WO2025011933A1

WO2025011933A1 - Washing method for removing proteinaceous stains

Info

Publication number: WO2025011933A1
Application number: PCT/EP2024/067635
Authority: WO
Inventors: Jürgen Carsten Franz KNÖTZEL; Carl Mikael BAUER
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2023-07-07
Filing date: 2024-06-24
Publication date: 2025-01-16
Anticipated expiration: 2026-01-07

Abstract

The present invention relates to washing methods for improved removal of proteinaceous stains on textiles and to use of chelating agents for improving proteinaceous stain removal.

Description

WASHING METHOD FOR REMOVING PROTEINACEOUS STAINS

REFERENCE TO A SEQUENCE LISTING

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

FIELD OF THE INVENTION

The present invention relates to washing methods for improved removal of proteinaceous stains on textiles and to use of chelating agents for improving proteinaceous stain removal

BACKGROUND OF THE INVENTION

One of the primary considerations for consumers when buying laundry detergent is performance on stain removal. Proteinaceous stains are among the most common and tenacious stains with examples including food stains such as meat, dairy, egg, cocoa, and some vegetables, outdoor stains such as grass and mud, and stains arising from bodily fluids such as blood, sweat, and sebum. Proteinaceous stains are susceptible to degradation by proteases, and proteases have become the technically and commercially most important enzyme class for strain removal in laundry detergents.

An increasing number of commercially available proteases used for laundry are engineered variants of naturally occurring wild type proteases. Protease variants have been described in the art with alterations relative to a parent protease resulting in improvements such as better wash performance, thermal stability, storage stability, and catalytic activity.

There is a continued need in the art for further improvements of proteinaceous stain removal. In particular, there is a need for further improving proteinaceous stain removal obtained with protease variants that are already highly optimized as a result of protein engineering.

SUMMARY OF THE INVENTION

The present inventors have identified a method for washing a textile that provides improved removal of proteinaceous stains. The method of the invention is based on the surprising finding that chelating agents and proteases acting in concert provide dramatically improved proteinaceous stain removal, especially when high levels of chelating agents are employed.

In a first aspect, the present invention relates to a method of washing a textile, comprising the steps of: a) obtaining a textile comprising a proteinaceous stain; b) treating the textile in a wash step, wherein the wash step comprises contacting the textile with a wash liquor, wherein the wash liquor is prepared by mixing a laundry detergent composition with water, and wherein the laundry detergent composition comprises a protease; and c) further treating the textile during the wash step with a chelating agent having affinity for calcium ions (Ca²⁺), wherein the chelating agent is added to the wash liquor, and wherein the chelating agent is present in the wash liquor at a concentration of at least 1 g/L; wherein the proteinaceous stain is at least partially removed from the textile.

In a second aspect, the present invention relates to a method of washing a textile, comprising the steps of: a) obtaining a textile comprising a proteinaceous stain; b) treating said textile in a wash step, wherein the wash step comprises contacting the textile with a wash liquor, wherein the wash liquor is prepared by mixing a laundry detergent composition with water, and wherein the laundry detergent composition comprises a protease; and c) treating said textile in a rinse step, wherein the rinse step comprises contacting the textile with a chelating liquor comprising a chelating agent at a concentration of at least 1 g/L, wherein the chelating liquor is prepared by mixing the chelating agent with water, and wherein the chelating agent has affinity for calcium ions (Ca²⁺); wherein the proteinaceous stain is at least partially removed from the textile.

DEFINITIONS

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

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

Chelating agent: The term “chelating agent” means an organic compound that is capable of binding and forming a complex with a metal ion such as, e.g., Ca²⁺, Mg²⁺, Cu²⁺, and Fe²⁺. Examples of chelating agents include carbonates (e.g., sodium carbonate), silicates (e.g., sodium silicate), phosphates (e.g., sodium phosphate), polyphosphates (e.g., sodium hexametaphosphate), phosphonates (e.g., HEDP, ATMP, or EDTMP), polycarboxylates (e.g., EDTA or NTA), citrates (e.g., sodium citrate), gluconic acid, polyacrylic acid, zeolites, and aminocarboxylates (e.g., MGDA or GLDA). In an aspect, the chelating agent is capable of binding divalent metal cations. In a preferred aspect, the chelating agent is capable of binding and forming a complex with Ca²⁺ and/or Mg²⁺. In a most preferred aspect, the chelating agent is capable of binding and forming a complext with Ca²⁺.

Chelating liquor: The term “chelating liquor” means a solution or mixture of water and a chelating agent. In one aspect, the chelating liquor comprises water and a chelating agent and is substantially free from anionic and non-ionic surfactants. In a preferred aspect, the chelating liquor comprises water and a chelating agent and is free from anionic and non-ionic surfactants. Protease: The term “protease” means an enzyme having peptidase activity (EC 3.4; also known as proteolytic activity or protease activity) that catalyzes the hydrolysis of peptide bonds. The EC 3.4 group includes several sub-groups, including EC 3.4.21 (serine endopeptidase), which further contains several sub-groups, including EC. 3.4.21.62 (subtilisin). For purpose of the present invention, protease activity may be determined according to Protease Activity Assay I or Protease Activity Assay II described in the Examples herein.

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

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

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

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

(Identical Deoxyribonucleotides x 100)/(Length of Alignment- Total Number of Gaps in Alignment)

Stain and soil: The term “stain” means a soiled or discolored spot present on a textile, and the term “soil” is understood as the actual substance that produces a stain upon contact with a textile. A textile may thus comprise both a stain and a soil producing said stain at the same time. The terms “proteinaceous stain” and “proteinaceous soil” means a stain and soil, respectively, that comprises a protein substrate that is susceptible to degradation by a protease. For purposes of the present invention, the term “stain” (e.g., a proteinaceous stain) encompasses both the stain itself and the soil (e.g., a proteinaceous soil) producing the stain, and the term “stain removal” encompasses both removal of the stain itself and of the soil producing the stain. The skilled person is aware of suitable proteinaceous stains to be removed. Examples include, but are not limited to, meat, dairy, egg, cocoa, grass, blood, and sebum stains. Textile: The term “textile” means any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and toweling. The textile may be cellulose-based such as natural cellulosics, including cotton, flax/linen, jute, ramie, sisal or coir, or manmade cellulosics (e.g., originating from wood pulp) including viscose/rayon, cellulose acetate fibers (tricell), lyocell, or blends thereof. The textile or fabric may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit, and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene, spandex/elastane, or blends thereof as well as blends of cellulose based and non-cellulose-based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fiber (e.g., polyamide fiber, acrylic fiber, polyester fiber, polyvinyl chloride fiber, polyurethane fiber, polyurea fiber, aramid fiber), and/or cellulose-containing fiber (e.g., rayon/viscose, ramie, flax/linen, jute, cellulose acetate fiber, lyocell). In the context of the present invention, the term “textile” also covers fabrics.

Wash liquor: The term “wash liquor” is defined herein as the solution or mixture of water and laundry detergent components. In one aspect, the wash liquor comprises water and one or more surfactants. In a preferred aspect, the wash liquor comprises water and one or more anionic and/or non-ionic surfactants.

CONVENTION FOR DESIGNATION OF VARIANTS

For purposes of the present invention and unless otherwise indicated, the polypeptide disclosed in SEQ ID NO:2 is used to determine the corresponding amino acid positions in another protease. The amino acid sequence of another protease is aligned with the polypeptide disclosed in SEQ ID NO:2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO:2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

In describing protease variants suitable for methods of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IIIPAC single letter or three letter amino acid abbreviation is employed.

Substitutions: For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”) or commas (“,”), e.g., “G205R+S411 F” or “G205R,S411 F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

Deletions: For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or “G195*+S411*”.

Insertions: For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by the addition of 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 thus be:

Multiple alterations: Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations: Where different alterations can be introduced at a position, the different alterations are separated by a slahs (“/”), e.g., “R170Y/E” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, “Y167G/A + R170G/A” designates the following variants: “Y167G+R170G”, “Y167G+R170A”, “Y167A+R170G”, and

“Y167A+R170A”.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified a method for washing a textile that provides improved removal of proteinaceous stains. The method of the invention is based on the surprising finding that chelating agents and proteases acting in concert provide a dramatically improved proteinaceous stain removal, especially when high levels of chelating agents are employed.

Chelating agents (also sometimes known as builders) are often included in laundry detergent compositions and are typically present in the wash liquor at a concentration of up to 0.3 g/L, e.g., from 0.01 g/L to 0.3 g/L. However, and as illustrated by the Examples herein, the improved strain removal is particularly pronounced when the level of chelating agent is higher than typical laundry detergent levels, e.g., when the chelating agent is applied at a concentration of at least 1 g/L, e.g., 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 20 g/L, 50 g/L, or more.

Without being bound by theory it is speculated that the higher levels of chelating agents sequester calcium ions (Ca²⁺) present in the wash liquor to an increased extent and thereby prevents formation of complexes comprising calcium ions and the peptide fragments arising from proteolytic degradation of the proteinaceous stain. By preventing complex formation and maintaining the peptide fragments in solution, the chelating agents decrease redepostioining of the peptide fragments onto the textile, which subsequently leads to improved stain removal.

Washing methods

In a second aspect, the present invention relates to a method of washing a textile, comprising the steps of: a) obtaining a textile comprising a proteinaceous stain; b) treating said textile in a wash step, wherein the wash step comprises contacting the textile with a wash liquor, wherein the wash liquor is prepared by mixing a laundry detergent composition with water, and wherein the laundry detergent composition comprises a protease; and c) treating said textile in a rinse step, wherein the rinse step comprises contacting the textile with a chelating liquor comprising a chelating agent at a concentration of at least 1 g/L, wherein the chelating liquor is prepared by mixing the chelating agent with water, wherein the chelating agent has affinity for calcium ions (Ca²⁺); wherein the proteinaceous stain is at least partially removed from the textile.

The steps of the method of the present invention may be conducted in an automatic washing machine, a manual wash operation, or a mixture thereof, preferably in an automatic washing machine.

The methods of the invention involve a step of obtaining a textile comprising a proteinaceous stain. The textile may be any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles). The textile may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and toweling.

In some embodiments, the textile is a cellulose-based textile such as a natural cellulosic including cotton, flax/linen, jute, ramie, sisal, and coir, or such as a manmade cellulosic (e.g., originating from wood pulp) including viscose/rayon, cellulose acetate fibers (tricell), lyocell, or blends thereof.

In some embodiments, the textile is a non-cellulose-based textile such as a natural polyamide including wool, camel, cashmere, mohair, rabbit, and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene, spandex/elastane, or blends thereof.

In some embodiments, the textile comprises a blend of cellulose based and non-cellulose- based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fiber (e.g., polyamide fiber, acrylic fiber, polyester fiber, polyvinyl chloride fiber, polyurethane fiber, polyurea fiber, or aramid fiber), and/or cellulose- containing fiber (e.g., rayon/viscose, ramie, flax/linen, jute, cellulose acetate fiber, or lyocell).

The textile comprises a proteinaceous stain to be removed. The skilled person is aware of suitable proteinaceous stains to be removed. Examples include, but are not limited to, meat, dairy, egg, cocoa, grass, blood, and sebum stains.

The methods of the invention further involve treating the textile with a wash liquor obtained by mixing a laundry detergent composition comprising a protease with water. Treatment with the wash liquor occurs in a wash step, which comprises contacting the textile with said wash liquor.

Preferably, the laundry detergent composition is a liquid laundry detergent composition, e.g., a regular, compact, or concentrated liquid laundry detergent composition. Alternatively, the laundry detergent composition may be in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, or a gel.

In one embodiment, the laundry detergent composition is a powder.

In one embodiment, the laundry detergent composition is a pouch having two or more compartments.

In one embodiment, the laundry detergent is a soap bar.

In one embodiment, the laundry detergent composition is a single unit dose format.

Suitable laundry detergent compositions are described in more detail below.

The wash liquor is prepared by mxing the laundry detergent composition with water. The wash liquor may be prepared by diluting the laundry detergent composition in water between 5 and 500-fold, preferably between 10 and 100-fold, more preferably between 10 and 50-fold, and most preferably between 10 and 20-fold. In some alternative embodiments, the laundry detergent composition is diluted between 100 and 1000-fold, preferably between 300 and 800-fold, most preferably between 400 and 700-fold.

In some embodiments, the water used to prepare the wash liquor has a hardness of 0 to 10 dH, e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 dH. In some embodiments, the water used to prepare the wash liquor has a hardness of 10 to 30 dH, e.g, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 dH, preferably from 10 to 20 dH.

The wash liquor may be at a temperature of between 5 °C and 90 °C, preferably between 10 °C and 60 °C, more preferably between 15 °C and 45 °C, most preferably between 15 °C and 40 °C.

The wash step may take between 5 minutes and 90 minutes, preferably between 5 minutes and 50 minutes, more preferably between 5 minutes and 40 minutes, even more preferably between 5 minutes and 30 minutes, even more preferably between 5 minutes and 20 minutes, most preferably between 6 minutes and 18 minutes, to complete.

The laundry detergent composition comprises a protease. The protease may be added to the laundry detergent composition in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, most preferably 0.1-10 mg of enzyme protein per liter of wash liquor. A suitable laundry detergent composition may for example include a protease in an amount of 0.0001-10%, such as 0.001- 7.5%, such as 0.1-5% of enzyme protein by weight of the composition.

The protease may be any suitable protease. The protease may be a serine endopeptidase (E.C. 3.4.21), preferably a subtilisin (E.C. 3.4.21.62), or a metalloendopeptidase (E.C. 3.4.24), preferably a thermolysin (E.C. 3.4.24.27) or a bacillolysin (E.C. 3.4.24.28).

The protease may be of any origin but is preferably of microbial origin such as bacterial or fungal origin. Although proteases suitable for liquid laundry detergent compositions may be obtained from a variety of organisms, including fungi such as Aspergillus, such proteases have generally been obtained from bacteria and in particular from Bacillus. Examples of Bacillus species from which suitable proteases have been derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus gibsonii.

The protease may also be an engineered or chemically modified variant of a naturally occurring (/.e., wild-type) protease, i.e., a protease variant

A suitable serine protease may for example be of the S1 family, such as trypsin, or the S8 family, such as a subtilisin.

Examples of trypsin-like proteases include the Fusarium protease described in WO 94/25583 and WO 2005/040372, and the chymotrypsin proteases derived from Cellumonas described in WO 2005/052161 and WO 2005/052146. Examples of subtilisins include subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN’, subtilisin 309 (/.e., Savinase), subtilisin 147, and subtilisin 168. Other useful subtilins are, e.g., those described in WO 93/93/18140, WO 01/16285, and WO 02/16547.

Examples of useful proteases are the protease variants described in WO 89/06279 WO 92/19729, WO 96/34946, WO 98/20115, WO 98/20116, WO 99/11768, WO 01/44452, WO 03/006602, WO 2004/003186, WO 2004/041979, WO 2007/006305, WO 2011/036263, WO 2014/207227, WO 2016/087617, and WO 2016/174234.

Preferred protease variants may, for example, comprise one or more of the mutations selected from the group consisting of: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, S85R, A96S, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, A120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Q200L, Y203W, S206G, L211Q, L211 D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, S253D, N255W, N255D, N255E, L256E, L256D, T268A, and R269H, wherein position numbers correspond to positions of the Bacillus lentus protease shown in SEQ ID NO:1. Protease variants having one or more of these mutations are preferably variants of the Bacillus lentus protease (Savinase®, also known as subtilisin 309) shown in SEQ ID NO:1 or of the Bacillus amyloliquefaciens protease (BPN’) shown in SEQ ID NO:2. Such protease variants preferably have a sequence identity of at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 or to SEQ ID NO:2.

Another protease of interest is the alkaline protease from Bacillus lentus DSM 5483, as described for example in WO 91/02792, and variants thereof which are described for example in WO 92/21760, WO 95/23221 , EP 1921147, EP 1921148, and WO 2016/096711.

The protease may alternatively be a variant of the TY-145 protease having SEQ ID NO:3, for example a variant comprising a substitution at one or more positions corresponding to positions 27, 109, 111 , 171 , 173, 174, 175, 180, 182, 184, 198, 199 and 297 of SEQ ID NO:3, wherein said protease variant has a sequence identity of at least 75%, e.g., at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:3. TY145 variants of interest are described in, e.g., WO 2015/014790, WO 2015/014803, WO 2015/014804, WO 2016/097350, WO 2016/097352, WO 2016/097357, and WO 2016/097354.

Examples of preferred proteases include:

1) variants of SEQ ID NO:1 comprising three or more, e.g., four or more, five or more, six or more, or seven, substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2, for example: 1) a variant comprising the substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E; ii) a variant comprising the substitutions S9E, N43R, N76D, N185E, S188E, Q191 N, A194P, Q206L, Y209W, S259D, and L262E; or iii) a variant comprising the substitutions S9E, N43R, N76D, G97D, V205I, Q206L, Y209W, A215K, S259D, N261W, and L262E;

2) a variant of the polypeptide of SEQ ID NO:1 comprising the insertion S99SE, wherein position numbers are based on the numbering of SEQ ID NO:2;

3) a variant of the polypeptide of SEQ ID NO:1 comprising the substitutions Y167A, R170S, and A194P, wherein position numbers are based on the numbering of SEQ ID NO:2;

4) a variant of the polypeptide of SEQ ID NO:1 comprising the substitutions S99D, S101 R/E, S103A, V104I, and G160S, wherein position numbers are based on the numbering of SEQ ID NO:2, for example: i) a variant comprising the substitutions S99D, S101 E, S103A, V104I, and G160S; ii) a variant comprising the substitutions S99D, S101 E, S103A, V104I, S156D, G160S, and L262E; iii) a variant comprising the substitutions S3T, V4I, S99D, S101 E, S103A, V104I, G160S, V205I; or iv) a variant comprising the substitutions S3T, V4I, S99D, S101 E, S103A, V104I, S156D, G160S, V205I, Q206L, Y209W, A215K, and L262E;

5) a variant of the polypeptide of SEQ ID NO:1 comprising the substitutions S3T, S9R, R19L, N62D, and A194P, wherein position numbers are based on the numbering of SEQ ID NO:2;

6) a variant of the polypeptide of SEQ ID NO:1 comprising the substitutions V68A and S106A, wherein position numbers are based on the numbering of SEQ ID NO:2;

7) a variant of the polypeptide of SEQ ID NO:1 comprising the substitutions S128L, P129A, and S130A, wherein position numbers are based on the numbering of SEQ ID NO:2; for example: i) a variant comprising the substitutions S87N, G118V, S128L, P129Q, and S130A; ii) a variant comprising the substitutions S87N, S101M, G118V, S128L, P129Q, and S130A; or iii) a variant comprising the substitutions N76D, S87R, G118R, S128L, P129Q, and S130A;

8) a variant of the polypeptide of SEQ ID NO:1 comprising the substitutions S101G, S103A, and V104I, wherein position numbers are based on the numbering of SEQ ID NO:2, for example: i) a variant comprising the substitutions S101G, S103A, V104I, G160D, A232V, Q236H, Q245R, N248D, and N252K; ii) a variant comprising the substitutions T22A, N62D, S101G, S103A, V104I, N116L, G160D, S188D, T213A, A232V, Q245R, N248D, and E271 F; iii) a variant comprising the substitutions T22R, S101G, S103A, V104I, A220V, and Q233R; or iv) a variant comprising the substitutions T22A, S101G, S103A, V104I, N116L, G160D, S188D, A232V, Q245R, N248D, and E271 F;

9) a variant of the polypeptide of SEQ ID NO:1 comprising the substitutions S3T, N43R, N76D, S87N, G118M, S128Q, N184E, V205I, Q206L, Y209W, S259D, N261W, and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2; and

10) a variant of the polypeptide of SEQ ID NO:2 comprising the substitutions S24G, S53G, S78N, S101 N, G128A/S, and Y217Q, wherein position numbers are based on the numbering of SEQ ID NO:2, for example a variant comprising the substitutions S24G, S53G, S78N, S101 N, G128S, and Y217Q;

11) a variant of the polypeptide of SEQ ID NO:3 comprising the substitutions S27K, N109K, S111 E, S171 E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199, and T297P, wherein position numbers are based on the numbering of SEQ ID NO:3;

12) a variant of the polypeptide of SEQ ID NO:4 comprising the substitutions A69S, T78N, T79I, G128S, A129P, G166Q, N185Q, A203V, N218S, and S259P, wherein position numbers are based on the numbering of SEQ ID NO:2; and

13) a variant of the polypeptide of SEQ ID NO:4 comprising the substitutions P9T, Q17H, T78N, T79I, N97D, Y104F, G128T, A129K, S130Q, G166Q, N185Q, A203V, G204E, and S259P, wherein position numbers are based on the numbering of SEQ ID NO:2.

Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase™, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Blaze Evity® 200T, Neutrase®, Everlase®, Esperase®, Progress® Uno, Progress® In, Progress Tough®, and Progress® Excel (Novozymes A/S), those sold under the tradename Maxatase™, Maxacai™, Maxapem®, Purafect®, Purafect Prime®, Purafect® Ox, Purafect® OxP, Puramax®, Purafast™, FN2™, FN3™, FN4ex™, Excellase®, Excellenz™ P1000, Excellenz™ P1250, Eraser™, Preferenz® P100, Preferenz® P110, Preferenz® P300, Preferenz® P400, Effectenz P1000™, Effectenz P1050™, Effectenz™ P2000, Properase®, Opticlean™, and Optimase® (IFF), BLAP (sequence shown in Figure 29 of US 5352604) and variants hereof (Henkel AG), and KAP (Bacillus alkalophilus subtilisin) from Kao.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions Y167A, R170S, and A194P, wherein position numbers are based on the numbering of SEQ ID NO:2. In a preferred embodiment, the protease comprises, consists essentially of, or consists of SEQ ID NO:1 with the substitutions Y167A, R170S, and A194P, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising three or more, e.g., four or more, five or more, six or more, or seven, substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2, such as: i) a variant comprising the substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E; ii) a variant comprising the substitutions S9E, N43R, N76D, N185E, S188E, Q191 N, A194P, Q206L, Y209W, S259D, and L262E; or iii) a variant comprising the substitutions S9E, N43R, N76D, G97D, V205I, Q206L, Y209W, A215K, S259D, N261W, and L262E.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions S9E, N43R, N76D, N185E, S188E, Q191 N, A194P, Q206L, Y209W, S259D, and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ I D NO: 1 and comprising the substitutions S9E, N43R, N76D, G97D, V205I , Q206L, Y209W, A215K, S259D, N261W, and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions S3T, S9R, R19L, N62D, and A194P, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions S3T, N43R, N76D, S87N, G118M, S128Q, N184E, V205I, Q206L, Y209W, S259D, N261W, and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions S99D, S101 E, S103A, V104I, and G160S, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions S99D, S101 E, S103A, V104I, S156D, G160S, and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions S3T, V4I, S99D, S101 E, S103A, V104I, G160S, V205I, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions S3T, V4I, S99D, S101 E, S103A, V104I, S156D, G160S, V205I, Q206L, Y209W, A215K, and L262E; wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to the polypeptide of SEQ ID NO:3 and comprising the substitutions S27K, N109K, S111 E, S171 E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199, and T297P, wherein position numbers are based on the numbering of SEQ ID NO:3.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to the polypeptide of SEQ ID NO:4 and comprising the substitutions A69S, T78N, T79I, G128S, A129P, G166Q, N185Q, A203V, N218S, and S259P, wherein position numbers are based on the numbering of SEQ ID NO:2.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to the polypeptide of SEQ ID NO:4 and comprising the substitutions P9T, Q17H, T78N, T79I, N97D, Y104F, G128T, A129K, S130Q, G166Q, N185Q, A203V, G204E, and S259P, wherein position numbers are based on the numbering of SEQ ID NO:2.

The protease may also be a metalloprotease (EC 3.4.24). Preferred metalloproteases include those of EC 3.4.24.27 (Thermolysin) and EC 3.4.24.28 (Bacillolysin). The metalloprotease may be from the M4 family (e.g., thermolysin), or alternatively from the M5, M7, or M8 families. Examples of metalloproteases include the neutral metalloproteases described in WO 2007/044993 such as those derived from Bacillus amyloliquefaciens, as well as, e.g., the metalloproteases described in WO 2015/158723 and WO 2016/075078.

In a preferred embodiment, the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:7. In a preferred embodiment, the protease comprises, consists essentially of, or consists of SEQ ID NO:7.

The protease may also be a glutamyl endopeptidase (EC 3.4.21.19). In a preferred embodiment, the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:8. In a preferred embodiment, the protease comprises, consists essentially of, or consists of SEQ ID NO:8.

The protease may also be trypsin-type protease with specificity for Arg and Lys residues. In a preferred embodiment, the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:9. In a preferred embodiment, the protease comprises, consists essentially of, or consists of SEQ ID NO:9. The methods of the invention further involve treating the textile with a chelating agent. Chelating agents suitable for the washing methods of the present invention are capable of binding and forming a complex with metal ions, preferably divalent metal cations, more preferably calcium ions (Ca²⁺) and/or magnesium ions (Mg²⁺), most preferably calcium ions (Ca²⁺). Alternatively stated, suitable chelating agents have affinity for metal ions, preferably divalent metal cations, more preferably calcium ions (Ca²⁺) and/or magnesium ions (Mg²⁺), most preferably calcium ions (Ca²⁺).

Examples of suitable chelating agents include carbonates (e.g., sodium carbonate), silicates (e.g., sodium silicate), phosphates (e.g., sodium phosphate), polyphosphates (e.g., sodium hexametaphosphate), phosphonates (e.g., HEDP, ATMP, or EDTMP), polycarboxylates (e.g., EDTA or NTA), citrates (e.g., sodium citrate), gluconic acid, polyacrylic acid, zeolites, and aminocarboxylates (e.g., MGDA or GLDA).

In one embodiment, the chelating agent is selected from the group consisting of citric acid or a salt thereof (e.g., sodium citrate), gluconic acid, glutamic acid-N,N-diacetic acid (GLDA), methylglycinediacetic acid (MGDA), and thylenediaminetetraacetic acid (EDTA), and mixtures thereof.

In a preferred embodiment, the chelating agent is citric acid or a salt thereof, preferably sodium citrate.

In a preferred embodiment, the chelating agent is an aminocarboxylate.

In a preferred embodiment, the chelating agent is gluconic acid.

In a preferred embodiment, the chelating agent is glutamic acid-N,N-diacetic acid (GLDA).

In a preferred embodiment, the chelating agent is methylglycinediacetic acid (MGDA).

The textile may generally be treated with a chelating agent at a concentration of 0.01 g/L to 100 g/L, preferably 0.05 g/L to 50 g/L, more preferably 0.1 g/L to 25 g/L, even more preferably 0.5 g/L to 15 g/L, most preferably 1 g/L to 10 g/L.

In one aspect, treatment of the textile with the chelating agent occurs during the wash step, wherein the chelating agent is added to the wash liquor. The chelating agent may be added to the wash liquor at the onset of the wash step in order for treatment of the textile with the laundry detergent composition comprising a protease and the chelating agent to occur at the same time throughout the duration of the wash step. Alternatively, the chelating agent may be added to the wash liquor after the onset of the wash step, preferably 5 min, 10 min, 15 min, 20 min, or 25 min after the onset. The chelating agent may be added as one single dose or as multiple, sequential doses.

When treatment of the textile with chelating agent occurs during the wash step, the wash liquor comprises the chelating agent at a concentration of at least 0.01 g/L, e.g., 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L, 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6, g/L 1.7 g/L, 1.8 g/L, 1.9 g/L, 2 g/L, 2.1 g/L, 2.2 g/L, 2.3 g/L, 2.4 g/L, 2.5 g/L, 2.6 g/L, 2.7 g/L, 2.8 g/L, 2.9 g/L, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, or more.

In a preferred embodiment, the wash liquor comprises the chelating agent at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more.

In a particularly preferred embodiment, the wash liquor comprises the chelating agent at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more, and the chelating agent is citric acid or a salt thereof, preferably sodium citrate.

In a particularly preferred embodiment, the wash liquor comprises the chelating agent at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more, and the chelating agent is an aminocarboxylate, preferably methylglycinediacetic acid (MGDA) or glutamic acid-N,N-diacetic acid (GLDA).

In one embodiment, the chelating agent is present in the wash liquor at a concentration of 0.01 g/L to 10 g/L, preferably 0.05 g/L to 5 g/L, most preferably 0.1 g/L to 1 g/L.

In one embodiment, the chelating agent is present in the wash liquour at a concentration of 0.05 g/L to 5 g/L, preferably 0.1 g/L to 1 g/L, wherein the chelating agent is citric acid or a salt thereof (preferably sodium citrate).

In one embodiment, the chelating agent is present in the wash liquour at a concentration of 0.05 g/L to 5 g/L, preferably 0.1 g/L to 1 g/L, wherein the chelating agent is glutamic acid-N,N- diacetic acid (GLDA).

In a preferred embodiment, the chelating agent is present in the wash liquor at a concentration of 1 g/L to 50 g/L, preferably 1 g/L to 25 g/L, most preferably 1 g/L to 10 g/L.

In a particularly preferred embodiment, the chelating agent is present in the wash liquour at a concentration of 1 g/L to 50 g/L, preferably 1 g/L to 25 g/L, most preferably 1 g/L to 10 g/L, wherein the chelating agent is citric acid or a salt thereof (preferably sodium citrate).

In a particularly preferred embodiment, the chelating agent is present in the wash liquour at a concentration of 1 g/L to 50 g/L, preferably 1 g/L to 25 g/L, most preferably 1 g/L to 10 g/L, wherein the chelating agent is an aminocarboxylate, preferably methylglycinediacetic acid (MGDA) or glutamic acid-N,N-diacetic acid (GLDA).

The chelating agent may be added to the wash liquor as a single dose or as multiple doses. In one embodiment, the chelating agent is added as multiple, sequential doses. In another aspect, treatment of the textile with the chelating agent occurs in a rinse step. In this aspect, treatment of the textile with a chelating agent occurs by contacting the textile with a chelating liquor obtained by mixing the chelating agent with water.

The chelating liquor is prepared by mixing the chelating agent with water The chelating liquor may be prepared by diluting the chelating liquor in water between 5 and 500-fold, preferably between 10 and 100-fold, more preferably between 10 and 50-fold, and most preferably between 10 and 20-fold.

In some embodiments, the water used to prepare the chelating liquor has a hardness of 0 to 10 dH, e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 dH. In some embodiments, the water used to prepare the chelating liquor has a hardness of 10 to 30 dH, e.g, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 dH, preferably from 10 to 20 dH.

The rinse step may take between 5 minutes and 90 minutes, preferably between 5 minutes and 50 minutes, more preferably between 5 minutes and 40 minutes, even more preferably between 5 minutes and 30 minutes, even more preferably between 5 minutes and 20 minutes, most preferably between 6 minutes and 18 minutes, to complete.

The chelating liquor may be applied at a temperature of between 5 °C and 90 °C, preferably between 10 °C and 60 °C, more preferably between 15 °C and 45 °C, most preferably between 15 °C and 40 °C.

When treatment of the textile with chelating agent occurs during the rinse step, the chelating liquor comprises the chelating agent at a concentration of at least 0.01 g/L, e.g., 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L, 1.1 g/L, 1.2 g/L, 1.3 g/L, 1.4 g/L, 1.5 g/L, 1.6, g/L 1.7 g/L, 1.8 g/L, 1.9 g/L, 2 g/L, 2.1 g/L, 2.2 g/L, 2.3 g/L, 2.4 g/L, 2.5 g/L, 2.6 g/L, 2.7 g/L, 2.8 g/L, 2.9 g/L, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, or more.

In a preferred embodiment, the chelating liquor comprises the chelating agent at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more.

In a particularly preferred embodiment, the chelating liquor comprises the chelating agent at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more, and the chelating agent is citric acid or a salt thereof (preferably sodium citrate).

In a particularly preferred embodiment, the chelating liquor comprises the chelating agent at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more, and the chelating agent is an aminocarboxylate, preferably methylglycinediacetic acid (MGDA) or glutamic acid-N,N-diacetic acid (GLDA).

In one embodiment, the chelating agent is present in the chelating liquor at a concentration of 0.05 g/L to 50 g/L, preferably 0.1 g/L to 25 g/L, more preferably 0.5 g/L to 15 g/L, more preferably 1 g/L to 10 g/L.

In one embodiment, the chelating agent is present in the chelating liquour at a concentration of 0.5 g/L to 15 g/L, preferably 1 g/L to 10 g/L, and the chelating agent is citric acid or a salt thereof (preferably sodium citrate).

In one embodiment, the chelating agent is present in the chelating liquour at a concentration of 0.5 g/L to 15 g/L, more preferably 1 g/L to 10 g/L, and the chelating agent is glutamic acid-N,N-diacetic acid (GLDA).

In a preferred embodiment, the chelating agent is present in the chelating liquor at a concentration of 1 g/L to 50 g/L, preferably 1 g/L to 25 g/L, most preferably 1 g/L to 10 g/L.

In a particularly preferred embodiment, the chelating agent is present in the chelating liquour at a concentration of 1 g/L to 50 g/L, preferably 1 g/L to 25 g/L, most preferably 1 g/L to 10 g/L, wherein the chelating agent is citric acid or a salt thereof (preferably sodium citrate).

In a particularly preferred embodiment, the chelating agent is present in the chelating liquour at a concentration of 1 g/L to 50 g/L, preferably 1 g/L to 25 g/L, most preferably 1 g/L to 10 g/L, wherein the chelating agent is an aminocarboxylate, preferably methylglycinediacetic acid (MGDA) or glutamic acid-N,N-diacetic acid (GLDA).

In one embodiment, the chelating liquor further comprises a cationic surfactant. Cationic surfactants help prevent build-up of friction and static electricity, resulting in softer and smoother textiles after washing. Some cationic surfactants also have biocidal activity. In a preferred embodiment, the cationic surfactant is an ester of a quaternary ammonium salt (/.e., an ester quat). In a most preferred embodiment, the cationic surfactant is selected from the group consisting of cetyltrimethylammonium bromide (CTAB), diethyl ester dimethyl ammonium chloride (DEEDMAC), dimethyldioctadecylammonium chloride (DSDMAC), dimethyldistearylammonium chloride (DSDMAC), triethanolamine quat (TEAQ), alkyldimethylethanolamine quat (ADMEAQ), and 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP).

In one embodiment, the chelating liquor comprises from 0.1 % to 20% w/w of cationic surfactant, preferably from 0.5% to 15% w/w, most preferably from 1% to 10% w/w.

The chelating liquor may be added as a single dose or as multiple doses. In one embodiment, the chelating liquor is added as multiple, sequential doses. Laundry Detergent Compositions

The methods of present invention involve treating a textile comprising a proteinaceous stain with a wash liquor obtained by mixing a laundry detergent composition with water, wherein the laundry detergent composition comprises a protease.

Suitable laundry detergent compositions may in addition to a protease comprise one or more detergent components and/or one or more additional enzymes.

In a preferred embodiment, the laundry detergent composition comprises one or more detergent components, in particular one or more non-naturally occurring detergent components.

In a preferred embodiment, the laundry detergent composition comprises one or more additional enzymes selected from the group consisting of amylases, arabinases, catalases, cellulases (e.g., endoglucanases), cutinases, DNases (e.g., phosphodiesterases), galactanases, haloperoxygenases, licheninases, lipases, mannanases, oxidases (e.g., laccases and/or peroxidases), pectinases, pectin lyases, peroxidases, xanthanases, xyloglucanases, xylanases, or any mixture thereof.

In one embodiment, the laundry detergent composition comprises at least two proteases such as a first protease and a second protease, wherein the first protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:1. In a preferred embodiment, the first protease comprises, consists essentially of, or consists of SEQ ID NO:1.

In one embodiment, the laundry detergent composition comprises at least two proteases such as a first protease and a second protease, wherein the first protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:5. In a preferred embodiment, the first protease comprises, consists essentially of, or consists of SEQ ID NO:5.

In one embodiment, the laundry detergent composition comprises at least two proteases such as a first protease and a second protease, wherein the first protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:6. In a preferred embodiment, the first protease comprises, consists essentially of, or consists of SEQ ID NO:6.

In one embodiment, the laundry detergent composition comprises at least two proteases such as a first protease and a second protease, wherein the first protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:7. In a preferred embodiment, the first protease comprises, consists essentially of, or consists of SEQ ID NO:7. In one embodiment, the laundry detergent composition comprises at least two proteases such as a first protease and a second protease, wherein the first protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:8. In a preferred embodiment, the first protease comprises, consists essentially of, or consists of SEQ ID NO:8.

In one embodiment, the laundry detergent composition comprises at least two proteases such as a first protease and a second protease, wherein the first protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:9. In a preferred embodiment, the first protease comprises, consists essentially of, or consists of SEQ ID NO:9.

Suitable laundry detergent compositions may be in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.

In a preferred embodiment, the laundry detergent composition is a liquid composition, in particular a regular, compact, or concentrated liquid composition.

In a preferred embodiment, the laundry detergent composition is a powder composition.

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

In a preferred embodiment, the laundry detergent composition is a single unit dose format.

The choice of additional components for the suitable laundry detergent compositions is within the skill of the artisan and includes conventional ingredients, including the exemplary nonlimiting components set forth below. The choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product.

In a particular embodiment, a suitable laundry detergent composition comprises a protease and one or more non-naturally occurring detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, antiredeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

In one embodiment, the protease may be added to the laundry detergent composition in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, in particular 0.1-10 mg of enzyme protein per liter of wash liquor. A suitable liquid laundry detergent composition may for example include a protease in an amount of 0.0001 %-10%, such as 0.001-7.5%, such as 0.1%-5% of enzyme protein by weight of the composition.

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

Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the person skilled in the art.

Surfactants

The laundry detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. Surfactants lower the surface tension in the detergent, which allows the stain being cleaned to be lifted and dispersed and then washed away. The surfactant(s) is typically present at a level of from about 0.1 % to 60% by weight, such as about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in detergents may be utilized. In a particular embodiment, the laundry detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants.

When included therein, the laundry detergent composition will usually contain from about 1 % to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof. When included therein, the laundry detergent composition will usually contain from about 0.5% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alklydimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

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

When included therein, the laundry detergent composition will usually contain from about 0% to about 10% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N- dimethylamine oxide and N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

When included therein, the laundry detergent composition will usually contain from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.

Builders and Co-Builders

Builders soften the wash water by removing the metal ions form the liquid. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with calcium and magnesium ions. Any builder and/or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS- 6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2’,2”-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof. The laundry detergent composition may contain 1-10% by weight, such as from about 1 % to about 5% by weight, of a detergent builder and/or co-builder, or a mixture thereof. The laundry detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid. Additional specific examples include 2,2’,2”-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N’-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid- N,N-diacetic acid (GLDA), 1-hydroxyethane-1 ,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentakis (methylenephosphonic acid) (DTPMPA or DTMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid- N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2- sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), a-alanine-N, N-diacetic acid (a-ALDA), serine-N, N-diacetic acid (SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diacetic acid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TLIDA) and sulfomethyl-N, N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylidenediamine-N, N’, N’-triacetate (HEDTA), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 2009/102854 and US 5,977,053.

Bleaching Systems

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

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

Some non-limiting examples of bleach catalysts that may be used in laundry detergent compositions suitable for the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1 ,4,7-trimethyl-1 ,4,7- triazacyclononane (Me3-TACN) or 1 ,2,4,7-tetramethyl-1 ,4,7-triazacyclononane (Me4-TACN), in particular Me3-TACN, such as the dinuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3- TACN)](PF6)2, and [2,2',2"-nitrilotris(ethane-1 ,2-diylazanylylidene-KN- methanylylidene)triphenolato-K3O]manganese(lll). The bleach catalysts may also be other metal compounds, such as iron or cobalt complexes.

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

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

Hydrotropes

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

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

Polymers

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

Fabric hueinq agents

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

Additional Enzymes

In addition to a protease, suitable laundry detergent compositions may comprise one or more enzymes such as an amylase, arabinase, catalase, cellulase (e.g., endoglucanase), cutinase, DNase (e.g., phosphodiesterase), galactanase, haloperoxygenase, licheninase, lipase, mannanase, oxidase (e.g., laccase and/or peroxidase), pectinase, pectin lyase, peroxidase, xanthanase, xyloglucanase, xylanase, or any mixture thereof.

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

Amylases

Suitable amylases include an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1 ,296,839.

Suitable amylases include amylases having SEQ ID NO:2 of WO 95/10603 or variants having 90% sequence identity to SEQ ID NO:3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424, and SEQ ID NO:4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 211 , 243, 264, 304, 305, 391 , 408, and 444.

Different suitable amylases include amylases having SEQ ID NO:6 in WO 02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having a deletion in positions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO:4 of WO 2006/066594 or variants having 90% sequence identity thereof.

Other examples are amylase variants such as those described in WO2011/098531 , WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ Amplify, and Amplify Prime™ (from Novozymes A/S), and Rapidase™, Purastar™/Effectenz™, Powerase™, Preferenz™ S1000, Preferenz™ S100 and Preferenz™ S110 (from Genencor International Inc./DuPont).

Cellulases

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases providing or maintaining whiteness and preventing redeposition or having color care benefits. Examples of such cellulases are cellulases 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 and W099/001544.

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

Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S) Carezyme Premium™ (Novozymes A/S), Celluclean ™ (Novozymes A/S), Celluclean Classic™ (Novozymes A/S), Cellusoft™ (Novozymes A/S), Whitezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Deoxyribonucleases (DNases)

The term “DNase” means a polypeptide with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. DNases are also known as phosphodiesterases (PDE). Correspondingly, ribonucleases (RNases) catalyzes the hydrolytic cleavage of phosphodiester linkages in the RNA backbone, thus degrading and RNA. There are two primary classifications based on the locus of activity. Exonucleases digest nucleic acids from the ends. Endonucleases act on regions in the middle of target molecules.

The nuclease is preferably a DNase, which is preferable is obtainable from a microorganism, preferably a fungus or a bacterium. A DNase which is obtainable from a species of Bacillus, such as Bacillus cibi, Bacillus subtilis, or Bacillus licheniformis, is particularly preferred. Examples of such DNases are described in WO 2011/098579, WQ2014/087011 and WO2 017/060475. A DNase obtainable from a species of Aspergillus, in particular Aspergillus oryzae, such as a DNase described in WO 2015/155350, is also particularly preferred.

Commercially available DNases include Pristine™ (Novozymes A/S).

Mannanases

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

A commercially available mannanase is Mannaway (Novozymes A/S). Oxidases and Peroxidases

Suitable oxidases and peroxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

A suitable peroxidase is preferably a peroxidase enzyme comprised by the enzyme classification EC 1.11.1.7, as set out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment derived therefrom, exhibiting peroxidase activity.

Suitable peroxidases also include a haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochlorite from chloride ions. The haloperoxidase may be a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containing haloperoxidase. In a preferred method the vanadate-containing haloperoxidase is combined with a source of chloride ion.

Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.

Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocinia, and Streptomyces, e.g., S. aureofaciens.

The haloperoxidase may be derivable from Curvularia sp., in particular from Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046, C. verruculosa CBS 147.63 as described in WO 97/04102, or C. verruculosa CBS 444.70 as described in WO 97/04102. The haloperoxidase may also be derivable from Drechslera, in particular from Drechslera hartlebii as described in WO 01/79459 or from Dendryphiella salina as described in WO 01/79458. The haloperoxidase may also be derivable from Phaeotrichoconis, in particular from Phaeotrichoconis crotalarie as described in WO 01/79461 . The haloperoxidase may also be derivable from Geniculosporium sp. as described in WO 01/79460.

Suitable oxidases include in particular any laccase enzyme comprised by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom exhibiting laccase activity, or a compound exhibiting a similar activity, such as a catechol oxidase (EC 1.10.3.1), an o- aminophenol oxidase (EC 1.10.3.4), or a bilirubin oxidase (EC 1.3.3.5).

Preferred laccase enzymes are enzymes of microbial origin. The enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts).

Suitable examples from fungi include a laccase derivable from a strain of Aspergillus, Neurospora (e.g., N. crassai), Podospora, Botrytis, Collybia, Pomes, Lentinus, Pleurotus, Trametes (e.g., T. villosa or T. versicolor), Rhizoctonia (e.g., R. solani), Coprinopsi (e.g., C. cinerea, C. comatus, C. friesii, or C. plicatilis), Psathyrella (e.g., P. condelleana), Panaeolus (e.g., P. papilionaceus), Myceliophthora (e.g., M. thermophila), Schytalidium (e.g., S. thermophilum), Polyporus (e.g., P. pinsitus), Phlebia (e.g., P. radiata, \i\iO 92/01046), or Coriolus (e.g., C. hirsutus, JP 2238885).

Suitable examples from bacteria include a laccase derivable from a strain of Bacillus.

A laccase derived from Coprinopsis or Myceliophthora is preferred, in particular a laccase derived from Coprinopsis cinerea, as disclosed in WO 97/08325, or from Myceliophthora thermophila, as disclosed in WO 95/33836.

Licheninases

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

Pectinases

Pectinases (pectolytic enzymes) can be classified according to their preferential substrate, highly methyl-esterified pectin or low methyl-esterified pectin and polygalacturonic acid (pectate), and their reaction mechanism, beta-elimination or hydrolysis. Pectinases can be mainly endoacting, cutting the polymer at random sites within the chain to give a mixture of oligomers, or they may be exo-acting, attacking from one end of the polymer and producing monomers or dimers. Several pectinase activities acting on the smooth regions of pectin are included in the classification of enzymes provided by the Enzyme Nomenclature (1992) such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67), exo-polygalacturonate lyase (EC 4.2.2.9) and exo-poly-alpha-galacturonosidase (EC 3.2.1.82).

Xyloglucanases

Xyloglucanases catalyze hydrolysis of xyloglucan. The reaction involves endo hydrolysis of 1 ,4-beta-D-glucosidic linkages in xyloglucan. For purposes of the present invention, xyloglucanase activity is determined using AZCL-xyloglucan (from Megazyme) as the reaction substrate. The assay can be performed in several ways, e as described in WO 01/62903. One unit of xyloglucanase activity (Xyloll) is defined by reference to the assay method described in WO 01/62903, page 60, lines 3-17. Microorganisms

The laundry detergent composition may comprise one or more microorganisms or microbes. Generally, any microorganism may be used in the detergent composition in any suitable amount or concentration. Microorganisms may be used as the only biologically active ingredient, but they may also be used in conjunction with one or more of the enzymes described above.

The purpose of adding microorganisms may, for example, be to reduce malodor as described in WO 2012/112718. Other purposes could include in situ production of desirable biological compounds, or inoculation/population of a locus with the microorganism(s) to competitively prevent other non-desirable microorganisms form populating the same locus (competitive exclusion).

The term “microorganism” generally means small organisms that are visible through a microscope. Microorganisms often exist as single cells or as colonies of cells. Some microorganisms may be multicellular. Microorganisms include prokaryotic (e.g., bacteria and archaea) and eukaryotic (e.g., some fungi, algae, protozoa) organisms. Examples of bacteria may be Gram-positive bacteria or Gram-negative bacteria including both vegetative cells and endospores. Examples of fungi may be yeasts, molds and mushrooms in the form of hyphae and/or spores. Herein, viruses may be considered microorganisms.

Microorganisms may be recombinant or non-recombinant. In some examples, the microorganisms may produce various substances (e.g., enzymes) that are useful for inclusion in detergent compositions. Extracts from microorganisms or fractions from the extracts may be used in the detergents. Media in which microorganisms are cultivated or extracts or fractions from the media may also be used in detergents. In some examples, specific of the microorganisms, substances produced by the microorganisms, extracts, media, and fractions thereof, may be specifically excluded from the detergents. In some examples, the microorganisms, or substances produced by, or extracted from, the microorganisms, may activate, enhance, preserve, prolong, and the like, detergent activity or components contained with detergents.

Generally, microorganisms may be cultivated using methods known in the art. The microorganisms may then be processed or formulated in various ways. In some examples, the microorganisms may be desiccated (e.g., lyophilized). In some examples, the microorganisms may be encapsulated (e.g., by spray drying). Many other treatments or formulations are possible. These treatments or preparations may facilitate retention of microorganism viability over time and/or in the presence of detergent components. In some examples, however, microorganisms in detergents may not be viable. The processed or formulated microorganisms may be added to detergent composition prior to or at the time of usage.

In one embodiment, the microorganism is a species of Bacillus, for example, at least one species of Bacillus selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus atrophaeus, Bacillus pumilus, Bacillus megaterium, or a combination thereof. In a preferred embodiment, the aforementioned Bacillus species are on an endospore form, which significantly improves the storage stability.

Adjunct materials

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

Dispersants: The laundry detergent composition, in particular in the form of a powder composition, may also contain dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are described in Powdered Detergents, Surfactant Science Series, volume 71 , Marcel Dekker, Inc., 1997.

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

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

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

Anti-redeposition agents: The laundry detergent composition may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose-based polymers described under soil release polymers above may also function as antiredeposition agents. Other suitable adjunct materials include, but are not limited to, anti-shrink agents, antiwrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.

Uses

The present invention further relates to the use of a chelating agent for improving proteinaceous stain removal of a protease during a laundry process.

Stain removal may be improved by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, or more, wherein the improvement is measured relative to the stain removal obtained with the same protease applied under the same conditions but without the chelating agent being present.

In a preferred embodiment, the chelating agent is an aminocarboxylate.

In a preferred embodiment, the chelating agent is gluconic acid.

The chelating agent may be applied at a concentration of 0.01 g/L to 100 g/L, preferably 0.05 g/L to 50 g/L, more preferably 0.1 g/L to 25 g/L, even more preferably 0.5 g/L to 15 g/L, most preferably 1 g/L to 10 g/L.

In one embodiment, the chelating agent is applied at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more.

In a preferred embodiment, the chelating agent is applied at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more, and the chelating agent is citric acid or a salt thereof (preferably sodium citrate).

In a preferred embodiment, the chelating agent is applied at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more, and the chelating agent is an aminocarboxylate, preferably methylglycinediacetic acid (MGDA) or glutamic acid-N,N-diacetic acid (GLDA).

Improved stain removal may be obtained with the following proteases:

1) variants of SEQ ID NO:1 comprising three or more, e.g., four or more, five or more, six or more, or seven, substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2, for example:

1) a variant comprising the substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E; ii) a variant comprising the substitutions S9E, N43R, N76D, N185E, S188E, Q191 N, A194P, Q206L, Y209W, S259D, and L262E; or iii) a variant comprising the substitutions S9E, N43R, N76D, G97D, V205I, Q206L, Y209W, A215K, S259D, N261W, and L262E;

5) a variant of the polypeptide of SEQ I D NO: 1 comprising the substitutions S3T, S9R, R19L, N62D, and A194P, wherein position numbers are based on the numbering of SEQ ID NO:2;

7) a variant of the polypeptide of SEQ ID NO:1 comprising the substitutions S128L, P129A, and S130A, wherein position numbers are based on the numbering of SEQ ID NO:2; for example: i) a variant comprising the substitutions S87N, G118V, S128L, P129Q, and S130A; ii) a variant comprising the substitutions S87N, S101M, G118V, S128L, P129Q, and S130A; or iii) a variant comprising the substitutions N76D, S87R, G118R, S128L, P129Q, and

S130A;

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising the substitutions Y167A, R170S, and A194P, wherein position numbers are based on the numbering of SEQ ID NO:2. In a preferred embodiment, the protease comprises, consists essentially of, or consists of SEQ ID NO:1 with the substitutions Y167A, R170S, and A194P, wherein position numbers are based on the numbering of SEQ ID NO:2. In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to SEQ ID NO:1 and comprising three or more, e.g., four or more, five or more, six or more, or seven, substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, wherein position numbers are based on the numbering of SEQ ID NO:2, such as: i) a variant comprising the substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W, and L262E; ii) a variant comprising the substitutions S9E, N43R, N76D, N185E, S188E, Q191 N, A194P, Q206L, Y209W, S259D, and L262E; or iii) a variant comprising the substitutions S9E, N43R, N76D, G97D, V205I, Q206L, Y209W, A215K, S259D, N261W, and L262E.

In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to the polypeptide of SEQ ID NO:3 and comprising the substitutions S27K, N109K, S111 E, S171 E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199, and T297P, wherein position numbers are based on the numbering of SEQ ID NO:3. In a preferred embodiment, the protease is a variant having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to the polypeptide of SEQ ID NO:4 and comprising the substitutions A69S, T78N, T79I, G128S, A129P, G166Q, N185Q, A203V, N218S, and S259P, wherein position numbers are based on the numbering of SEQ ID NO:2.

The protease may also be trypsin-type protease with specificity for Arg and Lys residues. In a preferred embodiment, the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:9. In a preferred embodiment, the protease comprises, consists essentially of, or consists of SEQ ID NO:9.

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

Materials and Methods

Protease Activity Assay I

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

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

Protease Activity Assay II

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

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

Terg-o-tometer (TOM) Wash Assay

The Terg-o-tometer (TOM) is a medium scale wash assay that can be applied to simultaneously test up to 16 different conditions at the same time. Briefly, it consists of 16 x 2 L metal beakers, each fitted with an agitator, which rotate in a back-and-forth manner at a controlled speed to simulate the agitation occurring in commercial top-loader washing machines. The beakers are partly submerged in thermostatic water baths where the temperature can be controlled. Each beaker was filled with 1 L detergent solution, and test swatches, ballast and enzymes are added to the requisite levels. After a timed wash period, the swatches are promptly removed from the beakers and rinsed thoroughly.

The swatches are then spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches are done using a DataColor Model 800V reflectance spectrophotometer. The measurements are made without UV in the incident light and remission (REM) at 460 nm is extracted. Measurements are made on unwashed and washed swatches. The test swatch to be measured is placed on top of another swatch of the same type and color.

The effect of a protease on each swatch is calculated by subtracting the remission value of the swatch washed without protease (blank) from the swatch washed together with protease. Differences in delta remission values of more than 0.5 (A > 0.5) are considered statistically significant, whereas differences in delta remission values between 0 and 0.5 (0 < A < 0.5) are considered on par.

Bottle Wash Assay

The Bottle wash is a small-scale wash assay that can be applied to simultaneously test up to 30 different conditions at the same time. Briefly, it consists of up to 30 x 60 ml polypropylene bottles (VWR Sample container Cat No.: 216-1809). Each bottle is filled with 25 ml of detergent solution, test swatches and enzymes to the requisite levels. To apply mechanical agitation that resembles full scale wash conditions, the bottles are put into a Wascator (Wascator FOM71 CLS; Electrolux Professional AB, 34180 Ljungby, Sweden). To conduct the wash, the Wascator is filled with 2 kg of clean cotton ballast, 25 Liters of water, and up to 30 bottles.

After a timed wash period, the swatches are promptly removed from the bottles and rinsed thoroughly. The swatches are then spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches are done using a DataColor Model 800V reflectance spectrophotometer. The measurements are made without UV in the incident light and remission (REM) at 460 nm is extracted. Measurements are made on unwashed and washed swatches. The test swatch to be measured is placed on top of another swatch of the same type and color.

Table 1 : Detergent composition 1 and test conditions for TOM wash assay

Table 2: Detergent composition 2 and test conditions for TOM wash assay

Table 3: Detergent composition 3 and test conditions for TOM wash assay

Table 4: Detergent composition 1 and test conditions for Bottle wash assay

Example 1 : Addition of chelator after wash during rinse

The Terg-o-tometer (TOM) wash assay was applied to test SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 in liquid laundry model detergent 1 , 2, and 3, respectively, as described above. After wash, conducted at a water hardness of 6 °dH or 15 °dH, a first short rinse of 2 min was applied in water in the same water hardness. Subsequently, the swatches were rinsed in a second rinse for 15 min, either in the corresponding water hardness of 6 °dH or 15 °dH of the wash, or in the corresponding water hardness of 6 °dH or 15 °dH with the addition of sodium citrate. 2.5 g/L of sodium citrate were added to the water with 6 °dH, and 10 g/L of sodium citrate were added to the water with 15 °dH. The swatches were rinsed in 1 L of 6 °dH or 15 °dH water, or 1 L of the sodium citrate solutions in the TOM beakers in the thermostatic water bath at 20 °C. Concluding, a last third rinse occurred in corresponding water hardness of 6 °dH or 15 °dH.

After the wash and rinse experiments, the swatches were spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. The remission (REM) at 460 nm was measured, and the delta remission was calculated versus the washes without addition of enzyme.

Table 5: Delta remission for the proteases SEQ ID NO:1 , 5, 6, and 7 on PC-10 (Pigment, Oil, Milk (POM)) swatches after TOM wash at different water hardness of 6 °dH or 15 °dH, and subsequent rinse in the absence or in the presence of sodium citrate

The data presented in Table 5 demonstrate that the soil removal from PC-10 expressed as delta intensity, is substantially improved with the addition of sodium citrate during rinse. This is true for the proteases SEQ ID NO:1 , 5, 6, and 7 in all detergent formulations, the liquid laundry model detergent 1 , 2, and 3 at both water hardness conditions. Table 6: Delta remission for the proteases SEQ ID NO:1 , 5, 6, and 7 on PC-03 (Chocolate milk with carbon black) swatches after TOM wash at different water hardness of 6 °dH or 15 °dH, and subsequent rinse in the absence or in the presence of sodium citrate

The data presented in Table 6 demonstrate that the soil removal from PC-03 expressed as delta intensity, is substantially improved with the addition of sodium citrate during rinse. This is true for the proteases SEQ ID NO:1 , 5, 6, and 7 in all detergent formulations, the liquid laundry model detergent 1 , 2, and 3 at both water hardness conditions.

Table 7: Delta remission for the proteases SEQ ID NO:1 , 5, 6, and 7 on C-S-07 (Grass, pure) swatches after TOM wash at different water hardness of 6 °dH or 15 °dH, and subsequent rinse in the absence or in the presence of sodium citrate

The data presented in Table 7 demonstrate that the soil removal from C-S-07 expressed as delta intensity, is substantially improved with the addition of sodium citrate during rinse. This is true for the proteases SEQ ID NO:1 , 5, 6, and 7 in all detergent formulations, the liquid laundry model detergent 1 , 2, and 3 at both water hardness conditions.

Table 8: Delta remission for the proteases SEQ ID NO:1 , 5, 6, and 7 on PC-05 (Blood, Milk, Ink (BMI)) swatches after TOM wash at a water hardness of 15 °dH, and subsequent rinse in the absence or in the presence of sodium citrate

The data presented in Table 8 demonstrate that the soil removal from PC-05 expressed as delta intensity, is substantially improved with the addition of sodium citrate during rinse. This is true for the proteases SEQ ID NO:1 , 5, 6, and 7 in liquid laundry model detergent formulations 1 and 2, at high water hardness condition of 15 °dH.

Table 9: Delta remission for the proteases SEQ ID NO:1 , 5, 6, and 7 on C-S-37 (Full egg with carbon black) swatches after TOM wash at a water hardness of 6 °dH, and subseguent rinse in the absence or in the presence of sodium citrate

The data presented in Table 9 demonstrate that the soil removal from C-S-37 expressed as delta intensity, is substantially improved with the addition of sodium citrate during rinse. This is true for the proteases SEQ ID NO:1 , 5, 6, and 7 in liquid laundry model detergent formulations 1 , 2, and 3, at a water hardness condition of 6 °dH. Example 2: Addition of chelator with the onset (start) of the wash

The Bottle wash assay was applied to test SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:9 in liquid laundry model detergent 1 , as described above. The washes were conducted at a water hardness of 15°dH. Washes were done without additional chelator and soil removal was compared to washes with the addition of 1 g/L GLDA (L-glutamic acid, N,N-diacetic acid, tetrasodium salt), and 1 g/L sodium citrate, respectively.

After the wash experiments, the swatches were spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. The remission (REM) at 460 nm is measured. The delta remission is calculated versus the washes without addition of enzyme.

Table 10: Delta remission for the proteases SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:9 on C-S-07 (Grass, pure) swatches after Bottle wash at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 10 demonstrate that the soil removal from C-S-07 expressed as delta intensity, is substantially improved with the addition of chelator, either 1 g/L GLDA, or 1 g/L sodium citrate with the start of the wash compared to the wash without additional chelator. This is true for the proteases SEQ ID NO:2, 5 and 6 in liquid laundry model detergent formulation 1 at a water hardness condition of 15 °dH.

Table 11 : Delta remission for the proteases SEQ ID NO:5 and SEQ ID NO:8 on PC-03 (Chocolate milk with carbon black) swatches after Bottle wash at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 11 demonstrate that the soil removal from PC-03 expressed as delta intensity, is substantially improved with the addition of chelator, either 1 g/L GLDA, or 1 g/L sodium citrate with the start of the wash compared to the wash without additional chelator. This is true for the proteases SEQ ID NO:5 and SEQ ID NO:8 in liquid laundry detergent model formulation 1 at a water hardness condition of 15 °dH.

Table 12: Delta remission for the proteases SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:9 on PC-05 (Blood, Milk, Ink (BMI)) swatches after Bottle wash at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 12 demonstrate that the soil removal from PC-03 expressed as delta intensity, is substantially improved with the addition of chelator, either 1 g/L GLDA, or 1 g/L sodium citrate with the start of the wash compared to the wash without additional chelator. This is true for the proteases SEQ ID NO:5, 8, and 9 in liquid laundry model detergent formulation 1 at a water hardness condition of 15 °dH.

The Terg-o-tometer (TOM) wash assay was applied to test SEQ ID NO:5 and SEQ ID NO:7 in liquid laundry model detergent 1 at a water hardness of 15 °dH. Washes were done without additional chelator and soil removal was compared to washes with the addition of 0.25 g/L, 0.5 g/L, and 1 g/L sodium citrate, respectively.

After the wash experiments, the swatches were spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. The remission (REM) at 460 nm is measured. The delta remission is calculated versus the washes without addition of enzyme. Table 13: Delta remission for the proteases SEQ ID NO:5 and SEQ ID NO:7 on PC-03 (Chocolate milk with carbon black) swatches after TOM wash at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 13 demonstrate that the soil removal from PC-03 expressed as delta intensity, is substantially improved with the addition of chelator, either 0.25 g/L sodium citrate, or 0.5 g/L sodium citrate, or 1 g/L sodium citrate with the start of the wash compared to the wash without additional chelator. This is true for the proteases SEQ ID NO:5 and SEQ ID NO:7 in liquid laundry detergent formulation 1 at a water hardness condition of 15 °dH.

Table 14: Delta remission for the proteases SEQ ID NO:5 and SEQ ID NO:7 on PC-10 (Pigment, Oil, Milk (POM)) swatches after TOM wash at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 14 demonstrate that the soil removal from PC-10 expressed as delta intensity, is substantially improved with the addition of chelator, either 0.25 g/L sodium citrate, or 0.5 g/L sodium citrate, or 1 g/L sodium citrate with the start of the wash compared to the wash without additional chelator. This is true for the proteases SEQ ID NO:5 and SEQ ID NO:7 in liquid laundry detergent formulation 1 at a water hardness condition of 15 °dH.

Example 3: Addition of chelator during wash

The Terg-o-tometer (TOM) wash assay was applied to test SEQ ID NO:5 and SEQ ID NO:7 in liquid laundry model detergent 1 at a water hardness of 15 °dH. Washes were done for 25 min without additional chelator and soil removal was compared to washes with the addition of 0.25 g/L, 0.5 g/L, and 1 g/L sodium citrate during wash, respectively. The sodium citrate was added after 10 min of the total wash time of 25 min.

Table 15: Delta remission for the proteases SEQ ID NO:5 and SEQ ID NO:7 on PC-03 (Chocolate milk with carbon black) swatches after TOM wash at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 15 demonstrate that the soil removal from PC-03 expressed as delta intensity, is substantially improved with the addition of chelator, either 0.25 g/L sodium citrate, or 0.5 g/L sodium citrate, or 1 g/L sodium citrate added 10 min after the start of the wash compared to the wash without additional chelator. This is true for the proteases SEQ ID NO:5 and SEQ ID NO:7 in liquid laundry detergent formulation 1 at a water hardness condition of 15 °dH.

Table 16: Delta remission for the proteases SEQ ID NO:2 and SEQ ID NO:4 on PC-10 (Pigment, Oil, Milk (POM)) swatches after TOM wash at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 16 demonstrate that the soil removal from PC-10 expressed as delta intensity, is substantially improved with the addition of chelator, either 0.25 g/L sodium citrate, or 0.5 g/L sodium citrate, or 1 g/L sodium citrate added 10 min after the start of the wash compared to the wash without additional chelator. This is true for the proteases SEQ ID NO:5 and SEQ ID NO:7 in liquid laundry detergent formulation 1 at a water hardness condition of 15 °dH.

Table 17: Delta remission for the proteases SEQ ID NO:5 and SEQ ID NO:7 on PC-05 (Blood, Milk, Ink (BMI)) swatches after TOM wash at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 17 demonstrate that the soil removal from PC-05 expressed as delta intensity, is substantially improved with the addition of chelator, either 0.25 g/L sodium citrate, or 0.5 g/L sodium citrate, or 1 g/L sodium citrate added 10 min after the start of the wash compared to the wash without additional chelator. This is true for the proteases SEQ ID NO:5 and SEQ ID NO:7 in liquid laundry detergent formulation 1 at a water hardness condition of 15 °dH.

Example 4: Sequential addition of chelator to the wash

The Terg-o-tometer (TOM) wash assay was applied to test SEQ ID NO:2 in liquid laundry model detergent 1 at a water hardness of 15 °dH. Washes were done for 25 min with additional chelator, and soil removal was compared between washes where the sodium citrate was added in one large portion after 10 min of the total wash time of 25 min, to washes where the sodium citrate was added in three to five smaller portions, every 2.5 minutes (after 10 min, 12.5 min, 15 min, 17.5 min, 20 min) up to the same total amount of chelator in the wash.

Table 18: Delta remission for the protease SEQ ID NO:5 on PC-03 (Chocolate milk with carbon black) swatches after TOM wash for 25 min at a water hardness of 15 °dH, and subsequent rinse in tap water

The data presented in Table 18 demonstrate that the soil removal from PC-03 expressed as delta intensity, is improved with the sequential addition of chelator compared to the addition of the chelator in one portion at the onset of the wash. Adding three times 0.1 g/L sodium citrate (3 x 0.1 g/L) after 10 min, 12.5 min, and 15 min of the wash, results in higher delta remission values compared to the addition of one time 0.3 g/L sodium citrate (1 x 0.3 g/L) after 10 min of wash. Adding 5 times 0.1 g/L sodium citrate (5 x 0.1 g/L) after 10 min, 12.5 min, 15 min, 17.5 min, and 20 min of the wash, results in higher delta remission values compared to the addition of one time 0.5 g/L sodium citrate (1 x 0.5 g/L) after 10 min of wash. This is true for the protease SEQ ID NO:5 at all dosages tested in liquid laundry detergent formulation 1 at a water hardness condition of 15 °dH.

Table 19: Delta remission for the protease SEQ ID NO:5 on PC-05 (Blood, Milk, Ink (BMI)), C-S- 07 (Grass, pure), and C-S-101 (Blood, slightly aged) swatches after TOM wash for 20 min at a water hardness of 15 °dH, and subseguent rinse in tap water

The data presented in Table 19 demonstrate that the soil removal from the three swatch types PC-05, C-S-07, and C-S-101 expressed as delta intensity, is improved with the sequential addition of chelator compared to the addition of the chelator in one portion at the onset of the wash. Adding 5 times 0.2 g/L (5 x 0.2 g/L) sodium citrate, with the start of the wash (0 min) and after 2.5 min, 5 min, 7.5 min, and 10 min of the wash, results in higher delta remission values compared to the addition of one time 1 g/L (1 x 1 g/L) sodium citrate at the onset of the wash (0 min). This is true for the protease SEQ ID NO:5 in liquid laundry detergent formulation 1 at a water hardness condition of 15 °dH. The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

1 . A method of washing a textile, comprising the steps of: a) obtaining a textile comprising a proteinaceous stain; b) treating the textile in a wash step, wherein the wash step comprises contacting the textile with a wash liquor, wherein the wash liquor is prepared by mixing a laundry detergent composition with water, and wherein the laundry detergent composition comprises a protease; and c) further treating the textile during the wash step with a chelating agent having affinity for calcium ions (Ca²⁺), wherein the chelating agent is added to the wash liquor, and wherein the chelating agent is present in the wash liquor at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more; wherein the proteinaceous stain is at least partially removed from the textile.

2. A method of washing a textile, comprising the steps of: a) obtaining a textile comprising a proteinaceous stain; b) treating said textile in a wash step, wherein the wash step comprises contacting the textile with a wash liquor, wherein the wash liquor is prepared by mixing a laundry detergent composition with water, and wherein the laundry detergent composition comprises a protease; and c) treating said textile in a rinse step, wherein the rinse step comprises contacting the textile with a chelating liquor comprising a chelating agent at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more, wherein the chelating liquor is prepared by mixing the chelating agent with water, and wherein the chelating agent has affinity for calcium ions (Ca²⁺); wherein the proteinacous stain is at least partially removed from the textile.

3. The method according to any one of the preceding claims, wherein the proteinaceous stain is a meat, dairy, egg, cocoa, grass, blood, or sebum stain.

4. The method according to any one of the preceding claims, wherein the laundry detergent composition is liquid laundry detergent composition, preferably in the form of a regular, compact, or concentrated liquid composition, or a single unit dose format.

5. The method according to any one of the preceding claims, wherein the protease is a serine endopeptidase (E.C. 3.4.21), preferably a subtilisin (E.C. 3.4.21.62).

6. The method according to claim 5, wherein the protease is selected from the group consisting of: a) a protease having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:1 ; preferably a protease comprising, consisting essentially of, or consisting of SEQ ID NO:1; b) a protease having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:5; preferably a protease comprising, consisting essentially of, or consisting of SEQ ID NO:5; and c) a protease having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:6; preferably a protease comprising, consisting essentially of, or consisting of SEQ ID NO:6.

7. The method according to any of claims 1-4, wherein the protease is a metalloprotease (EC 3.4.24), preferably a thermolysin (EC 3.4.24.27) or bacillolysin (EC 3.4.24.28).

8. The method according to claim 7, wherein the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:7; preferably the protease comprises, consists essentially of, or consists of SEQ ID NO:7.

9. The method according to any one of claims 1-4, wherein the protease is a glutamyl endopeptidase (EC 3.4.21.19).

10. The method according to claim 9, wherein the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:8; preferably the protease comprises, consists essentially of, or consists of SEQ ID NO:8.

11. The method according to any one of claims 1-4, wherein the protease is a trypsin-type protease with specificity for Arg and Lys residues.

12. The method according to claim 11 , wherein the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:9; preferably the protease comprises, consists essentially of, or consists of SEQ ID NO:9.

13. The method according to any one of the preceding claims, wherein the chelating agent is present at a concentration of 0.01 g/L to 100 g/L, preferably 0.05 g/L to 50 g/L, more preferably 0.1 g/L to 25 g/L, even more preferably 0.5 g/L to 15 g/L, most preferably 1 g/L to 10 g/L.

14. The method according to any one of the preceding claims, wherein the chelating agent is selected from the group consisting of citric acid or a salt thereof (e.g., sodium citrate), gluconic acid, glutamic acid-N,N-diacetic acid (GLDA), methylglycinediacetic acid (MGDA), ethylenediaminetetraacetic acid (EDTA), and mixtures thereof.

15. The method according to claim 14, wherein the chelating agent is citric acid or a salt thereof, preferably sodium citrate, or glutamic acid-N,N-diacetic acid (GLDA).

16. The method according to claim 1 , wherein the chelating liquor further comprises a cationic surfactant.

17. The method according to claim 16, wherein the cationic surfactant is an ester of a quaternary ammonium salt (/.e., an ester quat).

18. The method according to claim 16, wherein the cationic surfactant is selected from the group consisting of cetyltrimethylammonium bromide (CTAB), diethyl ester dimethyl ammonium chloride (DEEDMAC), dimethyldioctadecylammonium chloride (DSDMAC), dimethyldistearylammonium chloride (DSDMAC), triethanolamine quat (TEAQ), alkyldimethylethanolamine quat (ADMEAQ), and 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP).

19. The method according to any one of claims 16-18, wherein the chelating liquor comprises from 0.1 % to 20% w/w of cationic surfactant, preferably from 0.5% to 15% w/w, most preferably from 1 % to 10% w/w.

20. The method according to claim 1 , wherein the chelating agent is added to the wash liquor as a single dose or as multiple doses; preferably wherein the chelating agent is added to the wash liquor as multiple, sequential doses.

21. The method according to claim 2, wherein the chelating liquor is added as a single dose or as multiple doses; preferably wherein the chelating liquor is added as multiple, sequential doses.

22. Use of a chelating agent for improving proteinaceous stain removal of a protease during a laundry process.

23. The use according to claim 22, wherein stain removal is improved by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, or more, and wherein the improvement is measured relative to the stain removal obtained with said protease applied under the same conditions but without said chelating agent being present.

24. The use according to any one of claims 22-23, wherein the chelating agent is selected from the group consisting of citric acid or a salt thereof (e.g., sodium citrate), gluconic acid, glutamic acid-N,N-diacetic acid (GLDA), methylglycinediacetic acid (MGDA), and thylenediaminetetraacetic acid (EDTA), and mixtures thereof.

25. The use according to any of claims 22-24, wherein the chelating agent is applied at a concentration of at least 1 g/L, e.g., at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, at least 15 g/L, at least 16 g/L, at least 17 g/L, at least 18 g/L, at least 19 g/L, at least 20 g/L, or more.

26. The use according to any one of claims 22-25, wherein the protease is a serine endopeptidase (E.C. 3.4.21), preferably a subtilisin (E.C. 3.4.21.62).

27. The method according to claim 26, wherein the protease is selected from the group consisting of: a) a protease having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:1; preferably a protease comprising, consisting essentially of, or consisting of SEQ ID NO:1 ; b) a protease having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:5; preferably a protease comprising, consisting essentially of, or consisting of SEQ ID NO:5; and c) a protease having a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:6; preferably a protease comprising, consisting essentially of, or consisting of SEQ ID NO:6.

28. The use according to any one of claims 22-25, wherein the protease is a metalloprotease (EC 3.4.24), preferably a thermolysin (EC 3.4.24.27) or bacillolysin (EC 3.4.24.28).

29. The use according to claim 28, wherein the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:7; preferably the protease comprises, consists essentially of, or consists of SEQ ID NO:7.

30. The use according to any one of claims 22-25, wherein the protease is a glutamyl endopeptidase (EC 3.4.21.19).

31. The use according to claim 30, wherein the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:8; preferably the protease comprises, consists essentially of, or consists of SEQ ID NO:8.

32. The use according to any one of claims 22-25, wherein the protease is a trypsin-type protease with specificity for Arg and Lys residues.

33. The use according to claim 32, wherein the protease has a sequence identity of at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, to SEQ ID NO:9; preferably the protease comprises, consists essentially of, or consists of SEQ ID NO:9.