Detergent composition comprising two proteases

Abstract

The present invention relates to detergent compositions comprising a first protease and a second protease having different net charge characteristics, and novel protease variants.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of February 26, 2020, application number 202080013079.5, and invention name “Detergent composition containing two proteases”.

[0002] References to sequence listings

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

[0004] The present invention relates to detergent compositions comprising at least a first protease and a second protease having different net charge characteristics, which together provide enhanced cleaning performance relative to the individual enzymes; the use of the composition in cleaning processes, particularly for laundry; and novel protease variants. Background Art

[0005] Subtilisin is a serine protease from the S8 family, specifically from the S8A subfamily, as defined by the MEROPS database (https: / / www.ebi.ac.uk / merops / index.shtml). In the S8A subfamily, the key active site residues Asp, His and Ser are typically found in a motif that differs from those of the S8B subfamily.

[0006] In the detergent industry, enzymes have been used for decades in detergent compositions for laundry or hard surface cleaning (such as dishwashing). The enzymes used in such compositions include proteases, lipases, amylases, cellulases, mannosidases, and other enzymes, or mixtures thereof. Commercially, the most important enzyme is protease.

[0007] It is known that different proteases may behave differently on different types of stains. Attempts have been made to address this problem and improve washing performance by including two different proteases in a single detergent composition. Such compositions are disclosed in, for example, WO 2009 / 021867, WO 2014 / 177430, WO 2016 / 000970 and WO 2016 / 000971.

[0008] However, although some such compositions comprising two different proteases are disclosed in the patent literature, in practice, detergent compositions comprising two different proteases have not yet achieved any widespread commercial success.

[0009] Further, relatively few compositions comprising two proteases described in the patent literature are disclosed in the context of automatic dishwashing. In contrast, there has been no attention paid to developing detergent compositions with two different proteases adapted to laundry conditions and requirements.

[0010] Therefore, given that individual proteases used in currently available detergent compositions are not effective in removing all relevant protein-based stains in any given environment, particularly in a laundry environment, there remains a need for detergent compositions having enhanced protein stain removal properties.

[0011] The present invention meets this need by providing a detergent composition comprising at least two different proteases having different net formal charge characteristics, wherein the combination of the two proteases results in improved cleaning performance on individual stains and / or covers a wider range of stains compared to the individual enzymes. It is contemplated that the compositions of the present invention will be particularly useful as laundry detergent compositions, especially liquid laundry detergent compositions. Summary of the invention

[0012] The present invention relates to a detergent composition comprising at least a first protease and a second protease having different net charge characteristics.

[0013] In one embodiment, the present invention relates to a detergent composition comprising a first protease and a second protease, wherein:

[0014] 1) the first protease has a net formal charge of -1, -3, or -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1; or

[0015] 2) the first protease has a net formal charge of -3, -4, or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1, 0, or +1 relative to the protease of SEQ ID NO: 1.

[0016] In other embodiments, the detergent composition can comprise a first protease and a second protease, wherein: the first protease has a net formal charge of 0, 1, 2, 3, 4, or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +5, +4, +3, +2, +1, 0, or -1 relative to the protease of SEQ ID NO: 1; the first protease has a net formal charge of -3, -4, or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2 relative to the protease of SEQ ID NO: 1; or the first protease has a net formal charge of 0, -1, or -2 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2, +3, +4, or +5 relative to the protease of SEQ ID NO: 1.

[0017] The present invention also relates to the use of the compositions disclosed herein in cleaning processes, for example for laundry or hard surface cleaning (such as dishwashing), in particular for laundry; and to methods of cleaning using the compositions.

[0018] In another aspect, the present invention relates to novel protease variants having advantageous properties, for example, improved cleaning performance when such protease is used alone as a single protease or together with another protease in a detergent composition, wherein the protease is a variant of SEQ ID NO: 1 comprising substitutions at positions 97, 209 and 215.

[0019] Sequence review

[0020] SEQ ID NO: 1 is the sequence of a Savinase® protease polypeptide from Bacillus lentus.

[0021] SEQ ID NO: 2 is the sequence of the BPN' protease polypeptide from Bacillus amyloliquefaciens. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is an alignment of the amino acid sequences of subtilisin 309 (SEQ ID NO: 1) and subtilisin BPN' (SEQ ID NO: 2).

[0023] definition

[0024] Subtilase / Protease: The terms "subtilase" and "protease" are used interchangeably herein and refer to enzymes that hydrolyze peptide bonds in proteins. This includes any enzyme belonging to the EC 3.4 enzyme group (including each of its 13 subclasses), and in particular endopeptidases (EC 3.4.21). EC numbers refer to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including Supplements 1-5 as published in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650, respectively.

[0025] Protease activity: The term "protease activity" means proteolytic activity (EC 3.4), in particular endopeptidase activity (EC 3.4.21). There are several types of protease activity, the three main types of activity being: trypsin-like (wherein there is cleavage of an amide substrate after Arg or Lys at P1), chymotrypsin-like (wherein cleavage occurs after a hydrophobic amino acid at P1), and elastase-like (cleavage after Ala at P1). Protease activity can be determined according to the procedure described in WO2016 / 087619.

[0026] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0027] For purposes of the present invention, use as implemented in the Needle program of the EMBOSS bag (EMBOSS: The European Molecular Biology Open Software Suite [EMBOSS: European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16: 276-277) (preferred 5.0.0 version or updated version) Needleman-Wunsch (Needleman-Wunsch) algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Molecular Biology] 48: 443-453) to determine the sequence identity between two amino acid sequences. The parameter employed is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output (using non-simplified option (nobriefoption) to obtain) of Needle, which is labeled as "longest identity", is used as identity percentage and is calculated as follows:

[0028] (identical residues x 100) / (alignment length - total number of gaps in the alignment)

[0029] For purposes of the present invention, use the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same) implemented in the Needle program of the EMBOSS bag (EMBOSS: The European Molecular Biology Open Software Suite [EMBOSS: European Molecular Biology Open Software Suite], Rice et al., 2000, the same) (preferably 5.0.0 version or updated version) to determine the sequence identity between two deoxyribonucleotide sequences. The parameter employed is a gap opening penalty of 10, a gap extension penalty of 0.5 and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output (using the non-simplified option to obtain) of the Needle labeled "longest identity" is used as identity percentage and is calculated as follows:

[0030] (identical deoxyribonucleotides x 100) / (alignment length - total number of gaps in the alignment)

[0031] Variant: The term "variant" means a polypeptide having protease activity comprising an alteration (i.e., substitution, insertion, and / or deletion) at one or more positions. Substitution means replacing the amino acid occupying a position with a different amino acid, deletion means removing the amino acid occupying a position, and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0032] Variant naming conventions

[0033] For the purposes of the present invention, the polypeptide of SEQ ID NO: 2 is used to determine the corresponding amino acid residue numbering in the variant of SEQ ID NO: 1. The amino acid sequence of the variant of SEQ ID NO: 1 is aligned with SEQ ID NO: 2, and the amino acid position number corresponding to any amino acid residue in the polypeptide of SEQ ID NO: 1 is determined based on the alignment. For further information, see the paragraph "Numbering of amino acid positions / residues" below.

[0034] The identification of the corresponding amino acid residue in another subtilase can be determined by aligning the multiple polypeptide sequences using several computer programs using their corresponding default parameters, including but not limited to MUSCLE (Multiple Sequence Comparison by Log-Expectation; Version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (Version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology [Molecular Biology Methods] 537: 39-64 ; Katoh and Toh, 2010, Bioinformatics [Journal of History] 26: 1899-1900 ), and EMBOSS EMMA with ClustalW (version 1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680).

[0035] In describing variants of the present invention, the nomenclature described below has been adjusted for ease of reference. Accepted IUPAC single-letter or three-letter abbreviations for amino acids are used. The terms "alteration" or "mutation" are used interchangeably herein to refer to substitutions, insertions, and deletions.

[0036] replace . For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, the substitution of threonine at position 220 by alanine is represented as "T220A". Multiple substitutions can be separated by a plus sign ("+"), for example, "T220A + G229V" represents that threonine (T) at position 220 and glycine (G) at position 229 are replaced by alanine (A) and valine (V), respectively. Alternatively, multiple substitutions can be listed with separate mutations separated by spaces or commas. Alternative substitutions in a specific position can be represented by a slash (" / "). For example, the substitution of threonine in position 220 by alanine, valine, or leucine can be represented as "T220A / V / L". The "X" before the position means that any original amino acid at that position can be replaced. For example, X9E means that any amino acid residue other than E at position 9 is replaced by E.

[0037] Missing For amino acid deletions, the following nomenclature is used: original amino acid, position, * Thus, a deletion of the threonine at position 220 is represented as "T220*". Multiple deletions may be separated by a plus sign ("+"), e.g., "T220*+G229*", or alternatively may be separated by spaces or commas. An "X" used before a position number is used for substitutions as described above, e.g., "X131*" means that the amino acid residue at position 131 is deleted.

[0038] insert For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, the insertion of lysine after the threonine at position 220 is represented as "T220TK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after the threonine at position 220 is represented as "T220TKA".

[0039] In such cases, the inserted amino acid residue(s) are numbered by adding lowercase letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would therefore be:

[0040]

[0041] Multiple changes Variants containing multiple alterations are separated by plus signs ("+"), for example, "R170Y+G195E" represents substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple alterations may be listed as separate mutations separated by spaces or commas.

[0042] For example, a combination of substitution and insertion may be represented as follows: S99AD, which represents that the serine residue at position 99 is substituted with an alanine residue and an aspartic acid residue is inserted.

[0043] Different changes When different changes can be introduced at one position, the different changes can be separated by commas, for example, "R170Y,E" represents that the arginine at position 170 is replaced by tyrosine or glutamic acid. Therefore, "Y167G,A + R170G,A" represents the following variants:

[0044] “Y167G + R170G”, “Y167G + R170A”, “Y167A + R170G”, and “Y167A + R170A”.

[0045] Different changes at one position may also be indicated by a slash (" / "), such as "T220A / V / L", as explained above. Alternatively, different changes may be indicated by using brackets, such as R170 [Y,G].

[0046] The number of the amino acid position / residue. The numbering used herein is based on the numbering of SEQ ID NO: 2. Thus, the amino acid residues of SEQ ID NO: 1 are numbered based on the corresponding amino acid residues in SEQ ID NO: 2. Specifically, the numbering is based on the alignment in Table 1 of WO 89 / 06279, which shows an alignment of five proteases including the mature polypeptide of the subtilisin BPN' (BASBPN) sequence (sequence c in the table) and the mature polypeptide of subtilisin 309 (also known as Savinase®) (BLSAVI) from Bacillus lentus (sequence a in the table). Those skilled in the art will appreciate that position numbering for subtilisin 309 and other proteases in the patent literature is generally based on the corresponding position numbering of BPN' according to this alignment.

[0047] For reference purposes only Figure 1 , and Table 1 based on WO 89 / 06279 shows an alignment between SEQ ID NO: 1 and SEQ ID NO: 2, from which the position numbers corresponding to the positions of SEQ ID NO: 2 can be easily determined. DETAILED DESCRIPTION

[0048] The present invention relates to a detergent composition comprising at least a first protease and a second protease, wherein the first protease and the second protease have different net formal charges relative to the protease of SEQ ID NO: 1.

[0049] The term "net formal charge" or "net charge" refers to the net charge of an enzyme, wherein the amino acid residues aspartic acid (D) and glutamic acid (E) contribute one negative charge (-1), and the amino acid residues arginine (R) and lysine (K) contribute one positive charge (+1), wherein the charges of these residues in the first protease or the second protease are compared to the charges of the residues in the protease of SEQ ID NO: 1. This means that replacing a neutral amino acid (e.g., the neutral amino acid in SEQ ID NO: 1) with an acidic amino acid (D or E) will provide a negative charge, while replacing a neutral amino acid with a basic amino acid (R or K) will provide a positive charge. Similarly, replacing an acidic amino acid with a basic amino acid will provide two positive charges, while replacing a basic amino acid with an acidic amino acid will provide two negative charges.

[0050] As explained above, the net charge of the protease in the detergent composition of the present invention is expressed relative to the net charge of the protease of SEQ ID NO: 1. As can be seen from the sequence of SEQ ID NO: 1, this protease contains 5 Asp residues, 5 Glu residues, 8 Arg residues, and 5 Lys residues, so the net charge of SEQ ID NO: 1 is +3, and the first protease and the second protease of the present invention will be compared with this value. Therefore, the net charge of the protease in the composition of the present invention can be easily determined by counting the number of acidic amino acids and basic amino acids in the amino acid sequence and comparing the calculated net charge with the net charge of the protease of SEQ ID NO: 1. It is obvious that the net charge of the first protease and the second protease determined in this way is a function of the number of acidic (D and E) amino acids and basic (R and K) amino acid residues, and for the purposes of the present invention, is independent of the composition having any specific pH value.

[0051] The present invention particularly relates to a detergent composition comprising a first protease and a second protease, wherein

[0052] 1) the first protease has a net formal charge of -1, -3, or -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1; or

[0053] 2) the first protease has a net formal charge of -3, -4, or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1, 0, or +1 relative to the protease of SEQ ID NO: 1.

[0054] It will be apparent that the terms "first protease" and "second protease" are used herein to distinguish between two proteases having different net charges in a given composition, and that a protease having a given net formal charge may in some cases be considered a "first protease" or a "second protease" depending on the context of the particular composition in which it is contained. For example, a protease having a net formal charge of -1 may be referred to as a "first protease" when present in a composition comprising another protease having a net formal charge of -2, but may be a "second protease" when present in a composition comprising another protease having a net formal charge of -3, -4, or -5.

[0055] In one aspect, the invention thus relates to a detergent composition comprising a first protease and a second protease, wherein the first protease has a net formal charge of -1, -3 or -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1. In preferred embodiments of this aspect, the first protease has a net formal charge of -3 or -4.

[0056] In one embodiment, the first protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1.

[0057] In one embodiment, the first protease has a net formal charge of -3 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1.

[0058] In one embodiment, the first protease has a net formal charge of -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1.

[0059] In another aspect, the present invention relates to a detergent composition comprising a first protease and a second protease, wherein the first protease has a net formal charge of -3, -4 or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1, 0, or +1 relative to the protease of SEQ ID NO: 1.

[0060] In one embodiment, the first protease has a net formal charge of -3 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +1 relative to the protease of SEQ ID NO: 1.

[0061] In one embodiment, the first protease has a net formal charge of -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +1 relative to the protease of SEQ ID NO: 1.

[0062] In one embodiment, the first protease has a net formal charge of -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +1 relative to the protease of SEQ ID NO: 1.

[0063] In one embodiment, the first protease has a net formal charge of -3 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of 0 relative to the protease of SEQ ID NO: 1.

[0064] In one embodiment, the first protease has a net formal charge of -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of 0 relative to the protease of SEQ ID NO: 1.

[0065] In one embodiment, the first protease has a net formal charge of -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of 0 relative to the protease of SEQ ID NO: 1.

[0066] In one embodiment, the first protease has a net formal charge of -3 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1.

[0067] In one embodiment, the first protease has a net formal charge of -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1.

[0068] In one embodiment, the first protease has a net formal charge of -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1.

[0069] It will be apparent that the difference in net formal charge between the first and second proteases in the composition of the invention may vary, such that the net formal charge of the two proteases in the composition may differ by, for example, 1, 2, 3, 4, 5, 6 or 7.

[0070] In certain preferred embodiments, the detergent compositions of the present invention comprise a first protease and a second protease, each of the first protease and the second protease having a negative net formal charge, such as -1, -2, -3 or -4, relative to the protease of SEQ ID NO: 1, wherein the net formal charges of the first protease and the second protease differ from each other by 1 or 2. Specific examples of such compositions include compositions comprising:

[0071] a) a first protease having a net formal charge of -4 and a second protease having a net formal charge of -2 relative to the protease of SEQ ID NO: 1 (charge difference of 2);

[0072] b) a first protease having a net formal charge of -3 and a second protease having a net formal charge of -2 relative to the protease of SEQ ID NO: 1 (charge difference is 1); and

[0073] c) a first protease having a net formal charge of -3 and a second protease having a net formal charge of -1 relative to the protease of SEQ ID NO: 1 (charge difference of 2).

[0074] Combinations having two proteases having a net formal charge of -1, -2, -3 or -4 and a charge difference of 1 or 2 are contemplated to be particularly useful when formulated into liquid laundry detergent compositions.

[0075] As described above, it has been found that using a combination of at least two different proteases having different net formal charge characteristics can improve wash performance on individual stains and / or provide improved overall performance on a wider set of stains, such as in laundry detergent formulations, compared to the individual enzymes. The concept of using separate proteases of this different net formal charge allows the blend to be tailored to different wash conditions to achieve an optimal charge blend for improved performance on target stains.

[0076] In particular, it has been found that by using this concept, enhanced cleaning performance can be provided on certain difficult to remove stains (e.g., oily protein stains, such as sebum stains) without increasing the total protease content, while substantially maintaining or improving performance on other protein-based stains (such as cocoa stains). The overall result is improved cleaning performance on a wider set of stains using the same amount of enzyme protein.

[0077] In another aspect, the present invention relates to a detergent composition comprising a first protease and a second protease, wherein the first protease has a net formal charge of -3, -4 or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2 relative to the protease of SEQ ID NO: 1.

[0078] In a further aspect, the present invention is directed to a detergent composition comprising a first protease and a second protease, wherein the first protease has a net formal charge of 0, -1, or -2 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2, +3, +4, or +5 relative to the protease of SEQ ID NO: 1. It is contemplated that such compositions wherein one protease has a relatively positive net formal charge relative to the protease of SEQ ID NO: 1 will provide advantageous results in relatively high pH wash settings, such as in automatic dishwashing applications.

[0079] In one embodiment, the first protease has a net formal charge of -3 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2 relative to the protease of SEQ ID NO: 1.

[0080] In one embodiment, the first protease has a net formal charge of -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2 relative to the protease of SEQ ID NO: 1.

[0081] In one embodiment, the first protease has a net formal charge of -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2 relative to the protease of SEQ ID NO: 1.

[0082] In one embodiment, the first protease has a net formal charge of 0 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2 relative to the protease of SEQ ID NO: 1.

[0083] In one embodiment, the first protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2 relative to the protease of SEQ ID NO: 1.

[0084] In one embodiment, the first protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2 relative to the protease of SEQ ID NO: 1.

[0085] In one embodiment, the first protease has a net formal charge of 0 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +3 relative to the protease of SEQ ID NO: 1.

[0086] In one embodiment, the first protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +3 relative to the protease of SEQ ID NO: 1.

[0087] In one embodiment, the first protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +3 relative to the protease of SEQ ID NO: 1.

[0088] In one embodiment, the first protease has a net formal charge of 0 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +4 relative to the protease of SEQ ID NO: 1.

[0089] In one embodiment, the first protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +4 relative to the protease of SEQ ID NO: 1.

[0090] In one embodiment, the first protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +4 relative to the protease of SEQ ID NO: 1.

[0091] In one embodiment, the first protease has a net formal charge of 0 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +5 relative to the protease of SEQ ID NO: 1.

[0092] In one embodiment, the first protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +5 relative to the protease of SEQ ID NO: 1.

[0093] In one embodiment, the first protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +5 relative to the protease of SEQ ID NO: 1.

[0094] In any detergent composition of the present invention, the first protease and the second protease preferably have an amino acid sequence having at least 60% amino acid sequence identity with SEQ ID NO: 1, such as at least 70% or at least 80% sequence identity. Thus, the first protease may have at least 85% sequence identity with SEQ ID NO: 1, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity with SEQ ID NO: 1. Similarly, the second protease may have at least 85% sequence identity with SEQ ID NO: 1, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity with SEQ ID NO: 1. It will be understood that if such protease comprises any additional mutations relative to SEQ ID NO: 1 beyond those mutations specified herein, the protease should meet the relevant net formal charge criteria associated with SEQ ID NO: 1. The first protease and / or the second protease may also have an amino acid sequence comprising or consisting of SEQ ID NO: 1 with specific mutations as defined herein.

[0095] In one embodiment, the first protease has a net formal charge of -5, -4 or -3 and comprises one or more mutations introducing increased negative charge relative to SEQ ID NO: 1, the mutation being selected from the group consisting of S9E, K27H, K27M, *36D, N62D, N76D, N77D, G97D, S99D, S101E, H120D, N140D, S156D, N185E, S188E, G195E, K235L, K235M, K237M, S259D and L262E. Preferred substitutions introducing negative charge include N76D, G97D, S99D, S101E and S156D. In a preferred embodiment, the first protease comprises one or more mutations in these mutations, preferably three or more mutations in these mutations, for example four, five or six mutations in these mutations. In some embodiments, variants having four or more of these mutations that provide increased negative charge can be combined with one or more mutations (e.g., one or two mutations) that provide increased positive charge to produce a net formal charge of -5, -4, or -3 relative to the protease of SEQ ID NO: 1. Examples of such mutations that provide increased positive charge include, for example, S9R, N43R, N117R, A215K, and Q245R.

[0096] In one embodiment, the first protease thus comprises four or more mutations (e.g., four, five, six, or seven mutations) relative to SEQ ID NO: 1 that introduce an increased negative charge, combined with one or more mutations (e.g., one or two mutations) that introduce an increased positive charge, thereby resulting in a net formal charge of -5, -4 or -3 relative to the protease of SEQ ID NO: 1. Thus, the first protease can comprise four or more mutations introducing increased negative charge (e.g., selected from the group consisting of S9E, K27H, K27M, *36D, N62D, N76D, N77D, G97D, S99D, S101E, H120D, N140D, S156D, N185E, S188E, G195E, K235L, K235M, K237M, S259D and L262E) together with one or two mutations introducing increased positive charge (e.g., selected from the group consisting of S9R, N43R, N117R, A215K and Q245R), wherein the protease has a net formal charge of -5, -4 or -3 relative to the protease of SEQ ID NO: 1.

[0097] In all examples of this protease comprising variants of SEQ ID NO: 1 below and elsewhere herein, the position numbering is based on the numbering of SEQ ID NO: 2 as explained in the paragraph "Numbering of amino acid positions / residues" above.

[0098] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S99D + S101E + S103A + V104I + S156D + G160S + L262E, i.e. having a net formal charge of -4 relative to SEQ ID NO: 1.

[0099] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E, i.e. having a net formal charge of -4 relative to SEQ ID NO: 1.

[0100] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + K235M + Q245R + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0101] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0102] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M + Q245R + L262E, i.e. having a net formal charge of -4 relative to SEQ ID NO: 1.

[0103] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0104] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W + S259D + L262E, i.e. having a net formal charge of -5 relative to SEQ ID NO: 1.

[0105] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions *36D + N76D + H120D + G195E + K235L, ie having a net formal charge of -5 relative to SEQ ID NO: 1.

[0106] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + K27M + S99A + S101E + S103A + V104I + S156D + G160S + K237M + Q245R + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0107] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + K27M + S101E + S103A + V104I + S156D + G160S + K237M + Q245R + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0108] In one embodiment, the first protease is a variant of SEQ ID NO: 1, comprising the substitutions S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + A215K + K237M + Q245R + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0109] In one embodiment, the first protease is a variant of SEQ ID NO: 1, comprising the substitutions S9R + K27M + N43R + S99D + S101E + S103A + V104I + S156D + G160S + K237M + Q245R + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0110] In one embodiment, the first protease is a variant of SEQ ID NO: 1, comprising the substitutions S9R + N43R + S99D + S101E + S103A + V104I + S156D + G160S + N173D + K235M + Q245R + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0111] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + G97D + S156D + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0112] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101E + S156D + A172V + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0113] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + N140D + S156D + Y209W + A215K + L262E, i.e. having a net formal charge of -4 relative to SEQ ID NO: 1.

[0114] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + S101E + T180A + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0115] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + S101E + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0116] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + G97D + S101E + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0117] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + G97D + S101E + V177I + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0118] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + N76D + G97D + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0119] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + G97D + S156D + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0120] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N77D + G97D + S156D + Y209W + A215K + L262E, i.e. having a net formal charge of -4 relative to SEQ ID NO: 1.

[0121] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N76D + G97D + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0122] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N62D + G97D + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0123] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + G97D + S101E + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0124] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N76D + G97D + Y209W + A215K + L262E + A270T, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0125] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N76D + G97D + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0126] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N62D + G97D + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0127] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + K27M + G97D + Y209W + A215K + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0128] In one embodiment, the first protease is a variant of SEQ ID NO: 1, comprising the substitutions S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W + A215K + Q245R + S259D + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0129] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N43R + N76D + S188E + Q191N + A194P + Q206L + Y209W + A215K + S259D + L262E, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0130] In one embodiment, the first protease is a variant of SEQ ID NO: 1, comprising the substitutions S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W + A215K + S259D + L262Q, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0131] In one embodiment, the first protease is a variant of SEQ ID NO: 1, comprising the substitutions S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W + Q245R + S259D + L262Q, i.e. having a net formal charge of -3 relative to SEQ ID NO: 1.

[0132] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1 comprising the substitution K27M, i.e. having a net formal charge of -1 relative to SEQ ID NO: 1.

[0133] In one embodiment, the second protease is the polypeptide of SEQ ID NO: 1.

[0134] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1 comprising the mutation S99AD (i.e. S is replaced by A and D is inserted), i.e. having a net formal charge of -1 relative to SEQ ID NO: 1.

[0135] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K, ie having a net formal charge of 0 relative to SEQ ID NO: 1.

[0136] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising G97D + N117R + Y209W + A215K, ie having a net formal charge of +1 relative to SEQ ID NO: 1.

[0137] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + K27M + N43R + N76D + V205I + Q206L + Y209W + A215K + Q245R + S259D + N261W + L262E, i.e. having a net formal charge of 0 relative to SEQ ID NO: 1.

[0138] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + N43R + N76D + V205I + Q206L + Y209W + A215K + S259D + N261W + L262E, i.e. having a net formal charge of 0 relative to SEQ ID NO: 1.

[0139] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1, which comprises the substitutions S9R + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E, i.e., has a net formal charge of -1 relative to SEQ ID NO: 1.

[0140] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1, which comprises the substitutions S9R + S99A + S101E + S103A + V104I + S156D + G160S + A215K + K235M + Q245R + L262E, i.e., has a net formal charge of -1 relative to SEQ ID NO: 1.

[0141] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1, which comprises the substitutions S9R + K27M + N43R + S99A + S101E + S103A + V104I + S156D + G160S + A215K + K237M + Q245R + L262E, i.e., has a net formal charge of -1 relative to SEQ ID NO: 1.

[0142] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1, which comprises the substitutions S9R + K27M + N43R + S101E + S103A + V104I + S156D + G160S + A215K + K237M + Q245R + L262E, i.e., has a net formal charge of -1 relative to SEQ ID NO: 1.

[0143] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1 comprising the substitutions Y167A + R170S + A194P, i.e. having a net formal charge of -1 relative to SEQ ID NO: 1.

[0144] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + A15T + V68A + N218D + Q245R, ie having a net formal charge of +1 relative to SEQ ID NO: 1.

[0145] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + A15T + G61E + V68A + A194P + V205I + Q245R + N261D, i.e. having a net formal charge of 0 relative to SEQ ID NO: 1.

[0146] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + A15T + V68A + S99G + Q245R + N261D, i.e. having a net formal charge of +1 relative to SEQ ID NO: 1.

[0147] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + A15T + V68A + H120D + P131S + Q137H + Q245R, i.e. having a net formal charge of +1 relative to SEQ ID NO: 1.

[0148] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + A15T + V68A + H120N + P131S + Q137H + Q245M, i.e. having a net formal charge of +1 relative to SEQ ID NO: 1.

[0149] In one embodiment, the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + A15T + G61E + V68A + A98S + S99G + N218D + Q245R, i.e. having a net formal charge of 0 relative to SEQ ID NO: 1.

[0150] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1, which comprises the substitutions S3T + V4I + S99D + S101R + S103A + V104I + G160S + V205I + L217D, i.e., has a net formal charge of -1 relative to SEQ ID NO: 1.

[0151] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1, which comprises the substitutions S3T + V4I + S99D + S101R + S103A + V104I + G160S + A194P + V205I + L217D, i.e., has a net formal charge of -1 relative to SEQ ID NO: 1.

[0152] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K + L262E, i.e. having a net formal charge of -1 relative to SEQ ID NO: 1.

[0153] In one embodiment, the second protease (or the first protease, wherein the second protease has a net formal charge of -2) is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K, i.e. having a net formal charge of -1 relative to SEQ ID NO: 1.

[0154] Other suitable protease variants that can be used as the first protease or the second protease are those having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the proteases disclosed above, and wherein any additional mutations in the individual protease variants as described above result in the same net formal charge.

[0155] Illustrative, non-limiting examples of the invention having specific first and second proteases are provided below.

[0156] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S99D + S101E + S103A + V104I + S156D + G160S + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M.

[0157] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M.

[0158] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + K235M + Q245R + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitution K27M.

[0159] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M + Q245R + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitution K27M.

[0160] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S99D + S101E + S103A + V104I + S156D + G160S + L262E, and the second protease is the polypeptide of SEQ ID NO: 1.

[0161] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E, and the second protease is the polypeptide of SEQ ID NO: 1.

[0162] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M + Q245R + L262E, and the second protease is the polypeptide of SEQ ID NO: 1.

[0163] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S99D + S101E + S103A + V104I + S156D + G160S + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the mutation S99AD.

[0164] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the mutation S99AD.

[0165] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + K235M + Q245R + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the mutation S99AD.

[0166] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the mutation S99AD.

[0167] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M + Q245R + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the mutation S99AD.

[0168] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S99D + S101E + S103A + V104I + S156D + G160S + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K or G97D + N117R + Y209W + A215K.

[0169] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K or G97D + N117R + Y209W + A215K.

[0170] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W + S259D + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K or G97D + N117R + Y209W + A215K.

[0171] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the mutations *36D + N76D + H120D + G195E + K235L, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K or G97D + N117R + Y209W + A215K.

[0172] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitution K27M, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E.

[0173] In one embodiment, the first protease is the polypeptide of SEQ ID NO: 1 and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E.

[0174] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the mutation S99AD, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E.

[0175] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + N140D + S156D + Y209W + A215K + L262E, and the second protease is the polypeptide of SEQ ID NO: 1.

[0176] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + N140D + S156D + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K.

[0177] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + N140D + S156D + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K + L262E.

[0178] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N77D + G97D + S156D + Y209W + A215K + L262E, and the second protease is the polypeptide of SEQ ID NO: 1.

[0179] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N77D + G97D + S156D + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K.

[0180] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N77D + G97D + S156D + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K + L262E.

[0181] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + N117R + Y209W + A215K, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0182] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0183] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0184] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + N76D + G97D + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0185] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + G97D + S101E + V177I + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0186] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + G97D + S101E + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0187] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101E + S156D + A172V + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0188] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + N140D + S156D + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0189] In one embodiment, the first protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N77D + G97D + S156D + Y209W + A215K + L262E, and the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E.

[0190] In one embodiment, the first protease has a net formal charge of -1, -3, or -4 relative to the protease of SEQ ID NO: 1, the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1 and is a variant of SEQ ID NO: 1 having the substitutions G97D + S156D + Y209W + A215K + L262E or S9R + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E (preferably G97D + S156D + Y209W + A215K + L262E).

[0191] In one embodiment, the first protease has a net formal charge of -1, -3, or -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1, wherein the first protease is a variant of SEQ ID NO: 1 having a set of mutations selected from the group consisting of:

[0192] •K27M;

[0193] •S99AD;

[0194] •G97D + S156D + Y209W + A215K;

[0195] •G97D + Y209W + A215K + L262E;

[0196] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + K235M + Q245R +L262E;

[0197] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0198] •S9E + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E;

[0199] •N62D + N76D + G97D + Y209W + A215K + L262E;

[0200] •N62D + G97D + S101E + V177I + Y209W + A215K + L262E;

[0201] •N62D + G97D + S101E + Y209W + A215K + L262E;

[0202] •G97D + S101E + S156D + A172V + Y209W + A215K + L262E;

[0203] •S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0204] •K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E;

[0205] •S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M +Q245R + L262E;

[0206] • N76D + G97D + N140D + S156D + Y209W + A215K + L262E; and

[0207] •K27M + N77D + G97D + S156D + Y209W + A215K + L262E;

[0208] And wherein the second protease is preferably a variant of SEQ ID NO: 1 having the substitutions G97D + S156D + Y209W + A215K + L262E or S9R + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E (preferably G97D + S156D + Y209W + A215K + L262E).

[0209] In one embodiment, the first protease has a net formal charge of -1, -3, or -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1, wherein the second protease is a variant of SEQ ID NO: 1 having a set of mutations selected from the group consisting of:

[0210] •S87N + G97D + S156D + Y209W + A215K + L262E;

[0211] •G97D + S101G + S156D + Y209W + A215K + L262E;

[0212] •G97D + V104N + S156D + Y209W + A215K + L262E;

[0213] •G97D + G118V + S156D + Y209W + A215K + L262E;

[0214] •G97D + S156D + A194P + Y209W + A215K + L262E;

[0215] •S87N + G97D + S101G + S156D + Y209W + A215K + L262E;

[0216] •S87N + G97D + S101N + S156D + Y209W + A215K + L262E;

[0217] •S87N + G97D + V104N + S156D + Y209W + A215K + L262E;

[0218] •S87N + G97D + G118V + S156D + Y209W + A215K + L262E;

[0219] •S87N + G97D + S156D + A194P + Y209W + A215K + L262E;

[0220] •G97D + S101G + G118V + S156D + Y209W + A215K + L262E;

[0221] •G97D + S101G + S156D + A194P + Y209W + A215K + L262E; and

[0222] •G97D + S101N + V104N + S156D + Y209W + A215K + L262E.

[0223] When the first protease has a net formal charge of -3, -4, or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1, 0, or +1 relative to the protease of SEQ ID NO: 1, the first protease can be a variant of SEQ ID NO: 1 comprising a set of mutations selected from the group consisting of:

[0224] •S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0225] •K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E;

[0226] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + K235M + Q245R +L262E;

[0227] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0228] •S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M +Q245R + L262E;

[0229] •S9E + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E;

[0230] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +S259D + L262E;

[0231] •*36D + N76D + H120D + G195E + K235L;

[0232] •S9R + K27M + S99A + S101E + S103A + V104I + S156D + G160S + K237M +Q245R + L262E;

[0233] •S9R + K27M + S101E + S103A + V104I + S156D + G160S + K237M + Q245R +L262E;

[0234] •S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + A215K +K237M + Q245R + L262E;

[0235] <h2 style=";text-align:left;direction:ltr">•S9R + K27M + N43R + S99D + S101E + S103A + V104I + S156D + G160S +K237M + Q245R + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0236] <h2 style=";text-align:left;direction:ltr"> •S9R+N43R+S99D+S101E+S103A+V104I+S156D+G160S+N173D+K235M+Q245R+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0237] <h2 style=";text-align:left;direction:ltr"> •S9E + G97D + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0238] <h2 style=";text-align:left;direction:ltr"> •G97D + S101E + S156D + A172V + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0239] <h2 style=";text-align:left;direction:ltr"> •N76D + G97D + N140D + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0240] <h2 style=";text-align:left;direction:ltr"> •N76D+G97D+S101E+T180A+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0241] <h2 style=";text-align:left;direction:ltr"> •N76D + G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0242] <h2 style=";text-align:left;direction:ltr"> •N62D + G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0243] <h2 style=";text-align:left;direction:ltr"> •N62D + G97D + S101E + V177I + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0244] <h2 style=";text-align:left;direction:ltr"> •N62D + N76D + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0245] <h2 style=";text-align:left;direction:ltr"> •K27M+G97D+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0246] <h2 style=";text-align:left;direction:ltr"> •K27M+N77D+G97D+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0247] •K27M + N76D + G97D + Y209W + A215K + L262E;

[0248] •K27M + N62D + G97D + Y209W + A215K + L262E;

[0249] •S9E + G97D + S101E + Y209W + A215K + L262E;

[0250] •S9E + N76D + G97D + Y209W + A215K + L262E + A270T;

[0251] •S9E + N76D + G97D + Y209W + A215K + L262E;

[0252] •S9E + N62D + G97D + Y209W + A215K + L262E;

[0253] •S9E + K27M + G97D + Y209W + A215K + L262E;

[0254] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +A215K + Q245R + S259D + L262E;

[0255] •S9E + N43R + N76D + S188E + Q191N + A194P + Q206L + Y209W + A215K +S259D + L262E;

[0256] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +A215K + S259D + L262Q; and

[0257] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +Q245R + S259D + L262Q;

[0258] And the second protease can be the protease of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 comprising a set of mutations selected from the group consisting of:

[0259] •K27M;

[0260] •S99AD;

[0261] •G97D + Y209W + A215K;

[0262] •G97D + N117R + Y209W + A215K;

[0263] •S9R + K27M + N43R + N76D + V205I + Q206L + Y209W + A215K + Q245R +S259D + N261W + L262E;

[0264] •S9R + N43R + N76D + V205I + Q206L + Y209W + A215K + S259D + N261W +L262E;

[0265] •S9R + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E;

[0266] •S9R + S99A + S101E + S103A + V104I + S156D + G160S + A215K + K235M +Q245R + L262E;

[0267] •S9R + K27M + N43R + S99A + S101E + S103A + V104I + S156D + G160S +A215K + K237M + Q245R + L262E;

[0268] •S9R + K27M + N43R + S101E + S103A + V104I + S156D + G160S + A215K +K237M + Q245R + L262E;

[0269] •Y167A + R170S + A194P;

[0270] •S9R + A15T + V68A + N218D + Q245R;

[0271] •S9R + A15T + G61E + V68A + A194P + V205I + Q245R + N261D;

[0272] •S9R + A15T + V68A + S99G + Q245R + N261D;

[0273] •S9R + A15T + V68A + H120D + P131S + Q137H + Q245R;

[0274] •S9R + A15T + V68A + H120N + P131S + Q137H + Q245M;

[0275] •S9R + A15T + G61E + V68A + A98S + S99G + N218D + Q245R;

[0276] •S3T + V4I + S99D + S101R + S103A + V104I + G160S + V205I + L217D;

[0277] •S3T + V4I + S99D + S101R + S103A + V104I + G160S + A194P + V205I +L217D;

[0278] •G97D + Y209W + A215K + L262E; and

[0279] •G97D + S156D + Y209W + A215K.

[0280] In some preferred embodiments, when the first protease has a net formal charge of -3, -4 or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1, 0 or +1 relative to the protease of SEQ ID NO: 1, the first protease can be a variant of SEQ ID NO: 1 having a set of mutations selected from the group consisting of:

[0281] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +S259D + L262E;

[0282] •*36D + N76D + H120D + G195E + K235L;

[0283] •S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0284] •K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E;

[0285] •S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M +Q245R + L262E;

[0286] •N76D + G97D + N140D + S156D + Y209W + A215K + L262E;

[0287] •K27M + N77D + G97D + S156D + Y209W + A215K + L262E;

[0288] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + K235M + Q245R +L262E;

[0289] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0290] •S9E + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E;

[0291] •N62D + N76D + G97D + Y209W + A215K + L262E;

[0292] •N62D + G97D + S101E + V177I + Y209W + A215K + L262E;

[0293] • N62D + G97D + S101E + Y209W + A215K + L262E; and

[0294] •G97D + S101E + S156D + A172V + Y209W + A215K + L262E;

[0295] And the second protease can be the protease of SEQ ID NO: 1 or a variant thereof having a set of mutations selected from the group consisting of:

[0296] •K27M;

[0297] •S99AD;

[0298] •G97D + S156D + Y209W + A215K;

[0299] •G97D + Y209W + A215K + L262E;

[0300] •G97D + Y209W + A215K; and

[0301] •G97D + N117R + Y209W + A215K.

[0302] In aspects of the invention wherein the first protease has a net formal charge of 0, -1 or -2 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of +2, +3, +4 or +5 relative to the protease of SEQ ID NO: 1, the first protease having a net formal charge of 0, -1 or -2 can, for example, be selected from a polypeptide of SEQ ID NO: 1 and a variant of SEQ ID NO: 1 having a set of mutations selected from the group consisting of:

[0303] •G97D + Y209W + A215K

[0304] •S9R + K27M + N43R + N76D + V205I + Q206L + Y209W + A215K + Q245R +S259D + N261W + L262E

[0305] •S9R + N43R + N76D + V205I + Q206L + Y209W + A215K + S259D + N261W +L262E

[0306] •S9R + A15T + G61E + V68A + A194P + V205I + Q245R + N261D

[0307] •S9R + A15T + G61E + V68A + A98S + S99G + N218D + Q245R

[0308] •K27M

[0309] •S99AD

[0310] •S9R + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E

[0311] •S9R + S99A + S101E + S103A + V104I + S156D + G160S + A215K + K235M +Q245R + L262E

[0312] •S9R + K27M + N43R + S99A + S101E + S103A + V104I + S156D + G160S +A215K + K237M + Q245R + L262E

[0313] •S9R + K27M + N43R + S101E + S103A + V104I + S156D + G160S + A215K +K237M + Q245R + L262E

[0314] •Y167A + R170S + A194P

[0315] •S3T + V4I + S99D + S101R + S103A + V104I + G160S + V205I + L217D

[0316] •S3T + V4I + S99D + S101R + S103A + V104I + G160S + A194P + V205I +L217D

[0317] •G97D + Y209W + A215K + L262E

[0318] •G97D + S156D + Y209W + A215K

[0319] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0320] <h2 style=";text-align:left;direction:ltr"> •S9R+K27M+N43R+S101E+S103A+V104I+S156D+G160S+K237M+Q245R+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0321] <h2 style=";text-align:left;direction:ltr"> •S9R+K27M+N43R+S99D+S101E+S103A+V104I+S156D+G160S+A215K+K237M+Q245R+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0322] <h2 style=";text-align:left;direction:ltr"> •S9R+S99A+S101E+S103A+V104I+A151V+S156D+G160S+K235M+Q245R+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0323] <h2 style=";text-align:left;direction:ltr"> •S9R + S99A + S101E + S103A + V104I + S156D + G160S + K235M + Q245R +L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0324] <h2 style=";text-align:left;direction:ltr"> •S9R + S99D + S101E + S103A + V104I + S156D + G160S + A215K + K235M +Q245R + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0325] <h2 style=";text-align:left;direction:ltr"> •N62D + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0326] <h2 style=";text-align:left;direction:ltr"> •G97D + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0327] <h2 style=";text-align:left;direction:ltr"> •G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0328] <h2 style=";text-align:left;direction:ltr"> •N76D + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0329] <h2 style=";text-align:left;direction:ltr"> •K27M+G97D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0330] <h2 style=";text-align:left;direction:ltr"> •S9E + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0331] •S9R + N43R + N76D + N185E + Q191N + A194P + Q206L + Y209W + S259D +L262E; and

[0332] •S9E+N43R+N76D+V205I+Q206L+Y209W+Q245R+S259D+N261W+L262E.

[0333] In an additional aspect, the first protease can have a net formal charge of -2 relative to SEQ ID NO: 1, and the second protease can have a net formal charge of -1, 0, or +1 relative to SEQ ID NO: 1.

[0334] As an example of an embodiment of this aspect, the first protease can be a variant of SEQ ID NO: 1 comprising the substitutions S9R + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E, and the second protease can be a polypeptide of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 comprising the substitution K27M or comprising the mutation S99AD.

[0335] In one embodiment of the present invention, the detergent composition of the present invention does not comprise two proteases, wherein one protease is contained in a solid enzyme preparation and the other protease is contained in a liquid enzyme preparation.

[0336] In one embodiment, the detergent composition of the present invention does not contain the protease of SEQ ID NO: 8 disclosed in US 2017 / 0198243. In one embodiment, the detergent composition of the present invention does not contain the protease of SEQ ID NO: 9 disclosed in US 2017 / 0198243. In a further embodiment, the two proteases in the detergent composition of the present invention are not the protease of SEQ ID NO: 8 and the protease of SEQ ID NO: 9 disclosed in US 2017 / 0198243.

[0337] Preferably, the detergent composition of the present invention comprising the first protease and the second protease as defined above has improved washing performance compared to the same composition comprising the first protease or the second protease. The improved washing performance can be the improved performance to one or more individual stains and / or the improved overall performance to a group of stains (e.g., 2, 3, 4, 5 or more stains). Washing performance can be determined, for example, using the AMSA method described herein, for example, using the model detergent listed in Table 1. Performance can be tested on any stain suitable for intended application (e.g., laundry or automatic dishwashing). Examples of such stains are, for example, standard laundry stains PC-10 and / or PC-03.

[0338] Proteases having a relatively negative net formal charge (e.g., -4 or -3) compared to SEQ ID NO: 1 tend to be better at removing certain types of protein stains (e.g., cocoa-containing stains). On the other hand, proteases having a more positive, relatively neutral net formal charge (e.g., -1 or 0) tend to be better at removing other types of protein stains (e.g., sebum stains and other oily protein stains). Thus, surprisingly, the detergent compositions of the present invention comprising two proteases having different net formal charge characteristics can provide improved cleaning of different types of stains without increasing the total amount of proteases.

[0339] It is also surprising that using a mixture of two different proteases with different charge characteristics, rather than relying on a relatively neutral protease that is better at removing oily protein stains, results in significantly better removal of the oily PC-10 stain. The PC-10 stain (pigment, oil, milk) simulates a natural sebum stain containing protein, and oil / fats, thus providing a good indication of how a detergent composition performs on this difficult to remove natural stain.

[0340] In one embodiment, the detergent composition of the present invention comprising the first protease and the second protease has improved cleaning performance on PC-10 compared to the same composition comprising the first protease or the second protease.

[0341] In another embodiment, the detergent composition of the present invention comprising a first protease and a second protease has improved overall cleaning performance on PC-10 and PC-03 compared to the same composition comprising either the first protease or the second protease.

[0342] In one embodiment, the relative washing performance of the detergent composition of the present invention comprising the first protease and the second protease is improved by at least 1%, preferably at least 2%, at least 3%, at least 4%, or at least 5%, such as at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%, for example at least 15% or at least 20%, relative to the washing performance of the composition comprising the first protease or the second protease. This improved relative washing performance is preferably obtained at least on the PC-10 stain from the Center for Testmaterials (CFT). More preferably, this improved relative washing performance is the overall washing performance of the improvement of a group of stains comprising PC-10 and PC-03 (all from CFT) and optionally comprising one or more additional standard stains. Examples of such additional stains that may be used are one or more of CS-05S, CS-67, CS-75, CS-95, C-H151, C-H163, C-H252, CS-60 (all from Center for Testmaterials, CFT), and French Mustard WE5FSMWKC, 011 KC WC PCPE Bacon Grease, 028 KC WC PC PE Cheese Sauce, 080 KC WC PC PE Mayonnaise (all from Warwick Equest). Relative wash performance may be determined, for example, based on the sum of delta intensities as described in Example 1.

[0343] It will be appreciated that when comparing the cleaning performance of a composition of the present invention to a corresponding composition comprising only a single enzyme (ie either the first protease or the second protease), the compared compositions will contain the same total amount of protease protein (by weight).

[0344] In a further aspect, the present invention relates to novel protease variants having advantageous properties, wherein the protease is a variant of SEQ ID NO: 1 comprising substitutions at positions 97, 209 and 215. It has been found that such proteases not only have beneficial properties when used in detergent compositions with another protease having a different net formal charge (such as those disclosed herein), but also have beneficial properties when used alone, such as improved laundry performance.

[0345] In this regard, the invention relates to a protease variant comprising the substitutions X97D, X209W and X215K (e.g., G97D, Y209W and A215K), wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% with SEQ ID NO: 1. In some embodiments of this aspect of the invention, the protease variant preferably comprises one or more additional substitutions at one or more positions selected from positions 27, 62, 76, 77, 87, 101, 104, 117, 118, 140, 156, 172, 177, 194 and 262 (e.g., at one, two, three or four positions). Such additional substitutions particularly include X9E (e.g., S9E), X27M (e.g., K27M), X62D (e.g., N62D), X76D (e.g., N76D), X77D (e.g., N77D), X87N (e.g., S87N), X101E / G / N (e.g., S101E / G / N), X104N (e.g., V104N), X117R (e.g., N117R), X118V (e.g., G118V), X140D (e.g., N140D), X156D (e.g., S156D), X172V (e.g., A172V), X177I (e.g., V177I), X194P (e.g., A194P), and X262E (e.g., L262E). Specific embodiments of this aspect of the invention are provided below.

[0346] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 with this set of substitutions.

[0347] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0348] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0349] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + N117R + Y209W + A215K, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 with this set of substitutions.

[0350] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0351] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N77D + G97D + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0352] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + N76D + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0353] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + G97D + S101E + V177I + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0354] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + G97D + S101E + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0355] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + N140D + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0356] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101E + S156D + A172V + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0357] In an embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N62D + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0358] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + G97D + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0359] In an embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + G97D + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0360] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions N62D + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1.

[0361] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + S101E + T180A + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0362] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + S101E + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0363] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N76D + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0364] In an embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + N62D + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0365] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + G97D + S101E + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0366] In an embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N76D + G97D + Y209W + A215K + L262E + A270T, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0367] In an embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + N76D + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0368] In an embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + K27M + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0369] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101E + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0370] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions N76D + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0371] In an embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions K27M + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0372] In an embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S9E + G97D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0373] In one embodiment, the present invention relates to a protease which is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E and optionally at least one additional substitution selected from S87N, S101G / N, V104N, G118V, and A194P. Examples of such proteases are provided below.

[0374] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S87N + G97D + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0375] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101G + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0376] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101N + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0377] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + V104N + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% sequence identity to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0378] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + G118V + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0379] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + A194P + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0380] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S87N + G97D + S101G + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0381] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S87N + G97D + S101N + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0382] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S87N + G97D + V104N + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0383] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S87N + G97D + G118V + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0384] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions S87N + G97D + S156D + A194P + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0385] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101G + G118V + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0386] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101G + S156D + A194P + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0387] In one embodiment, the protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S101N + V104N + S156D + Y209W + A215K + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2, and the variant has protease activity and a sequence identity of at least 80%, such as at least 85%, at least 90%, or at least 95% but less than 100% to SEQ ID NO: 1. The protease may, for example, comprise or consist of SEQ ID NO: 1 having this set of substitutions.

[0388] It will be apparent from the above description and the examples below that in a detergent composition of the present invention comprising two proteases having different net charge characteristics, one or both proteases may be a variant of SEQ ID NO: 1 comprising substitutions G97D + Y209W + A215K and optionally other substitutions as described herein, as long as both proteases meet the relevant net formal charge criteria relative to SEQ ID NO: 1.

[0389] In addition to the amino acid changes specifically disclosed herein, the protease variants in the compositions of the present invention may comprise additional changes at one or more other positions. These additional changes may be minor, i.e., generally conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein and do not change the net formal charge as described herein; typically small deletions of 1-30 amino acids; or small amino-terminal extensions or carboxyl-terminal extensions.

[0390] Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not change specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979 in The Proteins, Academic Press, New York. Common conservative substitution groups include, but are not limited to, G=A=S, I=V=L=M, D=E, Y=F, and N=Q (wherein, for example, "G=A=S" means that these three amino acids can replace each other).

[0391] Alternatively, these amino acid changes have such a property that the physicochemical properties of the polypeptide are changed. For example, the amino acid changes can increase the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, and the like.

[0392] 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 resulting mutant molecules are tested for protease activity to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutations to putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0393] Detergent composition

[0394] The detergent compositions of the present invention may be in any convenient form, for example, a homogeneous tablet, a tablet having two or more layers, a sachet having one or more compartments, a conventional or compressed powder, granules, a paste, a gel, a bar, or a regular, compressed, or concentrated liquid.

[0395] In one embodiment, the first protease and the second protease as described elsewhere herein are both present in the same formulation, such as in the same liquid phase or solid phase. For example, in the case of a liquid formulation such as a liquid or gel form, the first protease and the second protease will both be present in the liquid phase or gel phase. Similarly, in the case of a solid formulation such as a powder, granule or tablet, the first protease and the second protease will both be present in the solid phase.

[0396] In a preferred embodiment, the detergent composition of the present invention is a laundry composition, particularly a liquid laundry composition. The use of two proteases with different net charge characteristics according to the present invention allows flexibility in incorporation into different liquid laundry detergent compositions, such as surfactant compositions and / or levels, and flexibility in use under different washing conditions (e.g., wash pH and / or water hardness).

[0397] In one embodiment, the present invention is directed to a detergent composition as described above, comprising at least a first protease and a second protease and further comprising one or more additional enzymes selected from the group consisting of amylases, catalases, cellulases (e.g., endoglucanases), cutinases, deoxyribonucleases, haloperoxidases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, xanthanases, lichenases, and xyloglucanases, or any mixture thereof.

[0398] The selection of additional components of the detergent composition is within the skill of the ordinary artisan and includes conventional ingredients, including the exemplary, non-limiting components listed below. For fabric care, the selection of components may include considerations such as the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning is to be performed, and the formulation of the detergent product.

[0399] In a specific embodiment, the detergent composition comprises a first protease and a second protease and one or more non-naturally occurring detergent components such as surfactants, hydrotropes, builders, co-builders, chelants or chelating agents, bleach systems or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfumes, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizing agents. The detergent composition typically comprises at least a surfactant and a builder.

[0400] In one embodiment, the protease may be added to the detergent composition in an amount corresponding to 0.01-200 mg enzyme protein / liter of washing liquor, preferably 0.05-50 mg enzyme protein / liter of washing liquor, in particular 0.1-10 mg enzyme protein / liter of washing liquor.

[0401] A granular composition for use in laundry may, for example, comprise 0.001% to 20%, such as 0.01% to 10%, such as 0.05% to 5%, of an enzyme protein by weight of the composition.

[0402] Automatic dishwashing (ADW) compositions may, for example, comprise 0.001%-30%, such as 0.01%-20%, such as 0.1%-15%, such as 0.5%-10% enzyme protein by weight of the composition.

[0403] Enzymes such as proteases can be stabilized using conventional stabilizers, for example, polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid, or boric acid derivatives such as aromatic borate esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid, and the composition can be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708, or the proteases can be stabilized using peptide aldehydes or ketones as described in WO 2005 / 105826 and WO 2009 / 118375.

[0404] The detergent composition can be formulated as a granular detergent for laundry. For example, such a detergent may, for example, comprise:

[0405] a) at least 0.01 mg protease per gram of composition,

[0406] b) preferably 5 to 50 wt % of anionic surfactant,

[0407] c) preferably 1 to 8 wt % of a nonionic surfactant,

[0408] d) preferably 5 to 40 wt% of a builder, such as a carbonate, zeolite, phosphate builder, calcium sequestering builder, or a complexing agent.

[0409] Although the components mentioned below are categorized by general headings according to specific functionality, this is not to be construed as limiting as a component may contain additional functionality as will be appreciated by one skilled in the art.

[0410] Surfactants

[0411] The detergent composition can comprise one or more surfactants, which can be anionic and / or cationic and / or nonionic and / or semipolar and / or zwitterionic or a mixture thereof. In a specific embodiment, the detergent composition comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. These one or more surfactants typically exist by weight at a level from about 0.1% to 60% (such as about 1% to about 40% or about 3% to about 20% or about 3% to about 10%). Select these one or more surfactants based on desired cleaning applications, and these surfactants include any one or more conventional surfactants as known in the art. Any surfactant for use in detergents as known in the art can be utilized. Surfactant reduces the surface tension in the detergent, which makes the stain of cleaning be raised and dispersed, and then washed away.

[0412] When included therein, the detergent will typically contain from about 1% to about 40%, such as from about 5% to about 30%, including from about 5% to about 15% or from about 20% to about 25% by weight of anionic surfactants. Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkanesulfonates, alpha-olefin sulfonates (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 alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic fatty acid methyl esters (α-SFMe or SES) (including methyl sulfonate (MES)), alkyl succinic acid or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soap, and combinations thereof.

[0413] When included therein, detergents will typically contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethylethanol quaternary ammonium (ADMEAQ), cetyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0414] When included therein, detergents will typically contain from about 0.2% to about 40% by weight of nonionic surfactant, for example from about 0.5% to about 30%, especially 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 nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (such as ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamide (FAM), fatty acid diethanolamide (FADA), ethoxylated fatty acid monoethanolamide (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA), or fatty acid glucamide (FAGA)), and products available under the SPAN and TWEEN trade names, and combinations thereof.

[0415] When included therein, the detergent will typically contain from about 0% to about 10% by weight of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs), such as alkyl dimethyl amine oxides), N-(coconut oil alkyl)-N,N-dimethyl amine oxides, and N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl) amine oxides, fatty acid alkanolamides, and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0416] When included therein, detergents will typically contain from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines, alkyl dimethyl betaines, sulfobetaines, and combinations thereof.

[0417] Builders and co-builders

[0418] The detergent composition can contain about 0-65% by weight, such as about 5% to about 45% detergent builder, or co-builder, or its mixture. In dishwashing detergents, the level of builder is typically 40%-65%, particularly 50%-65%. Builders and chelating agents are for example softened wash water by removing metal ions from liquid. Builders and / or co-builders can particularly be chelating agents that form water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in laundry detergents can 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"-nitrotriethanol), and carboxymethylinulin (CMI), and combinations thereof.

[0419] In a preferred embodiment, the detergent composition is free of phosphates.

[0420] The detergent composition can also contain 0-20% by weight, such as about 5% to about 10% detergent co-builder or its mixture. The detergent composition can include a single co-builder, or combine with a builder (e.g., zeolite builder). The limiting examples of co-builders include homopolymers or copolymers of polyacrylates, such as poly(acrylic acid) (PAA) or copolymerization (acrylic acid / maleic acid) (PAA / PMA). Further limiting examples include citrate, chelating agents (such as aminocarboxylates, aminopolycarboxylates, and phosphonates), and alkyl succinic acid, or alkenyl succinic 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), diethylenetriaminepenta(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 ... acid) (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), o-amino Benzoic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N'-triacetate (HEDTA), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotri(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or co-builders are described, for example, in WO 2009 / 102854 and US 5,977,053.

[0421] The first protease and the second protease of the present invention can also be formulated into a dishwashing composition, preferably an automatic dishwashing composition (ADW), comprising:

[0422] a) at least 0.01 mg of active protease, and

[0423] b) 10 wt % to 50 wt % of a builder, preferably selected from citric acid, methylglycine-N,N-diacetic acid (MGDA), and / or glutamic acid-N,N-diacetic acid (GLDA), and mixtures thereof, and

[0424] c) at least one bleaching component.

[0425] Bleaching system

[0426] The detergent may contain 0-50% by weight, such as about 0.1% to about 25% bleaching system. Bleach systems remove the fading often caused by oxidation, and many bleaches also have strong bactericidal properties and are used for disinfection and sterilization. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleach catalysts, photobleaches, bleach activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, preformed peracids, and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts (e.g., potassium persulfate (Oxone(R)), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems in combination with a peracid-forming bleach activator, which may comprise, for example, inorganic salts, including alkali metal salts such as sodium perborate (typically monohydrate or tetrahydrate), percarbonates, persulfates, perphosphates, persilicates.

[0427] The term bleach activator means herein a compound that reacts with a peroxide bleach (like hydrogen peroxide) to form a peracid. The peracid thus formed constitutes an activated bleach. Suitable bleach activators for use herein include those belonging to esteramides, imides or anhydride categories. Suitable examples are tetraacetylethylenediamine (TAED), 4-[(3,5,5-trimethylhexanoyl)oxy] sodium benzenesulfonate (ISONOBS), diperoxylauric acid, 4-(dodecyloxy) benzenesulfonate (LOBS), 4-(decanoyloxy) benzenesulfonate, 4-(decanoyloxy) benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A specific family of purpose bleach activators is disclosed in EP 624154 and particularly preferred in the family is triethyl acetyl citrate (ATC). ATC or short chain triglycerides like triacetin have the advantage of being environmentally friendly because they eventually degrade into citric acid and alcohol. In addition, triethyl acetyl citrate and triacetin have good hydrolytic stability in the product during storage and are efficient bleach activators. Finally, ATC provides good builder ability for laundry additives. Alternatively, the bleaching system may comprise peroxy acids of the amide, imide, or sulfone type, for example. The bleaching system may also comprise peracids such as 6-(phthalimido)perhexanoic acid (PAP). The bleaching system may also comprise a bleaching catalyst or accelerator.

[0428] Some non-limiting examples of bleach catalysts that can be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese collagen, cobalt-amine catalysts, and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), in particular Me3-TACN, such as binuclear manganese complexes [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2,2',2''-nitrilotri(ethane-1,2-diylazanyl-kappaN-methyl-ylidene)triphenol-kappa3O]manganese(III). These bleach catalysts can also be other metal compounds, such as iron or cobalt complexes.

[0429] In some embodiments, the bleaching component may be an organic catalyst selected from the group consisting of organic catalysts having the formula:

[0430]

[0431] (iii) and mixtures thereof; wherein each R 1is independently a branched alkyl group containing from 9 to 24 carbons or a straight chain alkyl group containing from 11 to 24 carbons. Preferably, each R 1 is independently a branched alkyl group containing from 9 to 18 carbons or a straight chain alkyl group containing from 11 to 18 carbons, more preferably, each R 1 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 in, for example, WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242. Suitable photobleaches can, for example, be sulfonated zinc phthalocyanine.

[0432] Water-soluble additives

[0433] Hydrotropes are compounds that dissolve hydrophobic compounds in aqueous solutions (or, conversely, polar substances in non-polar environments). Typically, hydrotropes have both hydrophilic and hydrophobic characteristics (so-called amphiphilicity, as known from surfactants); however, the molecular structure of hydrotropes is generally not conducive to spontaneous self-aggregation, see, for example, Hodgdon and Kaler, 2007, Current Opinion in Colloid & Interface Science 12: 121-128 for a review. Hydrotropes do not show a critical concentration, above which self-aggregation as found for surfactants and lipid formation of micelles, lamellae or other well-defined mesophases will occur. On the contrary, many hydrotropes show a continuous type of aggregation process, in which the size of the aggregates grows with increasing concentration. However, many hydrotropes change the phase behavior, stability, and colloidal properties of systems containing substances with polar and non-polar characteristics (including mixtures of water, oil, surfactants, and polymers). Hydrotropes are commonly used in various industries ranging from pharmaceuticals, personal care, food to technical applications. The use of hydrotropes in detergent compositions allows, for example, more concentrated surfactant formulations (such as in the process of compacting liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0434] The detergent may contain 0-5% by weight, such as about 0.5% to about 5%, or about 3% to about 5% hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols, and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0435] polymer

[0436] The detergent may contain 0-10% by weight (such as 0.5%-5%, 2%-5%, 0.5%-2% or 0.2%-1%) of the polymer. Any polymer known in the art for use in the detergent may be utilized. The polymer may act as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning, and / or defoaming properties. Some polymers may have more than one of the above-mentioned properties and / or more than one of the motifs mentioned below. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (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 oligoethylene glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethylene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfates. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the above mentioned polymers are also contemplated.

[0437] Fabric Toner

[0438] The detergent composition of the present invention can also include a fabric hueing agent, such as a dye or a pigment, which, when formulated in the detergent composition, can be deposited on the fabric when the fabric contacts the washing solution comprising the detergent composition, and thus changes the color of the fabric by absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. By contrast, when fabric hueing agents absorb at least part of the visible spectrum, they change the color of the surface. Suitable fabric hueing agents include dyes and dye-clay conjugates, and can 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 the following dyes falling into the Colour Index (CI) classification: direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet, and basic red, or mixtures thereof, such as described in WO 2005 / 003274, WO 2005 / 003275, WO 2005 / 003276, and EP 1876226 (incorporated herein by reference). The detergent composition preferably comprises from about 0.00003 wt.% to about 0.2 wt.%, from about 0.00008 wt.% to about 0.05 wt.%, or even from about 0.0001 wt.% to about 0.04 wt.% of fabric hueing agent. The composition may comprise from 0.0001 wt % to 0.2 wt. % of a fabric hueing agent, which may be particularly preferred when the composition is in the form of a unit dose bag. Suitable hueing agents are also disclosed in, for example, WO 2007 / 087257 and WO 2007 / 087243.

[0439] Additional enzymes

[0440] The detergent additive or detergent composition may comprise one or more additional enzymes, such as an amylase, an arabinase, a carbohydrase, a cellulase (e.g., an endoglucanase), a cutinase, a deoxyribonuclease, a galactanase, a haloperoxidase, a lipase, a mannanase, an oxidase (e.g., a laccase and / or a peroxidase), a pectinase, a pectin lyase, an additional protease, a xylanase, a xanthanase, or a xyloglucanase.

[0441] The properties of the enzyme or enzymes selected should be compatible with the selected detergent (eg, pH optimum, compatibility with other enzyme or non-enzyme ingredients, etc.).

[0442] Cellulase

[0443] Suitable cellulases include single components and mixtures of enzymes of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are also contemplated. The cellulases may for example be single component endo-1,4-β-glucanases (also referred to as endoglucanases), or mixtures of single component endo-1,4-β-glucanases.

[0444] Suitable cellulases include those from Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include those from Humicola insolens (US 4,435,307) or from Trichoderma, e.g., from T. reesei or T. viride. Other suitable cellulases are from Thielavia, e.g., Thielavia terrestris described in WO 96 / 29397 or fungal cellulases produced by Myceliophthora thermophila and Fusarium oxysporum as disclosed in US 5,648,263, US 5,691,178, US 5,776,757, WO 89 / 09259, and WO 91 / 17244. Cellulases from Bacillus are also relevant as described in WO 02 / 099091 and JP 2000210081. Suitable cellulases are alkaline or neutral cellulases with care benefits. Examples of cellulases are described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307.

[0445] Other cellulases are endo-β-1,4-glucanases having a sequence that is at least 97% identical to the amino acid sequence from position 1 to position 773 of SEQ ID NO: 2 of WO 2002 / 099091, or Family 44 xyloglucanases having a sequence that is at least 60% identical to positions 40-559 of SEQ ID NO: 2 of WO 2001 / 062903.

[0446] Commercially available cellulases include Carezyme®, Carezyme® Premium, Celluzyme®, Celluclean®, Celluclast®, Endolase®, Renozyme®, Whitezyme® Celluclean® Classic, Cellusoft® (Novozymes A / S), Puradax®, Puradax HA, and Puradax EG (available from Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

[0447] Mannanase

[0448] 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 from B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or Humicola insolens. Suitable mannanases are described in WO 1999 / 064619. Commercially available mannanases are Mannaway® (Novozymes A / S).

[0449] Protease

[0450] In addition to the first protease and the second protease disclosed herein, the composition can include one or more additional proteases, including those of bacteria, fungi, plants, viruses, or animal origin. Proteases of microbial origin are preferred. Proteases can be alkaline proteases, such as serine proteases or metalloproteases. Serine proteases can be, for example, serine proteases of the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteases can be, for example, thermolysin or another metalloprotease from, for example, the M4 family, such as those metalloproteases from the M5, M7, or M8 families.

[0451] Examples of metalloproteases are neutral metalloproteases as described in WO 2007 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0452] Suitable commercially available proteases include those sold under the following trade names: Alcalase®, Duralase Tm 、Durazym Tm , Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Neutrase®, Everlase®, Esperase®, Progress® Uno, and Progress® Excel (Novozymes A / S), those proteases sold under the trade names: Maxatase®, Maxacal®, Maxapem®, Purafect® TM , Purafect® Ox, Purafect® OxP, PurafectPrime®, Puramax®, FN2®, FN3®, FN4®, Excelllase®, Excellenz P1000 TM 、ExcellenzP1250 TM , Eraser®, Preferenz® P100, Preferenz® P110, Effectenz P1000 TM 、EffectenzP1050TM 、Effectenz P2000 TM , Purafast®, Properase®, Opticlean®, and Optimase® (Danisco / DuPont), Axapem TM (Gist-Brocases N.V.), BLAP (sequence shown in FIG. 29 of US5352604) and its variants (Henkel AG), and KAP (Bacillus alkalophilus subtilisin) from Kao Corporation.

[0453] Lipase and cutinase

[0454] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified mutant enzymes or protein engineered mutant enzymes are included. Examples include lipases from Thermomyces, e.g., T. lanuginosus (formerly named Humicola lanuginosa) as described in EP 258068 and EP 305216; cutinases from Humicola, e.g., Humicola insolens (WO 96 / 13580); lipases from strains of Pseudomonas (some of these now renamed Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), Pseudomonas species strain SD705 (WO 95 / 06720 and WO 95 / 06740); 96 / 27002), P. wisconsinensis (WO 96 / 12012); GDSL-type Streptomyces lipase (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 lipases from Streptomyces griseus (WO 2011 / 150157) and S. pristinaespiralis (WO 2012 / 137147).

[0455] 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.

[0456] Preferred commercial lipase products include Lipolase TM 、Lipex™、Lipolex TM and Lipoclean TM (Novozymes A / S), Lumafast (from Genencor), and Lipomax (from Gist-Brocades).

[0457] 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), acyltransferases from Mycobacterium smegmatis (WO 2005 / 056782), perhydrolases from the CE 7 family (WO 2009 / 067279), and variants of the Mycobacterium smegmatis perhydrolase, in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 2010 / 100028).

[0458] Amylase

[0459] Suitable amylases that can be used with the protease may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, for example, a special strain of Bacillus licheniformis (described in more detail in GB 1,296,839).

[0460] Suitable amylases include those having SEQ ID NO: 2 in WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO: 3. 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 having 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.

[0461] Different suitable amylases include those 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 deletions in positions 181 and 182 and substitutions in position 193.

[0462] Other suitable amylases are hybrid alpha-amylases comprising residues 1-33 of an alpha-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of a Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid alpha-amylase are those having a substitution, deletion, or insertion in one or 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 Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 are those having the following substitutions:

[0463] M197T;

[0464] H156Y + A181T + N190F + A209V + Q264S; or

[0465] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0466] Other suitable amylases are those 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 substitutions, deletions, or insertions in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those having deletions in positions R181 and G182, or positions H183 and G184.

[0467] Additional amylases that 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 substitutions, deletions, or insertions 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 with deletions in two positions selected from 181, 182, 183, and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. The most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 are those with deletions in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304, and 476.

[0468] Other amylases that can be used are those having SEQ ID NO: 2 of WO 2008 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 2008 / 153815, or variants thereof having 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 are those having substitutions, deletions, or insertions in one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0469] Further suitable amylases are those having SEQ ID NO: 2 of WO 2009 / 061380, or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having a C-terminal truncation, and / or a substitution, deletion, or insertion in one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO: 2 are those with substitutions in one or more of the following positions: Q87E, R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E, R, N272E, R, S243Q, A, E, D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or those with deletions in positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are those with the following substitutions:

[0470] N128C + K178L + T182G + Y305R + G475K;

[0471] N128C + K178L + T182G + F202Y + Y305R + D319T + G475K;

[0472] S125A + N128C + K178L + T182G + Y305R + G475K; or

[0473] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K,

[0474] wherein the variant is C-terminally truncated and optionally further comprises a substitution at position 243 and / or a deletion at position 180 and / or position 181.

[0475] Further suitable amylases are those having SEQ ID NO: 1 of WO 2013 / 184577, or variants thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 are those having substitutions, deletions, or insertions in one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, 1203, S241, R458, T459, D460, G476, and G477. More preferred variants of SEQ ID NO: 1 are those having substitutions in one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K, and G477K, and / or those having deletions in positions R178 and / or S179 or T180 and / or G181. The most preferred amylase variants of SEQ ID NO: 1 comprise the following substitutions:

[0476] E187P + I203Y + G476K

[0477] E187P + I203Y + R458N + T459S + D460T + G476K

[0478] and optionally further comprises a substitution at position 241 and / or a deletion at position 178 and / or position 179.

[0479] Further suitable amylases are those having SEQ ID NO: 1 of WO 2010 / 104675, or variants thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 are those having substitutions, deletions, or insertions in one or more of the following positions: N21, D97, V128, K177, R179, S180, I181, G182, M200, L204, E242, G477, and G478.

[0480] More preferred variants of SEQ ID NO: 1 are those having substitutions in one or more of the following positions: N21D, D97N, V128I, K177L, M200L, L204YF, E242QA, G477K, and G478K, and / or those having deletions in positions R179 and / or S180 or I181 and / or G182. The most preferred amylase variants of SEQ ID NO: 1 comprise substitutions N21D + D97N + V128I, and optionally further comprise substitutions at position 200 and / or deletions at position 180 and / or position 181.

[0481] Other suitable amylases are alpha-amylases having SEQ ID NO: 12 in WO 01 / 66712 or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions, or insertions in one or more of the following positions of SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions R118K, N195F, R320K, and R458K, and variants additionally having substitutions at one or more positions selected from the group consisting of: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, most preferably variants additionally having substitutions in all of these positions.

[0482] Other examples are those amylase variants described, for example, in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087. Commercially available amylases include Duramyl TM Termamyl TM 、Fungamyl TM 、Stainzyme TM 、Stainzyme Plus TM 、Natalase TM 、Liquozyme X、BAN TM, Amplify®, and Amplify® Prime (from Novozymes A / S), and Rapidase TM 、Purastar TM / Effect TM , Powerase, Preferenz S1000, Preferenz S100, and Preferenz S110 (from Genencor International Inc. / DuPont.

[0483] A preferred amylase is a variant of the amylase having SEQ ID NO: 13 in WO 2016 / 180748 having changes H1*+ N54S + V56T + K72R + G109A + F113Q + R116Q + W167F + Q172G + A174S + G182*+D183*+ G184T + N195F + V206L + K391A + P473R + G476K.

[0484] Another preferred amylase is a variant of the amylase having SEQ ID NO: 1 in WO 2013 / 001078 having changes D183* + G184* + W140Y + N195F + V206Y + Y243F + E260G + G304R + G476K.

[0485] Another preferred amylase is a variant of an amylase having SEQ ID NO: 1 in WO 2018 / 141707 having changes H1*+G7A + G109A + W140Y + G182* + D183* + N195F + V206Y + Y243F + E260G + N280S + G304R + E391A + G476K.

[0486] Further preferred amylases are variants of the amylase having SEQ ID NO: 1 in WO 2017 / 191160 having changes L202M + T246V.

[0487] Peroxidase / Oxidase

[0488] Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. Chemically modified mutants or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, for example from C. cinereus, and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0489] Commercially available peroxidases include Guardzyme™ (Novozymes A / S).

[0490] Deoxyribonuclease (DNase)

[0491] Suitable deoxyribonucleases (DNA enzymes) are any enzymes that catalyze the hydrolytic cleavage of the phosphodiester bonds in the DNA backbone to degrade the DNA. Preferably, bacterial DNA enzymes are preferably DNA enzymes obtainable from Bacillus species, particularly preferably DNA enzymes obtainable from Bacillus subtilis or Bacillus licheniformis. Examples of such DNA enzymes are described in WO 2011 / 098579 and WO 2014 / 087011.

[0492] Auxiliary materials

[0493] Can also utilize any detergent component used in laundry detergent as known in the art.Other optional detergent components include anticorrosive, shrink-proofing agent, anti-soil redeposition agent, wrinkle-resistant agent, bactericide, adhesive, corrosion inhibitor, disintegrating agent / disintegrating agent, dye, enzyme stabilizer (including boric acid, borate, CMC and / or polyol, such as propylene glycol), fabric conditioner (including clay), filler / processing aid, fluorescent whitening agent / optical brightener, foaming agent, foam (bubble) conditioning agent, spices, dirt suspension agent, softener, foam suppressant, dark inhibitor and wicking agent, alone or in combination.Can utilize any composition used in laundry detergent as known in the art.The selection of such composition is fully within the technical scope of technicians.

[0494] Dispersants: The detergent composition of the present invention may also contain a dispersant. In particular, powdered detergents may contain a dispersant. Suitable water-soluble organic materials include homopolymeric or copolymeric acids or their salts, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by not more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant science series, Volume 71, Marcel Dekker, 1997.

[0495] Dye transfer inhibitors :Detergent compositions of the present invention can also include one or more dye transfer inhibitors.Suitable polymer dye transfer inhibitors include but are not limited to polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazolidone and polyvinylimidazole or its mixture.When present in the subject composition, the dye transfer inhibitor can exist by the weight of the composition at a level from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3%.

[0496] Fluorescent brightener:The detergent composition of the present invention will preferably also contain additional components that can color the items being cleaned, such as fluorescent whitening agents or optical brighteners. When present, the level of brightener is preferably from about 0.01% to about 0.5%. Any fluorescent whitening agent suitable for use in laundry detergent compositions can be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-distyryl derivatives. Examples of fluorescent whitening agents of diaminostilbene-sulfonic acid derivative type include the following sodium salts: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazine-6-ylamino) stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-diphenylamino-s-triazine-6-ylamino) stilbene-2.2'-disulfonate, 4,4'-bis-(2-anilino-4 (N-methyl-N-2-hydroxy-ethylamino)-s-triazine- 6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, and 2-(distyryl-4"-naphthalene-1.,2':4,5)-1,2,3-triazole-2"-sulfonate. Preferred fluorescent brighteners are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene disulfonate. Tianlaibao CBS is the disodium salt of 2,2'-bis-(phenyl-styryl) disulfonate. It is also preferred that the fluorescent whitening agent is commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals in Mumbai, India. Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazoline and 7-aminoalkyl coumarin. Suitable fluorescent brightener levels include from about 0.01 wt.%, from 0.05 wt.%, from about 0.1 wt.% or even from about 0.2 wt.% lower levels to 0.5 wt.% or even 0.75 wt.% higher levels.

[0497] Soil Release PolymersThe detergent composition of the present invention can also include one or more soil release polymers, which help remove dirt from fabrics (such as cotton and polyester-based fabrics), particularly remove hydrophobic dirt from polyester-based fabrics. Soil release polymers can be, for example, polymers based on nonionic or anionic terephthalic acid, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, Powdered Detergents [powder detergent], Surfactant science series [surfactant science series], Vol. 71, Chapter 7, Marcel Dekker, Inc. Another type of soil release polymer is an amphiphilic alkoxylated oil-cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to the core structure. The core structure can include a polyalkyl imine structure or a polyalkanolamine structure, as described in detail in WO 2009 / 087523 (which is hereby incorporated by reference). In addition, random graft copolymers are suitable soil release polymers. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856 and WO 2006 / 113314 (which are hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, such as those described in EP 1867808 or WO 03 / 040279 (both of which are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulosic polymers include anion-modified cellulose, nonion-modified cellulose, cation-modified cellulose, zwitterionic-modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, ester carboxymethylcellulose, and mixtures thereof.

[0498] 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), polyvinyl pyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulose-based polymers described above under soil release polymers may also function as anti-redeposition agents.

[0499] Other suitable excipientsIncluding but not limited to shrink-proofing agents, anti-wrinkle agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, fragrances, pigments, foam suppressants, solvents, and structurants and / or structural elastic agents for liquid detergents.

[0500] Preparation of detergent products

[0501] The detergent enzymes (ie, the first and second proteases and optionally one or more additional enzymes) can be included in the detergent composition by adding a separate additive containing one or more enzymes, or by adding a combination additive comprising these enzymes.

[0502] As mentioned above, the detergent composition may be in the form of, for example, a powder, granules, tablet, sachet, paste, gel, or liquid.

[0503] pouch

[0504] The pouch (pod) can be configured as a single or multiple compartments and can be any form, any shape, and any material suitable for holding the composition, without allowing the composition to be released from the pouch before contact with water. The pouch is made of a water-soluble film, which includes an internal volume. The internal volume can be divided into the compartments of the pouch. The preferred film is a polymeric material, preferably a polymer formed into a film or sheet. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates, and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer in the film, such as PVA, is at least about 60%. The preferred average molecular weight typically will be about 20,000 to about 150,000. The film can also be a blended composition comprising a hydrolytically degradable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under the commodity reference number M8630, as sold by Chris Craft In. Prod., Gary, Indiana, USA) plus a plasticizer, like glycerol, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. The pouch can comprise, for example, a solid laundry detergent composition or a portion of a component and / or a liquid cleaning composition or a portion of a component separated by a water-soluble film. The compartment for the liquid component can be different in composition from the compartment containing the solid. See, for example, US 2009 / 0011970.

[0505] The detergent ingredients can be physically separated from each other by compartments in water-soluble sachets or in different layers of tablets, thereby avoiding undesirable storage interactions between components. Different dissolution profiles of each compartment can also lead to delayed dissolution of selected components in the wash liquor.

[0506] Liquids and gels

[0507] Non-unit dose liquid or gel detergents can be aqueous, typically containing at least 20% and up to 95% water by weight, 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 but not limited to alkanols, amines, glycols, ethers, and polyols can be included in the aqueous liquid or gel. The aqueous liquid or gel detergent can contain from 0-30% organic solvent.

[0508] Liquid or gel detergents may also be non-aqueous.

[0509] Granular detergent formulations

[0510] Enzyme in the form of granules is usually used in granular (powder) detergents, which comprises an enzyme-containing core and optional one or more coatings. The various methods for preparing the core are well-known in the art, and include for example a) spray-drying a solution containing a liquid enzyme, b) producing a layered product, wherein the enzyme is coated as a layer around a preformed inert core particle, for example using a fluidized bed device to carry out, c) the enzyme is absorbed on the surface of the preformed core and / or wherein, d) extrude an enzyme-containing paste, e) the enzyme-containing powder is suspended in molten wax and atomized to produce a granular product, f) mixing granulation is carried out by adding the enzyme-containing liquid to the dry powder composition of the granulation component, g) by grinding or pulverizing larger particles, pellets, etc., the enzyme-containing core is subjected to particle size reduction, and h) fluidized bed granulation. The enzyme-containing core can be dried (for example using a fluidized bed dryer or other known methods for drying particles in a feed or enzyme industry), resulting in a water content of typically 0.1%-10% w / w water.

[0511] The enzyme-containing core may optionally be provided with a coating to improve storage stability and / or reduce dust formation. One coating commonly used for enzyme granules for detergents is a salt coating, typically an inorganic salt coating, which may be applied, for example, with a solution of the salt using a fluid bed. Other coating materials that may be used are, for example, polyethylene glycol (PEG), methylhydroxy-propylcellulose (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.

[0512] For further information on enzyme granules and their preparation, see WO 2013 / 007594, and for example WO 2009 / 092699, EP 1705241, EP 1382668, WO 2007 / 001262, US 6,472,364, WO 2004 / 074419 and WO 2009 / 102854.

[0513] Formulation of enzymes in co-granules

[0514] Enzyme of the present invention can be formulated as granules, for example, formulated as co-granules in combination with one or more enzymes. Then, each enzyme will be present in a variety of granules, which ensure that the enzyme is more evenly distributed in the detergent. This also reduces the physical isolation of different enzymes caused by different particle sizes. The method for producing multi-enzyme co-granules for the detergent industry is disclosed in IP.com disclosure IPCOM000200739D.

[0515] Another example of formulation of enzymes by using co-granules is disclosed in WO 2013 / 188331, which relates to a detergent composition comprising: (a) a multi-enzyme co-granule, (b) less than 10 wt% of zeolite (on anhydrous basis), and (c) less than 10 wt% of phosphate (on anhydrous basis), wherein the enzyme co-granule comprises from 10 wt% to 98 wt% of a hygroscopic component, and the composition additionally comprises from 20 wt% to 80 wt% of a detergent hygroscopic component.

[0516] WO 2013 / 188331 also relates to a method for treating and / or cleaning a surface, preferably a textile surface, comprising the steps of: (i) contacting said surface in an aqueous wash liquor with a detergent composition as claimed and described herein, (ii) rinsing and / or drying the surface.

[0517] The multi-enzyme co-granule can comprise an enzyme of the invention and (a) one or more enzymes selected from the group consisting of first wash lipases, cleaning cellulases, xyloglucanases, perhydrolases, peroxidases, lipoxygenases, laccases, and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, proteases, care cellulases, cellobiose dehydrogenases, xylanases, phospholipases, esterases, cutinases, pectinases, mannanases, pectin lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pentosanases, lichenases, glucanases, arabinosidases, hyaluronidases, chondroitinase, amylases, and mixtures thereof.

[0518] Laundry soap bars

[0519] The polypeptide of the present invention can be added into laundry soap bar and used for hand washing laundry, fabric, and / or textile. The term laundry soap bar includes laundry bar, soap bar, combination bar (combo bar), synthetic detergent bar and detergent bar. The type of bar is usually distinguished by the type of surfactant they contain, and the term laundry soap bar includes those containing soap and / or synthetic soap from fatty acids. The laundry soap bar has a physical form that is solid rather than liquid, gel, or powder at room temperature. The term solid is defined as a physical form that does not change significantly over time, that is, if a solid object (e.g., laundry soap bar) is placed in a container, the solid object will not change in order to fill the container in which it is placed. The bar is typically in the form of a bar when it is solid, but it can also be other solid shapes such as round or oval.

[0520] Bar soaps for hand washing can be in the form of "soap bars" (oil-based) or "non-soap detergent" (NSD) bars. As the name implies, non-soap detergent bars are characterized by the lack of fatty acid soap ingredients from plant or animal sources, and are instead based on synthetic detergents such as LAS (linear alkylbenzene sulfonate).

[0521] Laundry bars comprising the first and second proteases of the present invention can be produced by methods conventionally known and used to produce soap bars and using conventional laundry bar manufacturing equipment such as, but not limited to, blenders, plodders (e.g., two-stage vacuum plodders), extruders, mills, cutters, labeling dies, cooling tunnels, and packaging machines.

[0522] use

[0523] The present invention also relates to methods for washing textiles and fabrics using the detergent compositions, including domestic laundry and industrial laundry applications. The present invention also relates to the use of the compositions of the present invention in cleaning processes such as laundry or hard surface cleaning such as dishwashing. In a preferred embodiment, the compositions of the present invention are designed for and used in laundry applications. In a further preferred embodiment, the composition may be in the form of a liquid laundry formulation.

[0524] The detergent compositions of the present invention may be formulated, for example, as hand or machine laundry detergent compositions including a laundry additive composition suitable for pre-treating stained fabrics, and a rinse added fabric softener composition, or as detergent compositions for general household hard surface cleaning operations, or for hand or machine dishwashing operations.

[0525] This cleaning process or textile care process can be, for example, a laundry process, a dishwashing process, or a hard surface (such as bathroom tiles, floors, desktops, drains, sinks, and washbasins) cleaning. The laundry process can be, for example, domestic laundry, but can also be industrial laundry. In addition, the present invention relates to a method for washing fabrics and / or clothing, wherein the method comprises treating fabrics with a washing solution containing a detergent composition of the present invention. For example, a cleaning process or textile care process can be carried out in machine washing or in manual washing. The washing solution can be, for example, an aqueous washing solution containing a detergent composition.

[0526] The present invention further relates to the use of a detergent composition in the process of removing protein stains. The protein stains can be stains such as food stains (e.g., baby food, cocoa, eggs, or milk), or other stains (such as sebum, blood, ink, or grass), or combinations thereof.

[0527] This aspect also relates to a method of cleaning, especially for cleaning fabrics or textiles, or for dishwashing, which method comprises contacting the detergent composition of this aspect with the fabrics / textiles or dishwashing under conditions suitable for cleaning the fabrics / textiles or dishwashing.

[0528] The first protease and the second protease in the composition according to this aspect and for use and cleaning methods thereof, may be any protease described herein.

[0529] Washing method

[0530] The present invention provides a method of cleaning, especially for cleaning fabrics or textiles (ie laundry), or for dishwashing, using a detergent composition of the present invention comprising a first protease and a second protease.

[0531] The cleaning method comprises contacting the object with a detergent composition comprising a first protease and a second protease under conditions suitable for cleaning the object. In a preferred embodiment, the detergent composition is used in a laundry process.

[0532] Another embodiment is directed to a method for removing stains from fabric or textiles, comprising contacting the fabric or textile with a composition of the present invention under conditions suitable for cleaning the object.

[0533] Another embodiment is directed to a method for removing stains from dishware comprising contacting the dishware with a composition of the present invention under conditions suitable for cleaning the object.

[0534] The composition can be used in a solution at a concentration of from about 100 ppm (preferably 500 ppm) to about 15000 ppm. The water temperature range is typically from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, and about 90°C. The water to fabric ratio is typically from about 1:1 to about 30:1.

[0535] The enzymes of the detergent compositions of the present invention can be stabilized using conventional stabilizers and protease inhibitors, for example, polyols (such as propylene glycol or glycerol, sugars or sugar alcohols), different salts (such as NaCl, KCl), lactic acid, formic acid, boric acid, or boric acid derivatives (e.g., aromatic borate), or phenylboronic acid derivatives (such as 4-formylphenylboronic acid), or peptide aldehydes (such as dipeptide aldehydes, tripeptide aldehydes, or tetrapeptide aldehydes), or aldehyde analogs (B1-B0-R form, wherein R is H, CH3, CX3, CHX2, or CH2X (X = halogen), B0 is a single amino acid residue (preferably with optionally substituted aliphatic or aromatic side chains), and B1 consists of one or more amino acid residues (preferably one, two, or three), optionally containing an N-terminal protecting group; or as described in WO 2009 / 118375, WO 98 / 13459), or proteinaceous protease inhibitors such as RASI, BASI, WASI (bifunctional alpha-amylase / subtilisin inhibitor of rice, barley, and wheat), or CI2, or SSI. The composition can be formulated, for example, as described in WO 92 / 19709, WO 92 / 19708, and US 6,472,364. In some embodiments, the enzymes employed herein are stabilized by water-soluble sources of zinc (II), calcium (II), and / or magnesium (II) ions, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II), and vanadium (IV)) present in the finished composition to provide such ions for the enzymes.

[0536] Production method

[0537] The first protease and the second protease may be produced by standard procedures well known in the art using cultivation of the host cells in a suitable nutrient medium.

[0538] The host cell can be any cell useful for recombinase production, for example, a prokaryotic or eukaryotic cell.

[0539] The prokaryotic host cell will typically be a Gram-positive or Gram-negative bacterium, such as a Gram-positive bacterium selected from the genera Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces; or a Gram-negative bacterium selected from the genera Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0540] The bacterial host cell can be, for example, a Bacillus cell selected from the group consisting of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0541] For information on suitable host cells, see, for example, WO 2017 / 207762.

[0542] For example, host cells can be cultured in a laboratory or industrial fermentor tank in a suitable substratum and under conditions that allow expression and / or separation of variants by shaking flask culture or small-scale or large-scale fermentation (comprising continuous, batch, fed-batch or solid-state fermentation).Use procedures as known in the art, cultivation occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts.Suitable substratum can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalogue of American Type Culture Collection).If variant is secreted into the nutrient medium, variant can be directly recovered from the substratum.If variant is not secreted, it can be recovered from cell lysate.

[0543] The variant can be detected using methods known in the art specific for variants having protease activity, and can be recovered and purified using methods known in the art. For further information, see, for example, WO 2017 / 207762.

[0544] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0545] Examples

[0546] Materials and Methods

[0547] Preparation and purification of peptides

[0548] Mutations were made and expression cassettes were introduced into Bacillus subtilis by standard methods known in the art. All DNA manipulations were performed by PCR using standard methods known to the skilled artisan (e.g., as described by Sambrook et al., Molecular Cloning; 3rd ed., 2001, Harbor Laboratory Press).

[0549] The recombinant Bacillus subtilis construct encoding the subtilase polypeptide was inoculated into complex medium (TBgly) and cultured at 37°C for 24 h. Overnight cultures were used to inoculate a rich medium (PS-1: 100 g / L sucrose (Danisco Catalog No. 109-0429), 40 g / L soybean hulls (soy flour), 10 g / L Na 2 HPO 4 •12H 2The shake flasks were incubated with 0.1 ml / L Dowfax 63N10 (Dow) and 30°C with shaking at 270 rpm for 4 days.

[0550] Purification of culture supernatant was performed as follows:

[0551] The culture was centrifuged at 26,000 x g for 20 minutes and the supernatant was carefully decanted from the pellet. The supernatant was filtered through a Nalgene 0.2 µm filter unit to remove remaining host cells. The pH of the 0.2 µm filtrate was adjusted to pH 8 with 3 M Tris base and the pH adjusted filtrate was applied to a 20 mM Tris / HCl, 1 mM CaCl 2 A MEP Hypercel column (Pall Corporation) was equilibrated in 5% HCl (pH 8.0). After washing the column with the equilibration buffer, the column was washed with 20 mM CH 3 COOH / NaOH, 1 mM CaCl 2 (pH 4.5) stepwise elution. Fractions from the column were analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9 and peak fractions were pooled. The pH of the pool from the MEP Hypercel column was adjusted using 20% ​​(v / v) CH 3 COOH or 3 M Tris base to pH 6, and the pH adjusted pool was diluted with deionized water to a concentration of 20 mM MES / NaOH, 2 mM CaCl 2 The diluted pool was applied to a 20 mM MES / NaOH, 2 mM CaCl 2 The protease variants were then loaded onto a SP-Sepharose® Fast Flow column (GE Healthcare) equilibrated in 5% equilibration buffer (pH 6.0). After washing the column with the equilibration buffer, the protease variants were loaded onto a linear NaCl gradient ( ) was eluted over five column volumes. Fractions from the column were analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9, and active fractions were analyzed by SDS-PAGE. Fractions where only one band was observed on a Coomassie-stained SDS-PAGE gel were pooled as purified preparations and used for further experiments.

[0552] Automatic Mechanical Stress Assay (AMSA) for Laundry

[0553] Experiments were conducted to evaluate the wash performance of selected protease variants in laundry detergent compositions. Protease was tested using automatic mechanical stress assay (AMSA). Using AMSA, the wash performance of a large number of small volume enzyme detergent solutions can be checked. The AMSA plate has many slots for test solutions and a lid for pressing the test sample to be washed (textile sample for testing laundry detergent) against the slot opening. During washing, the plate, test solution, test sample, and lid are shaken vigorously to contact the test solution with the soiled test sample, and mechanical stress is applied in a regular, periodic oscillating manner. For further description, see WO 02 / 42740, especially the "Specific Method Embodiment" paragraph on pages 23-24.

[0554] In this embodiment, the performance of enzyme variants and their blends is measured as the brightness of the color of a textile sample washed with a specific protease or protease blend. Brightness can be expressed as the intensity of light reflected from a textile sample when illuminated with white light. When the textile has stains, the intensity of reflected light is lower than the intensity of reflected light of a clean textile. Therefore, the intensity of reflected light can be used to measure the washing performance of proteases and protease blends.

[0555] Color measurements were performed using a professional flatbed scanner (Epson Expression 10000XL), which was used to capture images of the washed textile samples.

[0556] To extract light intensity values ​​from the scanned images, a specially designed software application (Novozymes Color Vector Analyzer) was used. The program retrieves the values ​​from the image and converts them into red, green, and blue (RGB) values. The intensity value (Int) is calculated by summing the RGB values ​​as a vector and then taking the length of the resulting vector:

[0557]

[0558] Standard textile parts were obtained from Center for Testmaterial BV, PO Box 120, 3133KT Vlaardingen, The Netherlands. The detergent used for the washing performance test was a liquid laundry model detergent. The composition of the detergent and other test parameters are given in the table below.

[0559] Table 1: Detergent compositions and test conditions for AMSA

[0560]

[0561] The proteases tested are listed in Table 2. These proteases are SEQ ID NO: 1 and variants thereof, with position numbering based on SEQ ID NO: 2 numbering, and with the indicated net formal charge relative to SEQ ID NO: 1.

[0562] Table 2: Proteases tested with charge combinations

[0563]

[0564] Example 1

[0565] The wash performance of different variants of SEQ ID NO: 1 having a net charge of -4 or -3 relative to SEQ ID NO: 1 with the mutations shown in Tables 3 and 4 below were tested alone in AMSA as described above ("single enzyme") or together with a variant of SEQ ID NO: 1 having a substitution K27M and a net charge of -1 relative to SEQ ID NO: 1. The wash performance of the K27M variant alone was also tested in AMSA.

[0566] For each enzyme concentration (0.25, 0.5, 1, and 2 mg enzyme protein / L as described above), the Δ intensity values ​​for the single enzymes and 1:1 mixtures were calculated as the intensity value (Int) for the test material washed with detergent containing the single enzyme or mixture minus the intensity value for the test material washed with detergent alone (i.e., without any enzyme). For each treatment (single enzyme or 1:1 mixture), the Δ intensity values ​​for each of the four enzyme concentrations were added together to obtain the sum of the Δ intensities for each treatment for each of the two stains, PC-10 and PC-03.

[0567] The Δ Intensity Sum of the 1:1 mixture was then compared to the Δ Intensity Sum of the individual enzymes in the mixture and the relative performance of the mixture compared to the individual enzymes was determined for PC-10 and PC-03 stains. The relative performance values ​​for the individual stains PC-10 and PC-03 expressed as a percentage relative performance to the individual enzymes in Tables 3 and 4 below were determined by dividing the Δ Intensity Sum of the mixture by the Δ Intensity Sum of the individual enzymes.

[0568] In Table 3, the mixtures are compared to individual enzymes having a net formal charge of -4 or -3 relative to SEQ ID NO: 1 (enzymes (a), (b), (c), (d) in the "Variant / Single Enzyme" column), while Table 4 compares the mixtures to the K27M variant alone.

[0569] Finally, to indicate the overall performance of the mixture relative to the single enzymes for a different set of protein-based stains, the last column in each table, "PC-03 + PC-10", provides the percentage relative performance of the mixture relative to the single enzymes calculated in the same manner as described above for the individual stains PC-10 and PC-03. However, in this case, the sum of the delta intensities of the mixture for PC-10 and PC-03 was added together and divided by the sum of the delta intensities of the single enzymes for PC-10 and PC-03.

[0570] Table 3: Relative performance of charge mixtures with variant K27M for single enzymes (a), (b), (c), (d)

[0571]

[0572] Table 4: Relative performance of charge mixtures with variant K27M versus variant K27M alone

[0573]

[0574] Example 2

[0575] The wash performance of different variants of SEQ ID NO: 1 having a net charge of -4 relative to SEQ ID NO: 1 with the mutations shown in Tables 5 and 6 below were tested alone ("single enzyme") or with SEQ ID NO: 1 (Savinase®) in AMSA as described above. The wash performance of SEQ ID NO: 1 alone was also tested in AMSA.

[0576] The delta intensity values ​​for each treatment on each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percent relative performance of the mixture compared to the single enzymes was calculated, and the overall performance of the mixture on a panel of stains (PC-03 + PC-10) was calculated compared to the single enzymes.

[0577] In Table 5, the mixtures are compared to single enzymes having a net formal charge of -4 relative to SEQ ID NO: 1 (enzymes (a), (b), (c) in the "Variant / Single Enzyme" column), while Table 6 compares the mixtures to SEQ ID NO: 1 alone.

[0578] Table 5: Relative performance of charge mixtures with SEQ ID NO: 1 against single enzymes (a), (b), (c)

[0579]

[0580] Table 6: Relative performance of charge mixtures with SEQ ID NO: 1 versus SEQ ID NO: 1 alone

[0581]

[0582] Example 3

[0583] The wash performance of different variants of SEQ ID NO: 1 with a net charge of -4 or -3 relative to SEQ ID NO: 1 having mutations as shown in Tables 7 and 8 below was tested alone ("single enzyme") in AMSA as described above, or with a variant of SEQ ID NO: 1 with mutation S99AD (net charge relative to SEQ ID NO: 1 was -1). The wash performance of the S99AD variant alone was also tested in AMSA.

[0584] The delta intensity values ​​for each treatment on each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percent relative performance of the mixture compared to the single enzymes was calculated, and the overall performance of the mixture on a panel of stains (PC-03 + PC-10) was calculated compared to the single enzymes.

[0585] In Table 7, the mixtures are compared to single enzymes having a net formal charge of 4 or 3 relative to SEQ ID NO: 1 (enzymes (a), (b), (c), (d), (e) in the "Variant / Single Enzyme" column), while Table 8 compares the mixtures to the individual S99AD variants.

[0586] Table 7: Relative performance of charge mixtures with variant S99AD for single enzymes (a), (b), (c), (d), (e)

[0587]

[0588] Table 8: Relative performance of charge mixtures with variant S99AD vs. S99AD alone

[0589]

[0590] Example 4

[0591] The wash performance of different variants of SEQ ID NO: 1 with a net charge of -5, -4, or -3 relative to SEQ ID NO: 1 having the mutations shown in Tables 9 and 10 below was tested alone ("single enzyme") in AMSA as described above, or with a variant of SEQ ID NO: 1 having the substitution G97D Y209W A215K (net charge relative to SEQ ID NO: 1 is 0). The wash performance of the G97D Y209W A215K variant alone was also tested in AMSA.

[0592] The delta intensity values ​​for each treatment on each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percent relative performance of the mixture compared to the single enzymes was calculated, and the overall performance of the mixture on a panel of stains (PC-03 + PC-10) was calculated compared to the single enzymes.

[0593] In Table 9, the mixtures are compared to single enzymes having a net formal charge of -5, -4, or -3 relative to SEQ ID NO: 1 (enzymes (a), (b), (c), (d) in the "Variant / Single Enzyme" column), while Table 10 compares the mixtures to the G97DY209W A215K variant alone.

[0594] Table 9: Relative charge mixtures with variants G97D Y209W A215K to single enzymes (a), (b), (c), (d) performance

[0595]

[0596] Table 10: Charge mixture with variant G97D Y209W A215K vs variant G97D Y209W alone A215K's Relative Performance

[0597]

[0598] Example 5

[0599] The wash performance of different variants of SEQ ID NO: 1 with a net charge of -5, -4, or -3 relative to SEQ ID NO: 1 having the mutations shown in Tables 11 and 12 below was tested alone ("single enzyme") in AMSA as described above, or with a variant of SEQ ID NO: 1 having the substitution G97D N117R Y209W A215K (net charge relative to SEQ ID NO: 1 was +1). The wash performance of the G97D N117R Y209W A215K variant alone was also tested in AMSA.

[0600] The delta intensity values ​​for each treatment on each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percent relative performance of the mixture compared to the single enzymes was calculated, and the overall performance of the mixture on a panel of stains (PC-03 + PC-10) was calculated compared to the single enzymes.

[0601] In Table 11, the mixtures are compared to single enzymes having a net formal charge of -5, -4 or -3 relative to SEQ ID NO: 1 (enzymes (a), (b), (c) in the "Variant / Single Enzyme" column), while Table 12 compares the mixtures to the G97D N117RY209W A215K variant alone.

[0602] Table 11: Charge mixtures with variants G97D N117R Y209W A215K for single enzymes (a), (b), (c) Relative performance

[0603]

[0604] Table 12: Charge mixture with variant G97D N117R Y209W A215K vs variant G97D N117R alone Relative performance of Y209W A215K

[0605]

[0606] The data presented in Examples 1 to 5 above show that the combination of a first protease having a net charge of -5, -4, or -3 compared to SEQ ID NO: 1 and a second protease having a net charge of -1, 0, or +1 compared to SEQ ID NO: 1 results in improved wash performance. In most cases, the wash performance of the mixture on PC-10 and PC-03 stains was greatly improved compared to the individual enzymes, thereby greatly improving the overall wash performance. In a few cases, the performance of the mixture on PC-03 was poor, but this was outweighed by the improved wash performance on PC-10, so the overall performance of the mixture on PC-03 + PC-10 was still better than the individual enzymes.

[0607] Example 6

[0608] The wash performance of SEQ ID NO: 1 or variants thereof having a net charge of 0 or -1 relative to SEQ ID NO: 1 with mutations as shown in Tables 13 and 14 below was tested alone in AMSA as described above, or with a variant having mutations S9R S99D S101E S103A V104I S156D G160S Q245R L262E relative to SEQ ID NO: 1 (net charge of -2 relative to SEQ ID NO: 1). The wash performance of the S9R S99D S101E S103A V104I S156D G160S Q245R L262E variant alone was also tested in AMSA.

[0609] The delta intensity values ​​for each treatment on each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percent relative performance of the mixture compared to the single enzymes was calculated, and the overall performance of the mixture on a panel of stains (PC-03 + PC-10) was calculated compared to the single enzymes.

[0610] In Table 13, the mixtures are compared to single enzymes having a net formal charge of -1 or 0 relative to SEQ ID NO: 1 (enzymes (a), (b), (c) in the "Variant / Single Enzyme" column), while Table 14 compares the mixtures to the individual variants S9R S99DS101E S103A V104I S156D G160S Q245R L262E.

[0611] Table 13: Charge Mixing with S9R S99D S101E S103A V104I S156D G160S Q245R L262E Relative performance of single enzymes (a), (b), and (c)

[0612]

[0613] Table 14: Charge mixture vs individual S9R S99D S101E S103A V104I S156D G160S Q245R L262E Relative Performance

[0614]

[0615] Example 7

[0616] The wash performance of SEQ ID NO: 1 or its variants having the mutations shown in Tables 15 and 16 below was tested alone ("single enzyme") in AMSA as described above, or with a variant of SEQ ID NO: 1 having the substitution N76D G97D N140D S156D Y209W A215K L262E (net charge relative to SEQ ID NO: 1 was -4). The wash performance of the N76D G97D N140D S156D Y209W A215K L262E variant alone was also tested in AMSA.

[0617] The delta intensity values ​​for each treatment on each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percent relative performance of the mixture compared to the single enzymes was calculated, and the overall performance of the mixture on a panel of stains (PC-03 + PC-10) was calculated compared to the single enzymes.

[0618] In Table 15, the mixtures are compared to the single enzymes (enzymes (a), (b), (c) listed in the "Variant / Single Enzyme" column), while Table 16 compares the mixtures to the individual N76D G97D N140D S156D Y209W A215K L262E variants.

[0619] Table 15: Charge mixtures with N76D G97D N140D S156D Y209W A215K L262E vs. single enzymes Relative performance of (a), (b), and (c)

[0620]

[0621] Table 16: Charge mixture versus individual variants N76D G97D N140D S156D Y209W A215K L262E Relative performance

[0622]

[0623] Example 8

[0624] The wash performance of SEQ ID NO: 1 or its variants having the mutations shown in Tables 17 and 18 below was tested alone ("single enzyme") in AMSA as described above, or with a variant of SEQ ID NO: 1 having the substitution K27M N77D G97D S156D Y209W A215K L262E (net charge relative to SEQ ID NO: 1 was -4). The wash performance of the K27M N77D G97D S156D Y209W A215K L262E variant alone was also tested in AMSA.

[0625] The delta intensity values ​​for each treatment on each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percent relative performance of the mixture compared to the single enzymes was calculated, and the overall performance of the mixture on a panel of stains (PC-03 + PC-10) was calculated compared to the single enzymes.

[0626] In Table 17, the mixtures are compared to the individual enzymes (enzymes (a), (b), (c) listed in the "Variant / Single Enzyme" column), while Table 18 compares the mixtures to the individual K27M N77D G97D S156D Y209W A215K L262E variants.

[0627] Table 17: Charge mixture vs single enzyme with K27M N77D G97D S156D Y209W A215K L262E Relative performance of (a), (b), and (c)

[0628]

[0629] Table 18: Comparison of charge mixtures to individual variants K27M N77D G97D S156D Y209W A215K L262E About performance

[0630]

[0631] Example 9

[0632] The wash performance of variants of SEQ ID NO: 1 having the mutations shown in Tables 19 and 20 below was tested alone ("single enzyme") in AMSA as described above, or with a variant of SEQ ID NO: 1 having the substitution G97D S156D Y209W A215K L262E (net charge relative to SEQ ID NO: 1 was -2). The wash performance of the G97D S156D Y209W A215KL262E variant alone was also tested in AMSA.

[0633] The delta intensity values ​​for each treatment on each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percent relative performance of the mixture compared to the single enzymes was calculated, and the overall performance of the mixture on a panel of stains (PC-03 + PC-10) was calculated compared to the single enzymes.

[0634] In Table 19, the mixtures are compared to single enzymes having a net formal charge of +1, -1, -3, or -4 (enzymes (a), (b), (c), (d), (e), (f), (g), (h), (i) listed in the "Variant / Single Enzyme" column), while Table 20 compares the mixtures to the individual variants G97D S156D Y209W A215K L262E.

[0635] Table 19: Charge mixtures with G97D S156D Y209W A215K L262E for single enzymes (a), (b), (c), Relative performance of (d), (e), (f), (g), (h), and (i)

[0636]

[0637] Table 20: Relative performance of charge mixtures versus individual variants G97D S156D Y209W A215K L262E

[0638]

[0639] Example 10

[0640] The wash performance of variants of SEQ ID NO: 1 comprising substitutions G97D + Y209W + A215K and at least one additional substitution as shown in Table 21 below was tested in the AMSA assay as described above and compared to the performance of the protease of SEQ ID NO: 1.

[0641] The delta intensity values ​​for each enzyme for each of the two stains PC-10 and PC-03 were determined as described in Example 1, the percentage relative performance of the enzyme compared to SEQ ID NO: 1 was calculated, and the overall performance of each enzyme on a set of stains (PC-03 + PC-10) compared to SEQ ID NO: 1 was calculated. In this example, only individual enzymes were tested. Table 21 shows that all tested variants comprising the substitutions G97D + Y209W + A215K exhibited improved wash performance relative to the reference protease having SEQ ID NO: 1.

[0642] Table 21: Relative performance of variants of SEQ ID NO: 1 compared to SEQ ID NO: 1

[0643]

[0644] Example 11

[0645] The wash performance of variants of SEQ ID NO: 1 comprising the substitutions G97D + Y209W + A215K and the additional substitutions shown in Table 22 below was tested in the AMSA assay as described above and compared to the performance of a variant of SEQ ID NO: 1 having the substitutions S99D + S101E + S103A + V104I + G160S as a reference. This example tested a variant of SEQ ID NO: 1 having the substitutions G97D + S156D + Y209W + A215K + L262E and at least one additional substitution selected from S87N, S101G / N, V104N, G118V, and A194P.

[0646] The Delta Intensity values ​​for each enzyme for each of the two stains PC-10 and PC-03 were determined as described in Example 1 and the percent relative performance of the enzyme compared to the reference protease was calculated. In this example, only the individual enzymes were tested.

[0647] Table 22: SEQ ID NO: 1 compared to SEQ ID NO: 1 + S99D S101E S103A V104I G160S Relative performance of variants

[0648]

[0649] Table 22 shows that all tested variants exhibited improved washing performance relative to the reference protease of SEQ ID NO: 1 + S99D + S101E + S103A + V104I + G160S (which itself exhibited good cleaning performance). For PC-03 stains, the tested variants had at least equivalent performance and in most cases improved performance relative to the reference protease, while for PC-10 stains, all tested variants had improved performance relative to the reference protease. As explained above, PC-10 stains simulate natural sebum stains, thus providing a good indication of the performance of enzyme detergent compositions for this difficult-to-remove natural stain.

[0650] The present disclosure also relates to the following embodiments:

[0651] 1. A detergent composition comprising a first protease and a second protease, wherein

[0652] 1) the first protease has a net formal charge of -1, -3 or -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1; or

[0653] 2) the first protease has a net formal charge of -3, -4 or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1, 0 or +1 relative to the protease of SEQ ID NO: 1.

[0654] 2. A detergent composition as described in embodiment 1, wherein the first protease has a net formal charge of -1, -3 or -4 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -2 relative to the protease of SEQ ID NO: 1, for example, wherein the first protease has a net formal charge of -3 or -4 relative to the protease of SEQ ID NO: 1.

[0655] 3. A detergent composition as described in embodiment 1, wherein the first protease has a net formal charge of -3, -4 or -5 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1, 0 or +1 relative to the protease of SEQ ID NO: 1, for example, wherein the first protease has a net formal charge of -3 relative to the protease of SEQ ID NO: 1, and the second protease has a net formal charge of -1 relative to the protease of SEQ ID NO: 1.

[0656] 4. A detergent composition as described in any of the preceding embodiments, comprising a first protease and a second protease, each of the first protease and the second protease having a net formal charge of -1, -2, -3 or -4 relative to the protease of SEQ ID NO: 1, and wherein the net formal charge of the first protease and the second protease differs from each other by 1 or 2.

[0657] 5. A detergent composition as described in embodiment 4, wherein the composition comprises:

[0658] a) a first protease having a net formal charge of -4 and a second protease having a net formal charge of -2 relative to the protease of SEQ ID NO: 1;

[0659] b) a first protease having a net formal charge of -3 and a second protease having a net formal charge of -2 relative to the protease of SEQ ID NO: 1; or

[0660] c) a first protease having a net formal charge of -3 and a second protease having a net formal charge of -1 relative to the protease of SEQ ID NO: 1.

[0661] 6. A detergent composition as described in any of embodiments 1-2 or 4-5, comprising a first protease having a net formal charge of -1, -3 or -4 relative to the protease of SEQ ID NO: 1 and a second protease having a net formal charge of -2 relative to the protease of SEQ ID NO: 1, wherein the second protease is a variant of SEQ ID NO: 1 comprising the substitutions G97D + S156D + Y209W + A215K + L262E or S9R + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E, wherein the position numbering is based on the numbering of SEQ ID NO: 2.

[0662] 7. A detergent composition as described in any of embodiments 1-2 or 4-6, comprising a first protease having a net formal charge of -1, -3 or -4 relative to the protease of SEQ ID NO: 1 and a second protease having a net formal charge of -2 relative to the protease of SEQ ID NO: 1, wherein the first protease is a variant of SEQ ID NO: 1 having a set of mutations selected from the group consisting of:

[0663] •K27M;

[0664] •S99AD;

[0665] •G97D + S156D + Y209W + A215K;

[0666] •G97D + Y209W + A215K + L262E;

[0667] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + K235M + Q245R +L262E;

[0668] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0669] •S9E + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E;

[0670] •N62D + N76D + G97D + Y209W + A215K + L262E;

[0671] •N62D + G97D + S101E + V177I + Y209W + A215K + L262E;

[0672] •N62D + G97D + S101E + Y209W + A215K + L262E;

[0673] •G97D + S101E + S156D + A172V + Y209W + A215K + L262E;

[0674] •S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0675] •K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E;

[0676] •S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M +Q245R + L262E;

[0677] • N76D + G97D + N140D + S156D + Y209W + A215K + L262E; and

[0678] •K27M + N77D + G97D + S156D + Y209W + A215K + L262E;

[0679] The position numbers are based on those of SEQ ID NO: 2.

[0680] 8. A detergent composition as described in any of embodiments 1 or 3-5, comprising a first protease having a net formal charge of -3, -4 or -5 relative to the protease of SEQ ID NO: 1 and a second protease having a net formal charge of -1, 0 or +1 relative to the protease of SEQ ID NO: 1, wherein the first protease is a variant of SEQ ID NO: 1 comprising a set of mutations selected from the group consisting of:

[0681] •S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0682] •K27H + S99D + S101E + S103A + V104I + S156D + G160S + Q245R + L262E;

[0683] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + K235M + Q245R +L262E;

[0684] •S9R + S99D + S101E + S103A + V104I + S156D + G160S + L262E;

[0685] •S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + K237M +Q245R + L262E;

[0686] •S9E + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E;

[0687] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +S259D + L262E;

[0688] •*36D + N76D + H120D + G195E + K235L;

[0689] •S9R + K27M + S99A + S101E + S103A + V104I + S156D + G160S + K237M +Q245R + L262E;

[0690] •S9R + K27M + S101E + S103A + V104I + S156D + G160S + K237M + Q245R +L262E;

[0691] •S9R + K27M + S99D + S101E + S103A + V104I + S156D + G160S + A215K +K237M + Q245R + L262E;

[0692] <h2 style=";text-align:left;direction:ltr">•S9R + K27M + N43R + S99D + S101E + S103A + V104I + S156D + G160S +K237M + Q245R + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0693] <h2 style=";text-align:left;direction:ltr"> •S9R+N43R+S99D+S101E+S103A+V104I+S156D+G160S+N173D+K235M+Q245R+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0694] <h2 style=";text-align:left;direction:ltr"> •S9E + G97D + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0695] <h2 style=";text-align:left;direction:ltr"> •G97D + S101E + S156D + A172V + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0696] <h2 style=";text-align:left;direction:ltr"> •N76D + G97D + N140D + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0697] <h2 style=";text-align:left;direction:ltr"> •N76D+G97D+S101E+T180A+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0698] <h2 style=";text-align:left;direction:ltr"> •N76D + G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0699] <h2 style=";text-align:left;direction:ltr"> •N62D + G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0700] <h2 style=";text-align:left;direction:ltr"> •N62D + G97D + S101E + V177I + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0701] <h2 style=";text-align:left;direction:ltr"> •N62D + N76D + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0702] <h2 style=";text-align:left;direction:ltr"> •K27M+G97D+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0703] <h2 style=";text-align:left;direction:ltr"> •K27M+N77D+G97D+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0704] •K27M + N76D + G97D + Y209W + A215K + L262E;

[0705] •K27M + N62D + G97D + Y209W + A215K + L262E;

[0706] •S9E + G97D + S101E + Y209W + A215K + L262E;

[0707] •S9E + N76D + G97D + Y209W + A215K + L262E + A270T;

[0708] •S9E + N76D + G97D + Y209W + A215K + L262E;

[0709] •S9E + N62D + G97D + Y209W + A215K + L262E;

[0710] •S9E + K27M + G97D + Y209W + A215K + L262E;

[0711] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +A215K + Q245R + S259D + L262E;

[0712] •S9E + N43R + N76D + S188E + Q191N + A194P + Q206L + Y209W + A215K +S259D + L262E;

[0713] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +A215K + S259D + L262Q; and

[0714] •S9E + N43R + N76D + N185E + S188E + Q191N + A194P + Q206L + Y209W +Q245R + S259D + L262Q;

[0715] The position numbers are based on those of SEQ ID NO: 2.

[0716] 9. A detergent composition as described in any of embodiments 1, 3-5 or 8, comprising a first protease having a net formal charge of -3, -4 or -5 relative to the protease of SEQ ID NO: 1 and a second protease having a net formal charge of -1, 0 or +1 relative to the protease of SEQ ID NO: 1, wherein the second protease is the protease of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 comprising a set of mutations selected from the group consisting of:

[0717] •K27M;

[0718] •S99AD;

[0719] •G97D + Y209W + A215K;

[0720] •G97D + N117R + Y209W + A215K;

[0721] •S9R + K27M + N43R + N76D + V205I + Q206L + Y209W + A215K + Q245R +S259D + N261W + L262E;

[0722] •S9R + N43R + N76D + V205I + Q206L + Y209W + A215K + S259D + N261W +L262E;

[0723] •S9R + N43R + N76D + V205I + Q206L + Y209W + S259D + N261W + L262E;

[0724] •S9R + S99A + S101E + S103A + V104I + S156D + G160S + A215K + K235M +Q245R + L262E;

[0725] •S9R + K27M + N43R + S99A + S101E + S103A + V104I + S156D + G160S +A215K + K237M + Q245R + L262E;

[0726] •S9R + K27M + N43R + S101E + S103A + V104I + S156D + G160S + A215K +K237M + Q245R + L262E;

[0727] •Y167A + R170S + A194P;

[0728] •S9R + A15T + V68A + N218D + Q245R;

[0729] •S9R + A15T + G61E + V68A + A194P + V205I + Q245R + N261D;

[0730] •S9R + A15T + V68A + S99G + Q245R + N261D;

[0731] •S9R + A15T + V68A + H120D + P131S + Q137H + Q245R;

[0732] •S9R + A15T + V68A + H120N + P131S + Q137H + Q245M;

[0733] •S9R + A15T + G61E + V68A + A98S + S99G + N218D + Q245R;

[0734] •S3T + V4I + S99D + S101R + S103A + V104I + G160S + V205I + L217D;

[0735] •S3T + V4I + S99D + S101R + S103A + V104I + G160S + A194P + V205I +L217D;

[0736] •G97D + Y209W + A215K + L262E; and

[0737] •G97D + S156D + Y209W + A215K;

[0738] The position numbers are based on those of SEQ ID NO: 2.

[0739] 10. A detergent composition as described in any of the preceding embodiments, wherein the first protease and the second protease each have an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1, such as at least 70%, at least 80%, at least 85%, or at least 90% sequence identity with SEQ ID NO: 1.

[0740] 11. The detergent composition of any one of the preceding embodiments, wherein the composition is a laundry detergent composition.

[0741] 12. The detergent composition of embodiment 11, wherein the composition is a liquid laundry detergent composition.

[0742] 13. A detergent composition as described in any of the preceding embodiments, wherein the composition has improved washing performance in laundry compared to the same composition comprising the first protease or the second protease; for example, wherein the composition has improved washing performance on PC-10 stains compared to the same composition comprising the first protease or the second protease, and / or wherein the composition has improved overall washing performance on a set of standard stains comprising at least PC-10 and PC-03 compared to the same composition comprising the first protease or the second protease.

[0743] 14. Use of a composition according to any one of embodiments 1 to 13 in a cleaning process, in particular for laundry.

[0744] 15. A method of cleaning, in particular for cleaning fabrics or textiles, comprising contacting the fabrics or textiles with a detergent composition as described in any one of embodiments 1 to 13 under conditions suitable for cleaning the fabrics or textiles.

[0745] 16. A protease variant comprising substitutions X97D, X209W and X215K, e.g., G97D, Y209W and A215K, wherein the position numbering is based on that of SEQ ID NO: 2, and the variant has protease activity and has a sequence identity of at least 80% but less than 100% to SEQ ID NO: 1.

[0746] 17. The protease variant of embodiment 16, wherein the protease is a variant of SEQ ID NO: 1 comprising a set of substitutions selected from the group consisting of:

[0747] G97D + Y209W + A215K;

[0748] G97D + S156D + Y209W + A215K; <h2 style=";text-align:left;direction:ltr">

[0749] <h2 style=";text-align:left;direction:ltr"> G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0750] <h2 style=";text-align:left;direction:ltr"> G97D + N117R + Y209W + A215K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0751] <h2 style=";text-align:left;direction:ltr"> S9E + G97D + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0752] <h2 style=";text-align:left;direction:ltr"> N62D + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0753] <h2 style=";text-align:left;direction:ltr"> G97D + S101E + S156D + A172V + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0754] <h2 style=";text-align:left;direction:ltr"> N76D + G97D + N140D + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0755] <h2 style=";text-align:left;direction:ltr"> N76D + G97D + S101E + T180A + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0756] <h2 style=";text-align:left;direction:ltr"> N76D + G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0757] <h2 style=";text-align:left;direction:ltr"> N62D + G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0758] <h2 style=";text-align:left;direction:ltr"> N62D + G97D + S101E + V177I + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0759] <h2 style=";text-align:left;direction:ltr"> N62D + N76D + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0760] <h2 style=";text-align:left;direction:ltr"> K27M+G97D+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0761] <h2 style=";text-align:left;direction:ltr"> K27M+N77D+G97D+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0762] <h2 style=";text-align:left;direction:ltr"> K27M+N76D+G97D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0763] <h2 style=";text-align:left;direction:ltr">K27M+N62D+G97D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0764] <h2 style=";text-align:left;direction:ltr"> S9E + G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0765] <h2 style=";text-align:left;direction:ltr"> S9E + N76D + G97D + Y209W + A215K + L262E + A270T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0766] <h2 style=";text-align:left;direction:ltr"> S9E+N76D+G97D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0767] <h2 style=";text-align:left;direction:ltr"> S9E+N62D+G97D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0768] <h2 style=";text-align:left;direction:ltr"> S9E+K27M+G97D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0769] <h2 style=";text-align:left;direction:ltr"> G97D + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0770] <h2 style=";text-align:left;direction:ltr"> G97D + S101E + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0771] <h2 style=";text-align:left;direction:ltr"> N76D + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0772] <h2 style=";text-align:left;direction:ltr"> K27M+G97D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0773] <h2 style=";text-align:left;direction:ltr"> S9E + G97D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0774] <h2 style=";text-align:left;direction:ltr"> S87N+G97D+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0775] <h2 style=";text-align:left;direction:ltr"> G97D + S101G + S156D + Y209W + A215K + L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0776] <h2 style=";text-align:left;direction:ltr"> G97D+S101N+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0777] <h2 style=";text-align:left;direction:ltr"> G97D+V104N+S156D+Y209W+A215K+L262E;<h2 style=";text-align:left;direction:ltr">

[0778] G97D + G118V + S156D + Y209W + A215K + L262E;

[0779] G97D + S156D + A194P + Y209W + A215K + L262E;

[0780] S87N + G97D + S101G + S156D + Y209W + A215K + L262E;

[0781] S87N + G97D + S101N + S156D + Y209W + A215K + L262E;

[0782] S87N + G97D + V104N + S156D + Y209W + A215K + L262E;

[0783] S87N + G97D + G118V + S156D + Y209W + A215K + L262E;

[0784] S87N + G97D + S156D + A194P + Y209W + A215K + L262E;

[0785] G97D + S101G + G118V + S156D + Y209W + A215K + L262E;

[0786] G97D + S101G + S156D + A194P + Y209W + A215K + L262E; and

[0787] G97D + S101N + V104N + S156D + Y209W + A215K + L262E;

[0788] The position numbers are based on those of SEQ ID NO: 2.

Claims

1. A protease variant comprising substitutions X97D, X209W and X215K (e.g., G97D, Y209W and A215K), wherein the position numbering is based on the numbering of SEQ ID NO: 2, and wherein the variant has protease activity and has a sequence identity of at least 80% but less than 100% to SEQ ID NO:

1.

2. A protease variant according to claim 1, which has a sequence identity of at least 85%, such as at least 90% or at least 95% but less than 100% with SEQ ID NO:

1.

3. The protease variant according to claim 1 or 2, further comprising one or more additional substitutions selected from the group consisting of: X9E (e.g., S9E), X27M (e.g., K27M), X62D (e.g., N62D), X76D (e.g., N76D), X77D (e.g., N77D), X87N (e.g., S87N), X101E / G / N (e.g., S101E / G / N), X104N (e.g., V104N), X117R (e.g., N117R), X118V (e.g., G118V), X140D (e.g., N140D), X156D (e.g., S156D), X172V (e.g., A172V), X177I (e.g., V177I), X194P (e.g., A194P), and X262E (e.g., L262E).

4. The protease variant according to any one of claims 1-3, wherein the protease variant is a variant of SEQ ID NO: 1 comprising a set of substitutions selected from the group consisting of: G97D+Y209W+A215K; G97D+S156D+Y209W+A215K; G97D+Y209W+A215K+L262E; G97D+N117R+Y209W+A215K; S9E+G97D+S156D+Y209W+A215K+L262E; N62D+G97D+Y209W+A215K+L262E; G97D+S101E+S156D+A172V+Y209W+A215K+L262E; N76D+G97D+N140D+S156D+Y209W+A215K+L262E; N76D+G97D+S101E+T180A+Y209W+A215K+L262E; N76D+G97D+S101E+Y209W+A215K+L262E; N62D+G97D+S101E+Y209W+A215K+L262E; N62D+G97D+S101E+V177I+Y209W+A215K+L262E; N62D+N76D+G97D+Y209W+A215K+L262E; K27M+G97D+S156D+Y209W+A215K+L262E; K27M+N77D+G97D+S156D+Y209W+A215K+L262E; K27M+N76D+G97D+Y209W+A215K+L262E; K27M+N62D+G97D+Y209W+A215K+L262E; S9E+G97D+S101E+Y209W+A215K+L262E; S9E+N76D+G97D+Y209W+A215K+L262E+A270T; S9E+N76D+G97D+Y209W+A215K+L262E; S9E+N62D+G97D+Y209W+A215K+L262E; S9E+K27M+G97D+Y209W+A215K+L262E; G97D+S156D+Y209W+A215K+L262E; G97D+S101E+Y209W+A215K+L262E; N76D+G97D+Y209W+A215K+L262E; K27M+G97D+Y209W+A215K+L262E; S9E+G97D+Y209W+A215K+L262E; S87N+G97D+S156D+Y209W+A215K+L262E; G97D+S101G+S156D+Y209W+A215K+L262E; G97D+S101N+S156D+Y209W+A215K+L262E; G97D+V104N+S156D+Y209W+A215K+L262E; G97D+G118V+S156D+Y209W+A215K+L262E; G97D+S156D+A194P+Y209W+A215K+L262E; S87N+G97D+S101G+S156D+Y209W+A215K+L262E; S87N+G97D+S101N+S156D+Y209W+A215K+L262E; S87N+G97D+V104N+S156D+Y209W+A215K+L262E; S87N+G97D+G118V+S156D+Y209W+A215K+L262E; S87N+G97D+S156D+A194P+Y209W+A215K+L262E; G97D+S101G+G118V+S156D+Y209W+A215K+L262E; G97D+S101G+S156D+A194P+Y209W+A215K+L262E; Japanese G97D+S101N+V104N+S156D+Y209W+A215K+L262E; Its location is based on SEQ ID NO:

2.

5. A detergent composition comprising the protease variant according to any one of claims 1 to 4.

6. A detergent composition according to claim 5 in the form of a homogeneous tablet, a tablet having two or more layers, a sachet having one or more compartments, a conventional or compressed powder, a granulate, a paste, a gel, a bar, or a regular, compressed or concentrated liquid.

7. The detergent composition according to claim 5 or 6, further comprising an amylase, an arabinase, a carbohydrase, a cellulase (e.g., an endoglucanase), a cutinase, a deoxyribonuclease, a galactanase, a haloperoxidase, a lipase, a mannanase, an oxidase (e.g., a laccase and / or a peroxidase), a pectinase, a pectin lyase, an additional protease, a xylanase, a xanthanase, a xyloglucanase, or any mixture thereof.

8. A detergent composition according to claim 7, wherein the additional protease has a different net formal charge.

9. Use of the protease variant according to any one of claims 1 to 4 in a cleaning process, preferably laundry.

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