Use of cellulase for removal of dust mites from textiles

Abstract

The present invention relates to detergent compositions comprising an enzyme capable of degrading cellulosic material and their use for removing dust mites from the surface of a textile. In one aspect, the enzyme capable of degrading cellulosic material is a GH45 cellulase. In a further aspect, the GH45 cellulase is a Thermobifida fusca endoglucanase.

Description

[0001] This application is a divisional application of Chinese patent application No. 202180044693.2, filed on June 16, 2021, entitled “Use of cellulase to remove dust mites from textiles”. Technical Field

[0002] This invention relates to the use of enzymes capable of degrading cellulose materials, and in particular the use of cellulase for removing dust mites from the surface of textiles.

[0003] References to sequence lists

[0004] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Background Technology

[0005] Dust mites are mites (a type of arachnid, such as spiders and scorpions). They are between 0.1 mm and 0.5 mm in size and live in all types of fibers, including carpets, clothing, upholstered sofas, and bed sheets. Over the past few decades, it has become well known that dust mites are highly allergenic and can trigger asthma and other allergy attacks. Attempts have been made to prevent exposure to allergens using organic or inorganic acaricides, detergents, or ultraviolet light. Specifically, US 5672362 A uses a solution of disodium tetraborate tetrahydrate (DOT) to kill dust mites. WO 9836640 discloses compositions containing benzyl benzoate and alcohol for controlling dust mites. KR 200287251 discloses a portable ultraviolet (UV) sterilizer for removing dust mites. However, so far, results have been disappointing, as acaricides or detergents may cause new allergy attacks. US 2004255983 discloses a method for removing dust mites by repeatedly washing products to the desired level. However, excessive washing cycles can compromise the integrity and safety of textiles, thus reducing their lifespan.

[0006] A new method is needed to remove dust mites from textile surfaces without introducing new allergens or reducing the lifespan of textiles. Summary of the Invention

[0007] In one aspect, the present invention relates to the use of one or more enzymes capable of degrading cellulose materials in a washing cycle to reduce dust mites from textiles. In another aspect, the enzyme is a cellulase or hemicellulase. In yet another aspect, the cellulase may be GH45 cellulase.

[0008] In another aspect, the present invention also relates to an enzyme composition for removing dust mites. The enzyme composition comprises an enzyme capable of degrading cellulose materials and can be used in any washing, cleaning, and / or fabric care method, including soaking methods, pretreatment methods, methods with a rinsing step (in which a separate rinsing agent and the composition may be added), and post-treatment methods.

[0009] In another aspect, the enzyme composition comprises one or more cellulases or hemicellulases. Additionally, the cellulase may be GH45 cellulase. In one aspect, the GH45 cellulase is an endoglucanase from *Thermothielavioides terrestris* (T. t).

[0010] In another aspect, the T. t endoglucanase is selected from the group consisting of:

[0011] (1) T. t endoglucanase, which contains or is composed of the mature polypeptide of SEQ ID NO: 2 or a fragment thereof;

[0012] (2) T. t endoglucanase, which comprises, or is composed of, an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 2;

[0013] (3) T. t endoglucanase, which is encoded by a polynucleotide comprising, or being composed of, a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO: 1;

[0014] (4) A T. t endoglucanase encoded by a polynucleotide that hybridizes to the coding sequence of the mature polypeptide of SEQ ID NO: 1 or its full-length complementary sequence under at least high-stress conditions and very high-stress conditions. In another aspect, the cellulase composition further comprises one or more detergent components or fabric softeners.

[0015] In another aspect, this application further includes a detergent composition containing an enzyme capable of degrading cellulose materials or an enzyme composition as mentioned above.

[0016] In another aspect, this application includes a method for reducing or removing dust mites, the method comprising:

[0017] (1) Washing or exposing textiles with a detergent composition containing an enzyme capable of degrading cellulose material or an enzyme composition containing an enzyme capable of degrading cellulose material, wherein the enzyme capable of degrading cellulose material comprises GH45 cellulase, wherein the GH45 cellulase is GH45 endoglucanase, and in another aspect, the endoglucanase is T. t endoglucanase;

[0018] (2) Rinse the textile as you see fit.

[0019] This invention provides the following:

[0020] 1. The use of enzymes capable of degrading cellulose materials in reducing / removing dust mites from textiles.

[0021] 2. The use as described in item 1, wherein the enzyme capable of degrading cellulose materials is GH45 cellulase.

[0022] 3. The use as described in item 1, wherein the enzyme capable of degrading cellulosic materials is any one or more GH45 cellulases selected from the table below:

[0023]

[0024] .

[0025] 4. The use as described in item 3, wherein the GH45 cellulase is an endoglucanase from *Leuconostoc mesenteroides*.

[0026] 5. The use as described in item 4, wherein the terrestrial thermophilic rhizobium endoglucanase is selected from the group consisting of:

[0027] (1) T. t endoglucanase, which contains or is composed of the mature polypeptide of SEQ ID NO: 2 or a fragment thereof;

[0028] (2) T. t endoglucanase, which comprises, or is composed of, an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 2;

[0029] (3) T. t endoglucanase, which is encoded by a polynucleotide comprising, or being composed of, a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO: 1;

[0030] (4) T. t endoglucanase, which is encoded by a polynucleotide that hybridizes with the mature polypeptide coding sequence of SEQ ID NO: 1 or its full-length complementary sequence under at least high-stress conditions and very high-stress conditions. In another respect, the cellulase composition further comprises one or more detergent components or fabric softeners.

[0031] 6. An enzyme composition for reducing or removing dust mites from textiles, the enzyme composition comprising an enzyme capable of degrading cellulose materials.

[0032] 7. The enzyme composition as described in item 6, wherein the enzyme capable of degrading cellulose material is GH45 cellulase.

[0033] 8. The enzyme composition as described in item 6, wherein the enzyme capable of degrading cellulosic materials is selected from the GH45 cellulase in the table below:

[0034]

[0035]

[0036] .

[0037] 9. The enzyme composition of item 8, wherein the GH45 cellulase is an endoglucanase from *Leuconostoc mesenteroides*.

[0038] 10. The enzyme composition of claim 9, wherein the terrestrial thermophilic rhizosphere endoglucanase is selected from the group consisting of:

[0039] (1) T. t endoglucanase, which contains or is composed of the mature polypeptide of SEQ ID NO: 2 or a fragment thereof;

[0040] (2) T. t endoglucanase, which comprises, or is composed of, an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 2;

[0041] (3) T. t endoglucanase, which is encoded by a polynucleotide comprising, or being composed of, a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO: 1;

[0042] (4) T. t endoglucanase, which is encoded by a polynucleotide that hybridizes to the mature polypeptide encoding sequence of SEQ ID NO: 1 or its full-length complementary sequence under at least high-strict conditions and very high-strict conditions; on the other hand, the cellulase composition further comprises one or more detergent components or fabric softeners.

[0043] 11. The enzyme composition of claim 6, further comprising at least one or more additional enzymes selected from the group consisting of: hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, keratinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, amylopectinase, tanninase, pentosanase, maranaase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, DNase, chlorophyllase, amylase, perhydrolase, peroxidase, xanthan gumase, and mixtures thereof.

[0044] 12. The enzyme composition of claim 11, wherein the at least one or more additional enzymes are selected from proteases, lipases, mannanases, pectin lyases, and amylases.

[0045] 13. A detergent composition for reducing or removing dust mites from textiles, the detergent composition comprising an enzyme composition, a surfactant, a builder, and a polymer as described in any one of items 6-12.

[0046] 14. The detergent composition of claim 13, wherein the surfactant is selected from the group consisting of anionic, cationic, nonionic, semipolar and zwitterionic surfactants.

[0047] 15. The detergent composition of claim 13 or 14, further comprising one or more components selected from the group consisting of: bleaching systems, bleaching activators, bleaching catalysts, silicates, and dyes.

[0048] 16. The detergent composition as described in any of the preceding items, wherein the composition is in the form of: powder, strip, uniform tablet, tablet having two or more layers, bag having one or more chambers, regular or compressed powder, granules, paste, gel, or regular, compressed or concentrated liquid.

[0049] 17. The detergent composition as described in any one of the preceding items, wherein the amount of enzyme composition used in the detergent composition corresponds to the following: 0.001-200 mg of protein per liter of detergent solution, such as 0.005-100 mg of protein, preferably 0.01-50 mg of protein, more preferably 0.05-20 mg of protein, and even more preferably 0.1-10 mg of protein.

[0050] 18. A method for reducing or removing dust mites from textiles, the method comprising:

[0051] (1) The textile is washed or exposed with any detergent composition as described in items 13-17 or with any enzyme composition as described in items 6-12;

[0052] (2) Rinse the textile as you see fit.

[0053] 19. The method of item 18, wherein the method is performed by hand or machine.

[0054] 20. The method of any one of items 18-19, wherein the method is a washing or cleaning method, or a dishwashing method. Attached Figure Description

[0055] Figure 1 An exemplary anti-pilling test is shown.

[0056] Figure 2 The results of T. t. endoglucanase on E-252PA fabric for dust mite resistance are shown.

[0057] Figure 3 The results of T. t. endoglucanase on CN-42 fabric for dust mite resistance are shown.

[0058] definition

[0059] Anti-pilling The term "anti-pilling" refers to the removal of lint from the surface of textiles and / or the prevention of lint formation on the surface of textiles.

[0060] Cellulase or cellulaseThe term “cellulase” or “cellulase” refers to one or more (e.g., several) enzymes that hydrolyze cellulose materials. Such enzymes include one or more endoglucanases, one or more cellobiases, one or more β-glucosidases, or combinations thereof. Two basic methods for measuring cellulose degrading activity include (1) measuring total cellulose degrading activity, and (2) measuring individual cellulose degrading activities (endoglucanase, cellobiase, and β-glucosidase), as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulose degrading activity is typically measured using insoluble substrates, including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common assay for total cellulose degrading activity is the filter paper assay using Whatman No. 1 filter paper as the substrate. This determination was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

[0061] For the purposes of this invention, cellulase activity is determined by measuring the increase in hydrolysis of cellulose material by one or more cellulases under the following conditions: cellulose (or other pretreated cellulose material) in pretreated corn stalks (PCS) with 1-50 mg cellulase protein / g of pretreated cellulose (PCS) added, for 3-7 days, compared to control hydrolysis without added cellulase protein. Typical conditions are: 1 ml reaction, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate (pH 5), 1 mM MnSO4, 50°C, 55°C, or 60°C, 72 hours, glycosylation analysis performed via an AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, California, USA). Other methods known in the art for measuring cellulase activity may also be applicable.

[0062] Cellulose materials:The term "cellulose material" refers to any material containing cellulose. The primary polysaccharide in the primary cell walls of biomass is cellulose, followed by hemicellulose, and then pectin. Secondary cell walls, formed after cell cessation of growth, also contain polysaccharides and are reinforced by polymeric lignin covalently cross-linked with hemicellulose. Cellulose is a homopolymer of dehydrated cellobiose and is therefore a linear β-(1-4)-D-glucan, while hemicellulose comprises a variety of compounds with a complex branched structure containing a series of substituents, such as xylan, xyloglucan, arabinoylxylan, and mannan. Although cellulose is generally polymorphic, it is found primarily in plant tissues as an insoluble crystalline matrix of parallel glucan chains. Hemicellulose is typically hydrogen-bonded to cellulose and other hemicelluloses, which helps stabilize the cell wall matrix.

[0063] Cellulose is commonly found in vegetable food products such as salads, tomatoes, spinach, cabbage, and grains.

[0064] Detergent components This document defines the term "detergent component" as meaning the type of chemicals that can be used in a detergent composition for, for example, laundry. Examples of detergent components are surfactants, builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric conditioners, foaming agents, defoaming agents, dyes, fragrances, darkening inhibitors, bactericides, fungicides, soil suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, one or more transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0065] Detergent composition:The term "detergent composition" refers to a composition used to remove unwanted compounds from a surface to be cleaned, such as a textile surface. This detergent composition can be used, for example, to clean textiles, for both household and industrial cleaning. These terms encompass any material / compound chosen for the form (e.g., liquid, gel, powder, granules, paste, or spray compositions) of the desired specific type of cleaning composition and product, and include, but are not limited to, detergent compositions (e.g., liquid and / or solid laundry detergents and delicate fabric detergents; fabric fresheners; fabric softeners; and textile and garment pre-treatments / pre-treatments). Detergent compositions may contain one or more enzymes, such as hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, keratinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, amylopectinase, tannic acidase, pentosanase, maranaase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, DNase, chlorophyllase, amylase, hydrolase, peroxidase, xanthan gumase, and mixtures thereof. The detergent composition may further comprise detergent components such as surfactants, builders, chelating agents or chelating reagents, bleaching systems or bleaching components, polymers, fabric conditioners, foaming agents, defoaming agents, dyes, fragrances, darkening inhibitors, bactericides, fungicides, dirt suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, one or more transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0066] Dust mites:

[0067] House dust mites (Dermatophagoides spp.) are tiny arachnid arthropods that thrive in warm (22°C-26°C) and humid (75%-80% humidity) environments. They live on the dander and fur of humans or animals (cats or dogs). There are 16 species of house dust mites in the home, including the European house dust mite, the American house dust mite, the Maynella house dust mite, and mites such as the acarid mite and the Tymite, which carry tropical claw mites and flour mites. Among these, the European house dust mite (Dermatophagoides pteronyssinus), the American house dust mite (Dermatophagoides farinae), and the tropical claw mite (Blomia tropicalis) are allergens. In particular, the European house dust mite (Dermatophagoides pteronyssinus) is a major allergen for asthma, sneezing, allergic conjunctivitis, and atopic dermatitis.

[0068] Endoglucanase: The term "endoglucanase" refers to endo-1,4-(1,3;1,4)-β-D-glucan-4-glucan hydrolase (EC3.2.1.4), which catalyzes the endo-hydrolysis of β-1,4-β-D-glycosidic bonds in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichen polysaccharides, mixed β-1,3-glucans such as cereal β-D-glucan or xyloglucan, and other plant materials containing cellulose components. Endoglucanase activity can be determined by measuring the decrease in substrate viscosity or the increase in reducing ends as determined by reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). For the purposes of this invention, endoglucanase activity was determined using carboxymethyl cellulose (CMC) as a substrate at pH 5 and 40°C, following the procedure described in Ghose, 1987, Pure and Appl. Chem. 59: 257-268.

[0069] Excerpt: The term "fragment" refers to a polypeptide that has one or more (e.g., several) amino acids missing from the amino and / or carboxyl termini of the major portion of a mature polypeptide; wherein the fragment has enzymatic activity. In one respect, the fragment contains at least 85% (e.g., at least 90%, or at least 95%) of the amino acid residues of the mature polypeptide containing the enzyme.

[0070] Hemicellulase or hemicellulase:The term “hemicellulase” or “hemicellulase” refers to one or more (e.g., several) enzymes that can hydrolyze hemicellulosic materials. See, for example, Sharlom, D. and Shoham, Y., Microbial hemicellulases, Current Opinion in Microbiology, 2003, 6(3): 219-228. Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to: acetylmannan esterase, acetylxylan esterase, arabinonanase, arabinofuranylase, coumarin esterase, ferulic esterase, galactosidase, glucuronidase, glucuronidase, mannanase, mannosidase, xylanase, and xylosidase. The substrates of these enzymes (hemicellulose) are heterogeneous groups of branched and linear polysaccharides that are linked to cellulose microfibrils in plant cell walls via hydrogen bonds, thus crosslinking them into a stable network. Hemicellulose is also covalently attached to lignin, forming a highly complex structure together with cellulose. The variable structure and organization of hemicellulose require the synergistic action of many enzymes to achieve its complete degradation. The catalytic modules of hemicellulases are glycosidases (GH) that hydrolyze glycosidic bonds, or carbohydrate esterases (CE) that hydrolyze ester bonds on the side groups of acetic acid or ferulic acid. These catalytic modules can be assigned to the GH and CE families based on their primary sequence homology. Some families (with generally similar folds) can be further grouped into clans, labeled with letters (e.g., GH-A). The most detailed and up-to-date classification of these and other carbohydrate-active enzymes is available in the Carbohydrate Active Enzymes (CAZy) database. Hemicellulase activity can be measured at suitable temperatures (e.g., 50°C, 55°C, or 60°C) and pH (e.g., 5.0 or 5.5) according to Ghose and Bisaria, 1987, Pure & Applied Chem, 59: 1739-1752.

[0071] Improved washing performance The term “improved washing performance” is defined herein as the increased washing performance exhibited by an enzyme relative to a similar wash without the enzyme, for example by increasing dust mite removal or increasing dust mite reduction.

[0072] Low stringency requirements:The term "low stringency conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 50°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0073] Mature polypeptides: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In this invention, based on the SignalP 3.0 program predicting that amino acids 1 to 21 of SEQ ID NO: 2 are a signal peptide (Bendtsen et al., 2004, J. Mol. Biol. [Journal of Molecular Biology] 340: 783-795), the mature polypeptide of SEQ ID NO: 2 is amino acids 22 to 300 of SEQ ID NO: 2.

[0074] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide with enzymatic activity. In one respect, based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6), the mature polypeptide coding sequence is nucleotides 64 to 858 of SEQ ID NO: 1, and nucleotides 1 to 63 of SEQ ID NO: 1 encode a signal peptide.

[0075] Strict conditions: The term "medium-strict conditions" refers to a standard DNA blotting procedure followed for probes at least 100 nucleotides in length, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0076] Medium to high stringent requirements: The term "medium-high stringent conditions" refers to a standard DNA blotting procedure followed for probes at least 100 nucleotides in length, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0077] Sequence identity The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0078] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine sequence identity between two amino acid sequences. This algorithm is implemented in the Niedle procedure using the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 5.0.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Niedle output labeled "Longest Identity" (obtained using the non-simplification option) is used as the identity percentage and calculated as follows:

[0079] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine sequence identity between two deoxyribonucleotide sequences. This algorithm is implemented in the Niedle procedure of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 5.0.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Niedle output labeled "Longest Identity" (obtained using the non-simplification option) is used as the identity percentage and calculated as follows:

[0080] textileThe term "textiles" means any textile material, including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material, fabrics made from these materials, and products made from these fabrics (e.g., clothing and other articles). The textile or fabric can be in the form of knitted fabrics, woven fabrics, denim, nonwoven fabrics, felt, yarns, and terry cloth. The textile can be cellulose-based, such as natural cellulose products including cotton, flax / linen, jute, ramie, sisal, or coconut fiber, or man-made cellulose (e.g., derived from wood pulp), including viscose / rayon, cellulose acetate fiber (tricell), lyocell, or blends thereof. Textiles or fabrics may also be non-cellulose-based, such as natural polyamides including wool, camel hair, cashmere, mohair, rabbit hair, and silk, or synthetic polymers such as nylon, aramid, polyester, acrylate, polypropylene, and 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 fibers with one or more accompanying materials such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) and / or cellulose-containing fibers (e.g., rayon / viscose fibers, ramie, flax / linen, jute, cellulose acetate fibers, lyocell fibers). Fabrics can be conventional washable garments, such as stained household clothes. When the term fabric or garment is used, the broader term textiles is intended to also be included.

[0081] Washing cycle: The term "wash cycle" is defined herein as a washing operation in which the textile is exposed to the washing liquid for a sustained period of time by circulating the washing liquid and optionally mechanically treating it, in order to clean the textile and finally remove excess washing liquid. Wash cycles may be repeated once, twice, three times, four times, five times, or even six times at the same or different temperatures. Afterward, the textile is typically rinsed and dried. One type of wash cycle may be a soaking step, in which the textile is kept immersed in the washing liquid for a period of time.

[0082] Detergent: The term "washing liquid" is intended to refer to a solution or mixture of water and detergent components that optionally include enzymes for use, such as in laundry.

[0083] Dishwashing detergent composition: The term "dishwashing detergent composition" refers to a composition that can be used to remove and / or reduce unwanted compounds from surfaces to be cleaned, such as the surface of dishes or surfaces present inside a dishwashing machine.

[0084] Dishwashing detergent compositions can be any composition intended to reduce and / or remove dirt from plates, tableware, jars, pots, cutlery, and all forms of compositions intended to reduce and / or remove dirt from the inner surfaces of dishwashing machines. This invention is not limited to any particular type of dishwashing detergent composition. These terms encompass any detergent component chosen for the desired type of cleaning composition and product form (e.g., liquid, gel, powder, strip, granule, paste, or spray composition). The detergent composition may contain enzymes other than the amylase and / or protease contained in the composition, such as one or more additional enzymes, such as protease, amylase, lipase, keratinase, cellulase, endoglucanase, xyloglucanase, pectinase, pectin lyase, xanthan gumase, peroxidase, halogenated peroxidase, catalase, and mannanase, or any mixture thereof; and detergent components.

[0085] Dishwasher: The term "warewashing machine" refers to any kind of washing machine that can be used for industrial or institutional warewashing. This term includes, but is not limited to, door warewashing machines, hood warewashing machines, conveyor belt warewashing machines, undercounter warewashing machines, glass washing machines, flight warewashing machines, can and pot warewashing machines, and appliance washing machines.

[0086] utensils The term "vessel" is intended to mean any form of tableware, kitchenware, tableware set, or tableware for the dining table, such as, but not limited to, pots, pans, drinking glasses, cups, knives, forks, spoons, porcelain, etc. Vessels can be made of any suitable material, such as metal, glass, rubber, plastic, PVC, acrylic, ceramic, porcelain, or porcelain.

[0087] Washing performance The term "washing performance" is used to describe the ability of an enzyme to remove or reduce dust mites present on surfaces or textiles being cleaned, for example, during a cleaning cycle.

[0088] Washing time: The term "washing time" is defined in this article as the time taken for a complete washing process; that is, the time taken for one or more washing cycles and one or more rinsing cycles to be combined. Detailed Implementation

[0089] This invention relates to the use of one or more enzymes capable of degrading cellulose materials in a detergent during a washing cycle to reduce dust mites from textiles. The inventors of this invention have discovered that by adding enzymes capable of degrading cellulose materials to a detergent, the number of dust mites attached to textiles can be significantly reduced. In one aspect, the enzyme is an enzyme composition comprising cellulase or hemicellulase.

[0090] In another aspect, the present invention relates to an enzyme composition for removing dust mites. The enzyme composition for removing dust mites comprises one or more cellulases or hemicellulases. The cellulase may be GH45 cellulase. Additionally, the GH45 cellulase may be GH45 endoglucanase. In another aspect, the cellulase is the endoglucanase of *Leuconostoc mesenteroides* of SEQ ID NO:2, or a cellulase comprising an amino acid sequence or composed thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0091] In another aspect, this application further includes a detergent composition containing an enzyme capable of degrading cellulose materials or an enzyme composition as mentioned above.

[0092] In another aspect, this application includes a method for removing dust mites, the method comprising:

[0093] (1) Washing or exposing textiles with a detergent composition containing an enzyme capable of degrading cellulose material or an enzyme composition containing an enzyme capable of degrading cellulose material, wherein the enzyme capable of degrading cellulose material comprises GH45 cellulase, wherein the GH45 cellulase is GH45 endoglucanase, and in another aspect, the endoglucanase is T. t endoglucanase;

[0094] (2) Rinse the textile as you see fit.

[0095] enzymes

[0096] An enzyme or enzyme composition containing cellulase or hemicellulase may further contain one or more enzymes.

[0097] Examples include proteases, lipases, keratinases, amylases, glycoses, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases such as laccases, and / or peroxidases.

[0098] Generally, the properties of one or more enzymes selected should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzyme or non-enzyme components, etc.), and the one or more enzymes should be present in an effective amount.

[0099] Cellulase

[0100] Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Humicola*, *Fusarium*, *Thielavia*, and *Acremonium*, such as fungal cellulases produced by *Humicola insolens*, *Myceliophthora thermophila*, and *Fusarium oxysporum* as disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259.

[0101] Particularly suitable cellulases are alkaline or neutral cellulases that offer color-care benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are, for instance, those cellulases 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 WO99 / 001544.

[0102] Other cellulases are endoglucanases having the following sequence, which has at least 97% identity with the amino acid sequence at positions 1 to 773 of SEQ ID NO: 2 of WO 2002 / 099091, or family 44 xyloglucanases having the following sequence, which has at least 60% identity with positions 40-559 of SEQ ID NO: 2 of WO 2001 / 062903.

[0103] Commercially available cellulases include Celluzyme™ and Carezyme™ (Novozymes A / S), Carezyme Premium™ (Novozymes), Celluclean™ (Novozymes), CellucleanClassic™ (Novozymes), Cellusoft™ (Novozymes), Whitezyme™ (Novozymes), Clazinase™ and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

[0104] GH45 cellulase

[0105] GH family 45 cellulases (formerly known as family K) function by converting anomeric conformations to produce α-D anosomes of oligosaccharides as products. It has been elucidated that, at the active site, one aspartic amino acid acts as a generalized acid and another acts as a generalized base.

[0106] The three-dimensional structures of family 45 enzymes have been elucidated (see, for example, the structure of *Pseudomonas spp.* in the following literature: Davies et al., 1996, *Acta Crystallographica Section D - Biological Crystallography* 52:7-17 Part 1). These enzymes contain a β-barrel structure with six strands attached to a seventh strand. This structure contains parallel and antiparallel β-chains. The active site is located in an open substrate-binding groove.

[0107] As used herein, the terms “GH45 cellulase,” “family 45 cellulase,” or “Cel45” refer to carbohydrate-active cellulases containing the catalytic domain of glycoside hydrolases family 45, as classified according to EC 3.2.1.4. This term encompasses carbohydrate-active enzymes that hydrolyze cellulose and cellulosic oligosaccharides using a conversion mechanism and possess one of two characteristic sequences near the catalytic aspartic amino acid: (i) a first conserved characteristic sequence A / S / T-T-R / N / T-Y / F / T-X-D-X-X-X-X-X-C / A-A / G / S-W / C and a second conserved characteristic sequence H / Q / D / N-F / L-D-I / L / F; or (ii) possessing the second conserved characteristic sequence H / Q / D / N-F / L-D-I / L / F but lacking the first conserved sequence. In one embodiment, the second conserved characteristic sequence is HFDI.

[0108] Table 1: Members of Family 45 Cellulase Subfamily B:

[0109]

[0110] Table 2: Members of Family 45 Cellulase Subfamily A:

[0111]

[0112]

[0113]

[0114]

[0115] In a preferred embodiment, GH45 cellulase is an endoglucanase from the terrestrial thermophilic rhizosphere mold.

[0116] In another aspect, the T. t endoglucanase is selected from the group consisting of:

[0117] (1) T. t endoglucanase, which contains or is composed of the mature polypeptide of SEQ ID NO: 1 or a fragment thereof;

[0118] (2) T. t endoglucanase, which comprises, or is composed of, an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 1;

[0119] (3) T. t endoglucanase, which is encoded by a polynucleotide comprising, or being composed of, a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO: 2.

[0120] (4) T. t endoglucanase, which is encoded by a polynucleotide that hybridizes with the coding sequence of the mature polypeptide of SEQ ID NO: 2 or its full-length complementary sequence under at least high-strict conditions and very high-strict conditions.

[0121] Detergent composition

[0122] Enzymes capable of degrading cellulosic materials may be present in laundry detergent compositions containing at least one or more additional enzymes. These additional enzymes may be selected from the group consisting of: hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, keratinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, amylopectinase, tanninase, pentosanase, maranaase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, DNase, chlorophyllase, amylase, hydrolase, peroxidase, xanthan gumase, and mixtures thereof. In one embodiment of the invention, at least one or more additional enzymes are selected from protease, lipase, mannanase, pectin lyase, and amylase.

[0123] The detergent composition contains a surfactant, which can be selected from the group consisting of anionic, cationic, nonionic, semi-polar, and facultative zwitterionic surfactants. Additionally, other detergent components (such as builders and polymers) can be included in the detergent composition. When improving the absorbency of textiles, the benefit is that items (such as towels) can absorb more water when used to dry skin or surfaces.

[0124] The present invention further includes a method for removing dust mites, the method comprising subjecting textiles to enzymes capable of degrading cellulose materials, or to an enzyme composition comprising enzymes capable of degrading cellulose materials.

[0125] On one hand, methods for removing dust mites include:

[0126] (3) Washing or exposing textiles with a detergent composition containing an enzyme capable of degrading cellulose materials or an enzyme composition containing an enzyme capable of degrading cellulose materials, wherein the enzyme capable of degrading cellulose materials comprises GH45 cellulase, wherein the GH45 cellulase is GH45 endoglucanase, or in another aspect, the endoglucanase is T. t endoglucanase.

[0127] (4) Rinse the textile as you see fit.

[0128] The detergent composition may be, for example, a laundry detergent composition, and may be in the following forms: powder, strip, uniform tablet, tablet having two or more layers, bag having one or more chambers, regular or compressed powder, granules, paste, gel, or regular, compressed or concentrated liquid. The composition may be a powder or granules, wherein the acidic material is coated as an outer layer on the powder or granules.

[0129] Mannanase

[0130] Suitable mannanases include those of bacterial or fungal origin, including chemically or genetically modified mutants. Mannanases can be basic mannanases of families 5 or 26. They can be wild-type from the genera *Bacillus* or *Pythium*, particularly from *Bacillus agaradhaerens*, *Bacillus licheniformis*, *Bacillus halodurans*, *Bacillus clausii*, or *H. insolens*. Suitable mannanases are described in WO 1999 / 064619. Commercially available mannanases are from Mannaway (Novozymes).

[0131] Peroxidase / oxidase

[0132] Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, such as those from *C. cinereus*, and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include Guardzyme™ (Novozymes). Peroxidases according to the invention also include halogenated peroxidases, such as chloride peroxidase, bromoperoxidase, and compounds exhibiting chloride peroxidase or bromoperoxidase activity. Halogenated peroxidases are classified according to their specificity for halide ions. Chloride peroxidase (EC1.11.1.10) catalyzes the formation of hypochlorite from chloride ions.

[0133] In the embodiments, the halogenated peroxidase of the present invention is a chloride peroxidase. Preferably, the halogenated peroxidase is a vanadium halide peroxidase, i.e., a vanadate-containing halogenated peroxidase. In a preferred method of the present invention, the vanadate-containing halogenated peroxidase is combined with a chloride ion source.

[0134] Halogenated peroxidases have been isolated from many different fungi, particularly from the dematiaceous hyphomycete fungal group, such as Caldariomyces (e.g., C. fumago), Alternaria, Curvularia (e.g., C. verruculosa and C. inaequalis), Drechslera, Ulocladium, and Botrytis.

[0135] Halogenated peroxidases have also been isolated from bacteria such as Pseudomonas (e.g., P. pyrrocinia) and Streptomyces (e.g., Streptomyces aureofaciens).

[0136] In preferred embodiments, the halogenated peroxidase may be derived from species of the genus *Curvularia*, particularly *Curvularia verruculosa* or *Curvularia inaequalis*, such as *Curvularia inaequalis* CBS 102.42 described in WO 95 / 27046; or *Curvularia verruculosa* CBS 147.63 or *Curvularia verruculosa* CBS 444.70 described in WO 97 / 04102; or from species of the genus *Drechslerahartlebii* described in WO 01 / 79459, *Dendryphiella salina* described in WO 01 / 79458, *Phaeotrichoconis crotalarie* described in WO 01 / 79461, or *Geniculosporium* described in WO 01 / 79460.

[0137] The oxidases according to the invention specifically include any laccase or fragment thereof exhibiting laccase activity or exhibiting similar activity as defined in enzyme classification EC 1.10.3.2, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5).

[0138] protease

[0139] Suitable proteases include those of bacterial, fungal, plant, viral, or animal origin, such as those of plant or microbial origin. Microbial origin is preferred. This includes chemically modified mutants or protein-engineered mutants. It can be an alkaline protease, such as a serine protease or a metalloproteinase. Serine proteases can be, for example, serine proteases of the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteinases can be, for example, thermophilic bacterial proteases from the M4 family or other metalloproteinases, such as those from the M5, M7, or M8 families.

[0140] The term "subtilisinase" refers to the serine protease subgroup according to Siezen et al., Protein Engineering, 4(1991) 719-737 and Siezen et al., Protein Science, 6(1997) 501-523. Serine proteases are a subgroup of proteases characterized by the presence of a serine residue at its active site that forms a covalent adduct with the substrate. Subtilisinases can be classified into six subclasses: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanathionine antibiotic peptidase family, the Kexin family, and the Pyrolysin family.

[0141] Examples of subtilisinases are those derived from the genus Bacillus, such as Bacillus lentus, B. alkalophilus, Bacillus subtilis, B. amyloliquefaciens, Bacillus pumilus, and Bacillus gibsonii as described in US 7262042 and WO 09 / 021867; and Bacillus subtilis protease, Bacillus subtilis protease Novo, Bacillus subtilis protease Carlsberg, Bacillus licheniformis, Bacillus subtilis protease BPN', Bacillus subtilis protease 309, Bacillus subtilis protease 147, and Bacillus subtilis protease 168 as described in WO 89 / 06279, and protease PD138 as described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024, and WO 02 / 016547. Examples of trypsin-like proteases are trypsin (e.g., from porcine or bovine sources) and Fusarium proteases (described in WO 89 / 06270, WO 94 / 25583, and WO 05 / 040372), as well as chymotrypsin derived from Cellumonas (described in WO 05 / 052161 and WO 05 / 052146).

[0142] Another preferred protease is an alkaline protease from Bacillus tarda DSM 5483 (as described in, for example, WO95 / 23221) and its variants (described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148).

[0143] Examples of metalloproteinases are neutral metalloproteinases as described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0144] Examples of useful proteases are the variants described in the following: WO 92 / 19729, WO 96 / 034946, WO98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263, WO 11 / 036264, particularly variants with substitutions at one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274, numbered using BPN'. More preferably, the subtilisase variant may contain the following mutations: S3T, V4I, S9R, A15T, K27R, 36D, V68A, N76D, N87S,R 97E, A98S, S99G,D,A, S99AD, S101G,M,R, S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (numbered using BPN').

[0145] Suitable commercially available proteases include those sold under the following trade names: Alcalase®, Duralase Tm Durazym TmRelase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primease®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, and Esperase® (Novozymes), and those sold under the following trade names: Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Opticlean®, and Optimase® (Danisco / DuPont), Axapem TM (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (Bacillus alkalophilus protease) from Kao Corporation.

[0146] Lipase and keratinase

[0147] Suitable lipases and keratins include those of bacterial or fungal origin. This includes chemically modified mutant enzymes or protein-engineered mutant enzymes. Examples include lipases from the genus *Thermomyces*, such as those from *T. lanuginosus* (formerly named *Humicola lanuginosa*) as described in EP258068 and EP 305216; keratinases from the genus *Humicola*, such as *H. insolens* (WO 96 / 13580); and strains from the genus *Pseudomonas* (some of which are now renamed *Burkholderia*), such as *P. alcaligenes* or *P. pseudoalcaligenes* (EP 218272), *P. cepacia* (EP 331376), and *Pseudomonas* strain SD705 (WO 95 / 06720 and WO 06720). Lipases from *Pseudomonas wisconsinensis* (WO 96 / 27002), *Streptomyces* lipase (WO 10 / 065455), *Magnaporthe grisea* lipase (WO 10 / 107560), *Pseudomonas mendocina* lipase (US 5,389,536), *Thermobifidafusca* lipase (WO 11 / 084412), *Geobacillus stearothermophilus* lipase (WO 11 / 084417), *Bacillus subtilis* lipase (WO 11 / 084599), and *Streptomyces* lipase. Lipases from *Streptomyces griseus* (WO 11 / 150157) and *S. pristinaespiralis* (WO 12 / 137147).

[0148] Other examples are lipase variants, such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508, and WO 09 / 109500.

[0149] Preferred commercially available lipase products include Lipolase TM Lipex™; Lipolex TM and Lipoclean TM (Novozymes), Lumafast (originally from Genencor), and Lipomax (originally from Gist-Brocades).

[0150] Other examples include lipases sometimes called acyltransferases or perhydrolases, such as an acyltransferase homologous to Candida antarctica lipase A (WO 10 / 111143), an acyltransferase from Mycobacterium smegmatis (WO 05 / 56782), a perhydrolase from the CE 7 family (WO 09 / 67279), and variants of Mycobacterium smegmatis perhydrolases (particularly the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd) (WO 10 / 100028).

[0151] amylase

[0152] Suitable amylases that can be used with the enzyme preparations of the present invention can be α-amylases or glucosylamylases and can be of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Amylases include, for example, α-amylases obtained from specific strains of Bacillus, such as Bacillus licheniformis (described in more detail in GB 1,296,839).

[0153] Suitable amylases include the amylase having SEQ ID NO: 2 in WO 95 / 10603 or a variant thereof having 90% sequence identity with SEQ ID NO: 3. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and SEQ ID NO: 4 in WO 99 / 019467, such variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444.

[0154] Suitable amylases include the amylase having SEQ ID NO: 6 in WO 02 / 010355 or its variants having 90% sequence identity with SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those with deletions at positions 181 and 182 and substitutions at position 193.

[0155] Other suitable amylases are hybrid α-amylases comprising residues 1-33 of the α-amylase derived from *Bacillus amyloliquefaciens* as shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of the α-amylase derived from *Bacillus licheniformis* as shown in SEQ ID NO: 4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid α-amylase are those with substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred hybrid α-amylase comprising residues 1-33 of the α-amylase derived from Bacillus amyloliquefaciens as shown in WO 2006 / 066594 and residues 36-483 of the α-amylase of SEQ ID NO: 4 is a variant having the following substitutions:

[0156] M197T;

[0157] H156Y+A181T+N190F+A209V+Q264S; or

[0158] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0159] Other suitable amylases are those having SEQ ID NO: 6 in WO 99 / 019467 or variants thereof having 90% sequence identity with SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those having deletions at positions R181 and G182, or positions H183 and G184.

[0160] Other amylases that may be used are those of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96 / 023873, or variants thereof that have 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those that have substitutions, deletions or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476 (numbered using SEQ ID 2 of WO 96 / 023873). More preferred variants are those with deletions at two positions selected from 181, 182, 183, and 184 (e.g., 181 and 182, 182 and 183, or positions 183 and 184). The most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 are those with deletions at positions 183 and 184 and substitutions at one or more positions 140, 195, 206, 243, 260, 304, and 476.

[0161] Other amylases that may be used are those having SEQ ID NO: 2 of WO 08 / 153815, SEQ ID NO: 10 of WO 01 / 66712, or variants thereof having 90% sequence identity with SEQ ID NO: 2 of WO 08 / 153815, or variants thereof having 90% sequence identity with SEQ ID NO: 10 of WO 01 / 66712. Preferred variants of SEQ ID NO: 10 of WO 01 / 66712 are those having substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0162] Other suitable amylases are the amylase of SEQ ID NO: 2 having WO 09 / 061380 or its variants having 90% sequence identity with SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those with C-terminal truncation, and / or substitution, deletion, or insertion at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO: 2 are those having substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or those having deletions at positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are those having the following substitutions:

[0163] N128C+K178L+T182G+Y305R+G475K;

[0164] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0165] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0166] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein these variants are C-terminated and optionally further include a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0167] Other suitable amylases are the amylase of SEQ ID NO: 1 having WO 13184577 or its variants having 90% sequence identity with SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 are those with substitutions, deletions, or insertions at one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476, and G477. More preferred variants of SEQ ID NO: 1 are those having substitutions at one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K, and G477K, and / or those having deletions at positions R178 and / or S179 or T180 and / or G181. Most preferred amylase variants of SEQ ID NO: 1 are those having the following substitutions:

[0168] E187P+I203Y+G476K

[0169] E187P+I203Y+R458N+T459S+D460T+G476K

[0170] These variants optionally include a substitution at position 241 and / or a deletion at positions 178 and / or 179.

[0171] Other suitable amylases are the amylase of SEQ ID NO: 1 having WO 10104675 or its variants having 90% sequence identity with SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 are those with substitutions, deletions, or insertions at one or more of the following positions: N21, D97, V128, K177, R179, S180, I181, G182, M200, L204, E242, G477, and G478. More preferred variants of SEQ ID NO: 1 are those with substitutions at one or more of the following positions: N21D, D97N, V128I, K177L, M200L, L204YF, E242QA, G477K, and G478K, and / or those with deletions at positions R179 and / or S180 or I181 and / or G182. The most preferred amylase variant of SEQ ID NO: 1 is those with the following substitutions:

[0172] N21D+D97N+V128I

[0173] These variants optionally include a substitution at position 200 and / or a deletion at positions 180 and / or 181.

[0174] Other suitable amylases are α-amylases having SEQ ID NO: 12 in WO 01 / 66712 or variants having at least 90% sequence identity with SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions for R118K, N195F, R320K, and R458K, as well as variants having substitutions at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with the most preferred being variants having substitutions at all of these positions.

[0175] Other examples are amylase variants, such as those described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0176] Commercially available amylase is Duramyl TM Termonyl TM Fungaly TM Stainzyme TM StainzymePlus TM Natalase TM Liquozyme X and BAN TM (From Novozymes) and Rapidase TM Purastar TM / Effectenz TM Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genentech International / DuPont).

[0177] enzyme concentration

[0178] In one embodiment of the invention, the enzyme of the invention capable of degrading cellulose materials may be used in a detergent composition in amounts corresponding to: 0.001-200 mg of protein per liter of detergent solution, such as 0.005-100 mg of protein, preferably 0.01-50 mg of protein, more preferably 0.05-20 mg of protein, and even more preferably 0.1-10 mg of protein.

[0179] One or more enzymes in the detergent compositions of the present invention can be stabilized using conventional stabilizers, such as polyols, such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, such as aromatic borate esters, or phenyl boric acid derivatives, such as 4-formylphenylboronic acid, and the compositions can be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708.

[0180] surfactants

[0181] Laundry detergent compositions may contain one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semipolar and / or zwitterionic, or mixtures thereof.

[0182] In certain embodiments, the detergent composition comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. The one or more surfactants are typically present at levels ranging from about 0.1% to 60% by weight, for example, from about 1% to about 40%, or from about 3% to about 20%, or from about 3% to about 10%. The one or more surfactants are selected based on the desired cleaning application, and the one or more surfactants may include any one or more conventional surfactants known in the art.

[0183] When included therein, the detergent will typically contain from about 1% to about 40% by weight of anionic surfactant, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 15% to about 20%, or from about 20% to about 25% of anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates and disulfonates, alkyl sulfates (AS) (such as sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), and alcohols. Ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfo fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters or fatty acid salts (soaps) of sulfosuccinic acids, and combinations thereof.

[0184] When included therein, the detergent will typically contain from about 1% to about 40% by weight of a cationic surfactant, for example from about 0.5% to about 30%, particularly from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12%, or from about 10% to about 12%. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quaternary ammonium compounds, and combinations thereof.

[0185] When included therein, the detergent will typically contain from about 0.2% to about 40% by weight of a nonionic surfactant, for example from about 0.5% to about 30%, particularly from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12%, or from about 10% to about 12%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (such as ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA) or fatty acid glucosamide (FAGA)), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0186] When included therein, the detergent will typically contain from about 0% to about 20% by weight of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs), such as alkyl dimethylamine oxides, N-(cocoylalkyl)-N,N-dimethylamine oxides, and N-(butter-alkyl)-N,N-bis(2-hydroxyethyl)amine oxides, and combinations thereof.

[0187] When included therein, the detergent will typically contain about 0% to about 20% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines, such as alkyldimethyl betaine, sulfobetaine, and combinations thereof.

[0188] The polypeptides of the present invention can also be incorporated into detergent formulations disclosed in WO 97 / 07202, which is hereby incorporated by reference.

[0189] Builders and co-builders

[0190] Detergent compositions may contain about 0% to 10% (e.g., about 0.1% to about 5%) of a builder or co-builder, or a mixture thereof, by weight. In fabric softeners, the level of the builder is typically 0% to 1%, particularly 0% to 0.5%. The builder and / or co-builder may be chelating agents, particularly, that form water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in fabric softeners may be used. Non-limiting examples of detergent 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-aminoethanol (MEA), diethanolamine (DEA, also known as 2,2'-iminodiethanol), triethanolamine (TEA, also known as 2,2',2''-nitrotriethanol), and (carboxymethyl)inulin (CMI), and combinations thereof.

[0191] The detergent composition may also contain 0%-5% by weight, such as about 0% to about 2%, of a detergent co-adjuvant. The detergent composition may include a co-adjuvant alone, or in combination with an auxiliary agent such as a zeolite auxiliary agent. Non-limiting examples of co-adjuvants include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers of (acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents (e.g., aminocarboxylates, aminopolycarboxylates, and phosphonates), and alkyl succinic acids or alkenyl succinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-dibutanoic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta(methylenephosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), and iminodisuccinic acid. N-(2-sulfomethyl)-aspartic acid (IDA), N-(2-sulfoethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), o-amino acid N,N-diacetic acid (ANDA), sulfanilamide-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), sulfonyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N''-triacetate (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations thereof and their salts. Further exemplary builders and / or co-builders are described, for example, in WO 09 / 102854 and US 5977053.

[0192] polymer

[0193] The detergent may contain 0-10% (e.g., 0.5%-5%, 2%-5%, 0.5%-2%, or 0.2%-1%) of a polymer by weight. Any polymer known in the art for use in fabric softeners may be used. The polymer may function as a co-adjuvant as mentioned above, or may provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, defoaming properties, fragrance encapsulation, and lubrication. Some polymers may have more than one of the properties mentioned above and / or more than one of the motifs mentioned below. Exemplary polymers include polyquaternium salts, melamine polymers, siloxanes, silicones, (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 polycarboxylic acid esters (e.g., PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers), hydrophobically modified CMC (HM-CMC), copolymers of terephthalic acid and oligomeric polyethylene glycol, copolymers of poly(ethylene terephthalate) and poly(ethylene oxyterephthalate) (PET-POET), PVP, poly(vinylimidazolium) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazolium (PVPVI). Other exemplary polymers include sulfonated polycarboxylate esters, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the polymers mentioned above are also considered.

[0194] auxiliary materials

[0195] Any detergent components known in the art for use in fabric softeners may also be used. Other optional fabric softener components include solvents (including isopropanol, propylene glycol, alkanes / cycloalkanes), anti-shrinkage agents, anti-redeposition agents, anti-wrinkle agents, bactericides, preservatives (including benzisothiazolinone, methylisothiazolinone, and / or lactic acid), binders, dyes, enzyme stabilizers (including boric acid, borates, CMC, and / or polyols such as propylene glycol), emulsion stabilizers, defoamers (including dimethicone), and skin conditioning agents (including glyceryl caprylic / capric acid, ethylhexyl stearate, or coconut oil), used alone or in combination. Any ingredients known in the art for use in fabric softeners may be used. The selection of such ingredients is entirely within the skill of a person skilled in the art.

[0196] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0197] Anti-redeposition agent

[0198] The detergent compositions of the present invention may further include one or more anti-redeposition agents, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulose-based polymers described above under the category of dirt-releasing polymers may also function as anti-redeposition agents.

[0199] Preparation of detergent products

[0200] The detergent compositions of the present invention can be in any conventional form, such as strips, uniform tablets, tablets having two or more layers, bags having one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or concentrated liquids.

[0201] The bag can be configured as a single chamber or multiple chambers. It can have any form, shape, and material suitable for containing the composition, for example, preventing the composition from being released from the bag before contact with water. The bag is made of a water-soluble membrane containing an internal volume. This internal volume can be divided into chambers of the bag. Preferred membranes are polymeric materials, preferably polymers forming films or sheets. Preferred polymers, copolymers, or derivatives thereof are selected polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylate, most preferably polyvinyl alcohol copolymers, and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. Preferred average molecular weights will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under Trade Reference No. M8630, as sold by MonoSol LLC, Indiana, USA), plus plasticizers such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. The bag may contain a solid laundry cleaning composition or portions thereof and / or a liquid cleaning composition or portions thereof separated by a water-soluble membrane. Chambers for liquid components may differ in composition from those containing solids: US 2009 / 0011970 A1.

[0202] Detergent components can be physically separated from each other by compartments in different layers of water-soluble pouches or tablets. This avoids undesirable storage interactions between components. Furthermore, the different dissolution profiles of each compartment in the detergent solution can cause delayed dissolution of the selected components.

[0203] Non-unit dosage liquid or gel detergents may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70%, about 65%, about 55%, about 45%, or about 35%. Other types of liquids, including but not limited to alkanols, amines, glycols, ethers, and polyols, may be included in aqueous liquids or gels. Aqueous liquid or gel detergents may contain from 0% to 30% organic solvents.

[0204] Liquid or gel detergents can be non-aqueous.

[0205] Enzyme formulations in co-particles

[0206] The enzymes of the present invention can be formulated as granules, for example, as co-granules combining one or more enzymes. Each enzyme will then be present in multiple granules, ensuring a more uniform distribution of the enzyme in the detergent. This also reduces the physical isolation of different enzymes due to varying particle sizes. A method for producing multi-enzyme co-granules for the detergent industry is disclosed in IP.com disclosure IPCOM000200739D.

[0207] Another example of a formulation using co-particle enzymes is disclosed in WO 2013 / 188331, which relates to a detergent composition comprising: (a) a multi-enzyme co-particle; (b) less than 10 wt zeolite (anhydrous); and (c) less than 10 wt phosphate (anhydrous), wherein the enzyme co-particle comprises a sink component of 10 wt% to 98 wt%, and the composition further comprises a detergent sink component of 20 wt% to 80 wt%.

[0208] WO 2013 / 188331 also relates to a method of treating and / or cleaning a surface (preferably a fabric surface), the method comprising the steps of: (i) contacting the surface in an aqueous detergent solution with a detergent composition as claimed and described herein, and (ii) rinsing and / or drying the surface.

[0209] The multi-enzyme co-particle may comprise the enzymes of the present invention and (a) one or more enzymes selected from the group consisting of: first-wash lipase, cleansing cellulase, xyloglucanase, hydrolase, peroxidase, lipoxygenase, laccase, and mixtures thereof; and (b) one or more enzymes selected from the group consisting of: hemicellulase, protease, nursing cellulase, cellobiose dehydrogenase, xylanase, phospholipase, esterase, keratinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase, ligninase, amylopectinase, tanninase, pentosanase, licheninase, glucanase, arabinosidase, hyaluronidase, chondroitinase, amylase, and mixtures thereof.

[0210] Example

[0211] Washing method

[0212] The compositions of the present invention can be used in virtually any washing, cleaning and / or fabric care method, including but not limited to soaking methods, pretreatment methods, methods with a rinsing step (in which separate rinsing agents and compositions may be added), and posttreatment methods.

[0213] The method described herein involves contacting the fabric with the washing solution in a conventional manner, as illustrated below. Conventional laundry methods treat soiled fabrics with an aqueous liquid in which an effective amount of laundry detergent and / or fabric care composition is dissolved or dispersed. The method of the present invention is conveniently carried out during the cleaning process. The cleaning method is preferably carried out at a temperature between 5°C and 95°C, particularly between 10°C and 60°C. The pH of the treatment solution is 6-12, preferably 7-10.

[0214] The following examples are intended to illustrate the compositions of the invention, but are not necessarily intended to limit or otherwise restrict the scope of the invention. In the detergent compositions, the enzyme level is expressed by the amount of pure enzymes based on the weight of the total composition, and unless otherwise stated, the detergent component level is expressed by the weight of the total composition.

[0215] Materials and methods

[0216] Test enzyme:

[0217] Endoglucanase of *Leuconostoc mesenteroides* obtained according to WO 2012 / 089024;

[0218] Fabric:

[0219] CN-42, knitted cotton, purchased from the Center for Testmaterials BV (CFT), Netherlands;

[0220] E-252, striped colored cotton, purchased from the BV Centre for Testing Materials, Netherlands;

[0221] E-252PA, striped colored cotton, is derived from the E-252 mentioned above. It is prepared by pre-treating E-252 in a Wascator automatic washing machine at 40°C for 12 hours to create visible pilling on the surface.

[0222] Standard Detergent A:

[0223] Standard detergent A is prepared using the following ingredients:

[0224]

[0225] method:

[0226] Washing method

[0227] Terg-O-tometer (TOM) washing measurement

[0228] The Terg-O-tometer (TOM) is a medium-sized standard washing system that can be used to simultaneously test 16 different washing conditions. The TOM is essentially a large, temperature-controlled water bath in which up to 16 open metal beakers are immersed. Each beaker constitutes a small top-loading washer, and during the experiment, each beaker contains a solution of a specific detergent / enzyme system to test the properties of cotton fabrics. A rotating stirring arm is used to obtain the desired mechanical stress. The arm agitates the liquid within each beaker. Because the TOM beakers are lidless, samples can be retrieved during TOM experiments, and information can be analyzed online.

[0229] Equipment: A water bath with 16 steel beakers, each equipped with a rotary stirrer, and each beaker containing 1000 mL of detergent solution. Set the temperature to 40°C. Fill the water bath with water. Set the rotation speed to 200 rpm / min.

[0230] 1. Set the temperature in the Terg-O-Tometer to 40°C and start the rotation in the water bath. Maintain the temperature at 40 + / - 0.5°C.

[0231] 2. Prepare a washing solution by adding the required amount of detergent to the solution, and place it in a tank at 40 + / - 0.5°C and 14 dH with magnetic stirring for 10 min. The washing solution should be used within 30 to 60 min after preparation.

[0232] 3. Add 1000 ml of washing solution to the TOM beaker.

[0233] 4. Start stirring at 200 rpm and optionally add the enzyme to the beaker.

[0234] 5. Spread the fabric into the beaker, then add the ballast load.

[0235] 6. Start time measurement when small pieces of fabric and ballast are added to the beaker.

[0236] 7. Wash for 70 minutes.

[0237] 8. Stop stirring.

[0238] 9. Transfer the washing load from the TOM beaker to a sieve and rinse with cold tap water.

[0239] 10. Separate the test material from the ballast load. Transfer the test material to a 5 L beaker containing cold tap water while it is in flowing water.

[0240] 11. Set the timer to 5 minutes.

[0241] 12. Gently squeeze out the water with your hand and place the small test fabric sample on a tray covered with a sheet of paper. Add another sheet of paper on top of the fabric sample.

[0242] 13. Place the small fabric sample in a drum dryer to dry, and then measure it using the Color Eye as described below.

[0243] Anti-pilling effect test

[0244] E-252PA was measured using a Color Eye device (CE7000 / CE7000A, Largo AB). The L value quantifies the anti-pilling benefit. L indicates the white / black variation from 0 to 100, with a decrease in L indicating an increase in black (a decrease in white) and an increase in L indicating an increase in white (a decrease in black). Therefore, the lower the L value, the better the anti-pilling effect.

[0245] CN-42 was evaluated by a sensory panel, with 8-12 panel members assessing its pilling resistance. Higher scores indicated better performance of CN-42.

[0246] Anti-dust mite experiment:

[0247] The test procedures and materials are as follows:

[0248] Dust mites: Dermatophagoides farina Hughhes.

[0249] Steps to test the effectiveness of dust mite reduction:

[0250] 1. Sample preparation: Cut CN-42 into 6 pieces The samples were 6 cm in size and washed using the previous washing method. Each sample was then cut into a circle with a diameter of 58 mm.

[0251] 2. Prevention test procedure: Place a sponge with a side length of 200 mm and a height of 10 mm into a plastic container with a lid (side length of 200-300 mm, height of 50-100 mm, with a ventilation hole in the middle, diameter of 50 ± 10 mm, covered with a PTFE membrane, which is fixed to the plastic container lid with tape), and then inject a saturated NaCl solution into the sponge (immerse the sponge in the saturated NaCl solution).

[0252] 3. Use 7 culture dishes, placing one in the center and the other 6 in the outer area. Bridge every two adjacent dishes with Scotch tape. Then secure all 7 dishes to the plate.

[0253] 4. Place 3 test samples and 3 reference samples into the 6 surrounding petri dishes. Each test sample should be adjacent to one reference sample. Place 0.05 g of dust mite feed in each petri dish (see [link to petri dish]). Figure 1 ).

[0254] 5. Place 2000 ± 200 live dust mites into the center of the petri dish.

[0255] 6. Transfer the plate with the petri dish on top to the sponge in the container. Cover with the lid. Place the container in a temperature and humidity chamber at 25°C ± 2°C and 75% ± 5% humidity.

[0256] 7. After incubation for 24 hours, remove the sample and count the number of dust mites using a dissecting lens.

[0257] Example 1. Fabric treatment in the presence of cellulase

[0258] 2 g of standard detergent A was added to the Terg-o-tometer washing assay. *Leuconostoc mesenteroides*, SEQ ID NO:2, was used in this study. The enzyme protein concentration in the detergent was 0.005%. Each detergent was diluted with water to 1 L. Ballast was required to fill the washed items to 40 g.

[0259] Size 8 An 8 cm² fabric sample of E-252PA was used to evaluate the anti-pilling effect of the test enzyme. This fabric was mixed with standard detergent A diluted for the Terg-o-tometer washing assay and washed for 3 cycles at 40°C for 70 min. The value is used to represent the anti-pilling effect. The results are as follows: Figure 2 As shown. Figure 2 The results showed that better anti-pilling performance was achieved in the presence of cellulase compared to those without cellulase treatment (blank). The L value decreased from greater than 34.1 to approximately 27.9.

[0260] Size 10 A 10 cm² fabric sample of CN-42 was used to evaluate its effectiveness against dust mites. This fabric was mixed with standard detergent A diluted for the Terg-o-tometer washing test and washed for 3 cycles at 40°C for 70 min. Figure 3 The results of the anti-dust mite experiment are shown compared to the experiment without cellulase treatment (blank). From Figure 3 As can be seen, the number of dust mites has decreased to one-third of the blank (from 157 to 48).

[0261]

[0262]

[0263]

Claims

1. Use of an enzyme capable of degrading cellulose materials in reducing dust mite attachment to textiles, wherein the textiles are textiles washed with the enzyme capable of degrading cellulose materials, and wherein the enzyme has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 2, wherein preferably, the enzyme has at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 2 and is derived from *Leuconostoc mesenteroides*.

2. An enzyme composition for reducing dust mite attachment to textiles, the enzyme composition comprising an enzyme capable of degrading cellulose materials, wherein the enzyme has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 2, wherein preferably, the enzyme has at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 2 and is derived from *Leuconostoc mesenteroides*.

3. The enzyme composition of claim 2, further comprising at least one or more additional enzymes selected from the group consisting of: hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, keratinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, amylopectinase, tanninase, pentosanase, maranaase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, DNase, chlorophyllase, amylase, perhydrolase, peroxidase, xanthan gumase, and mixtures thereof.

4. The enzyme composition of claim 2 or 3, wherein the at least one or more additional enzymes are selected from proteases, lipases, mannanases, pectin lyases, and amylases.

5. A detergent composition for reducing dust mite attachment to textiles, the detergent composition comprising an enzyme composition, a surfactant, a detergent builder, and a polymer as described in any one of claims 2-4.

6. The detergent composition of claim 5, wherein the surfactant is selected from the group consisting of anionic, cationic, nonionic, semipolar and zwitterionic surfactants.

7. The detergent composition of claim 5 or 6, further comprising one or more components selected from the group consisting of: bleaching systems, bleaching activators, bleaching catalysts, silicates, and dyes.

8. The detergent composition of claim 5 or 6, wherein the composition is in the form of: powder, strip, uniform tablet, tablet having two or more layers, bag having one or more chambers, regular or compressed powder, granules, paste, gel, or regular, compressed or concentrated liquid.

9. The detergent composition of claim 5 or 6, wherein the amount of enzyme composition used in the detergent composition corresponds to the following: 0.001-200 mg of protein per liter of detergent solution, such as 0.005-100 mg of protein, preferably 0.01-50 mg of protein, more preferably 0.05-20 mg of protein, and even more preferably 0.1-10 mg of protein.

10. A method for reducing dust mites adhering to textiles, the method comprising: (1) The textile is washed or exposed with any of the detergent compositions as described in claims 5-9 or with any of the enzyme compositions as described in claims 2-4; (2) Rinse the textile as you see fit.

11. The method of claim 10, wherein the method is performed by hand or machine.

12. The method of claim 10 or 11, wherein the method is a washing or dishwashing method.

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