance, is obtained.

[0205] The surfactant(s) is typically present at a level of from 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 surfactant of the present invention may preferably be present in concentrations such that it exists mainly in micellar form, i.e. at concentrations above the critical micelle concentration in the main wash. The term “critical micelle concentration” as used herein, refers to a micelle concentration of a surfactant or blend of surfactants and is determined by a number of factors such as pH, temperature, ionic strength etc. To determine whether a surfactant or blend of surfactants is present in the main wash solution in concentrations at or above the critical micelle concentration a number of different methods may be used. The term “micelle” as used herein, refers to a spontaneously and reversibly formation of water-soluble aggregates of amphiphilic molecules, such as surfactants, in an aqueous solution.

[0206] In an embodiment, the concentration of anionic surfactant is at least 3 wt%, such as at least 4 wt%, such as at least 5 wt%, such as at least 6 wt%, such as at least 7 wt%, such as at least 8 wt%, of the detergent composition. It is to be understood that the concentration of the anionic surfactant in the detergent composition may be the same, but once the detergent composition is used in, e.g., a laundry process, the concentration of the anionic surfactant is changed and may change during the wash cycle. Preferably, the concentration of the anionic surfactant or mixture of surfactants in, e.g., a laundry process should be present in the main washat concentrations above the critical micelle concentration, meaning that the surfactants are mainly found in micellar form in the main wash.

[0207] In an embodiment, the concentration of the anionic surfactant is between 2 wt% and 14 wt% of the composition, such as between 3 wt% and 13 wt%, such as between 5 wt% and 12 wt% of the composition.

[0208] Detergent composition

[0209] The detergent compositions of the invention may further comprise one or more detergent components and / or one or more additional enzymes. In a preferred embodiment, the detergent composition comprises one or more detergent components, in particular one or more non-naturally occurring detergent components.

[0210] In one embodiment, the detergent composition comprising a protease variant and an anionic surfactant as described above further comprises one or more additional enzymes selected from the group consisting of amylases, catalases, cutinases, cellulases, DNases, endoglucanases, haloperoxygenases, lipases, mannanases, lechinase, pectinases, pectin lyases, peroxidases, proteases, xanthanases, xyloglucanases, and any mixture thereof.

[0211] In an embodiment, the detergent composition according to the invention has improved wash performance. In an embodiment, the detergent composition according to the invention has improved stain removal, preferably wherein the stain is a proteinaceous stain.

[0212] In an embodiment, the detergent composition according to the invention has improved wash performance as determined in Example 1 herein. In a preferred embodiment, the detergent composition of the invention has improved wash performance as determined according to Example 1 herein compared to a detergent composition comprising a protease according to SEQ ID NO:8.

[0213] The detergent composition may be in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.

[0214] In one preferred embodiment, the detergent composition is a liquid composition.

[0215] In one preferred embodiment, the detergent composition is a powder composition.

[0216] In one preferred embodiment, the detergent composition is a laundry soap bar.

[0217] The invention also relates to use of a detergent composition of the present invention in a cleaning process, such as laundry or hard surface cleaning such as dishwashing.

[0218] The choice of additional components for a detergent composition is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below. The choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product.In a particular embodiment, a detergent composition comprises one or more non-naturally occurring detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0219] In one embodiment, the protease is added to the detergent composition in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, in particular 0.1-10 mg of enzyme protein per liter of wash liquor.

[0220] An automatic dish wash (ADW) composition may for example include 0.001 %-30%, such as 0.01%-20%, such as 0.1-15%, such as 0.5-10% of enzyme protein by weight of the composition.

[0221] A granulated composition for laundry may for example include 0.001 %-20%, such as 0.01 %-10%, such as 0.05%-5% of enzyme protein by weight of the composition.

[0222] A liquid composition for laundry may for example include 0.0001 %-10%, such as 0.001-7%, such as 0.1%-5% of enzyme protein by weight of the composition.

[0223] The enzymes may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708 or the variants according to the invention may be stabilized using peptide aldehydes or ketones such as described in WO 2005 / 105826 and WO 2009 / 118375.

[0224] The detergent composition be formulated as a liquid laundry composition such as a liquid laundry compositions composition comprising:

[0225] a1) at least 0.01 mg of protease variant per liter detergent,

[0226] b) 2 wt% to 60 wt% of at least one anionic surfactant, and

[0227] c) 5 wt% to 50 wt% of at least one builder.

[0228] The detergent composition may be formulated into a granular detergent for laundry. Such detergent may comprise;

[0229] a1) at least 0.01 mg of protease variant per gram of composition,

[0230] b) anionic surfactant, preferably 5 wt % to 50 wt %,

[0231] c) nonionic surfactant, preferably 1 wt % to 8 wt %, and

[0232] d) builder, preferably 5 wt % to 40 wt %, such as carbonates, zeolites, phosphate builder, calcium sequestering builders or complexing agents.Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the person skilled in the art.

[0233] Liquid detergent composition

[0234] The liquid detergent composition may comprise a microcapsule of the invention, and thus form part of, any detergent composition in any form, such as liquid and powder detergents, and soap and detergent bars.

[0235] In one embodiment, the invention is directed to liquid detergent compositions comprising a microcapsule, as described above, in combination with one or more additional cleaning composition components.

[0236] The microcapsule, as described above, may be added to the liquid detergent composition in an amount corresponding to from 0.0001% to 5% (w / w) enzyme protein (AEP); preferably from 0.001% to 5%, more preferably from 0.005% to 5%, more preferably from 0.005% to 4%, more preferably from 0.005% to 3%, more preferably from 0.005% to 2%, even more preferably from 0.01% to 2%, and most preferably from 0.01% to 1% (w / w) enzyme protein.

[0237] The liquid detergent composition has a physical form, which is not solid (or gas). It may be a pourable liquid, a paste, a pourable gel or a non-pourable gel. It may be either isotropic or structured, preferably isotropic. It may be a formulation useful for washing in automatic washing machines or for hand washing. It may also be a personal care product, such as a shampoo, toothpaste, or a hand soap.

[0238] The liquid detergent composition may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to 70% water, up to 50% water, up to 40% water, up to 30% water, or up to 20% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid detergent. An aqueous liquid detergent may contain from 0-30% organic solvent. A liquid detergent may even be nonaqueous, wherein the water content is below 10%, preferably below 5%.

[0239] Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

[0240] The detergent composition may take the form of a unit dose product. A unit dose product is the packaging of a single dose in a non-reusable container. It is increasingly used in detergents for laundry. A detergent unit dose product is the packaging (e.g., in a pouch made from a water-soluble film) of the amount of detergent used for a single wash.

[0241] Pouches can be of any form, shape and material which is suitable for holding the composition, e.g., without allowing the release of the composition from the pouch prior to watercontact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be a blend composition comprising hydrolytically degradable and water-soluble polymer blends such as polyactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticizers like glycerol, ethylene glycerol, Propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and / or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids (see e.g., US 2009 / 0011970).

[0242] The choice of detergent components may include, for textile care, the consideration of the type of textile to be cleaned, the type and / or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.

[0243] The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.

[0244] Surfactants

[0245] In addition to the anionic surfactant of the invention, the detergent composition may comprise one or more further surfactants, which may be anionic and / or cationic and / or non-ionic and / or semi-polar and / or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant(s) is typically present at a level of from 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 surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in detergents may be utilized. Surfactants lower the surface tension in the detergent, which allows the stain being cleaned to be lifted and dispersed and then washed away.When included therein, the detergent will usually contain from about 1% to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, ether carboxylates, such as alkyl ether carboxylates, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soap, and combinations thereof.

[0246] When included therein, the detergent will usually contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alklydimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0247] When included therein, the detergent will usually contain from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0248] When included therein the detergent will usually contain from about 0.01 to about 10 % by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamine oxides, in particular N-(coco alkyl)-N,N-dimethylamine oxide and N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, and combinations thereof.When included therein the detergent will usually contain from about 0.01 % to about 10 % by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.

[0249] Additional bio-based surfactants may be used, e.g., wherein the surfactant is a sugar-based non-ionic surfactant which may be a hexyl-p-D-maltopyranoside, thiomaltopyranoside or a cyclic-maltopyranoside, such as described in EP2516606 B1. Other biosurfactants may include rhamnolipids and sophorolipids.

[0250] Builders and Co-Builders

[0251] The detergent composition may contain about 0-65% by weight, such as about 5% to about 45% of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typically 40-65%, particularly 50-65%. Builders and chelators soften, e.g., the wash water by removing the metal ions form the liquid. The builder and / or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2’,2”-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.

[0252] The detergent composition may also contain 0-20% by weight, such as about 5% to about 10%, of a detergent co-builder, or a mixture thereof. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid. 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), ethylenediaminetetra-(methylenephosphonic 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-methyliminodiaceticacid (MIDA), a-alanine-N, N-diacetic acid (a-ALDA), serine-N, N-diacetic acid (SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diacetic acid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TLIDA) and sulfomethyl-N, N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylidenediamine-N, N’, N’-triacetate (HEDTA), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or co-builders are described in, e.g., WO 2009 / 102854 and US 5,977,053.

[0253] Bleaching Systems

[0254] The detergent composition may contain 0-50% by weight, such as about 0.1% to about 25%, of a bleaching system. Bleach systems remove discolor often by oxidation, and many bleaches also have strong bactericidal properties, and are used for disinfecting and sterilizing. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra- hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator.

[0255] The term bleach activator is meant herein as a compound which reacts with peroxygen bleach like hydrogen peroxide to form a peracid. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters amides, imides or anhydrides. Suitable examples are tetracetylethylene diamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxy dodecanoic acid, 4-(dodecanoyloxy) benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4- (decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A particular family of bleach activators of interest was disclosed in EP 624154 and particularly preferred in that family is acetyl triethyl citrate (ATC). ATC or a short chain triglyceride like triacetin has the advantage that it is environmentally friendly as it eventually degrades into citric acid and alcohol. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product upon storage and are efficient bleach activators. Finally, ATC provides a good building capacity to the laundry additive. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching systemmay also comprise peracids such as 6-(phthahmido)peroxyhexanoic acid (PAP). The bleaching system may also include a bleach catalyst or a booster.

[0256] Some non-limiting examples of bleach catalysts that may 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), in particular 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-diylazanylylidene-KN-methanylylidene)triphenolato-K3O]manganese(lll). The bleach catalysts may also be other metal compounds, such as iron or cobalt complexes.

[0257] In some embodiments, the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formula:

[0258]

[0259] (iii) and mixtures thereof; wherein each R1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl. Other exemplary bleaching systems are described, e.g., in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242. Suitable photobleaches may for example be sulfonated zinc phthalocyanine.

[0260] Hydrotropes

[0261] A hydrotrope is a compound that solubilizes hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and hydrophobic characters (so-called amphiphilic properties as known from surfactants); however, the molecular structures of hydrotropes generally do not favour spontaneous self-aggregation, see, e.g., review by Hodgdon and Kaier, 2007, Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming micellar, lamellar orother well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care and food to technical applications. Use of hydrotropes in detergent compositions allows for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.

[0262] The detergent composition may contain 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0263] Polymers

[0264] The detergent composition may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and / or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and / or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed in, e.g., WO 2006 / 130575. Salts of the above-mentioned polymers are also contemplated.Fabric hueinq agents

[0265] The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when the fabric is contacted with a wash liquor comprising the detergent compositions and thus altering the tint of the fabric through absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Color Index (C.l.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO 2005 / 003274, WO 2005 / 003275, WO 2005 / 003276 and EP 1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt. % to about 0.2 wt. %, from about 0.00008 wt. % to about 0.05 wt. %, or even from about 0.0001 wt. % to about 0.04 wt. % fabric hueing agent. The composition may comprise from 0.0001 wt % to 0.2 wt. % fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g., WO 2007 / 087257 and WO 2007 / 087243.

[0266] Additional Enzymes

[0267] A detergent composition of the invention may comprise one or more additional enzymes such as an amylase, arabinase, carbohydrase, cellulase, cutinase, DNase, endoglucanase, galactanase, haloperoxygenase, lipase, mannanase, oxidase, e.g., laccase and / or peroxidase, pectinase, pectin lyase, proteasexylanase, xanthanase, or xyloglucanase.

[0268] The properties of the selected enzyme(s) should be compatible with the other components of the detergent composition and consider, e.g., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.

[0269] DNases

[0270] Suitable deoxyribonucleases (DNases) include any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. There are two primary classifications based on the locus of activity. Exonucleases digest nucleic acids from the ends. Endonucleases act on regions in the middle of target molecules. The DNase is preferable obtainable from a microorganism, preferably a fungus or bacterium. In particular, a DNase which is obtainable from a species of Bacillus is preferred; in particular a DNase which is obtainable from Bacillus ci bi, Metabacillus indicus, Bacillus subtilis or Bacillus licheniformis is preferred. Examplesof such DNases are described in WO 2011 / 098579, WO 2014 / 087011 and WO 2017 / 060475. Particularly preferred is also a DNase obtainable from a species of Aspergillus, in particular a DNase which is obtainable from Aspergillus oryzae, such as a DNase described in WO 2015 / 155350.

[0271] Cellulases

[0272] The term “cellulase” means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. The terms “cellulase” and the expression “polypeptide having cellulase activity” are used interchangeably. Cellulases may be selected from the group consisting of cellulases belonging to GH5, GH44, GH45, EC 3.2.1.4, EC 3.2.1.21, EC 3.2.1.91 and EC 3.2.1.172. Such enzymes include endoglucanase(s) (e.g., EC 3.2.1.4), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.

[0273] Suitable cellulases include mono-component and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein engineered mutants are also contemplated. The cellulase may for example be a mono-component or a mixture of mono-component endo-1,4-beta-glucanase also referred to as endoglucanase.

[0274] Suitable cellulases include those from the genera Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include a fungal cellulase from Humicola insolens (US 4,435,307) or from Trichoderma, e.g., T. reesei or T. viride. Other suitable cellulases are from Thielavia, e.g., Thielavia terrestris as described inWO 96 / 29397, or the fungal cellulases produced from Myceliophthora thermophila and Fusarium oxysporum disclosed in US 5,648,263, US 5,691 ,178, US 5,776,757, WO 89 / 09259 and WO 91 / 17244. Also relevant are cellulases from Bacillus as described in WO 02 / 099091 and JP 2000210081. Suitable cellulases are alkaline or neutral cellulases having care benefits. Examples of cellulases are described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307.

[0275] Other cellulases are endo-beta-1 ,4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO:2 of WO 2002 / 099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60% identity to positions 40-559 of SEQ ID NO:2 of WO 2001 / 062903.

[0276] Yet another group of suitable cellulases comprise a stabilized linker between the core and the CBM. Particularly useful are such cellulase having at least 80% identity to SEQ ID NO:397, SEQ ID NO:398 or SEQ ID NO:399 of WO 2023 / 061928.

[0277] Commercially available cellulases include Carezyme®, Carezyme® Premium, Celluzyme®, Carezyme Elite®, Celluclean®, Celluclast®, Endolase®, Renozyme®, Whitezyme® Celluclean® Classic, and Cellusoft® (Novozymes A / S); Puradax®, Puradax HA, Puradax EG,Revitalenz 1000, Revitalenz 200, and Revitalenz 2000 (Dupont Industrial Biosciences); KAC-500(B)™ (Kao Corporation); and Biotouch DCL and Biotouch FLX1 (AB Enzymes).

[0278] The two basic approaches for measuring cellulolytic enzyme activity include: (1) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., 2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman No. 1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, PureAppl. Chem. 59: 257-68).

[0279] Proteases

[0280] In some embodiments, the detergent compositions of the invention comprise one or more additional proteases including those of bacterial, fungal, plant, viral or animal origin, e.g., vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as subtilisin. A metalloprotease may for example be a thermolysin from, e.g., family M4 or other metalloprotease such as those from M5, M7 or M8 families.

[0281] Examples of metalloproteases are the neutral metalloproteases as described in WO 2007 / 044993 (Genencor I nt.) such as those derived from Bacillus amyloliquefaciens.

[0282] Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, Esperase®, Progress® Excel, Progress® Key, and Progress® Uno (Novozymes A / S), those sold under the tradename Maxatase®, Maxacai®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Opticlean®, Optimase®, Preferenz® P200, Preferenz® P300, and Preferenz® P400 (DuPont / IFF), Axapem™ (Gist-Brocades N.V.), BI_AP (sequence shown in Figure 29 of US5352604) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

[0283] Lipases and Cutinases

[0284] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g., from T. lanuginosus (previously named Humicola lanuginosa) as describedin EP 258068 and EP 305216, cutinase from Humicola, e.g., H. insolens (WO 96 / 13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95 / 06720 & WO 96 / 27002), P. wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 2010 / 065455), cutinase from Magnaporthe grisea (WO 2010 / 107560), cutinase from Pseudomonas mendocina (US 5,389,536), lipase from Thermobifida fusca (WO 2011 / 084412), Geobacillus stearothermophilus lipase (WO 2011 / 084417), lipase from Bacillus subtilis (WO 2011 / 084599), and lipase from Streptomyces griseus (WO 2011 / 150157) and S. pristinaespiralis (WO 2012 / 137147).

[0285] Other examples are lipase variants such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 2007 / 87508 and WO 2009 / 109500.

[0286] Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A / S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).

[0287] Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g., acyltransferases with homology to Candida antarctica lipase A (WO 2010 / 111143), acyltransferase from Mycobacterium smegmatis (WO 2005 / 056782), perhydrolases from the CE 7 family (WO 2009 / 067279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 2010 / 100028).

[0288] Amylases

[0289] In some embodiments, the detergent compositions of the invention comprise an amylase. Suitable amylases which can be used together with the variants of the invention may be an alphaamylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1,296,839.

[0290] Suitable amylases include amylases having SEQ ID NO:2 in WO 95 / 10603 or variants having 90% sequence identity to SEQ ID NO:3 thereof. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and SEQ ID NO:4 of WO 99 / 19467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.Different suitable amylases include amylases having SEQ ID NO:6 in WO 02 / 10355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having a deletion in positions 181 and 182 and a substitution in position 193.

[0291] Other amylases which are suitable are hybrid alpha-amylases comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO:4 of WO 2006 / 066594 or variants having 90% sequence identity thereof. Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO:4 are those having the substitutions:

[0292] M197T;

[0293] H 156Y+A 181 T+ N 190F+A209V+Q264S; or

[0294] G48A+T49I +G 107A+ H 156Y+A 181 T+ N 190F+ 1201 F+A209V+Q264S.

[0295] Other suitable amylases are amylases having the sequence of SEQ ID NO:6 in WO 99 / 19467 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.

[0296] Additional amylases which can be used are those having SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:7 of WO 96 / 23873 or variants thereof having 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7. Preferred variants of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96 / 23873 for numbering. More preferred variants are those having a deletion in two positions selected from 181, 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. Most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0297] Other amylases which can be used are amylases having SEQ ID NO:2 of WO 2008 / 153815, SEQ ID NQ:10 in WO 01 / 66712 or variants thereof having 90% sequence identity to SEQ ID NO:2 of WO 2008 / 153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NQ:10 in WO 01 / 66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.Further suitable amylases are amylases having SEQ ID NO:2 of WO 2009 / 061380 or variants having 90% sequence identity to SEQ ID NO:2 thereof. Preferred variants of SEQ ID NO:2 are those having a truncation of the C-terminus and / or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO:2 are those having the substitution in one of 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 deletion in position R180 and / or S181 or of T182 and / or G183. Most preferred amylase variants of SEQ ID NO:2 are those having the substitutions:

[0298] N 128C+K178L+T182G+Y305R+G475K;

[0299] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0300] S125A+N 128C+K178L+T182G+Y305R+G475K; or S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K,

[0301] wherein the variants are C-terminally truncated and optionally further comprise a substitution at position 243 and / or a deletion at position 180 and / or position 181.

[0302] Further suitable amylases are amylases having SEQ ID NO:1 of WO 2013 / 184577 or variants having 90% sequence identity to SEQ ID NO:1 thereof. Preferred variants of SEQ ID NO:1 are those having a substitution, a deletion or an insertion in one of more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO:1 are those having the substitution in one of more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K and / or a deletion in position R178 and / or S179 or of T180 and / or G181. Most preferred amylase variants of SEQ ID NO:1 comprise the substitutions:

[0303] E187P+I203Y+G476K

[0304] E187P+I203Y+R458N+T459S+D460T+G476K

[0305] and optionally further comprise a substitution at position 241 and / or a deletion at position 178 and / or position 179.

[0306] Further suitable amylases are amylases having SEQ ID NO:1 of WO 2010 / 104675 or variants having 90% sequence identity to SEQ ID NO:1 thereof. Preferred variants of SEQ ID NO:1 are those having a substitution, a deletion or an insertion in one of more of the following positions: N21, D97, V128 K177, R179, S180, 1181, G182, M200, L204, E242, G477 and G478.

[0307] More preferred variants of SEQ ID NO:1 are those having the substitution in one of more of the following positions: N21D, D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and / or a deletion in position R179 and / or S180 or of 1181 and / or G182. Most preferred amylase variants of SEQ ID NO:1 comprise the substitutions N21D+D97N+V128I, and optionallyfurther comprise a substitution at position 200 and / or a deletion at position 180 and / or position 181.

[0308] Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO 01 / 66712 or a variant having at least 90% sequence identity to SEQ ID NO:12. Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO:12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.

[0309] Other examples are amylase variants such as those described in WO 2011 / 098531 , WO 2013 / 001078 and WO 2013 / 001087. Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (from Novozymes A / S), and Rapidase™, Purastar™ / Effectenz™, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. / DuPont).

[0310] Mannanases

[0311] Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. The mannanase may be an alkaline mannanase of Family 5 or 26. It may be a wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 1999 / 064619. A commercially available mannanase is Mannaway (Novozymes A / S).

[0312] Licheninases

[0313] Suitable licheninases (lichenases) include enzymes that catalyse the hydrolysis of the beta-1, 4-glucosidic bonds to give beta-glucans. Licheninases (or lichenases) (e.g. EC 3.2.1.73) hydrolyse (1,4)-beta-D-glucosidic linkages in beta-D-glucans containing (1,3)- and (1,4)-bonds and can act on lichenin and cereal beta-D-glucans, but not on beta-D-glucans containing only 1,3- or 1,4-bonds. Examples of such licheninases are described in patent application WO 2017 / 097866 and in WO 2017 / 129754.

[0314] Peroxidases / Oxidases

[0315] Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0316] Commercially available peroxidases include Guardzyme™ (Novozymes A / S).

[0317] Other materials

[0318] Any detergent components known in the art for use in detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, and / or polyols such as propylene glycol), fabric conditioners including clays, fillers / processing aids, fluorescent whitening agents / optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.

[0319] Adjunct materials

[0320] Any detergent components known in the art for use in laundry detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and / or polyols such as propylene glycol), fabric conditioners including clays, fillers / processing aids, fluorescent whitening agents / optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.

[0321] Dispersants: The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant Science Series, volume 71, Marcel Dekker, Inc., 1997.

[0322] Dye Transfer Inhibiting Agents: The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinyhmidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the composition.

[0323] Fluorescent whitening agent: The detergent compositions of the present invention may preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01 % to about 05%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2,2'-disulphonate; 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2'-disulphonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate, 4,4'-bis-(4-phenyl-2, 1 ,3-triazol-2-yl)stilbene-2,2'-disulphonate; 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate and 2-(stilbyl-4"-naptho-1.,2':4,5)-1,2,3-trizole-2"-sulphonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbene disulphonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7-alkylaminocoumarins. Suitable fluorescent brightener levels include lower levels of from about 0.01, from 0.05, from about 0.1 or even from about 0.2 wt. % to upper levels of 0.5 or even 0.75 wt. %.

[0324] Soil release polymers: The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalate based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Another type of soil release polymers is amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009 / 087523 (hereby incorporated byreference). Furthermore, random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856 and WO 2006 / 113314 (hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose derivatives such as those described in EP 1867808 or WO 03 / 040279 (both are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.

[0325] Anti-redeposition agents: The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.

[0326] Other suitable adjunct materials include, but are not limited to, anti-shrink agents, antiwrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and / or structure elasticizing agents.

[0327] Formulation of Detergent Products

[0328] The protease variant and anionic surfactant, and optionally further enzyme(s), may be included in the detergent compositions of the invention by adding separate additives containing one or more of these actives, or by adding a combined additive comprising all of these actives. A detergent additive comprising one or more enzymes can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations include granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

[0329] The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. Other detergent formulation forms include layers (same or different phases), pouches, as well as forms for machine dosing unit.Pouches can be configured as single or multiple compartments. It can be of any form, shape and material which is suitable for hold the composition, e.g., without allowing the release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. The inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials, preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected from polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. The preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blend compositions comprising hydrolytically degradable and water-soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod, of Gary, Indiana, US) plus plasticizers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry detergent composition or part components and / or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids. See, e.g., US 2009 / 0011970.

[0330] Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

[0331] A liquid or gel detergent which is not unit dosed may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous.

[0332] Laundry Soap Bars

[0333] The detergent compositions of the invention may be formulated as 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, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and / or synthetic soaps. The laundry soap bar has a physical form which is solid and thus not a liquid, gel or powder at room temperature.The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adductor hemiacetal adduct), boric acid, borate, 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 a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na+, K+, or NH4+and the organic anion may be for example formate, acetate, citrate, or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.

[0334] The laundry soap bar may also contain complexing agents such as EDTA and HEDP, perfumes and / or different type of fillers, surfactants, e.g., anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and / or other compounds known in the art.

[0335] The laundry soap bar may be processed in conventional laundry soap bar making equipment such as, but not limited to, mixers, plodders, e.g., a two-stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers. A premix containing a soap, a protease, and anionic surfactant, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and the mixture is then plodded. The protease and anionic surfactant and optional additional enzymes may be added in turn or at the same time, and the protease may be added together with a protease inhibitor, for example a protease inhibitor in liquid form. Besides the mixing step and the plodding step, the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and / or wrapping.

[0336] Granular detergent formulations

[0337] Enzymes such as variants of the present invention in the form of granules, comprising an enzyme-containing core and optionally one or more coatings, are commonly used in granular (powder) detergents. Various methods for preparing the core are well-known in the art and include, for example, a) spray drying of a liquid enzyme-containing solution, b) production of layered products with an enzyme coated as a layer around a pre-formed inert core particle, e.g. using a fluid bed apparatus, c) absorbing an enzyme onto and / or into the surface of a pre-formed core, d) extrusion of an enzyme-containing paste, e) suspending an enzyme-containing powder in molten wax and atomization to result in prilled products, f) mixer granulation by adding an enzymecontaining liquid to a dry powder composition of granulation components, g) size reduction of enzyme-containing cores by milling or crushing of larger particles, pellets, etc., and h) fluid bedgranulation. The enzyme-containing cores may be dried, e.g., using a fluid bed drier or other known methods for drying granules in the feed or enzyme industry, to result in a water content of typically 0.1 -10% w / w water.

[0338] The enzyme-containing cores are optionally provided with a coating to improve storage stability and / or to reduce dust formation. One type of coating that is often used for enzyme granulates for detergents is a salt coating, typically an inorganic salt coating, which may, e.g., be applied as a solution of the salt using a fluid bed. Other coating materials that may be used are, for example, polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). The granules may contain more than one coating, for example a salt coating followed by an additional coating of a material such as PEG, MHPC or PVA.

[0339] The present invention thus also relates to enzyme granules / particles comprising the protease variant and an anionic surfactant of the invention. In an embodiment, the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core.

[0340] The core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.

[0341] The core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.

[0342] The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate. The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and / or an acidic buffer component, typically as a homogenous blend.

[0343] The core may include an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.

[0344] The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.

[0345] The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).

[0346] The coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%. The amount may be at most 100%, 70%, 50%, 40% or 30%.

[0347] The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit it is encapsulating / enclosing has few or none uncoated areas. The layer or coating should, in particular, be homogeneous in thickness.

[0348] The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0349] A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.

[0350] To provide acceptable protection, the salt coating is preferably at least 0.1 pm thick, e.g., at least 0.5 pm, at least 1 pm, at least 2 pm, at least 4 pm, at least 5 pm, or at least 8 pm. In a particular embodiment, the thickness of the salt coating is below 100 pm, such as below 60 pm, or below 40 pm.

[0351] The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.

[0352] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.

[0353] The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.

[0354] The salt in the coating may have a constant humidity at 20 °C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0355] Specific examples of suitable salts are NaCI (CH2o°c=76%), Na2CO3 (CH2o°c=92%), NaNO3(CH2O"C=73%), Na2HPO4(CH2o"c=95%), Na3PO4(CH25°c=92%), NH4CI (CH2o°c= 79.5%), (NH4)2HPO4(CH2O"C = 93,0%), NH4H2PO4(CH20°c = 93.1%), (NH4)2SO4(CH2o°c=81.1%), KOI(CH2O°C=85%), K2HPO4 (CH2O°C=92%), KH2PO4(CH20°C=96.5%), KNO3(CH20°C=93.5%), Na2SO4(CH20°C=93%), K2SO4(CH20°C=98%), KHSO4(CH20°C=86%), MgSO4(CH20°c=90%), ZnSO4(CH2O°C=9O%) and sodium citrate (CH25°c=86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2and magnesium acetate.

[0356] The salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO47H2O), zinc sulfate heptahydrate (ZnSO47H2O), sodium phosphate dibasic heptahydrate (Na2HPO4-7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0357] Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.

[0358] The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.

[0359] The granule may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0360] The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and / or high shear granulation.

[0361] Methods for preparing the core can be found in the Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.,

[0362] (a) Spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71; page 140-142; Marcel Dekker).

[0363] (b) Layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheresto the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97 / 23606.

[0364] (c) Absorbed core particles, wherein rather than coating the enzyme as a layer around the core, the enzyme is absorbed onto and / or into the surface of the core. Such a process is described in WO 97 / 39116.

[0365] (d) Extrusion or pelletized products, wherein an enzyme-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71; pages 140-142; Marcel Dekker).

[0366] (e) Prilled products, wherein an enzyme-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomizer, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71; page 140-142; Marcel Dekker). The product obtained is one wherein the enzyme is uniformly distributed throughout an inert material instead of being concentrated on its surface. U.S. Patent Nos. 4,016,040 and 4,713,245 describe this technique.

[0367] (f) Mixer granulation products, wherein an enzyme-containing liquid is added to a dry powder composition of conventional granulating components. The liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme. Such a process is described in U.S. Patent No.

[0368] 4,106,991 and related documents EP 170360, EP 304332, EP 304331, WO 90 / 09440 and WO 90 / 09428. In a particular product of this process, various high-shear mixers can be used as granulators. Granulates consisting of enzyme, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to produce a so-called T-granulate. Reinforced particles are more robust, and release less enzymatic dust.

[0369] (g) Size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0370] (h) Fluid bed granulation. Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray dropletsget wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.

[0371] (i) The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzymes, it is important the cores comprising the enzyme contain a low amount of water before coating with the salt. If water sensitive enzymes are coated with a salt before excessive water is removed, it will be trapped within the core and may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1-10% w / w water.

[0372] Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos.

[0373] 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art.

[0374] The granulate may further one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.

[0375] Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013 / 188331.

[0376] The enzyme may also be a protected enzyme prepared according to the method disclosed in EP 238,216.

[0377] In an embodiment, the granule further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof.

[0378] For further information on enzyme granules and production thereof, see WO 2013 / 007594 as well as, e.g., WO 2009 / 092699, EP 1705241, EP 1382668, WO 2007 / 001262, US 6,472,364, WO 2004 / 074419, and WO 2009 / 102854.

[0379] Uses

[0380] The present invention is also directed to methods for using the detergent compositions of the invention in laundering of textile and fabrics, such as household laundry washing and industrial laundry washing.The invention is also directed to methods for using the detergent compositions of the invention in cleaning hard surfaces such as floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dishwashing).

[0381] A detergent composition of the present invention may be formulated, for example, as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition or be formulated as a detergent composition for use in general household hard surface cleaning operations or be formulated for hand or machine dishwashing operations.

[0382] The cleaning process or the textile care process may for example be a laundry process, a dishwashing process, or cleaning of hard surfaces such as bathroom tiles, floors, tabletops, drains, sinks and washbasins. Laundry processes can for example be household laundering but may also be industrial laundering. Furthermore, the invention relates to a process for laundering of fabrics and / or garments, where the process comprises treating fabrics with a washing solution containing a detergent composition and at least one protease variant of the invention. The cleaning process or a textile care process can for example be carried out in a machine washing or manually. The washing solution can for example be an aqueous washing solution containing a detergent composition.

[0383] In one aspect, the detergent compositions of the invention are used in a cleaning process, e.g., a laundry process, that comprises a short wash cycle, typically a wash cycle of not more than about 30 minutes, such as not more than about 20 minutes, e.g., not more than about 15 minutes or not more than about 10 minutes. It has surprisingly been found that the protease variants of the invention are remarkably effective in short wash cycles lasting, for example, only about 10-20 minutes. This may be useful in, e.g., top-loading washing machines that often have short wash cycles or for hand-washing of laundry.

[0384] In another aspect, the detergent compositions of the invention are used in a cleaning process, e.g., a laundry process, where the wash water is used for more than one portion of laundry. In this case, the wash water containing a detergent composition of the invention may be used in a first wash cycle for a first portion of laundry, and then reused one or more times for additional wash cycles with new portions of laundry. It has been found that detergents containing a protease variant of the invention are able to substantially maintain cleaning performance on protease-sensitive stains even after three wash cycles or more. This may for example be useful for laundry washed by hand and / or in regions with water scarcity.

[0385] The last few years there has been an increasing interest in replacing components in detergents that are derived from petrochemicals with renewable biological components such as enzymes and polypeptides without compromising the wash performance. When the components of detergent compositions change, new enzyme activities or new enzymes having alternative and / or improved properties compared to the previously used detergent enzymes such asproteases, lipases and amylases may be needed to achieve a similar or improved wash performance when compared to the traditional detergent compositions.

[0386] The invention further concerns the use of detergent compositions of the invention in a proteinaceous stain removing process. The proteinaceous stains may be stains such as food stains, e.g., baby food, cocoa, egg or milk, or other stains such as sebum, blood, ink or grass, or a combination hereof.

[0387] Washing Method

[0388] The present invention provides a method of cleaning a fabric, dishware, or a hard surface with a detergent composition of the invention.

[0389] The method of cleaning comprises contacting an object with a detergent composition of the invention under conditions suitable for cleaning the object.

[0390] In a preferred embodiment the detergent composition is used in a laundry or a dish wash process.

[0391] Preferably, the detergent composition of the invention is used in a laundry process. Another embodiment relates to a method for removing stains from fabric or dishware which comprises contacting the fabric or dishware with a detergent composition of the invention under conditions suitable for cleaning the object. In the method of cleaning of the invention, the object being cleaned may be any suitable object such as a textile or a hard surface such as dishware or a floor, table, wall, etc.

[0392] Also contemplated are methods of treating fabrics (e.g., to desize a textile) using a detergent composition of the invention. The detergent composition can be used in any fabrictreating method which is well known in the art (see, e.g., US 6,077,316). For example, in one aspect, the feel and appearance of a fabric is improved by a method comprising contacting the fabric with a detergent composition for the invention. In one aspect, the fabric is treated with the solution under pressure.

[0393] The detergent compositions of the present invention are suited for use in laundry and hard surface applications, including dishwashing. Accordingly, the present invention includes a method for laundering a fabric or washing dishware, comprising contacting the fabric / dishware to be cleaned with a solution comprising the detergent composition according to the invention. The fabric may comprise any fabric capable of being laundered in normal consumer use conditions. The dishware may comprise any dishware such as crockery, cutlery, ceramics, plastics such as melamine, metals, china, glass and acrylics. The solution preferably has a pH from about 5.5 to about 11.5. The compositions may be employed at concentrations from about 100 ppm, preferably 500 ppm to about 15,000 ppm in solution. The water temperatures typically range from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C,about 75°C, about 80°C, about 85°C and about 90°C. The water to fabric ratio is typically from about 1:1 to about 30:1.

[0394] The enzymes of the detergent composition of the invention may be stabilized using conventional stabilizing agents and protease inhibitors, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, different salts such as NaCI, KCI, lactic acid, formic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, or a peptide aldehyde such as di-, tri- or tetrapeptide aldehydes or aldehyde analogues (either of the form B1-B0-R wherein, R is H, CH3, CX3, CHX2, or CH2X (X=halogen), BO is a single amino acid residue (preferably with an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (preferably one, two or three), optionally comprising an N-terminal protection group, or as described in WO 2009 / 118375, WO 98 / 13459) ora protease inhibitor of the protein type such as RASI, BASI, WASI (bifunctional alpha-amylase / subtilisin inhibitors of rice, barley and wheat) or CI2 or SSI. The composition may be formulated as described in, e.g., \NQ) 92 / 19709, WO 92 / 19708 and US 6,472,364. In some embodiments, the enzymes employed herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II) and / or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II), and oxovanadium (IV)).

[0395] The detergent compositions provided herein are typically formulated such that, during use in aqueous cleaning operations, the wash water has a pH of from about 5.0 to about 12.5, such as from about 5.0 to about 11.5, or from about 6.0 to about 10.5. In some embodiments, granular or liquid laundry products are formulated to have a pH from about 6 to about 8. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0396] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0397] EXAMPLES

[0398] Materials & Methods

[0399] Protease Activity Assay I

[0400] The proteolytic activity of a variant of the invention can be determined by a method employing the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and it is a blocked peptide which can becleaved by endo-proteases. Following proteolytic cleavage, a free pNA molecule having a yellow color is liberated and can be measured by visible spectrophotometry at wavelength 405 nm. The Suc-AAPF-PNA substrate may be purchased from Bachem.

[0401] A sample containing the protease variant to be analyzed is diluted in residual activity buffer (100 mM Tris, pH 8.6). The assay is performed by transferring 30 pl of diluted enzyme samples to 96 well microtiter plate and adding 70 pl substrate working solution (0.72 mg / ml in 100 mM Tris, pH 8.6). The solution is mixed at room temperature and absorption at 405 nm is measured over time, e.g., every 20 sec. over 5 minutes. The slope (absorbance per minute) of the timedependent absorption curve is directly proportional to proteolytic activity.

[0402] Protease Activity Assay II

[0403] The proteolytic activity of a detergent composition comprising a variant of the invention can be determined by a method employing N,N-dimethyl casein (DMC) as substrate. By hydrolysis of peptide bonds, carboxylic acids and primary amines are produced. The produced amines then react under alkaline conditions with 2,4,6-tri-nitrobenzene-sulphonic acid (TNBS, Sigma) to form a colored complex which can be measured at 405 nm.

[0404] A detergent sample containing a variant of the invention is dissolved in 0.08 M sodium sulfite buffer and stirred for 10 minutes, after which the sample is filtered (Whatman filter no. 54 or similar). Sample dilutions are made using buffer (0.05 M boric acid + 0.16 M sodium sulfite + 0.15 M potassium chloride + 0.0225% (w / v) Brij® L23, pH 9.00). Reagents, including 1) 3.2 g / L DMC substrate + 0.1 M sodium dihydrogen phosphate monohydrate + 0.07 M Borax + 0.02% (w / v) Brij® L23, pH 8.00, 2) 0.1% TNBS and 3) 0.1% TNBS + 0.4% DSAA are employed in running the analysis using a Konelab 30 Analyzer (ThermoFisher Scientific) according to the assay parameters outlined in Table 19. Activity values may then be calculated based on a standard curve.

[0405] Terg-o-tometer (TOM) Wash Assay

[0406] The Terg-o-tometer (TOM) is a medium scale wash assay that can be applied different conditions. Briefly, it consists of metal beakers, each fitted with an agitator, which rotate in a back-and-forth manner at a controlled speed to simulate the agitation occurring in commercial toploader washing machines. The beakers are partly submerged in thermostatic water baths where the temperature can be controlled. Each beaker was filled with 1 L detergent solution, and test swatches, ballast and enzymes are added to the requisite levels. After a timed wash period, the swatches are promptly removed from the beakers and rinsed thoroughly. The swatches are then spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches are done using a photograph was taken using a DigiEye® Imaging System (Verivide,UK) and remission (REM) at 460 nm is extracted. The effect of a protease on each swatch is calculated by subtracting the remission value of the swatch washed without protease (blank) from the swatch washed together with protease.

[0407] Table 1: TOM wash experimental conditions

[0408] Detergent European liquid model detergent

[0409] Detergent dosage 2.5 g / L

[0410] Test solution volume 1 L

[0411] pH 8.0

[0412] Wash time 20 minutes

[0413] Agitation 120 rpm

[0414] Temperature 20 °C

[0415] Water hardness 14 °dH (4:1 :7.5) (Ca2+:Mg2+:HCO3’)

[0416] Protease concentration in test

[0417] 24 nM

[0418] solution

[0419] 2 swatches (5 cm x 5 cm) of each stain per TOM beaker: C-03 Chocolate milk with carbon black, C-S-07 Grass, Test material pure, EMPA-117 EH Blood, milk, ink, extra heat-treated from Center for Testmaterials B.V., Stoomloggerweg 11, 3133 KT Vlaardingen, The Netherlands.

[0420]

[0421] The European model detergent was produced by mixing water, propylene glycol, triethanolamine and sodium hydroxide. Then topped palm kernel fatty acid, Na-LAS, SLES and AEO were added in portions and stirred for few hours. Trisodium citrate dihydrate, DTPMP Na7 and 2-phenoxyethanol were added and pH adjusted within target after overnight stirring at room temperature. Commercial raw materials were used and dosage of raw material was adjusted with the purity of the individual ingredients to achieve the listed active content. Emulsogen Col 100 (Clariant) was added separately to the dissolved detergent solution.

[0422] Table 2. European liquid model detergent composition

[0423] Weight % of active Ingredient

[0424] ingredient

[0425] Sodium linear alkylbenzene sulfonate (Na-LAS) 12.0%

[0426]

[0427] Sodium lauryl ether sulfate (SLES) 4.1% Alcohol ethoxylate with 7 mol EO (AEO) 12.0% Topped Palm Kernel Fatty Acid 3.0% Sodium hydroxide 0.5% Ethanol 6.0% Propylene glycol (MPG) 2.0% Triethanolamine (TEA) 2.0% Trisodium citrate dihydrate 3.9% Diethylenetriaminepentakis(methylene)pentakis(phosphonic

[0428] 1.6% acid), heptasodium salt (DTPMP Na7)

[0429] 2-Phenoxyethanol 0.5% Water (demineralized) Add to 100% pH target 8.4

[0430]

[0431] Example 1 : Wash performance in Terg-o-tometer (TOM) assay

[0432] Wash experiments were performed in order to assess the wash performance of the protease variants in a laundry model detergent with the surfactant Emulsogen Col 100.

[0433] The proteases tested were:

[0434] Protease 1: SEQ ID NO: 8.

[0435] Protease 2: SEQ ID NO: 9.

[0436] Table 3. Remission values on C-03, C-S-07 and EMPA-117 EH swatches after TOM wash with different proteases and detergents formulations

[0437] Protease Detergent C-03 C-S-07 EMPA-117 EH No protease Ell liquid model detergent 28.0 31.8 12.6 Protease 1 Ell liquid model detergent 31.5 39.4 25.6 Protease 2 Ell liquid model detergent 32.2 38.4 26.8

[0438] Ell liquid model detergent + 5 29.4 30.8 12.5 No Protease

[0439] wt% Emulsogen COL 100

[0440] Ell liquid model detergent + 5 30.9 39.2 27.3 Protease 1

[0441] wt% Emulsogen COL 100

[0442] EU liquid model detergent + 5 32.3 40.6 28.8 Protease 2

[0443] wt% Emulsogen COL 100

[0444]

[0445] The invention is further defined by the following numbered paragraphs:

[0446] 1. A detergent composition comprising:

[0447] a protease variant of a parent protease, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the protease variant has protease activity; and

[0448] an anionic surfactant.

[0449] 2. The detergent composition of paragraph 1, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 95 of SEQ ID NO:1 with Ala, Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Asp.

[0450] 3. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 209 of SEQ ID NO:1 with Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Lys.

[0451] 4. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 9 of SEQ ID NO:1 with Ala, Arg, Asn, Cys, Gin, Glu, Gly, His, lie, Leu, Met, Phe, Trp, Tyr, or Vai, preferably with Glu.

[0452] 5. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 42 of SEQ ID NO:1 with Ala, Arg, Cys, Gin, Glu, His, lie, Leu, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, preferably with Arg.

[0453] 6. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 74 of SEQ ID NO:1 with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Asp.7. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 199 of SEQ ID NO:1 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, orTyr, preferably with lie.

[0454] 8. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 200 of SEQ ID NO:1 with Ala, Arg, Asn, Asp, Cys, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Trp, or Vai, preferably with Leu.

[0455] 9. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 203 of SEQ ID NO:1 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Trp.

[0456] 10. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 253 of SEQ ID NO:1 with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Trp, Tyr, or Vai, preferably with Asp.

[0457] 11. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 255 of SEQ ID NO:1 with Ala, Arg, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Pro, Thr, Trp, Tyr, or Vai, preferably with Trp.

[0458] 12. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises a substitution of the amino acid residue at a position corresponding to position 256 of SEQ ID NO:1 with Ala, Arg, Asn, Cys, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Glu.

[0459] 13. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least three substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0460] 14. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least four substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.15. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least five substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0461] 16. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least six substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0462] 17. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least seven substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0463] 18. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least eight substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0464] 19. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises nine substitutions at positions corresponding to each of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.

[0465] 20. The detergent composition according to any of the preceding paragraphs, wherein the total number of substitutions in the protease variant compared to the parent protease is 5-20, e.g., 5-15 or 5-10, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions. 21. The detergent composition according to any of the preceding paragraphs, wherein the total number of substitutions in the protease variant compared to the parent protease is 5-11, such as 5, 6, 7, 8, 9, 10, or 11 substitutions.

[0466] 21a. The detergent composition according to any of paragraphs 1-21, wherein the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1.

[0467] 21b. The detergent composition according to any of paragraphs 1-21, wherein the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.22. The detergent composition according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:1.

[0468] 23. The detergent composition according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:2.

[0469] 24. The detergent composition according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:3.

[0470] 25. The detergent composition according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:4.

[0471] 26. The detergent composition according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:5.

[0472] 27. The detergent composition according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:6.

[0473] 28. The detergent composition according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:7.

[0474] 29. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises substitutions corresponding to G95D and A209Kof SEQ ID NO:1 and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.30. The detergent composition according to any of the preceding paragraphs, which comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprises substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

[0475] 30a. The detergent composition according to any of the preceding paragraphs, wherein the protease variant comprises further substitutions at one or more other positions corresponding to positions 60 (e.g., N60D), 97 (e.g., S97D), 99 (e.g., S99E), 116 (e.g., G116N), and 246 (e.g., N246L) of SEQ ID NO:1.

[0476] 31. The detergent composition according to any of the preceding paragraphs, wherein the protease variant further comprises one or more substitutions selected from the group consisting of substitutions corresponding to N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L of SEQ ID NO:1.

[0477] 32. The detergent composition according to any of the preceding paragraphs, wherein the anionic surfactant is selected from the group consisting of: ether carboxylates, such as alkyl ether carboxylates, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.

[0478] 33. The detergent composition according to any of the preceding paragraphs, wherein the anionic surfactant is an ether carboxylate according to the general Formula (I):

[0479] R-O-(OCH2CH2)n-CH2-COOX Formula (I)

[0480] wherein

[0481] R represents a linear or branched C4-C22 alkyl or alkenyl group;

[0482] n represents a number from 1-20; and

[0483] X represents a hydrogen, sodium, potassium, ammonium and / or alkanolammonium. 34. The detergent composition according to any of the preceding paragraphs, wherein the anionic surfactant is a carboxylate anionic surfactant.35. The detergent composition according to any of the preceding paragraphs, wherein the anionic surfactant is an ether carboxylate surfactant, preferably an alkyl ether carboxylate surfactant.

[0484] 36. The detergent composition according to any of the preceding paragraphs, wherein the anionic surfactant is a Cs- to Cis-alkyl ether carboxylate, preferably Ci6- to Cis-alkyl ether carboxylate.

[0485] 37. The detergent composition according to any of the preceding paragraphs, wherein the anionic surfactant is an ether carboxylate selected from the group of oleyl-O-(OCH2CH2)s-CH2-COOH or oleyl-O-(OCH2CH2) -CH2-COOH, preferably wherein the anionic surfactant is oleyl-O-(OCH2CH2)IO-CH2-COOH.

[0486] 38. The detergent composition according to any of the paragraphs 1-32, wherein the anionic surfactant is selected from linear alkylbenzene sulfonate (LAS) or alcohol ether sulfate (AEOS).

[0487] 39. The detergent composition according to any of the preceding paragraphs, wherein the detergent composition comprises one or more anionic surfactants.

[0488] 40. The detergent composition according to any of the preceding paragraphs, wherein the detergent composition comprises a mixture of two or more surfactants.

[0489] 41. The detergent composition according to any of the preceding paragraphs, wherein the detergent composition comprises a mixture of one or more non-ionic surfactants and one or more anionic surfactants.

[0490] 42. The detergent composition according to any of the preceding paragraphs, wherein:

[0491] the protease variant comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprises substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1, wherein position numbering is based on the numbering of SEQ ID NO:1, and wherein the protease variant has protease activity; and the anionic surfactant is an ether carboxylate surfactant, preferably an alkyl ether carboxylate surfactant.

[0492] 43. The detergent composition according to the preceding paragraph, wherein the alkyl ether carboxylate is selected from the group of oleyl-O-(OCH2CH2)5-CH2-COOH or oleyl-O-(OCH2CH2)IO-CH2-COOH, preferably oleyl-0-(OCH2CH2)io-CH2-COOH.

[0493] 44. The detergent composition according to any of the preceding paragraphs, wherein the detergent composition exhibits improved wash performance compared to a detergent composition comprising the parent protease.45. The detergent composition according to any of the preceding paragraphs, wherein the detergent composition exhibits improved wash performance compared to a detergent composition comprising an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ I D NO: 1.

[0494] 46. The detergent composition according to any of the preceding paragraphs, wherein the detergent composition exhibits improved stain removal, preferably wherein the stain is a proteinaceous stain.

[0495] 47. The detergent composition according to any of the preceding paragraphs further comprising one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases, mannanases, lechinase, RNase, DNAse, or any mixture thereof.

[0496] 48. The detergent composition according to any of the preceding paragraphs further comprising one or more additional components selected from the group consisting of stabilizing agents, additional surfactants, hydrotopes, builders, co-builders, chelating agents, bleaching systems, bleach activators, polymers and fabric-hueing agents.

[0497] 49. The detergent composition according to any of the preceding paragraphs comprising an additional surfactant, wherein the surfactant is selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants and amphoteric surfactants.

[0498] 50. The detergent composition according to any of the preceding paragraphs, wherein the detergent 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 regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.

[0499] 51. The detergent composition according to any of the preceding paragraphs wherein said detergent composition is a liquid laundry detergent composition, a powder laundry detergent composition, a liquid dishwash detergent composition, or a powder dishwash detergent composition.

[0500] 52. The detergent composition according to paragraph 51, wherein said composition is a liquid or powder laundry detergent composition.

[0501] 53. The detergent composition according to paragraph 51 , wherein said composition is a liquid or powder automatic dishwashing (ADW) detergent composition.

[0502] 54. The detergent composition according to paragraph 51 , wherein said composition is a liquid or powder manual dishwashing detergent composition.55. Use of a detergent composition according to any of the preceding paragraphs in a domestic or industrial cleaning process.

[0503] 56. The use according to paragraph 55 for cleaning of fabric, for example laundry.

[0504] 57. The use according to paragraph 55 for hard surface cleaning, for example dishwashing. 58. A method of cleaning an object comprising contacting said object with a detergent composition according to any of paragraphs 1-54 under conditions suitable for cleaning the object.

[0505] 59. The method according to paragraph 58, wherein the object is a fabric or textile.

[0506] 60. The method according to paragraph 58 or 59, wherein the method is performed by hand or using a washing machine.

[0507] 61. The method according to paragraph 58, wherein the object is dishware.

Claims

CLAIMS1. A detergent composition comprising:a protease variant of a parent protease, wherein the protease variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1; wherein position numbering is based on the numbering of SEQ ID NO:1; wherein the protease variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold; and wherein the protease variant has protease activity; andan anionic surfactant.

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

3. The detergent composition according to any of claims 1-2, wherein the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; preferably wherein the parent protease is SEQ ID NO:1.

4. The detergent composition according to any of the preceding claims, wherein the protease variant comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprises at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

735. The detergent composition according to any of the preceding claims, wherein the protease variant comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprises substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.

6. The detergent composition according to any of the preceding claims, wherein the protease variant further comprises a substitution at one or more positions selected from the group consisting of positions 60, 97, 99, 116, and 246 of SEQ ID NO:1.

7. The detergent composition according to any of the preceding claims, wherein the protease variant comprises one or more further substitutions selected from the group consisting of N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L of SEQ ID NO:1.

8. The detergent composition according to any of the preceding claims, wherein the anionic surfactant is selected from the group consisting of: ether carboxylates, such as alkyl ether carboxylates, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.

9. The detergent composition according to any of the preceding claims, wherein the anionic surfactant is an ether carboxylate according to the general Formula (I):R-O-(OCH2CH2)n-CH2-COOX Formula (I)whereinR represents a linear or branched C4-C22 alkyl or alkenyl group;n represents a number from 1-20; andX represents a hydrogen, sodium, potassium, ammonium and / or alkanolammonium.

10. The detergent composition according to any of the preceding claims, wherein the anionic surfactant is an ether carboxylate surfactant, preferably an alkyl ether carboxylate surfactant.

11. The detergent composition according to any of the preceding claims, wherein the anionic surfactant is an ether carboxylate selected from the group of oleyl-O-(OCH2CH2)5-CH2-COOH or74oleyl-O-(OCH2CH2) -CH2-COOH, preferably wherein the anionic surfactant is oleyl-O-(OCH2CH2)IO-CH2-COOH.

12. The detergent composition according to any of the preceding claims, wherein the detergent composition exhibits improved wash performance compared to a detergent composition comprising the parent protease or an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1.

13. The detergent composition according to any of the preceding claims, wherein the detergent 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 regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.

14. A method of cleaning an object, comprising contacting the object with a detergent composition according to any of claims 1-13 under conditions suitable for cleaning the object; preferably wherein the object is a fabric, dishware, or a hard surface; most preferably wherein the object is a fabric.

15. Use of a detergent composition according to any of claims 1-13 in a cleaning process, preferably laundry or hard surface cleaning.

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