WO2022083538A1 - Use of polypeptide, detergent composition and cleaning method - Google Patents

Use of polypeptide, detergent composition and cleaning method Download PDF

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Publication number
WO2022083538A1
WO2022083538A1 PCT/CN2021/124368 CN2021124368W WO2022083538A1 WO 2022083538 A1 WO2022083538 A1 WO 2022083538A1 CN 2021124368 W CN2021124368 W CN 2021124368W WO 2022083538 A1 WO2022083538 A1 WO 2022083538A1
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Prior art keywords
polypeptide
seq
dnase
detergent composition
acid
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PCT/CN2021/124368
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French (fr)
Inventor
Qiang Zhao
Cheng Zhang
Zezhen Zhang
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Novozymes A/S
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Publication of WO2022083538A1 publication Critical patent/WO2022083538A1/en

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/0005—Other compounding ingredients characterised by their effect
    • C11D3/0068—Deodorant compositions
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • C11D2111/12—

Definitions

  • the present invention relates to the use of a polypeptide having deoxyribonuclease (DNase) activity for preventing, reducing and/or removing malodor from a porous item, a detergent composition comprising said polypeptide as well as a method for cleaning a porous item soiled with a biofilm and/or a food stain.
  • DNase deoxyribonuclease
  • Porous items such as kitchen sponges for washing dishware may become sticky and smelly after use. These porous items often have a porous structure and a rough surface. Stains e.g. minced meat can be stuck into the porous structure or stick to the rough surface and thus are difficult to remove. As a result, microorganisms may grow quickly on the porous items and produce biofilms and bad odor etc. The biofilms may be sticky and may furthermore adhere more soil/food stains due to the sticky nature. As a result, the porous item becomes more and more smelly and sticky, and serves as a source of bad odor.
  • the invention relates to the use of a polypeptide having deoxyribonuclease (DNase) activity for preventing, reducing and/or removing malodor from a porous item.
  • DNase deoxyribonuclease
  • it further concerns the use of said polypeptide having DNase activity for preventing, reducing and/or removing stickiness from the porous item.
  • the invention concerns a detergent composition
  • a detergent composition comprising a polypeptide having DNase activity and a surfactant, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from a porous item.
  • said polypeptide having DNase activity is capable of preventing, reducing and/or removing stickiness from the porous item.
  • the detergent composition further comprises a protease.
  • the present invention further relates to a method for cleaning a porous item soiled with a biofilm and/or a food stain, comprising steps of:
  • 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 towelling.
  • the textile may be cellulose based such as natural cellulosics, including cotton, flax/linen, jute, ramie, sisal or coir or manmade cellulosics (e.g.
  • 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 and spandex/elastane, or blends thereof as well as blends of cellulose based and non-cellulose based fibers.
  • 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 and spandex/elastane, or blends thereof as well as blends of cellulose based and non-cellulose based fibers.
  • blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fiber (e.g.
  • Porous item means any material having a porous structure which may be used for scrubbing or scratching a surface of a subject to facilitate cleaning.
  • the porous item may be manmade e.g. a sponge pad, or a natural porous item such as a cylindrical sponge of a mature luffa (used to clean dishware in some countries) , or an artificial porous item that simulates the luffa sponge structure (e.g. a luffa kitchen scrubber pad) .
  • the porous item may include a layer of other materials such as a textile mesh to provide a rougher and stiffer surface to scrub sticky and stiff stains off a subject e.g. a dish plate.
  • the porous item is a kitchen sponge made with two layers, i.e., one layer is a sponge and the other layer is a felt.
  • Biofilm A biofilm is any group of microorganisms in which cells stick to each other or stick to a surface, such as a textile, dishware or hard surface or other kind of surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS) .
  • EPS extracellular polymeric substance
  • Biofilm EPS is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces.
  • the microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single cells that may float or swim in a liquid medium.
  • Bacteria living in a biofilm usually have significantly different properties from planktonic bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways.
  • One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community.
  • Biofilm producing bacteria can be found among the following species: Acinetobacter sp., Aeromicrobium sp., Brevundimonas sp., Microbacterium sp., Micrococcus luteus, Pseudomonas sp., Staphylococcus epidermidis, and Stenotrophomonas sp.
  • Detergent components is defined herein to mean the types of chemicals which can be used in detergent compositions.
  • detergent components are alkalis, surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants and solubilizers.
  • the detergent composition may further contain one or more enzymes such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof.
  • enzymes such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof.
  • the detergent composition may further contain detergent components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase (s) , hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.
  • detergent components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase (s) , hydrolytic enzymes, oxido reduct
  • cleaning relates generally to both household cleaning (e.g. clothes laundering or dishwashing etc. ) and industrial cleaning or laundry. In the context of the present invention, it refers to the process of treating items (e.g. textiles or porous materials such as kitchen sponges) with a solution containing e.g. a cleaning or detergent composition of the present invention.
  • the cleaning process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand, e.g. hand laundry or dishwashing.
  • Wash liquor is defined herein as the solution or mixture of water and detergent components optionally including the polypeptide or the enzyme of the invention.
  • Wash performance One way of measuring the wash performance in the present invention is by the color change of a BTB solution, which is described in detail in Example 3 below.
  • Another way of measuring the wash performance in the present invention is to evaluate the size and the amount of the visible residual stain particles left on the surface of the washed porous item by e.g. naked eyes.
  • the term “bacterial DNase” or “polypeptide having DNase activity obtained from a bacterial source” or “polypeptide is of bacterial origin” thus refers to a DNase encoded by and thus directly derivable from the genome of a bacterial species, where the bacterial species has not been subjected to a genetic modification introducing recombinant DNA encoding said DNase.
  • the nucleotide sequence encoding the bacterial polypeptide having DNase activity is a sequence naturally in the genetic background of a bacterial species.
  • the bacterial polypeptide having DNase activity encoding by such sequence may also be referred to a wildtype DNase (or parent DNase) .
  • fungal in relation to polypeptide (such as an enzyme, e.g. a DNase) refers to a polypeptide encoded by and thus directly derivable from the genome of a fungus, where such fungus has not been genetically modified to encode said polypeptide, e.g. by introducing the encoding sequence in the genome by recombinant DNA technology.
  • polypeptide such as an enzyme, e.g. a DNase
  • Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA.
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • DNase means a polypeptide/enzyme with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA.
  • DNase activity is determined according to the procedure described in the Assay I.
  • Sequence identity The relatedness between two amino acid sequences is described by the parameter “sequence identity” .
  • host cell means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • Isolated means a substance in a form or environment that does not occur in nature.
  • isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance) .
  • An isolated substance may be present in a fermentation broth sample; e.g. a host cell may be genetically modified to express the polypeptide of the invention. The fermentation broth from that host cell will comprise the isolated polypeptide.
  • Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
  • the mature polypeptide is amino acids 38 to 243 of SEQ ID NO: 1, and amino acids 1 to 22 of SEQ ID NO: 1 are a signal peptide and amino acids 23 to 37 of SEQ ID NO: 1 are a propeptide. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
  • a mature polypeptides contains up to 206 (such as 204) consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., amino acids 38 to 243 of SEQ ID NO: 1 or amino acids 1 to 206 of SEQ ID NO: 2 or amino acids 1 to 204 of SEQ ID NO: 3) , or up to 204 amino acid residues (e.g., amino acids 40 to 243 of SEQ ID NO: 1) .
  • the mature polypeptide consists of the of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
  • the mature polypeptide comprises or consists of the consecutive amino acid residues 1 to 182 of SEQ ID NO: 4.
  • Nucleic acid construct means a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
  • variant means a polypeptide/enzyme having same activity as the parent enzyme comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position; and
  • an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
  • a variant of an identified DNase has the enzymatic activity of the parent, i.e.
  • the deoxyribonuclease activity of the variant is increased with reference to the parent DNase, e.g. the mature polypeptide of an enzyme having deoxyribonuclease activity is selected from the group consisting of an enzyme comprising or consisting of the mature polypeptide of SEQ ID NO: 1, an enzyme comprising or consisting of the sequence set forth in SEQ ID NO: 2, an enzyme comprising or consisting of the sequence set forth in SEQ ID NO: 3, an enzyme comprising or consisting of the mature polypeptide of SEQ ID NO: 4.
  • polypeptides having deoxyribonuclease (DNase) activity can be used for preventing/reducing or removing malodor from porous items such as kitchen sponges used for scrubbing and cleaning dishware.
  • Food stains may be stuck into the porous structure or stick to the rough surface of a porous item (e.g. a kitchen sponge) and thus are difficult to remove.
  • some microorganisms may grow quickly on the porous item and produce biofilms and bad odor etc.
  • the biofilms may be sticky and may furthermore adhere more soil/food stains due to the sticky nature.
  • the porous item becomes more and more smelly and sticky, and serves as a source of bad odor.
  • the commercial detergent compositions available on the market do not provide efficient solutions for the above issues.
  • the present invention aims to solve at least one of the above problems by providing a use of a polypeptide having DNase activity for preventing, reducing or removing malodor from a porous item soiled with e.g. a biofilm and/or a food stain.
  • the polypeptide having DNase activity relates to the use of said polypeptide for preventing, reducing or removing the stickiness of the porous item.
  • the polypeptide having DNase activity is selected from the group consisting of: a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the polypeptide having DNase activity is obtained from a fungal source, e.g. Aspergillus oryzae.
  • the polypeptide having DNase activity is obtained from a bacterial source, e.g. Bacillus cibi.
  • the porous item comprises a sponge, for example, is a kitchen sponge used for scrubbing a surface of e.g. a soiled dishware.
  • a kitchen sponge used for scrubbing a surface of e.g. a soiled dishware.
  • Any kitchen sponge available in the market e.g. a microfiber kitchen mesh sponge or a luffa kitchen sponge etc. ) may be used in the present invention.
  • the present invention further concerns a detergent composition comprising said polypeptide having DNase activity and a surfactant.
  • the present detergent composition can be used for preventing, reducing or removing malodor from a porous item, and for preventing, reducing or removing the stickiness of the porous item.
  • the present detergent composition overcomes the problems (e.g. malodour and/stickiness issues associated with a porous item) of the prior art.
  • a surfactant e.g. a nonionic surfactant in a detergent composition comprising said polypeptide is that the wash performance is improved.
  • the detergent composition further comprises one or more other detergent components selected from the group consisting of flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric hueing agents, anti-foaming agents, dispersants, processing aids, and/or pigments.
  • other detergent components selected from the group consisting of flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors
  • the detergent composition further comprises a protease, which is of animal, vegetable or microbial origin.
  • the protease may be chemically modified or protein engineered.
  • the protease can be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease.
  • the protease has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%sequence identity to the mature polypeptide shown in SEQ ID NO: 5.
  • the protease is the mature form of the polypeptide of SEQ ID NO: 5 with 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 of WO 2016/001449.
  • proteases are as described in the “additional enzymes” section set forth below. These proteases may be prepared by any suitable methods known in the art.
  • the present invention further concerns a method for cleaning a porous item soiled with a biofilm and/or a food stain, comprising the steps of: a) contacting the porous item with a wash liquor comprising a polypeptide having DNase activity or a detergent composition comprising a polypeptide having DNase activity and a surfactant; and b) optionally rinsing the porous item, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from the porous item. Further, the polypeptide having DNase activity is preferably capable of preventing, reducing and/or removing stickiness from the porous item.
  • the concentration of the DNase (the polypeptide having DNase activity) of the present invention is typically in the range of 0.005-200 ppm enzyme protein, such as in the range of 0.008-150, in the range of 0.01-100, in the range of 0.05-80, in the range of 0.1-50, in the range of 0.2-30, in the range of 0.5-20 ppm enzyme protein, in the range of 1-10 ppm enzyme protein, or in the range of 1.5-5 ppm enzyme protein in the wash liquor.
  • the DNase of the present invention may be added to a detergent composition in an amount corresponding to at least 0.002 mg of DNase protein, such as at least 0.005 mg of DNase protein, at least 0.08 mg of DNase protein, at least 0.1 mg of DNase protein, at least 0.2mg of DNase protein, at least 1 mg of protein, at least 5 mg of protein, at least 10 mg of protein, at least 15 mg of protein, at least 20 mg of protein, at least 25 mg of protein, at least 30 mg of protein, at least 50 mg of protein, per gram of detergent composition.
  • DNase protein such as at least 0.005 mg of DNase protein, at least 0.08 mg of DNase protein, at least 0.1 mg of DNase protein, at least 0.2mg of DNase protein, at least 1 mg of protein, at least 5 mg of protein, at least 10 mg of protein, at least 15 mg of protein, at least 20 mg of protein, at least 25 mg of protein, at least 30 mg of protein, at least 50 mg of protein, per gram of detergent composition.
  • the detergent composition may comprise at least 0.01%DNase protein, at least 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.8%, 1.0%, 2.0%, or 5.0%of DNase protein.
  • the DNase of the detergent composition of the invention 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, WO92/19709 and WO92/19708.
  • 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
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric
  • a polypeptide of the present invention may also be incorporated in the detergent compositions disclosed in WO97/07202, which is hereby incorporated by reference.
  • the detergent composition of the present invention may comprise one or more additional enzymes such as a protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, nuclease, oxidase, e.g., a laccase, and/or peroxidase.
  • additional enzymes such as a protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, nuclease, oxidase, e.g., a laccase, and/or peroxidase.
  • the properties of the selected enzyme (s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc. ) , and the enzyme (s) should be present in effective amounts.
  • Suitable proteases may be of any origin, but are preferably of bacterial or fungal origin, optionally in the form of protein engineered or chemically modified mutants.
  • the protease may be an alkaline protease, such as a serine protease or a metalloprotease.
  • a serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as a subtilisin.
  • a metalloprotease may for example be a thermolysin, e.g. from the M4 family, or another metalloprotease such as those from the M5, M7 or M8 families.
  • subtilases refers to a sub-group of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523.
  • Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate.
  • the subtilases may be divided into six subdivisions, the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • proteases suitable for detergent use may be obtained from a variety of organisms, including fungi such as Aspergillus
  • detergent proteases have generally been obtained from bacteria and in particular from Bacillus.
  • Bacillus species from which subtilases have been derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus gibsonii.
  • Particular subtilisins include subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and e.g. protease PD138 (described in WO 93/18140) .
  • Other useful proteases are e.g. those described in WO 01/16285 and WO 02/16547.
  • trypsin-like proteases examples 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.
  • 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.
  • proteases examples include 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, V
  • Protease variants having one or more of these mutations are preferably variants of the Bacillus lentus protease (also known as subtilisin 309) shown in SEQ ID NO: 1 of WO 2016/001449 or of the Bacillus amyloliquefaciens protease (BPN’) shown in SEQ ID NO: 2 of WO 2016/001449.
  • Bacillus lentus protease also known as subtilisin 309
  • BPN Bacillus amyloliquefaciens protease
  • Such protease variants preferably have at least 80%sequence identity to SEQ ID NO: 1 or to SEQ ID NO: 2 of WO 2016/001449.
  • 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 TY145 protease having SEQ ID NO: 1 of WO 2004/067737, 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: 1 of WO 2004/067737, wherein said protease variant has a sequence identity of at least 75%but less than 100%to SEQ ID NO: 1 of WO 2004/067737.
  • 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.
  • proteases examples include:
  • variants of SEQ ID NO: 1 of WO 2016/001449 comprising two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, for example a variant with the substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E, or with 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 of WO 2016/001449;
  • Suitable commercially available protease enzymes include those sold under the trade names Duralase TM , Durazym TM , Ultra, Ultra, Primase TM , Ultra, Ultra, Pro, Blaze 100T, Blaze 125T, Blaze 150T, Blaze 200T, Uno, In and Excel (Novozymes A/S) , those sold under the tradename Maxatase TM , Maxacal TM , Ox, OxP, FN2 TM , FN3 TM , FN4 exTM , Excellenz TM P1000, Excellenz TM P1250, Eraser TM , P100, Purafect Prime, Preferenz P110 TM , Effectenz P1000 TM , Effectenz P1050 TM , Ox, Effectenz TM P2000, Purafast TM , Opticlean TM and (Danisco/DuPont) , BLAP (sequence shown in Figure 29 of US 5352604) and variant
  • Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580) , lipase from strains of Pseudomonas (some of these now renamed to Burkholderia) , e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272) , P.
  • Thermomyces e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216
  • cutinase from Humicola e.g. H. insolens (WO96/135
  • lipase variants such as those described in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381, WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.
  • Preferred commercial lipase products include include Lipolase TM , Lipex TM ; Lipolex TM and Lipoclean TM (Novozymes A/S) , Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades) .
  • lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/111143) , acyltransferase from Mycobacterium smegmatis (WO05/56782) , perhydrolases from the CE 7 family (WO09/67279) , and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10/100028) .
  • Suitable amylases which can be used together with the DNase of the invention may be 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 in WO 95/10603 or variants having 90%sequence identity to SEQ ID NO: 3 thereof.
  • Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, 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.
  • 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.
  • 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.
  • Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264.
  • hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:
  • amylases which are suitable are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90%sequence identity to SEQ ID NO: 6.
  • Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.
  • Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.
  • Additional amylases which can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90%sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7.
  • Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96/023873 for numbering.
  • More preferred variants are those having a deletion in two positions selected from 181, 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184.
  • Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.
  • amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90%sequence identity to SEQ ID NO: 10 in WO 01/66712.
  • Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.
  • amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90%sequence identity to SEQ ID NO: 2 thereof.
  • Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475.
  • More preferred variants of SEQ ID NO: 2 are those having the substitution in one of more of the following positions: Q87E, R, Q98R, S125A, N128C, T131 I, T165I, K178L, T182G, M201 L, F202Y, N225E, R, N272E, R, S243Q, A, E, D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180 and/or S181 or of T182 and/or G183.
  • Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:
  • variants are C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.
  • amylases having SEQ ID NO: 1 of WO13184577 or variants having 90%sequence identity to SEQ ID NO: 1 thereof.
  • Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477.
  • More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K and/or deletion in position R178 and/or S179 or of T180 and/or G181.
  • Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
  • variants optionally further comprise a substitution at position 241 and/or a deletion at position 178 and/or position 179.
  • amylases having SEQ ID NO: 1 of WO10104675 or variants having 90%sequence identity to SEQ ID NO: 1 thereof.
  • Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: N21, D97, V128 K177, R179, S180, I181, G182, M200, L204, E242, G477 and G478.
  • More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: N21D, D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion in position R179 and/or S180 or of I181 and/or G182.
  • Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
  • variants optionally further comprise a substitution at position 200 and/or a deletion at position 180 and/or position 181.
  • amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90%sequence identity to SEQ ID NO: 12.
  • Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: 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.
  • Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.
  • amylase variants such as those described in WO2011/098531, WO2013/001078 and WO2013/001087.
  • amylases are Duramyl TM , Termamyl TM , Fungamyl TM , Stainzyme TM , Stainzyme Plus TM , Natalase TM , Liquozyme X and BAN TM (from Novozymes A/S) , and Rapidase TM , Purastar TM /Effectenz TM , Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. /DuPont) .
  • Suitable cellulases include mono-component and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein engineered mutants are also contemplated.
  • the cellulase may for example be a mono-component or a mixture of mono-component endo-1, 4-beta-glucanase also referred to as endoglucanase.
  • Suitable cellulases include those from the genera Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium.
  • Exemplary cellulases include a fungal cellulase from Humicola insolens (US 4, 435, 307) or from Trichoderma, e.g. T. reesei or T. viride.
  • Other suitable cellulases are from Thielavia e.g.
  • Thielavia terrestris as described in WO 96/29397 or the fungal cellulases produced from Myceliophthora thermophila and Fusarium oxysporum disclosed in US 5, 648, 263, US 5, 691, 178, US 5, 776, 757, WO 89/09259 and WO 91/17244.
  • cellulases from Bacillus as described in WO 02/099091 and JP 2000210081. Suitable cellulases are alkaline or neutral cellulases having care benefits. Examples of cellulases are described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • 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.
  • 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.
  • cellulases include Premium, Classic, (Novozymes A/S) , Puradax HA, and Puradax EG (available from Genencor International Inc. ) and KAC-500 (B) TM (Kao Corporation) .
  • 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) .
  • Suitable peroxidases/oxidases 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 TM (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.
  • IUBMB International Union of Biochemistry and Molecular Biology
  • 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.
  • 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.
  • Caldariomyces e.g., C. fumago
  • Alternaria Curvularia
  • 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 Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102; or from Drechslera hartlebii as described in WO 01/79459, Dendryphiella salina as described in WO 01/79458, Phaeotrichoconis crotalarie as described in WO 01/79461, or 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. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P.
  • papilionaceus Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporous, e.g., P. pinsitus, Phlebia, e.g., P. radiata (WO 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.
  • the protease (s) may be stabilized using compounds that act by temporarily reducing the proteolytic activity (reversible inhibitors) .
  • composition of the invention may also include a protease inhibitor/stabilizer, which is a reversible inhibitor of protease activity, e.g., serine protease activity.
  • a protease inhibitor/stabilizer which is a reversible inhibitor of protease activity, e.g., serine protease activity.
  • the protease inhibitor is a (reversible) subtilisin protease inhibitor.
  • the protease inhibitor may be a peptide aldehyde, boric acid, or a boronic acid; or a derivative of any of these.
  • the protease inhibitor may be a boronic acid or a derivative thereof; preferably, a phenylboronic acid or a derivative thereof.
  • the phenyl boronic acid derivative is of the following formula:
  • R is selected from the group consisting of hydrogen, hydroxy, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl and substituted C1-C6 alkenyl.
  • R is hydrogen, CH 3 , CH 3 CH 2 or CH 3 CH 2 CH 2 .
  • the protease inhibitor (phenyl boronic acid derivative) is 4-formyl-phenyl boronic acid (4-FPBA) .
  • the protease inhibitor is selected from the group consisting of thiophene-2 boronic acid, thiophene-3 boronic acid, acetamidophenyl boronic acid, benzofuran-2 boronic acid, naphtalene-1 boronic acid, naphtalene-2 boronic acid, 2-FPBA, 3-FBPA, 4-FPBA, 1-thianthrene boronic acid, 4-dibenzofuran boronic acid, 5-methylthiophene-2 boronic, acid, thionaphtrene boronic acid, furan-2 boronic acid, furan-3 boronic acid, 4, 4 biphenyl-diborinic acid, 6-hydroxy-2-naphtalene, 4- (methylthio) phenyl boronic acid, 4 (trimethyl-silyl) phenyl boronic acid, 3-bromothiophene boronic acid, 4-methylthiophene boronic acid, 2-naphtyl
  • boronic acid derivatives suitable as protease inhibitors in the detergent composition are described in US 4, 963, 655, US 5, 159, 060, WO 95/12655, WO 95/29223, WO 92/19707, WO 94/04653, WO 94/04654, US 5442100, US 5488157 and US 5472628.
  • the protease stabilizer may have the formula: P-A-L-B-B0-R*wherein:
  • A is absent if L is absent, or is 1 or 2 amino acid residues connected to L via the N-terminal; thus, A may represent A1 or A2-A1, where A2 and A1 each represent one amino acid residue;
  • R is independently selected from the group consisting of C 1-6 alkyl, C 6-10 aryl or C 7-10 arylalkyl, optionally substituted with one or more, identical or different, substituents R’;
  • R is a C 1-6 alkyl group
  • P is selected from the group consisting of hydrogen, or -if L is absent -an N-terminal protection group;
  • B0 may be a single amino acid residue with L-or D-configuration, which is connected to H via the C-terminal of the amino acid.
  • B0 are the D-or L-form of arginine (Arg) , 3, 4-dihydroxyphenylalanine, isoleucine (Ile) , leucine (Leu) , methionine (Met) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , m-tyrosine, p-tyrosine (Tyr) and valine (Val) .
  • a particular embodiment is when B0 is leucine, methionine, phenylalanine, p-tyrosine, or valine. Paticularly preferred is p-tyrosine.
  • B1 which is connected to B0 via the C-terminal of the amino acid, may be an aliphatic, hydrophobic and/or neutral amino acid.
  • B1 are alanine (Ala) , cysteine (Cys) , glycine (GIy) , isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , proline (Pro) , serine (Ser) , threonine (Thr) and valine (VaI) .
  • Particular examples of B1 are alanine, glycine, isoleucine, leucine and valine. A particular embodiment is when B1 is alanine, glycine, or valine.
  • B2 if present, is connected to B1 via the C-terminal of the amino acid, and may be an aliphatic, hydrophobic, neutral and/or polar amino acid.
  • B2 are alanine (Ala) , arginine (Arg) , capreomycidine (Cpd) , cysteine (Cys) , glycine (GIy) , isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , proline (Pro) , serine (Ser) , threonine (Thr) , and valine (VaI) .
  • B2 are alanine, arginine, capreomycidine, glycine, isoleucine, leucine, phenylalanine and valine.
  • a particular embodiment is when B2 is arginine, glycine, leucine, phenylalanine, or valine.
  • B3 if present, is connected to B2 via the C-terminal of the amino acid, and may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid.
  • B3 isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , phenylglycine, tyrosine (Tyr) , tryptophan (Trp) and valine (VaI) .
  • Particular examples of B3 are leucine, phenylalanine, tyrosine, and tryptophan.
  • A1 if present, is connected to L via the N-terminal of the amino acid, and may be an aliphatic, aromatic, hydrophobic, neutral and/or polar amino acid.
  • Examples of A1 are alanine (Ala) , arginine (Arg) , capreomycidine (Cpd) , glycine (GIy) , isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , threonine (Thr) , tyrosine (Tyr) , tryptophan (Trp) and valine (VaI) .
  • A1 are alanine, arginine, glycine, leucine, phenylalanine, tyrosine, tryptophan and valine.
  • B2 is leucine, phenylalanine, tyrosine or tryptophan.
  • A2 if present, is connected to A1 via the N-terminal of the amino acid, and may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid.
  • A2 are arginine (Arg) , isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , phenylglycine, Tyrosine (Tyr) , tryptophan (Trp) and valine (VaI) .
  • Particular examples of A2 are phenylalanine and tyrosine.
  • the N-terminal protection group P may be selected from formyl, acetyl (Ac) , benzoyl (Bz) , trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups such as fluorenylmethyloxycarbonyl (Fmoc) , methoxycarbonyl (Moc) , (fluoromethoxy) carbonyl, benzyloxycarbonyl (Cbz) , t-butyloxycarbonyl (Boc) and adamantyloxycarbonyl; p-methoxybenzyl carbonyl, benzyl (Bn) , p-methoxybenzyl (PMB) , p-methoxyphenyl (PMP) , methoxyacetyl, methylamino carbonyl, methylsulfonyl, ethylsulfonyl, benzylsulf
  • Suitable peptide aldehydes are described in WO94/04651, WO95/25791, WO98/13458, WO98/13459, WO98/13460, WO98/13461, WO98/13462, WO07/141736, WO07/145963, WO09/118375, WO10/055052 and WO11/036153.
  • the peptide aldehyde may be Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF 3 , Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF 3 , Moc-Val-Ala-Leu-CF 3 , Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Phe-CF 3 , Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-T
  • the protease stabilizer may be a hydrosulfite adduct of the peptide aldehyde or ketone described above, e.g., as described in WO 2013/004636.
  • the adduct may have the formula P-A-L-B-N(H) -CHR-CH (OH) -SO 3 M, wherein P, A, L, B, and R are defined as above, and M is H or an alkali metal, preferably Na or K.
  • An aqueous solution of the hydrosulfite adduct may be prepared by reacting the corresponding peptide aldehyde with an aqueous solution of sodium bisulfite (sodium hydrogen sulfite, NaHSO 3 ) ; potassium bisulfite (KHSO 3 ) by known methods, e.g., as described in WO 98/47523; US 6, 500, 802; US 5, 436, 229; J. Am. Chem. Soc. (1978) 100, 1228; Org. Synth., Coll. vol. 7: 361.
  • sodium bisulfite sodium hydrogen sulfite
  • KHSO 3 potassium bisulfite
  • Particularly preferred peptide aldehyde protease stabilizers have the formula P-B3-B2-B1-B0-H, or a hydrosulfite adduct having the formula P-B3-B2-B1-N (H) -CHR-CHOH-SO 3 M, wherein
  • B1 and B2 are independently single amino acid residues
  • iv) B3 is a single amino acid residue, or is absent
  • R is independently selected from the group consisting of C 1-6 alkyl, C 6-10 aryl or C 7-10 arylalkyl optionally substituted with one or more, identical or different, substituents R’;
  • R is a C 1-6 alkyl group
  • P is an N-terminal protection group, preferably methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz) ;
  • ix) M is H or an alkali metal, preferably Na or K.
  • the peptide aldehyde protease stabilizer has the formula P-B2-B1-B0-H or an adduct having the formula P-B2-B1-N (H) -CHR-CHOH-SO 3 M, wherein
  • B1 and B2 are independently single amino acid residues
  • R is independently selected from the group consisting of C 1-6 alkyl, C 6-10 aryl or C 7-10 arylalkyl optionally substituted with one or more, identical or different, substituents R’;
  • R is a C 1-6 alkyl group
  • P is an N-terminal protection group, preferably methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz) ;
  • M is H or an alkali metal, preferably Na or K.
  • B0, B1, B2, B3, and P are as described above.
  • P is preferably acetyl, methoxycarbonyl, benzyloxycarbonyl, methylamino carbonyl, methylsulfonyl, benzylsulfonyl and benzylphosphoramidyl.
  • P is preferably acetyl, methoxycarbonyl, methylsulfonyl, ethylsulfonyl and methylphosphoramidyl.
  • the molar ratio of the above-mentioned peptide aldehydes (or hydrosulfite adducts) to the protease may be at least 1: 1 or 1.5: 1, and it may be less than 1000: 1, more preferred less than 500: 1, even more preferred from 100: 1 to 2: 1 or from 20: 1 to 2: 1, or most preferred, the molar ratio is from 10: 1 to 2: 1.
  • Formate salts e.g., sodium formate
  • formic acid have also shown good effects as inhibitor of protease activity. Formate can be used synergistically with the above-mentioned protease inhibitors, as shown in WO 2013/004635.
  • the formate salts may be present in the composition in an amount of at least 0.1%w/w or 0.5%w/w, e.g., at least 1.0%, at least 1.2%or at least 1.5%. The amount is typically below 5%w/w, below 4%or below 3%.
  • the protease is a metalloprotease and the inhibitor is a metalloprotease inhibitor, e.g., a protein hydrolysate based inhibitor (e.g., as described in WO 2008/134343) .
  • a metalloprotease inhibitor e.g., a protein hydrolysate based inhibitor (e.g., as described in WO 2008/134343) .
  • the invention is directed to detergent compositions comprising a polypeptide having DNase activity of the present invention in combination with one or more additional cleaning composition components such as a surfactant.
  • additional cleaning composition components such as a surfactant.
  • additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
  • the 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.
  • the detergent composition includes a surfactant system (comprising more than one surfactant) e.g. a mixture of one or more nonionic surfactants and one or more anionic surfactants.
  • the detergent comprises at least one anionic surfactant than at least one non-ionic surfactant, the weight ratio of anionic to nonionic surfactant may be from 10: 1 to 1: 10.
  • the amount of anionic surfactant is higher than the amount of non-ionic surfactant e.g.
  • the weight ratio of anionic to non-ionic surfactant may be from 10: 1 to 1.1: 1 or from 5: 1 to 1.5: 1.
  • the amount of anionic to non-ionic surfactant may also be equal and the weight ratios 1: 1.
  • the amount of non-ionic surfactant is higher than the amount of anionic surfactant and the weight ratio may be 1: 10 to 1: 1.1.
  • the weight ratio of anionic to non-ionic surfactant is from 10: 1 to 1: 10, such as from 5: 1 to 1: 5, or from 5: 1 to 1: 1.2.
  • the weight fraction of non-ionic surfactant to anionic surfactant is from 0 to 0.5 or 0 to 0.2 thus non-ionic surfactant can be present or absent if the weight fraction is 0, but if non-ionic surfactant is present, then the weight fraction of the nonionic surfactant is preferably at most 50%or at most 20%of the total weight of anionic surfactant and non-ionic surfactant.
  • Light duty detergent usually comprises more nonionic than anionic surfactant and there the fraction of non-ionic surfactant to anionic surfactant is preferably from 0.5 to 0.9.
  • the total weight of surfactant (s) is typically present at a level of from about 0.1%to about 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 may include any conventional surfactant (s) known in the art.
  • the detergent When included therein the detergent will usually contain from about 1%to about 40%by weight of an 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 an anionic surfactant.
  • Non-limiting examples of anionic surfactants include sulfates and sulfonates, typically available as sodium or potassium salts or salts of monoethanolamine (MEA, 2-aminoethan-1-ol) or triethanolamine (TEA, 2, 2', 2”-nitrilotriethan-1-ol) ; in particular, linear alkylbenzenesulfonates (LAS) , isomers of LAS such as branched alkylbenzenesulfonates (BABS) and phenylalkanesulfonates; olefin sulfonates, in particular alpha-olefinsulfonates (AOS) ; alkyl sulfates (AS) , in particular fatty alcohol sulfates (FAS) , i.e., primary alcohol sulfates (PAS) such as dodecyl sulfate; alcohol ethersulfates (AES or AEOS or FES, also known as alcohol
  • the detergent When included therein the detergent will usually contain from about 1%to about 40%by weight of a cationic 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%, from about 8%to about 12% or from about 10%to about 12%.
  • a cationic 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%, from about 8%to about 12% or from about 10%to about 12%.
  • Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ) , cetyltrimethylammonium bromide (CTAB) , dimethyldistearylammonium chloride (DSDMAC) , and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.
  • ADMEAQ alkyldimethylethanolamine quat
  • CTAB cetyltrimethylammonium bromide
  • DMDMAC dimethyldistearylammonium chloride
  • AQA alkoxylated quaternary ammonium
  • the detergent When included therein the detergent will usually contain from about 0.2%to about 40%by weight of a nonionic 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%, from about 8%to about 12%, or from about 10%to about 12%.
  • a nonionic surfactant include alcohol ethoxylates (AE or AEO) e.g.
  • AEO-7 alcohol propoxylates, in particular propoxylated fatty alcohols (PFA) , ethoxylated and propoxylated alcohols, alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters (in particular methyl ester ethoxylates, MEE) , alkylpolyglycosides (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 (glucamides, GA, or fatty acid glucamides, FAGA) , as well as products available under the trade names SPAN and T
  • the detergent When included therein the detergent will usually contain from about 0.01 to about 10 %by weight of a semipolar surfactant.
  • semipolar surfactants include amine oxides (AO) such as alkyldimethylamine oxides, in particular N- (coco alkyl) -N, N-dimethylamine oxide and N-(tallow-alkyl) -N, N-bis (2-hydroxyethyl) amine oxide, and combinations thereof.
  • AO amine oxides
  • the detergent When included therein the detergent will usually contain from about 0.01 %to about 10 %by weight of a zwitterionic surfactant.
  • zwitterionic surfactants include betaines such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.
  • bio-based surfactants may be used e.g. wherein the surfactant is a sugar-based non-ionic surfactant which may be a hexyl- ⁇ -D-maltopyranoside, thiomaltopyranoside or a cyclic-maltopyranoside, such as described in EP2516606 B1.
  • the surfactant is a sugar-based non-ionic surfactant which may be a hexyl- ⁇ -D-maltopyranoside, thiomaltopyranoside or a cyclic-maltopyranoside, such as described in EP2516606 B1.
  • a hydrotrope is a compound that solubilizes hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment) .
  • hydrotropes have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants) ; however the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by Hodgdon and Kaler (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 miceller, lamellar or other well defined meso-phases.
  • hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases.
  • many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers.
  • Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications.
  • Use of hydrotropes in detergent compositions allow 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 detergent may contain 0-10%by weight, for example 0-5%by weight, such as about 0.5 to about 5%, or about 3%to about 5%, of a hydrotrope.
  • Any hydrotrope known in the art for use in detergents may be utilized.
  • Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluene sulfonate (STS) , sodium xylene sulfonate (SXS) , sodium cumene sulfonate (SCS) , sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.
  • the cleaning composition may contain 0-50%by weight, such as 1-40%, such as 1-30%, such as about 1%to about 20%, of a bleaching system.
  • a bleaching system Any oxygen-based bleaching system comprising components known in the art for use in cleaning detergents may be utilized. Suitable bleaching system components include sources of hydrogen peroxide; peracids and sources of peracids (bleach activators) ; and bleach catalysts or boosters.
  • Suitable sources of hydrogen peroxide are inorganic persalts, including alkali metal salts such as sodium percarbonate and sodium perborates (usually mono-or tetrahydrate) , and hydrogen peroxide ⁇ urea (1/1) .
  • Peracids may be (a) incorporated directly as preformed peracids or (b) formed in situ in the wash liquor from hydrogen peroxide and a bleach activator (perhydrolysis) or (c) formed in situ in the wash liquor from hydrogen peroxide and a perhydrolase and a suitable substrate for the latter, e.g., an ester.
  • Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids such as peroxybenzoic acid and its ring-substituted derivatives, peroxy- ⁇ -naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, ⁇ -phthalimidoperoxycaproic acid [phthalimidoperoxyhexanoic acid (PAP) ] , and o-carboxybenzamidoperoxycaproic acid; aliphatic and aromatic diperoxydicarboxylic acids such as diperoxydodecanedioic acid, diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, 2-decyldiperoxybutanedioic acid, and diperoxyphthalic, -isophthalic and -terephthalic acids; perimidic acids; peroxymonosulfuric acid; peroxydisulfuric acid;
  • Suitable bleach activators include those belonging to the class of esters, amides, imides, nitriles or anhydrides and, where applicable, salts thereof. Suitable examples are tetraacetylethylenediamine (TAED) , sodium 4- [ (3, 5, 5-trimethylhexanoyl) oxy] benzene-1-sulfonate (ISONOBS) , sodium 4- (dodecanoyloxy) benzene-1-sulfonate (LOBS) , sodium 4-(decanoyloxy) benzene-1-sulfonate, 4- (decanoyloxy) benzoic acid (DOBA) , sodium 4-(nonanoyloxy) benzene-1-sulfonate (NOBS) , and/or those disclosed in WO98/17767.
  • TAED tetraacetylethylenediamine
  • ISONOBS sodium 4- [ (3, 5, 5-trimethylhexanoyl) oxy]
  • ATC acetyl triethyl citrate
  • ATC or a short chain triglyceride like triacetin has the advantage that they are environmentally friendly.
  • acetyl triethyl citrate and triacetin have good hydrolytical stability in the product upon storage and are efficient bleach activators.
  • ATC is multifunctional, as the citrate released in the perhydrolysis reaction may function as a builder.
  • the bleaching system may also include a bleach catalyst or booster.
  • bleach catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1, 4, 7-trimethyl-1, 4, 7-triazacyclononane (Me3-TACN) or 1, 2, 4, 7-tetramethyl-1, 4, 7-triazacyclononane (Me4-TACN) , in particular Me3-TACN, such as the dinuclear manganese complex [ (Me3-TACN) Mn (O) 3Mn (Me3-TACN) ] (PF6) 2, and [2, 2', 2”-nitrilotris (ethane-1, 2-diylazanylylidene- ⁇ N-methanylylidene) triphenolato- ⁇ 3O] manganese (III) .
  • the bleach catalyst include manganes
  • an organic bleach catalyst or bleach booster may be used having one of the following formulae:
  • 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, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl.
  • Suitable photobleaches may for example be sulfonated zinc or aluminium phthalocyanines.
  • the detergent composition may contain about 0-65%by weight, such as about 5%to about 50%, 20-60%of a detergent builder or co-builder, or a mixture thereof.
  • the level of builder is typically in the range 40-65%, particularly in the range 50-65%.
  • the builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in cleaning 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 Clariant) , ethanolamines such as 2-aminoethan-1-ol (MEA) , diethanolamine (DEA, also known as 2, 2'-iminodiethan-1-ol) , triethanolamine (TEA, also known as 2, 2', 2”-nitrilotriethan-1-ol) , and (carboxymethyl) inulin (CMI) , and combinations thereof.
  • zeolites such as 2-aminoethan-1-ol (MEA) , diethanolamine (DEA, also known as 2, 2'-iminodiethan-1-ol) , triethanolamine (TEA, also known as 2, 2', 2”-nitrilotrie
  • the detergent composition may also contain from about 0-50%by weight, such as about 5%to about 30%, of a detergent co-builder.
  • the detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder.
  • 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.
  • NTA 2, 2’, 2” -nitrilotriacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • IDS iminodisuccinic acid
  • EDDS ethylenediamine-N, N’-disuccinic acid
  • MGDA methylglycinediacetic acid
  • GLDA glutamic acid-N, N-diacetic acid
  • HEDP ethylenediaminetetramethylenetetrakis
  • EDTMPA diethylenetriaminepentamethylenepentakis (phosphonic acid)
  • DTMPA or DTPMPA N- (2-hydroxyethyl) iminodiacetic acid
  • EDG 2, 2’, 2” -nitrilotriacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • IDS iminodisuccinic acid
  • the detergent composition may contain 0.005-10%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.
  • Exemplary polymers include (carboxymethyl) cellulose (CMC) , poly (vinyl alcohol) (PVA) , poly (ethyleneglycol) or poly (ethylene oxide) (PEG or PEO) , ethoxylated poly (ethyleneimine) , (carboxymethyl) inulin (CMI) , carboxylate polymers and d, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) , silicones, copolycarboxylates such as polyacrylates, maleic/acrylic acid copolymers, acrylate/styrene copolymers, poly (aspartic) acipolymers of terephthalic acid and oligomeric glycols, copolymers of poly (ethylene terephthalate) and poly (oxyethene terephthalate) (PET-POET) , poly (vinylpyrrolidone) (PVP) , poly (vinylim
  • Suitable examples include PVP-K15, PVP-K30, ChromaBond S-400, ChromaBond S-403E and Chromabond S-100 from Ashland Aqualon, and HP 165, HP 50 (Dispersing agent) , HP 53 (Dispersing agent) , HP 59 (Dispersing agent) , HP 56 (dye transfer inhibitor) , HP 66 K (dye transfer inhibitor) from BASF.
  • Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate.
  • Particularly preferred polymer is ethoxylated homopolymer HP 20 from BASF, which helps to prevent redeposition of soil in the wash liquor.
  • Further exemplary polymers include sulfonated polycarboxylates, ethylene oxide-propylene oxide copolymers (PEO-PPO) , copolymers of PEG with and vinyl acetate, and diquaternium ethoxy sulfate or quaternized sulfated ethoxylated hexamethylenediamine.
  • PEO-PPO ethylene oxide-propylene oxide copolymers
  • Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.
  • adjunct materials may include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrates/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.
  • the choice of such ingredients is well within the skill of the artisan.
  • the detergent compositions of the present invention can also contain dispersants.
  • powdered detergents may comprise dispersants.
  • Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.
  • Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.
  • the detergent compositions of the present invention may also include one or more dye transfer inhibiting agents.
  • Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
  • 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.
  • the detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01%to about 0.5%.
  • Any fluorescent whitening agent suitable for use in a cleaning 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-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.
  • diaminostilbene-sulfonic 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'-disulfonate, 4, 4'-bis- (2, 4-dianilino-s-triazin-6-ylamino) stilbene-2.2'-disulfonate, 4, 4'-bis- (2-anilino-4- (N-methyl-N-2-hydroxy-ethylamino) -s-triazin-6-ylamino) stilbene-2, 2'-disulfonate, 4, 4'-bis- (4-phenyl-1, 2, 3-triazol-2-yl) stilbene-2, 2'-disulfonate and sodium 5- (2H-naphtho [1, 2-d] [1, 2, 3] triazol-2-yl) -2- [ (E) -2-phenylvinyl)
  • 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-2, 2'-disulfonate.
  • Tinopal CBS is the disodium salt of 2, 2'-bis- (phenyl-styryl) -disulfonate.
  • fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India.
  • Other fluorescers suitable for use in the invention include the 1-3-diaryl pyrazolines and the 7-alkylaminocoumarins.
  • Suitable fluorescent brightener levels include lower levels of from about 0.01, from 0.05, from about 0.1 or even from about 0.2 wt %to upper levels of 0.5 or even 0.75 wt%.
  • the detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics.
  • the soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.
  • Another type of soil release polymers are 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) .
  • random graft co-polymers are suitable soil release polymers. Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference) .
  • Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in EP 1867808 or WO 2003/040279 (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.
  • the detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC) , polyvinyl alcohol (PVA) , polyvinylpyrrolidone (PVP) , polyoxyethylene and/or polyethyleneglycol (PEG) , homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines.
  • CMC carboxymethylcellulose
  • PVA polyvinyl alcohol
  • PVP polyvinylpyrrolidone
  • PEG polyethyleneglycol
  • homopolymers of acrylic acid copolymers of acrylic acid and maleic acid
  • ethoxylated polyethyleneimines ethoxylated polyethyleneimines.
  • the cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
  • the detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • Pouches can be configured as single or multicompartments. They can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact.
  • the pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch.
  • Preferred films are polymeric materials preferably polymers which are formed into a film or sheet.
  • Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC) .
  • the level of polymer in the film for example PVA is at least about 60%.
  • Preferred average molecular weight will typically be about 20,000 to about 150,000.
  • Films can also be of blended compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticisers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof.
  • the pouches can comprise a solid cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water soluble film.
  • the compartment for liquid components can be different in composition than compartments containing solids: US2009/0011970 A1.
  • Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
  • a liquid or gel detergent which is not unit dosed, may be aqueous, typically containing at least 20%by weight and up to 95%water, such as up to about 70%water, up to about 65%water, up to about 55%water, up to about 45%water, up to about 35%water.
  • Other types of liquids including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel.
  • An aqueous liquid or gel detergent may contain from 0-30%organic solvent.
  • a liquid or gel detergent may be non-aqueous.
  • the enzyme of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles.
  • laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars.
  • the types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps.
  • the laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature.
  • the term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in.
  • the bar is a solid typically in bar form but can be in other solid shapes such as round or oval.
  • the laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct) , boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na + , K + or NH 4 + and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.
  • protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or
  • the laundry soap bar may also contain complexing agents like EDTA and HEDP, perfumes and/or different type of fillers, surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
  • the laundry soap bar may be processed in conventional laundry soap bar making equipment such as but not limited to: mixers, plodders, e.g a two stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers.
  • the invention is not limited to preparing the laundry soap bars by any single method.
  • the premix of the invention may be added to the soap at different stages of the process.
  • the premix containing a soap, a DNase of the present invention, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and and the mixture is then plodded.
  • the DNase of the present invention and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form.
  • the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.
  • the enzyme of the invention may be formulated as a granule for example as a co-granule that combines one or more enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of enzymes in the detergent. This also reduces the physical segregation of different enzymes due to different particle sizes.
  • Methods for producing multi-enzyme co-granulates for the detergent industry are disclosed in the IP. com disclosure IPCOM000200739D.
  • WO 2013/188331 Another example of formulation of enzymes by the use of co-granulates are disclosed in WO 2013/188331, which relates to a detergent composition comprising (a) a multi-enzyme co-granule; (b) less than 10 wt zeolite (anhydrous basis) ; and (c) less than 10 wt phosphate salt (anhydrous basis) , wherein said enzyme co-granule comprises from 10 to 98 wt%moisture sink component and the composition additionally comprises from 20 to 80 wt%detergent moisture sink component.
  • WO 2013/188331 also relates to a method of treating and/or cleaning a surface, preferably a fabric surface comprising the steps of (i) contacting said surface with the detergent composition as claimed and described herein in an aqueous wash liquor, (ii) rinsing and/or drying the surface.
  • the multi-enzyme co-granule may comprise an enzyme of the invention and (a) one or more enzymes selected from the group consisting of first-wash lipases, cleaning cellulases, xyloglucanases, perhydrolases, peroxidases, lipoxygenases, laccases and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, proteases, care cellulases, cellobiose dehydrogenases, xylanases, phospho lipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, amy
  • DNase deoxyribonuclease
  • polypeptide having DNase activity is capable of preventing, reducing or removing stickiness of the porous item.
  • polypeptide having DNase activity is selected from the group consisting of: a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • polypeptide having DNase activity is from Aspergillus or Bacillus.
  • polypeptide having DNase activity is from Aspergillus oryzae or Bacillus cibi.
  • a detergent composition comprising a polypeptide having deoxyribonuclease (DNase) activity and a surfactant, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from a porous item.
  • DNase deoxyribonuclease
  • polypeptide having DNase activity is selected from the group consisting of: a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the detergent composition further comprises one or more components selected from the group consisting of flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric huing agents, anti-foaming agents, dispersants, processing aids, and/or pigments.
  • the detergent composition according to paragraph 16 wherein the enzyme is selected from the group consisting of proteases, amylases, lipases, phospholipases, esterases, cellulases, xylanases, mannanases, pullulanases, malanases, ⁇ -glucanases, arabinosidases, peroxidases, xanthanase and mixtures thereof.
  • the enzyme is selected from the group consisting of proteases, amylases, lipases, phospholipases, esterases, cellulases, xylanases, mannanases, pullulanases, malanases, ⁇ -glucanases, arabinosidases, peroxidases, xanthanase and mixtures thereof.
  • a method for cleaning a porous item soiled with a biofilm and/or a food stain comprising steps of:
  • polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from the porous item.
  • DNase activity was determined on DNase Test Agar with Methyl Green (BD, Franklin Lakes, NJ, USA) , which was prepared according to the manual from supplier. Briefly, 21 g of agar was dissolved in 500 ml water and then autoclaved for 15 min at 121°C. Autoclaved agar was temperated to 48°C in water bath, and 20 ml of agar was poured into petri dishes and allowed to solidify by incubation o/n at room temperature. On solidified agar plates, 5 ⁇ l of enzyme solutions are added, and DNase activity are observed as colorless zones around the spotted enzyme solutions.
  • Methyl Green Methyl Green
  • Aspergillus oryzae DNase mature form of the polypeptide of SEQ ID NO: 1 (i.e. SEQ ID NO: 1 in WO 2017/060518) .
  • Bacillus Protease mature form of the polypeptide of SEQ ID NO: 5 with 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 of WO 2016/001449.
  • Fairy Original (detergent 1) a commercial hand dish wash detergent bought from P&G, containing 20.5%Sulphates, 3.0%Nonionics, 62.5%Water, 0.80%Ethanol, 0.10%Phenoxyethanol, 6.0%Amine Oxides, etc.
  • Natural Dish Liquid Free &Clear (detergent 2) : a commercial hand dish wash detergent purchased from Seventh Generation, containing 79.5%water, 16.2%sodium lauryl sulfate, 2.0%Glycerin, 2.11%auramine oxide, 0.07%caprylyl/myristyl glucoside, 0.08%magnesium chloride, 0.05%citric acid, 0.03%Benzisothiazolinone, 0.04%Methylisothiazolinone.
  • 20 g of defatted beef was ground together with 20 g of defatted pork in a meat grinder (BRAUN Power Plus 1300, using minimal filter pore) for two rounds.
  • the defatted pork or beef was prepared by cutting off the fatty part with a kitchen knife.
  • 3 g of the minced meat mixture was spread evenly on a dish plate by using a massage cushion (Inomata Round Shampoo Brush) .
  • the plate soiled with the minced meat mixture was dried overnight at room temperature followed by heating at 140°C for 2 hours in an oven (Thermo Electron LED GmbH, Thermo Scientific) .
  • the soiled pate was cooled down to room temperature before the wash test.
  • One soiled plate was soaked for 2 min in the wash liquor of detergent only, detergent with DNase, detergent with protease, or detergent with both DNase and protease, as prepared above. Then the soiled plate was scrubbed with the green or yellow side of the kitchen sponge (3M G6215, the green side is made with a felt and the yellow side is made with a sponge) until substantially all stain was removed away from the soiled plate.
  • yellow side i.e. the softer side
  • the green side (i.e. the rougher and stiffer side) of the kitchen sponge was used to scrub meat stains and fish stains, as these two stains are harder to remove.
  • the same sponge was used to scrub one more plate which was soiled with the same stain and soaked for 2 min in the same wash liquor. Then the sponge was rinsed under running tap water along with 5 times squeezing. 3 repetitions were carried out for each stain type.
  • each kitchen sponge was photographed for subsequent visual evaluation of residual stain particles left on the sponge (more specifically on the side for scrubbing stains) . Afterwards, each kitchen sponge was put into a Ziplock plastic bag and sealed for the following tests.
  • the rinsed sponge was sealed in a Ziplock plastic bag and incubated overnight at room temperature. Microorganisms will grow during incubation and produce bad odor (malodor) .
  • the malodor of each sponge after incubation was measured by POLFA, an odor sensor device bought from KALMOR company. For detecting malodor, the Ziplock bag was opened slightly to let the sensor of POLFA sense the sponge for about 400 seconds at which the malodor level reached plateau.
  • the malodor value was recorded by POLFA device automatically and can be read from the display window of POLFA device.
  • the malodor removal benefit of enzyme (s) was evaluated by malodor reduction percentage (%) , which is calculated by following formula.
  • V1 malodor value at 400 seconds of the sponge washed with detergent without enzyme (i.e. detergent only)
  • V2 malodor value at 400 seconds of the sponge washed with detergent plus enzyme
  • Each panelist was presented with four sponge samples from the four test conditions (i.e., detergent only, detergent+protease, detergent+DNase, detergent+DNase+protease) in a random order and was asked to rank the four sponges according to the intensity of their malodor. Then the four sponge samples were placed in the order from the weakest to the strongest malodor and were scored by the six panelists. Score range was from 1 to 4. A score of 1, 2, 3 and 4 indicates minor, small, medium and big difference in terms of malodor intensity, respectively.
  • the sensory odor value for the first sample (i.e. the one having the weakest malodor among the four samples) was preset as 1.
  • the fourth sponge samples were scored in the same way.
  • the average sensory odor value for sponges washed for egg stain and meat stain are summarized in below table 2. A higher average sensory odor value represents a stronger malodor felt by the panel group.
  • LB liquid (10g/L Tryptone, 5 g/L yeast extract and 10g/L NaCl) was 1: 1 diluted with Mili Q water. 10g/L lactose and 0.17g/L Bromothymol Blue was added to the diluted LB liquid and stirred for 10 mins by using magnetic stirring apparatus (IKAMAG, rdeq-fs-0133) . The pH of the resulted solution was adjusted to 8.6 with 1 M NaOH to obtain a BTB solution having a blue color.
  • IKAMAG magnetic stirring apparatus
  • the BTB solution can change color with the change of acidity, i.e., the BTB solution shows a blue color when it is alkaline, a yellow color at an acidic pH, and a green color at a neutral pH.
  • the BTB solution containing sponges washed by detergent without enzyme changed from blue to green gradually and after 8 hours became a totally yellow-orange color.
  • the BTB solution showed a bluish green color.
  • those washed by protease a yellowish green color was observed.
  • the size of the residual particles washed with enzyme (s) were in general smaller than those washed without enzyme (s) .
  • one of the possible mechanisms for the observed malodor removal benefit of the DNase might be that the DNase of the present invention disrupted more stains, especially those associated with bad odors such as biofilms, into smaller particles which can be rinsed away easily from the porous/rough surface of the porous items.
  • the DNase of the present invention disrupted more stains, especially those associated with bad odors such as biofilms, into smaller particles which can be rinsed away easily from the porous/rough surface of the porous items.

Abstract

Provided are the use of a polypeptide having deoxyribonuclease (DNase) activity for preventing, reducing and/or removing malodor from a porous item, a detergent composition comprising said polypeptide and a method for cleaning a porous item soiled with a biofilm and/or a food stain.

Description

USE OF POLYPEPTIDE, DETERGENT COMPOSITION AND CLEANING METHOD
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 the use of a polypeptide having deoxyribonuclease (DNase) activity for preventing, reducing and/or removing malodor from a porous item, a detergent composition comprising said polypeptide as well as a method for cleaning a porous item soiled with a biofilm and/or a food stain.
Background of Invention
Porous items such as kitchen sponges for washing dishware may become sticky and smelly after use. These porous items often have a porous structure and a rough surface. Stains e.g. minced meat can be stuck into the porous structure or stick to the rough surface and thus are difficult to remove. As a result, microorganisms may grow quickly on the porous items and produce biofilms and bad odor etc. The biofilms may be sticky and may furthermore adhere more soil/food stains due to the sticky nature. As a result, the porous item becomes more and more smelly and sticky, and serves as a source of bad odor.
International patent application no. WO 2017/060518 discloses a laundering method, where a DNase of fungal origin is used for washing textile (clothing) . Compared to textile (clothing) , porous items used for e.g. washing dishware have a quite different texture and are used in a different environment/situation. The commercial detergent compositions available on the market do not provide efficient solutions for such issues.
Summary of the Invention
The invention relates to the use of a polypeptide having deoxyribonuclease (DNase) activity for preventing, reducing and/or removing malodor from a porous item. In one aspect of the invention, it further concerns the use of said polypeptide having DNase activity for preventing, reducing and/or removing stickiness from the porous item.
In addition, the invention concerns a detergent composition comprising a polypeptide having DNase activity and a surfactant, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from a porous item. In one aspect of the invention, said polypeptide having DNase activity is capable of preventing, reducing and/or removing stickiness from  the porous item. In one embodiment of the invention, the detergent composition further comprises a protease.
The present invention further relates to a method for cleaning a porous item soiled with a biofilm and/or a food stain, comprising steps of:
a) Contacting the porous item with a wash liquor comprising a polypeptide having DNase activity or a detergent composition comprising a polypeptide having DNase activity and a surfactant; and
b) Optionally rinsing the porous item,
wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from the porous item. In one aspect of the invention, said polypeptide having DNase activity is further capable of preventing, reducing and/or removing stickiness from a porous item.
Definitions
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 towelling. 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 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 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) . Fabric may be conventional washable laundry, for example stained household laundry. When the term fabric or garment is used it is intended to include the broader term textiles as well. In the context of the present invention, the term “textile” also covers fabrics.
Porous item: The term “porous item” means any material having a porous structure which may be used for scrubbing or scratching a surface of a subject to facilitate cleaning. The porous item may be manmade e.g. a sponge pad, or a natural porous item such as a cylindrical sponge of a mature luffa (used to clean dishware in some countries) , or an artificial porous item that simulates  the luffa sponge structure (e.g. a luffa kitchen scrubber pad) . The porous item may include a layer of other materials such as a textile mesh to provide a rougher and stiffer surface to scrub sticky and stiff stains off a subject e.g. a dish plate. In one embodiment of the present invention, the porous item is a kitchen sponge made with two layers, i.e., one layer is a sponge and the other layer is a felt.
Biofilm: A biofilm is any group of microorganisms in which cells stick to each other or stick to a surface, such as a textile, dishware or hard surface or other kind of surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS) . Biofilm EPS is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces. The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single cells that may float or swim in a liquid medium.
Bacteria living in a biofilm usually have significantly different properties from planktonic bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community.
Biofilm producing bacteria can be found among the following species: Acinetobacter sp., Aeromicrobium sp., Brevundimonas sp., Microbacterium sp., Micrococcus luteus, Pseudomonas sp., Staphylococcus epidermidis, and Stenotrophomonas sp.
Detergent components: The term “detergent components” is defined herein to mean the types of chemicals which can be used in detergent compositions. Examples of detergent components are alkalis, surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants and solubilizers.
Detergent composition: The term “detergent composition” refers to compositions that find use in the removal of undesired compounds from items to be cleaned. The detergent composition may be used to e.g. clean items for both household cleaning and industrial cleaning. The terms encompass any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid detergents and fine fabric detergents; fabric fresheners; fabric softeners; and textile and laundry pre-spotters/pretreatment) . In addition to said polypeptide having DNase activity of the present invention, the detergent composition may further contain one or more enzymes such as proteases, amylases,  lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof. The detergent composition may further contain detergent components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase (s) , hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.
Cleaning: The term “cleaning” relates generally to both household cleaning (e.g. clothes laundering or dishwashing etc. ) and industrial cleaning or laundry. In the context of the present invention, it refers to the process of treating items (e.g. textiles or porous materials such as kitchen sponges) with a solution containing e.g. a cleaning or detergent composition of the present invention. The cleaning process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand, e.g. hand laundry or dishwashing.
Wash liquor: The term “wash liquor” is defined herein as the solution or mixture of water and detergent components optionally including the polypeptide or the enzyme of the invention.
Wash performance: One way of measuring the wash performance in the present invention is by the color change of a BTB solution, which is described in detail in Example 3 below.
Another way of measuring the wash performance in the present invention is to evaluate the size and the amount of the visible residual stain particles left on the surface of the washed porous item by e.g. naked eyes.
Malodor: The term “malodor” refers to an odor which is not desired on cleaned items. The cleaned item should smell fresh and clean without malodor adhered to the item. One example of malodor is compounds with an unpleasant smell, which may be produced by microorganisms. Another example is unpleasant smells can be sweat or body odor adhered to an item which has been in contact with human or animal. Another example of malodor can be the odor from spices, which sticks to items for example curry or other exotic spices which smell strongly. For the purpose of the present invention, the malodor level may be measured according to Example 1 or Example 3 set forth below.
Deep cleaning: The term “deep cleaning” means disruption or removal of biosoils e.g. a food stain, a biofilm or components of a biofilm such as polysaccharides, proteins, DNA, soil or other components present in the biofilm, or a mixture thereof.
Enzyme Detergency Benefit: The term “enzyme detergency benefit” is defined herein as the advantageous effect an enzyme may add to a detergent compared to the same detergent without the enzyme. Important detergency benefits which can be provided by enzymes are stain removal with no  or very little visible soils after washing and/or cleaning, prevention or reduction of redeposition of soils released in the washing process (an effect that also is termed anti-redeposition) , restoring fully or partly the whiteness of textiles which originally were white but after repeated use and wash have obtained a greyish or yellowish appearance (an effect that also is termed whitening) .
Textile care benefit: Textile care benefits, which are not directly related to catalytic stain removal or prevention of redeposition of soils, are also important for enzyme detergency benefits. Examples of such textile care benefits are prevention or reduction of dye transfer from one textile to another textile or another part of the same textile (an effect that is also termed dye transfer inhibition or anti-backstaining) , removal of protruding or broken fibers from a textile surface to decrease pilling tendencies or remove already existing pills or fuzz (an effect that also is termed anti-pilling) , improvement of the textile-softness, color clarification of the textile and removal of particulate soils which are trapped in the fibers of the textile. Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyze the formation of bleaching component such as hydrogen peroxide or other peroxides or other bleaching species.
Bacterial: In the context of the present invention, the term “bacterial” in relation to polypeptide (such as an enzyme, e.g. a DNase) refers to a polypeptide encoded by and thus directly derivable from the genome of a bacteria, where such bacteria has not been genetically modified to encode said polypeptide, e.g. by introducing the encoding sequence in the genome by recombinant DNA technology. In the context of the present invention, the term “bacterial DNase” or “polypeptide having DNase activity obtained from a bacterial source” or “polypeptide is of bacterial origin” thus refers to a DNase encoded by and thus directly derivable from the genome of a bacterial species, where the bacterial species has not been subjected to a genetic modification introducing recombinant DNA encoding said DNase. Thus, the nucleotide sequence encoding the bacterial polypeptide having DNase activity is a sequence naturally in the genetic background of a bacterial species. The bacterial polypeptide having DNase activity encoding by such sequence may also be referred to a wildtype DNase (or parent DNase) . In a further aspect, the invention provides polypeptides having DNase activity, wherein said polypeptides are substantially homologous to a bacterial DNase. In the context of the present invention, the term “substantially homologous” denotes a polypeptide having DNase activity which is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, and most preferably at least 99%identical to the amino acid sequence of a selected bacterial DNase.
Fungal: In the context of the present invention the term “fungal” in relation to polypeptide (such as an enzyme, e.g. a DNase) refers to a polypeptide encoded by and thus directly derivable from the genome of a fungus, where such fungus has not been genetically modified to encode said polypeptide, e.g. by introducing the encoding sequence in the genome by recombinant DNA  technology. In the context of the present invention, the term “fungal DNase” or “polypeptide having DNase activity obtained from a fungal source” or “polypeptide is of fungal origin” thus refers to a DNase encoded by and thus directly derivable from the genome of a fungal species, where the fungal species has not been subjected to a genetic modification introducing recombinant DNA encoding said DNase. Thus, the nucleotide sequence encoding the fungal polypeptide having DNase activity is a sequence naturally in the genetic background of a fungal species. The fungal polypeptide having DNase activity encoding by such sequence may also be referred to a wildtype DNase (or parent DNase) . In a further aspect, the invention provides polypeptides having DNase activity, wherein said polypeptides are substantially homologous to a fungal DNase. In the context of the present invention, the term “substantially homologous” denotes a polypeptide having DNase activity which is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, and most preferably at least 99%identical to the amino acid sequence of a selected fungal DNase.
Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
DNase: The term “DNase” means a polypeptide/enzyme with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. For purposes of the present invention, DNase activity is determined according to the procedure described in the Assay I. In one embodiment of the present invention, the DNase activity of a polypeptide having DNase activity is at least 105%, e.g., at least 110%, at least 120%, at least 130%, at least 140%, at least 160%, at least 170%, at least 180%, or at least 200%with reference to the DNase activity of the mature polypeptide of SEQ ID NO: 1, an enzyme comprising or consisting of the sequence set forth in SEQ ID NO: 2, an enzyme comprising or consisting of the sequence set forth in SEQ ID NO: 3, or an enzyme comprising or consisting of the mature polypeptide of SEQ ID NO: 4. The DNase of the present invention may be prepared according to the method described in WO 2015/155350 or WO 2017/060518. Other methods known in the art may also be used to obtain the DNase of the present invention.
Sequence identity: The relatedness between two amino acid 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) 
Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Isolated: The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance) . An isolated substance may be present in a fermentation broth sample; e.g. a host cell may be genetically modified to express the polypeptide of the invention. The fermentation broth from that host cell will comprise the isolated polypeptide.
Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one embodiment, the mature polypeptide is amino acids 38 to 243 of SEQ ID NO: 1, and amino acids 1 to 22 of SEQ ID NO: 1 are a signal peptide and amino acids 23 to 37 of SEQ ID NO: 1 are a propeptide. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide. In one embodiment, a mature polypeptides contains up to 206 (such as 204) consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (e.g.,  amino acids 38 to 243 of SEQ ID NO: 1 or amino acids 1 to 206 of SEQ ID NO: 2 or amino acids 1 to 204 of SEQ ID NO: 3) , or up to 204 amino acid residues (e.g., amino acids 40 to 243 of SEQ ID NO: 1) . In another embodiment, the mature polypeptide consists of the of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In yet another embodiment, the mature polypeptide comprises or consists of the consecutive amino acid residues 1 to 182 of SEQ ID NO: 4.
Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
Variant: The term “variant” means a polypeptide/enzyme having same activity as the parent enzyme comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. In the context of the present invention, a variant of an identified DNase has the enzymatic activity of the parent, i.e. the capacity of catalyzing the hydrolytic cleavage of phosphodiester linkages in the DNA backbone (deoxyribonuclease activity) . In one embodiment, the deoxyribonuclease activity of the variant is increased with reference to the parent DNase, e.g. the mature polypeptide of an enzyme having deoxyribonuclease activity is selected from the group consisting of an enzyme comprising or consisting of the mature polypeptide of SEQ ID NO: 1, an enzyme comprising or consisting of the sequence set forth in SEQ ID NO: 2, an enzyme comprising or consisting of the sequence set forth in SEQ ID NO: 3, an enzyme comprising or consisting of the mature polypeptide of SEQ ID NO: 4.
Detailed Description of the Invention
The inventors have found that polypeptides having deoxyribonuclease (DNase) activity can be used for preventing/reducing or removing malodor from porous items such as kitchen sponges used for scrubbing and cleaning dishware. Food stains may be stuck into the porous structure or stick to the rough surface of a porous item (e.g. a kitchen sponge) and thus are difficult to remove. As a result, some microorganisms may grow quickly on the porous item and produce biofilms and bad odor etc. The biofilms may be sticky and may furthermore adhere more soil/food stains due to the sticky nature. As a result, the porous item becomes more and more smelly and sticky, and serves as a source of bad odor. The commercial detergent compositions available on the market do not provide efficient solutions for the above issues.
The present invention aims to solve at least one of the above problems by providing a use  of a polypeptide having DNase activity for preventing, reducing or removing malodor from a porous item soiled with e.g. a biofilm and/or a food stain. In one embodiment of the invention the polypeptide having DNase activity relates to the use of said polypeptide for preventing, reducing or removing the stickiness of the porous item.
In one embodiment, the polypeptide having DNase activity is selected from the group consisting of: a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment of the invention, the polypeptide having DNase activity is obtained from a fungal source, e.g. Aspergillus oryzae. In another embodiment, the polypeptide having DNase activity is obtained from a bacterial source, e.g. Bacillus cibi.
In one embodiment of the invention, the porous item comprises a sponge, for example, is a kitchen sponge used for scrubbing a surface of e.g. a soiled dishware. Any kitchen sponge available in the market (e.g. a microfiber kitchen mesh sponge or a luffa kitchen sponge etc. ) may be used in the present invention.
The present invention further concerns a detergent composition comprising said polypeptide having DNase activity and a surfactant. The present detergent composition can be used for preventing, reducing or removing malodor from a porous item, and for preventing, reducing or removing the stickiness of the porous item. The present detergent composition overcomes the problems (e.g. malodour and/stickiness issues associated with a porous item) of the prior art. By including a surfactant e.g. a nonionic surfactant in a detergent composition comprising said polypeptide is that the wash performance is improved.
In one embodiment of the invention, the detergent composition further comprises one or more other detergent components selected from the group consisting of flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric hueing agents, anti-foaming agents, dispersants, processing aids, and/or pigments.
In one embodiment of the invention, the detergent composition further comprises a protease, which is of animal, vegetable or microbial origin. The protease may be chemically modified or protein engineered. The protease can be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease.
In one embodiment of the invention, the protease has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%sequence identity to the mature polypeptide shown in SEQ ID NO: 5.
In another embodiment of the invention, the protease is the mature form of the polypeptide of SEQ ID NO: 5 with 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 of WO 2016/001449.
Other suitable proteases are as described in the “additional enzymes” section set forth below. These proteases may be prepared by any suitable methods known in the art.
The present invention further concerns a method for cleaning a porous item soiled with a biofilm and/or a food stain, comprising the steps of: a) contacting the porous item with a wash liquor comprising a polypeptide having DNase activity or a detergent composition comprising a polypeptide having DNase activity and a surfactant; and b) optionally rinsing the porous item, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from the porous item. Further, the polypeptide having DNase activity is preferably capable of preventing, reducing and/or removing stickiness from the porous item.
Enzyme of the present invention
The concentration of the DNase (the polypeptide having DNase activity) of the present invention is typically in the range of 0.005-200 ppm enzyme protein, such as in the range of 0.008-150, in the range of 0.01-100, in the range of 0.05-80, in the range of 0.1-50, in the range of 0.2-30, in the range of 0.5-20 ppm enzyme protein, in the range of 1-10 ppm enzyme protein, or in the range of 1.5-5 ppm enzyme protein in the wash liquor.
The DNase of the present invention may be added to a detergent composition in an amount corresponding to at least 0.002 mg of DNase protein, such as at least 0.005 mg of DNase protein, at least 0.08 mg of DNase protein, at least 0.1 mg of DNase protein, at least 0.2mg of DNase protein, at least 1 mg of protein, at least 5 mg of protein, at least 10 mg of protein, at least 15 mg of protein, at least 20 mg of protein, at least 25 mg of protein, at least 30 mg of protein, at least 50 mg of protein, per gram of detergent composition. Or, the detergent composition may comprise at least 0.01%DNase protein, at least 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.8%, 1.0%, 2.0%, or 5.0%of DNase protein.
The DNase of the detergent composition of the invention 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, WO92/19709 and WO92/19708.
A polypeptide of the present invention may also be incorporated in the detergent compositions disclosed in WO97/07202, which is hereby incorporated by reference.
Additional Enzymes
The detergent composition of the present invention may comprise one or more additional enzymes such as a protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, nuclease, oxidase, e.g., a laccase, and/or peroxidase.
In general the properties of the selected enzyme (s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc. ) , and the enzyme (s) should be present in effective amounts.
Proteases
Suitable proteases may be of any origin, but are preferably of bacterial or fungal origin, optionally in the form of protein engineered or chemically modified mutants. The protease may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as a subtilisin. A metalloprotease may for example be a thermolysin, e.g. from the M4 family, or another metalloprotease such as those from the M5, M7 or M8 families.
The term "subtilases" refers to a sub-group of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into six subdivisions, the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
Although proteases suitable for detergent use may be obtained from a variety of organisms, including fungi such as Aspergillus, detergent proteases have generally been obtained from bacteria and in particular from Bacillus. Examples of Bacillus species from which subtilases have been derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus gibsonii. Particular subtilisins include subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and e.g. protease PD138 (described in WO 93/18140) . Other useful proteases are e.g. those described in WO 01/16285 and WO 02/16547.
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 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.
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, L211D, 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 of WO 2016/001449. Protease variants having one or more of these mutations are preferably variants of the Bacillus lentus protease (
Figure PCTCN2021124368-appb-000001
also known as subtilisin 309) shown in SEQ ID NO: 1 of WO 2016/001449 or of the Bacillus amyloliquefaciens protease (BPN’) shown in SEQ ID NO: 2 of WO 2016/001449. Such protease variants preferably have at least 80%sequence identity to SEQ ID NO: 1 or to SEQ ID NO: 2 of WO 2016/001449.
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 TY145 protease having SEQ ID NO: 1 of WO 2004/067737, 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: 1 of WO 2004/067737, wherein said protease variant has a sequence identity of at least 75%but less than 100%to SEQ ID NO: 1 of WO 2004/067737. 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:
(a) variants of SEQ ID NO: 1 of WO 2016/001449 comprising two or more substitutions selected from the group consisting of S9E, N43R, N76D, Q206L, Y209W, S259D and L262E, for example a variant with the substitutions S9E, N43R, N76D, V205I, Q206L, Y209W, S259D, N261W and L262E, or with 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 of WO 2016/001449;
(b) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the mutation S99SE, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(c) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the mutation S99AD, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(d) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the substitutions Y167A+R170S+A194P, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(e) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the substitutions S9R+A15T+V68A+N218D+Q245R, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(f) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the substitutions S9R+A15T+G61E+V68A+A194P+V205I+Q245R+N261D, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(g) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the substitutions S99D+S101 R/E+S103A+V104I+G160S; for example a variant of SEQ ID NO: 1 of WO 2016/001449 with the substitutions S3T+V4I+S99D+S101E+S103A+V104I+G160S+V205I, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(h) a variant of the polypeptide of SEQ ID NO: 2 of WO 2016/001449 with the substitutions S24G+S53G+S78N+S101 N+G128A/S+Y217Q, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(i) the polypeptide disclosed in GENESEQP under accession number BER84782, corresponding to SEQ ID NO: 302 in WO 2017/210295;
(j) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the substitutions S99D+S101E+S103A+V104I+S156D+G160S+L262E, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(k) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the substitutions S9R+A15T+G61E+V68A+N76D+S99G+N218D+Q245R, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449;
(l) a variant of the polypeptide of SEQ ID NO: 1 of WO 2016/001449 with the substitutions V68A+S106A, wherein position numbers are based on the numbering of SEQ ID NO: 2 of WO 2016/001449; and
(m) a variant of the polypeptide of SEQ ID NO: 1 of WO 2004/067737 with the substitutions S27K+N109K+S111E+S171E+S173P+G174K+S175P+F180Y+G182A+L184F+Q198E+N199+T297P, wherein position numbers are based on the numbering of SEQ ID NO: 1 of WO 2004/067737.
Suitable commercially available protease enzymes include those sold under the trade names 
Figure PCTCN2021124368-appb-000002
Duralase TM, Durazym TM, 
Figure PCTCN2021124368-appb-000003
Ultra, 
Figure PCTCN2021124368-appb-000004
Ultra, Primase TM, 
Figure PCTCN2021124368-appb-000005
Ultra, 
Figure PCTCN2021124368-appb-000006
Figure PCTCN2021124368-appb-000007
Ultra, 
Figure PCTCN2021124368-appb-000008
Pro, Blaze 
Figure PCTCN2021124368-appb-000009
100T, Blaze 
Figure PCTCN2021124368-appb-000010
125T, Blaze 
Figure PCTCN2021124368-appb-000011
150T, Blaze 
Figure PCTCN2021124368-appb-000012
200T, 
Figure PCTCN2021124368-appb-000013
Uno, 
Figure PCTCN2021124368-appb-000014
In and 
Figure PCTCN2021124368-appb-000015
Excel (Novozymes A/S) , those sold under the tradename Maxatase TM, Maxacal TM, 
Figure PCTCN2021124368-appb-000016
Ox, 
Figure PCTCN2021124368-appb-000017
OxP, 
Figure PCTCN2021124368-appb-000018
FN2 TM, FN3 TM, FN4 exTM, 
Figure PCTCN2021124368-appb-000019
Excellenz TM P1000, Excellenz TM P1250, Eraser TM, 
Figure PCTCN2021124368-appb-000020
P100, Purafect Prime, Preferenz P110 TM, Effectenz P1000 TM, 
Figure PCTCN2021124368-appb-000021
Effectenz P1050 TM, 
Figure PCTCN2021124368-appb-000022
Ox, Effectenz  TM P2000, Purafast TM, 
Figure PCTCN2021124368-appb-000023
Opticlean TM and 
Figure PCTCN2021124368-appb-000024
 (Danisco/DuPont) , BLAP (sequence shown in Figure 29 of US 5352604) and variants hereof (Henkel AG) , and KAP (Bacillus alkalophilus subtilisin) from Kao.
Lipases and Cutinases
Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580) , lipase from strains of Pseudomonas (some of these now renamed to Burkholderia) , e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272) , P. cepacia (EP331376) , P. sp. strain SD705 (WO95/06720 &WO96/27002) , P. wisconsinensis (WO96/12012) , GDSL-type Streptomyces lipases (WO10/065455) , cutinase from Magnaporthe grisea (WO10/107560) , cutinase from Pseudomonas mendocina (US5,389,536) , lipase from Thermobifida fusca (WO11/084412) , Geobacillus stearothermophilus lipase (WO11/084417) , lipase from Bacillus subtilis (WO11/084599) , and lipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis (WO12/137147) .
Other examples are lipase variants such as those described in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381, WO95/22615,  WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.
Preferred commercial lipase products include include Lipolase TM, Lipex TM; Lipolex TM and Lipoclean TM (Novozymes A/S) , Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades) .
Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/111143) , acyltransferase from Mycobacterium smegmatis (WO05/56782) , perhydrolases from the CE 7 family (WO09/67279) , and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10/100028) .
Amylases
Suitable amylases which can be used together with the DNase of the invention may be 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 in WO 95/10603 or variants having 90%sequence identity to SEQ ID NO: 3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, 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. Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:
M197T;
H156Y+A181T+N190F+A209V+Q264S; or
G48A+T49I+G107A+H156Y+A181T+N190F+I201 F+A209V+Q264S.
Further amylases which are suitable are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90%sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.
Additional amylases which can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90%sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96/023873 for numbering. More preferred variants are those having a deletion in two positions selected from 181, 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.
Other amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90%sequence identity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.
Further suitable amylases are amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90%sequence identity to SEQ ID NO: 2 thereof. Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 are those having the substitution in one of more of the following positions: Q87E, R, Q98R, S125A, N128C, T131 I, T165I, K178L, T182G, M201 L, F202Y, N225E, R, N272E, R, S243Q, A, E, D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180 and/or S181 or of T182 and/or G183. Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:
N128C+K178L+T182G+Y305R+G475K;
N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;
S125A+N128C+K178L+T182G+Y305R+G475K; or
S125A+N128C+T131 I+T165I+K178L+T182G+Y305R+G475K wherein the variants are C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.
Further suitable amylases are amylases having SEQ ID NO: 1 of WO13184577 or variants having 90%sequence identity to SEQ ID NO: 1 thereof. Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K and/or deletion in position R178 and/or S179 or of T180 and/or G181. Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
E187P+I203Y+G476K
E187P+I203Y+R458N+T459S+D460T+G476K wherein the variants optionally further comprise a substitution at position 241 and/or a deletion at position 178 and/or position 179.
Further suitable amylases are amylases having SEQ ID NO: 1 of WO10104675 or variants having 90%sequence identity to SEQ ID NO: 1 thereof. Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: N21, D97, V128 K177, R179, S180, I181, G182, M200, L204, E242, G477 and G478. More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: N21D, D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion in position R179 and/or S180 or of I181 and/or G182. Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
N21D+D97N+V128I
wherein the variants optionally further comprise a substitution at position 200 and/or a deletion at position 180 and/or position 181.
Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90%sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: 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. Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182,  G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.
Other examples are amylase variants such as those described in WO2011/098531, WO2013/001078 and WO2013/001087.
Commercially available amylases are Duramyl TM, Termamyl TM, Fungamyl TM, Stainzyme  TM, Stainzyme Plus TM, Natalase TM, Liquozyme X and BAN TM (from Novozymes A/S) , and Rapidase TM , Purastar TM/Effectenz TM, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. /DuPont) .
Cellulases
Suitable cellulases include mono-component and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein engineered mutants are also contemplated. The cellulase may for example be a mono-component or a mixture of mono-component endo-1, 4-beta-glucanase also referred to as endoglucanase.
Suitable cellulases include those from the genera Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include a fungal cellulase from Humicola insolens (US 4, 435, 307) or from Trichoderma, e.g. T. reesei or T. viride. Other suitable cellulases are from Thielavia e.g. Thielavia terrestris as described in WO 96/29397 or the fungal cellulases produced from Myceliophthora thermophila and Fusarium oxysporum disclosed in US 5, 648, 263, US 5, 691, 178, US 5, 776, 757, WO 89/09259 and WO 91/17244. Also relevant are cellulases from Bacillus as described in WO 02/099091 and JP 2000210081. Suitable cellulases are alkaline or neutral cellulases having care benefits. Examples of cellulases are described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5, 457, 046, US 5, 686, 593, US 5, 763, 254, WO 95/24471, WO 98/12307.
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
Figure PCTCN2021124368-appb-000025
Premium, 
Figure PCTCN2021124368-appb-000026
Figure PCTCN2021124368-appb-000027
Classic, 
Figure PCTCN2021124368-appb-000028
(Novozymes A/S) , 
Figure PCTCN2021124368-appb-000029
Puradax HA, and Puradax EG (available from Genencor International Inc. ) and KAC-500 (B)  TM (Kao Corporation) .
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) .
Peroxidases/Oxidases
Suitable peroxidases/oxidases 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 TM (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 Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102; or from Drechslera hartlebii as described in WO 01/79459, Dendryphiella salina as described in WO 01/79458, Phaeotrichoconis crotalarie as described in WO 01/79461, or 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. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporous, e.g., P. pinsitus, Phlebia, e.g., P. radiata (WO 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.
Protease stabilizers/inhibitors
The protease (s) , as described above, may be stabilized using compounds that act by temporarily reducing the proteolytic activity (reversible inhibitors) .
Thus, the composition of the invention may also include a protease inhibitor/stabilizer, which is a reversible inhibitor of protease activity, e.g., serine protease activity. Preferably, the protease inhibitor is a (reversible) subtilisin protease inhibitor. In particular, the protease inhibitor may be a peptide aldehyde, boric acid, or a boronic acid; or a derivative of any of these.
Boronic acids
The protease inhibitor may be a boronic acid or a derivative thereof; preferably, a phenylboronic acid or a derivative thereof. In an embodiment of the invention, the phenyl boronic acid derivative is of the following formula:
Figure PCTCN2021124368-appb-000030
wherein R is selected from the group consisting of hydrogen, hydroxy, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl and substituted C1-C6 alkenyl. Preferably, R is hydrogen, CH 3, CH 3CH 2 or CH 3CH 2CH 2.
In a preferred embodiment, the protease inhibitor (phenyl boronic acid derivative) is 4-formyl-phenyl boronic acid (4-FPBA) .
In another particular embodiment, the protease inhibitor is selected from the group consisting of thiophene-2 boronic acid, thiophene-3 boronic acid, acetamidophenyl boronic acid, benzofuran-2 boronic acid, naphtalene-1 boronic acid, naphtalene-2 boronic acid, 2-FPBA, 3-FBPA, 4-FPBA, 1-thianthrene boronic acid, 4-dibenzofuran boronic acid, 5-methylthiophene-2 boronic, acid, thionaphtrene boronic acid, furan-2 boronic acid, furan-3 boronic acid, 4, 4 biphenyl-diborinic acid, 6-hydroxy-2-naphtalene, 4- (methylthio) phenyl boronic acid, 4 (trimethyl-silyl) phenyl boronic acid, 3-bromothiophene boronic acid, 4-methylthiophene boronic acid, 2-naphtyl boronic acid, 5-bromothiphene boronic acid, 5-chlorothiophene boronic acid, dimethylthiophene boronic acid, 2-bromophenyl boronic acid, 3-chlorophenyl boronic acid, 3-methoxy-2-thiophene, p-methyl-phenylethyl boronic acid, 2-thianthrene boronic acid, di-benzothiophene boronic acid, 4-carboxyphenyl boronic acid, 9-anthryl boronic acid, 3, 5 dichlorophenyl boronic, acid, diphenyl boronic acidanhydride, o-chlorophenyl boronic acid, p-chlorophenyl boronic acid, m-bromophenyl boronic acid, p-bromophenyl boronic acid, p-flourophenyl boronic acid, p-tolyl boronic acid, o-tolyl boronic acid, octyl boronic acid, 1, 3, 5 trimethylphenyl boronic acid, 3-chloro-4-flourophenyl boronic acid, 3-aminophenyl boronic acid, 3, 5-bis- (triflouromethyl) phenyl boronic acid, 2, 4 dichlorophenyl boronic acid, and 4-methoxyphenyl boronic acid.
Further boronic acid derivatives suitable as protease inhibitors in the detergent composition are described in US 4, 963, 655, US 5, 159, 060, WO 95/12655, WO 95/29223, WO 92/19707, WO 94/04653, WO 94/04654, US 5442100, US 5488157 and US 5472628.
Peptide aldehyde or ketone
The protease stabilizer may have the formula: P-A-L-B-B0-R*wherein:
R* is H (hydrogen) , CH 3, CX 3, CHX 2, or CH 2X, wherein X is a halogen atom, particularly F (fluorine) ; preferably, R*=H (so the stabilizer is a peptide aldehyde with the formula P-A-L-B-B0-H) ;
L is either absent or a linker group of the formula -C (=O) -, -C (=O) -C (=O) -, -C (=S) -, -C (=S) -C (=S) -, or -C (=S) -C (=O) -;
A is absent if L is absent, or is 1 or 2 amino acid residues connected to L via the N-terminal; thus, A may represent A1 or A2-A1, where A2 and A1 each represent one amino acid residue;
B may be 1, 2, or 3 amino acid residues; thus, B may represent B1, B2-B1, or B3-B2-B1, which is connected to B0 via the C-terminal, where B3, B2, and B1 each represent one amino acid  residue; B0 is a single amino acid residue with L-or D-configuration of the formula –NH-CH (R) -C (=O) -;
R is independently selected from the group consisting of C 1-6 alkyl, C 6-10 aryl or C 7-10 arylalkyl, optionally substituted with one or more, identical or different, substituents R’;
R’ is independently selected from the group consisting of halogen, -OH, -OR”, -SH, -SR”, -NH 2, -NHR”, -NR” 2, -CO 2H, -CONH 2, -CONHR”, -CONR” 2, -NHC (=N) NH 2;
R” is a C 1-6 alkyl group; and
P is selected from the group consisting of hydrogen, or -if L is absent -an N-terminal protection group;
B0 may be a single amino acid residue with L-or D-configuration, which is connected to H via the C-terminal of the amino acid. B0 has the formula –NH-CH (R) -C (=O) -, wherein R is a C 1-6 alkyl, C 6-10 aryl or C 7-10 arylalkyl side chain, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl or benzyl, and wherein R may be optionally substituted with one or more, identical or different, substituents R’. Particular examples of B0 are the D-or L-form of arginine (Arg) , 3, 4-dihydroxyphenylalanine, isoleucine (Ile) , leucine (Leu) , methionine (Met) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , m-tyrosine, p-tyrosine (Tyr) and valine (Val) . A particular embodiment is when B0 is leucine, methionine, phenylalanine, p-tyrosine, or valine. Paticularly preferred is p-tyrosine.
B1, which is connected to B0 via the C-terminal of the amino acid, may be an aliphatic, hydrophobic and/or neutral amino acid. Examples of B1 are alanine (Ala) , cysteine (Cys) , glycine (GIy) , isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , proline (Pro) , serine (Ser) , threonine (Thr) and valine (VaI) . Particular examples of B1 are alanine, glycine, isoleucine, leucine and valine. A particular embodiment is when B1 is alanine, glycine, or valine.
B2, if present, is connected to B1 via the C-terminal of the amino acid, and may be an aliphatic, hydrophobic, neutral and/or polar amino acid. Examples of B2 are alanine (Ala) , arginine (Arg) , capreomycidine (Cpd) , cysteine (Cys) , glycine (GIy) , isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , proline (Pro) , serine (Ser) , threonine (Thr) , and valine (VaI) . Particular examples of B2 are alanine, arginine, capreomycidine, glycine, isoleucine, leucine, phenylalanine and valine. A particular embodiment is when B2 is arginine, glycine, leucine, phenylalanine, or valine.
B3, if present, is connected to B2 via the C-terminal of the amino acid, and may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid. Examples of B3 are isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , phenylglycine, tyrosine (Tyr) , tryptophan (Trp) and valine (VaI) . Particular examples of B3 are leucine, phenylalanine, tyrosine, and tryptophan.
The linker group L may be absent or selected from the group consisting of -C (=O) -, -C (=O) -C (=O) -, -C (=S) -, -C (=S) -C (=S) -or -C (=S) -C (=O) -. Particular embodiments of the invention are when L is absent or L is a carbonyl group -C (=O) -.
A1, if present, is connected to L via the N-terminal of the amino acid, and may be an aliphatic, aromatic, hydrophobic, neutral and/or polar amino acid. Examples of A1 are alanine (Ala) , arginine (Arg) , capreomycidine (Cpd) , glycine (GIy) , isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , threonine (Thr) , tyrosine (Tyr) , tryptophan (Trp) and valine (VaI) . Particular examples of A1 are alanine, arginine, glycine, leucine, phenylalanine, tyrosine, tryptophan and valine. A particular embodiment is when B2 is leucine, phenylalanine, tyrosine or tryptophan.
A2, if present, is connected to A1 via the N-terminal of the amino acid, and may be a large, aliphatic, aromatic, hydrophobic and/or neutral amino acid. Examples of A2 are arginine (Arg) , isoleucine (Ile) , leucine (Leu) , norleucine (Nle) , norvaline (Nva) , phenylalanine (Phe) , phenylglycine, Tyrosine (Tyr) , tryptophan (Trp) and valine (VaI) . Particular examples of A2 are phenylalanine and tyrosine.
The N-terminal protection group P (if present) may be selected from formyl, acetyl (Ac) , benzoyl (Bz) , trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups such as fluorenylmethyloxycarbonyl (Fmoc) , methoxycarbonyl (Moc) , (fluoromethoxy) carbonyl, benzyloxycarbonyl (Cbz) , t-butyloxycarbonyl (Boc) and adamantyloxycarbonyl; p-methoxybenzyl carbonyl, benzyl (Bn) , p-methoxybenzyl (PMB) , p-methoxyphenyl (PMP) , methoxyacetyl, methylamino carbonyl, methylsulfonyl, ethylsulfonyl, benzylsulfonyl, methylphosphoramidyl (MeOP (OH) (=O) ) and benzylphosphoramidyl (PhCH 2OP (OH) (=O) ) .
Suitable peptide aldehydes are described in WO94/04651, WO95/25791, WO98/13458, WO98/13459, WO98/13460, WO98/13461, WO98/13462, WO07/141736, WO07/145963, WO09/118375, WO10/055052 and WO11/036153. More particularly, the peptide aldehyde may be Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF 3, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF 3, Moc-Val-Ala-Leu-CF 3, Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Phe-CF 3, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu-H, MeNCO-Val-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Phe-H, MeSO 2-Phe-Gly-Ala-Leu-H, MeSO 2-Val-Ala-Leu-H, PhCH 2O-P (OH) (O) -Val-Ala-Leu-H, EtSO 2-Phe-Gly-Ala-Leu-H, PhCH 2SO 2-Val-Ala-Leu-H, PhCH 2O-P (OH) (O) -Leu-Ala-Leu-H, PhCH 2O-P (OH) (O) -Phe-Ala-Leu-H, or MeO-P (OH) (O) -Leu-Gly-Ala-Leu-H. A preferred stabilizer for use in the liquid composition of the invention is Cbz-Gly-Ala-Tyr-H, or a hydrosulfite adduct thereof, wherein Cbz is benzyloxycarbonyl.
Further examples of such peptide aldehydes include α-MAPI, β-MAPI, Phe-C (=O) -Arg-Val-Tyr-H, Phe-C (=O) -Gly-Gly-Tyr-H, Phe-C (=O) -Gly-Ala-Phe-H, Phe-C (=O) -Gly-Ala-Tyr-H, Phe-C (=O) -Gly-Ala-L-H, Phe-C (=O) -Gly-Ala-Nva-H, Phe-C (=O) -Gly-Ala-Nle-H, Tyr-C (=O) -Arg-Val-Tyr-H, Tyr-C (=O) -Gly-Ala-Tyr-H, Phe-C (=S) -Arg-Val-Phe-H, Phe-C (=S) -Arg-Val-Tyr-H, Phe-C (=S) -Gly-Ala-Tyr-H, Antipain, GE20372A, GE20372B, Chymostatin A, Chymostatin B, and Chymostatin C.
The protease stabilizer may be a hydrosulfite adduct of the peptide aldehyde or ketone described above, e.g., as described in WO 2013/004636. The adduct may have the formula P-A-L-B-N(H) -CHR-CH (OH) -SO 3M, wherein P, A, L, B, and R are defined as above, and M is H or an alkali metal, preferably Na or K.
An aqueous solution of the hydrosulfite adduct may be prepared by reacting the corresponding peptide aldehyde with an aqueous solution of sodium bisulfite (sodium hydrogen sulfite, NaHSO 3) ; potassium bisulfite (KHSO 3) by known methods, e.g., as described in WO 98/47523; US 6, 500, 802; US 5, 436, 229; J. Am. Chem. Soc. (1978) 100, 1228; Org. Synth., Coll. vol. 7: 361.
Particularly preferred peptide aldehyde protease stabilizers have the formula P-B3-B2-B1-B0-H, or a hydrosulfite adduct having the formula P-B3-B2-B1-N (H) -CHR-CHOH-SO 3M, wherein
i) H is hydrogen;
ii) B0 is a single amino acid residue with L-or D-configuration of the formula -NH-CH (R) -C (=O) -;
iii) B1 and B2 are independently single amino acid residues;
iv) B3 is a single amino acid residue, or is absent;
v) R is independently selected from the group consisting of C 1-6 alkyl, C 6-10 aryl or C 7-10 arylalkyl optionally substituted with one or more, identical or different, substituents R’;
vi) R’ is independently selected from the group consisting of halogen, -OH, -OR”, -SH, -SR”, -NH 2, -NHR”, -NR” 2, -CO 2H, -CONH 2, -CONHR”, -CONR” 2, -NHC (=N) NH 2;
vii) R” is a C 1-6 alkyl group;
viii) P is an N-terminal protection group, preferably methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz) ; and
ix) M is H or an alkali metal, preferably Na or K.
In an even more preferred embodiment, the peptide aldehyde protease stabilizer has the formula P-B2-B1-B0-H or an adduct having the formula P-B2-B1-N (H) -CHR-CHOH-SO 3M, wherein
i) H is hydrogen;
ii) B0 is a single amino acid residue with L-or D-configuration of the formula -NH-CH (R) -C (=O) -;
iii) B1 and B2 are independently single amino acid residues;
iv) R is independently selected from the group consisting of C 1-6 alkyl, C 6-10 aryl or C 7-10 arylalkyl optionally substituted with one or more, identical or different, substituents R’;
v) R’ is independently selected from the group consisting of halogen, -OH, -OR”, -SH, -SR”, -NH 2, -NHR”, -NR” 2, -CO 2H, -CONH 2, -CONHR”, -CONR” 2, -NHC (=N) NH 2;
vi) R” is a C 1-6 alkyl group;
vii) P is an N-terminal protection group, preferably methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz) ; and
viii) M is H or an alkali metal, preferably Na or K.
Preferred embodiments of B0, B1, B2, B3, and P are as described above.
When the peptide aldehyde has the formula P-B3-B2-B1-B0-H, or a hydrosulfite adduct thereof, P is preferably acetyl, methoxycarbonyl, benzyloxycarbonyl, methylamino carbonyl, methylsulfonyl, benzylsulfonyl and benzylphosphoramidyl.
When the peptide aldehyde has the formula P-B2-B1-B0-H, or a hydrosulfite adduct thereof, P is preferably acetyl, methoxycarbonyl, methylsulfonyl, ethylsulfonyl and methylphosphoramidyl.
The molar ratio of the above-mentioned peptide aldehydes (or hydrosulfite adducts) to the protease may be at least 1: 1 or 1.5: 1, and it may be less than 1000: 1, more preferred less than 500: 1, even more preferred from 100: 1 to 2: 1 or from 20: 1 to 2: 1, or most preferred, the molar ratio is from 10: 1 to 2: 1.
Formate salts (e.g., sodium formate) and formic acid have also shown good effects as inhibitor of protease activity. Formate can be used synergistically with the above-mentioned protease inhibitors, as shown in WO 2013/004635. The formate salts may be present in the composition in an amount of at least 0.1%w/w or 0.5%w/w, e.g., at least 1.0%, at least 1.2%or at least 1.5%. The amount is typically below 5%w/w, below 4%or below 3%.
In an embodiment, the protease is a metalloprotease and the inhibitor is a metalloprotease inhibitor, e.g., a protein hydrolysate based inhibitor (e.g., as described in WO 2008/134343) .
Detergent compositions
In one embodiment, the invention is directed to detergent compositions comprising a polypeptide having DNase activity of the present invention in combination with one or more additional cleaning composition components such as a surfactant. The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
Surfactants
The 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. In a particular embodiment, the detergent composition includes a surfactant system (comprising more than one  surfactant) e.g. a mixture of one or more nonionic surfactants and one or more anionic surfactants. In one embodiment the detergent comprises at least one anionic surfactant than at least one non-ionic surfactant, the weight ratio of anionic to nonionic surfactant may be from 10: 1 to 1: 10. In one embodiment the amount of anionic surfactant is higher than the amount of non-ionic surfactant e.g. the weight ratio of anionic to non-ionic surfactant may be from 10: 1 to 1.1: 1 or from 5: 1 to 1.5: 1. The amount of anionic to non-ionic surfactant may also be equal and the weight ratios 1: 1. In one embodiment the amount of non-ionic surfactant is higher than the amount of anionic surfactant and the weight ratio may be 1: 10 to 1: 1.1. Preferably the weight ratio of anionic to non-ionic surfactant is from 10: 1 to 1: 10, such as from 5: 1 to 1: 5, or from 5: 1 to 1: 1.2. Preferably, the weight fraction of non-ionic surfactant to anionic surfactant is from 0 to 0.5 or 0 to 0.2 thus non-ionic surfactant can be present or absent if the weight fraction is 0, but if non-ionic surfactant is present, then the weight fraction of the nonionic surfactant is preferably at most 50%or at most 20%of the total weight of anionic surfactant and non-ionic surfactant. Light duty detergent usually comprises more nonionic than anionic surfactant and there the fraction of non-ionic surfactant to anionic surfactant is preferably from 0.5 to 0.9. The total weight of surfactant (s) is typically present at a level of from about 0.1%to about 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 may include any conventional surfactant (s) known in the art. When included therein the detergent will usually contain from about 1%to about 40%by weight of an 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 an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, typically available as sodium or potassium salts or salts of monoethanolamine (MEA, 2-aminoethan-1-ol) or triethanolamine (TEA, 2, 2', 2”-nitrilotriethan-1-ol) ; in particular, linear alkylbenzenesulfonates (LAS) , isomers of LAS such as branched alkylbenzenesulfonates (BABS) and phenylalkanesulfonates; olefin sulfonates, in particular alpha-olefinsulfonates (AOS) ; alkyl sulfates (AS) , in particular fatty alcohol sulfates (FAS) , i.e., primary alcohol sulfates (PAS) such as dodecyl sulfate; alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates) ; paraffin sulfonates (PS) including alkane-1-sulfonates and secondary alkanesulfonates (SAS) ; ester sulfonates, including sulfonated fatty acid glycerol esters and alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES or MES) ; alkyl-or alkenylsuccinic acids such as dodecenyl/tetradecenyl succinic acid (DTSA) ; diesters and monoesters of sulfosuccinic acid; fatty acid derivatives of amino acids. Furthermore, salts of fatty acids (soaps) may be included.
When included therein the detergent will usually contain from about 1%to about 40%by weight of a cationic 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%, from about 8%to about 12% or from about 10%to about 12%. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ) , cetyltrimethylammonium bromide (CTAB) , dimethyldistearylammonium chloride (DSDMAC) , and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.
When included therein the detergent will usually contain from about 0.2%to about 40%by weight of a nonionic 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%, 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) e.g. the AEO-series such as AEO-7, alcohol propoxylates, in particular propoxylated fatty alcohols (PFA) , ethoxylated and propoxylated alcohols, alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters (in particular methyl ester ethoxylates, MEE) , alkylpolyglycosides (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 (glucamides, GA, or fatty acid glucamides, FAGA) , as well as products available under the trade names SPAN and TWEEN, and combinations thereof.
When included therein the detergent will usually contain from about 0.01 to about 10 %by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamine oxides, in particular N- (coco alkyl) -N, N-dimethylamine oxide and N-(tallow-alkyl) -N, N-bis (2-hydroxyethyl) amine oxide, and combinations thereof.
When included therein the detergent will usually contain from about 0.01 %to about 10 %by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.
Additional bio-based surfactants may be used e.g. wherein the surfactant is a sugar-based non-ionic surfactant which may be a hexyl-β-D-maltopyranoside, thiomaltopyranoside or a cyclic-maltopyranoside, such as described in EP2516606 B1.
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 a hydrophobic character (so-called amphiphilic properties as known from surfactants) ; however the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by Hodgdon and Kaler (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 miceller, 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, personal care, food, to technical applications. Use of hydrotropes in detergent compositions allow 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 detergent may contain 0-10%by weight, for example 0-5%by weight, such as about 0.5 to about 5%, or about 3%to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluene sulfonate (STS) , sodium xylene sulfonate (SXS) , sodium cumene sulfonate (SCS) , sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.
Bleaching Systems
The cleaning composition may contain 0-50%by weight, such as 1-40%, such as 1-30%, such as about 1%to about 20%, of a bleaching system. Any oxygen-based bleaching system comprising components known in the art for use in cleaning detergents may be utilized. Suitable bleaching system components include sources of hydrogen peroxide; peracids and sources of peracids (bleach activators) ; and bleach catalysts or boosters.
Sources of hydrogen peroxide:
Suitable sources of hydrogen peroxide are inorganic persalts, including alkali metal salts such as sodium percarbonate and sodium perborates (usually mono-or tetrahydrate) , and hydrogen peroxide―urea (1/1) .
Sources of peracids:
Peracids may be (a) incorporated directly as preformed peracids or (b) formed in situ in the wash liquor from hydrogen peroxide and a bleach activator (perhydrolysis) or (c) formed in situ in the wash liquor from hydrogen peroxide and a perhydrolase and a suitable substrate for the latter, e.g., an ester.
a) Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids such as peroxybenzoic acid and its ring-substituted derivatives, peroxy-α-naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid [phthalimidoperoxyhexanoic acid (PAP) ] , and o-carboxybenzamidoperoxycaproic acid; aliphatic and aromatic  diperoxydicarboxylic acids such as diperoxydodecanedioic acid, diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, 2-decyldiperoxybutanedioic acid, and diperoxyphthalic, -isophthalic and -terephthalic acids; perimidic acids; peroxymonosulfuric acid; peroxydisulfuric acid; peroxyphosphoric acid; peroxysilicic acid; and mixtures of said compounds. It is understood that the peracids mentioned may in some cases be best added as suitable salts, such as alkali metal salts (e.g., 
Figure PCTCN2021124368-appb-000031
) or alkaline earth-metal salts.
b) Suitable bleach activators include those belonging to the class of esters, amides, imides, nitriles or anhydrides and, where applicable, salts thereof. Suitable examples are tetraacetylethylenediamine (TAED) , sodium 4- [ (3, 5, 5-trimethylhexanoyl) oxy] benzene-1-sulfonate (ISONOBS) , sodium 4- (dodecanoyloxy) benzene-1-sulfonate (LOBS) , sodium 4-(decanoyloxy) benzene-1-sulfonate, 4- (decanoyloxy) benzoic acid (DOBA) , sodium 4-(nonanoyloxy) benzene-1-sulfonate (NOBS) , and/or those disclosed in WO98/17767. A particular family of bleach activators of interest was disclosed in EP624154 and particularly preferred in that family is acetyl triethyl citrate (ATC) . ATC or a short chain triglyceride like triacetin has the advantage that they are environmentally friendly. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytical stability in the product upon storage and are efficient bleach activators. Finally, ATC is multifunctional, as the citrate released in the perhydrolysis reaction may function as a builder.
Bleach catalysts and boosters
The bleaching system may also include a bleach catalyst or booster.
Some non-limiting examples of bleach catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1, 4, 7-trimethyl-1, 4, 7-triazacyclononane (Me3-TACN) or 1, 2, 4, 7-tetramethyl-1, 4, 7-triazacyclononane (Me4-TACN) , in particular Me3-TACN, such as the dinuclear manganese complex [ (Me3-TACN) Mn (O) 3Mn (Me3-TACN) ] (PF6) 2, and [2, 2', 2”-nitrilotris (ethane-1, 2-diylazanylylidene-κN-methanylylidene) triphenolato-κ3O] manganese (III) . The bleach catalysts may also be other metal compounds; such as iron or cobalt complexes.
In some embodiments, where a source of a peracid is included, an organic bleach catalyst or bleach booster may be used having one of the following formulae:
Figure PCTCN2021124368-appb-000032
(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, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl.
Other exemplary bleaching systems are described, e.g. in WO2007/087258, WO2007/087244, WO2007/087259, EP1867708 (Vitamin K) and WO2007/087242.
Suitable photobleaches may for example be sulfonated zinc or aluminium phthalocyanines.
Builders and Co-Builders
The detergent composition may contain about 0-65%by weight, such as about 5%to about 50%, 20-60%of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typically in the range 40-65%, particularly in the range 50-65%. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in cleaning 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 Clariant) , ethanolamines such as 2-aminoethan-1-ol (MEA) , diethanolamine (DEA, also known as 2, 2'-iminodiethan-1-ol) , triethanolamine (TEA, also known as 2, 2', 2”-nitrilotriethan-1-ol) , and (carboxymethyl) inulin (CMI) , and combinations thereof.
The detergent composition may also contain from about 0-50%by weight, such as about 5%to about 30%, of a detergent co-builder. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly (acrylic acid) (PAA) or copoly (acrylic acid/maleic acid) (PAA/PMA) . Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl-or alkenylsuccinic acid. Additional specific examples include 2, 2’, 2” -nitrilotriacetic acid (NTA) , ethylenediaminetetraacetic acid  (EDTA) , diethylenetriaminepentaacetic acid (DTPA) , iminodisuccinic acid (IDS) , ethylenediamine-N, N’-disuccinic acid (EDDS) , methylglycinediacetic acid (MGDA) , glutamic acid-N, N-diacetic acid (GLDA) , 1-hydroxyethane-1, 1-diylbis (phosphonic acid (HEDP) , ethylenediaminetetramethylenetetrakis (phosphonic acid) (EDTMPA) , diethylenetriaminepentamethylenepentakis (phosphonic acid) (DTMPA or DTPMPA) , N- (2-hydroxyethyl) iminodiacetic acid (EDG) , aspartic acid-N-monoacetic acid (ASMA) , aspartic acid-N, N-diacetic acid (ASDA) , aspartic acid-N-monopropionic acid (ASMP) , iminodisuccinic acid (IDA) , N- (2-sulfomethyl) aspartic acid (SMAS) , N- (2-sulfoethyl) aspartic acid (SEAS) , N- (2-sulfomethyl) glutamic acid (SMGL) , N- (2-sulfoethyl) glutamic acid (SEGL) , N-methyliminodiacetic acid (MIDA) , α-alanine-N, N-diacetic acid (α-ALDA) , serine-N, N-diacetic acid (SEDA) , isoserine-N, N-diacetic acid (ISDA) , phenylalanine-N, N-diacetic acid (PHDA) , anthranilic acid-N, N-diacetic acid (ANDA) , sulfanilic acid-N, N-diacetic acid (SLDA) , taurine-N, N-diacetic acid (TUDA) and sulfomethyl-N, N-diacetic acid (SMDA) , N- (2-hydroxyethyl) ethylenediamine-N, N’, N”-triacetic acid (HEDTA) , diethanolglycine (DEG) , aminotrimethylenetris (phosphonic acid) (ATMP) , and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 09/102854, US 5977053.
Polymers
The detergent composition may contain 0.005-10%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. Exemplary polymers include (carboxymethyl) cellulose (CMC) , poly (vinyl alcohol) (PVA) , poly (ethyleneglycol) or poly (ethylene oxide) (PEG or PEO) , ethoxylated poly (ethyleneimine) , (carboxymethyl) inulin (CMI) , carboxylate polymers and d, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) , silicones, copolycarboxylates such as polyacrylates, maleic/acrylic acid copolymers, acrylate/styrene copolymers, poly (aspartic) acipolymers of terephthalic acid and oligomeric glycols, copolymers of poly (ethylene terephthalate) and poly (oxyethene terephthalate) (PET-POET) , poly (vinylpyrrolidone) (PVP) , poly (vinylimidazole) (PVI) , poly (vinylpyridine-N-oxide) (PVPO or PVPNO) and copoly (vinylimidazole/vinylpyrrolidone) (PVPVI) . Suitable examples include PVP-K15, PVP-K30, ChromaBond S-400, ChromaBond S-403E and Chromabond S-100 from Ashland Aqualon, and 
Figure PCTCN2021124368-appb-000033
HP 165, 
Figure PCTCN2021124368-appb-000034
HP 50 (Dispersing agent) , 
Figure PCTCN2021124368-appb-000035
HP 53 (Dispersing agent) , 
Figure PCTCN2021124368-appb-000036
HP 59 (Dispersing agent) , 
Figure PCTCN2021124368-appb-000037
HP 56 (dye transfer inhibitor) , 
Figure PCTCN2021124368-appb-000038
HP 66 K (dye transfer inhibitor) from BASF. Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Particularly preferred polymer is ethoxylated homopolymer
Figure PCTCN2021124368-appb-000039
HP 20 from BASF, which helps to prevent redeposition  of soil in the wash liquor. Further exemplary polymers include sulfonated polycarboxylates, ethylene oxide-propylene oxide copolymers (PEO-PPO) , copolymers of PEG with and vinyl acetate, and diquaternium ethoxy sulfate or quaternized sulfated ethoxylated hexamethylenediamine. Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.
Adjunct materials
Any detergent adjunct components known in the art may also be utilized. Exemplary adjunct materials may include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrates/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. The choice of such ingredients is well within the skill of the artisan.
Dispersants
The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.
Dye Transfer Inhibiting Agents
The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones 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 detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical  brighteners. Where present the brightener is preferably at a level of about 0.01%to about 0.5%. Any fluorescent whitening agent suitable for use in a cleaning 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-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulfonic 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'-disulfonate, 4, 4'-bis- (2, 4-dianilino-s-triazin-6-ylamino) stilbene-2.2'-disulfonate, 4, 4'-bis- (2-anilino-4- (N-methyl-N-2-hydroxy-ethylamino) -s-triazin-6-ylamino) stilbene-2, 2'-disulfonate, 4, 4'-bis- (4-phenyl-1, 2, 3-triazol-2-yl) stilbene-2, 2'-disulfonate and sodium 5- (2H-naphtho [1, 2-d] [1, 2, 3] triazol-2-yl) -2- [ (E) -2-phenylvinyl] benzenesulfonate. 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-2, 2'-disulfonate. Tinopal CBS is the disodium salt of 2, 2'-bis- (phenyl-styryl) -disulfonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the 1-3-diaryl pyrazolines and the 7-alkylaminocoumarins.
Suitable fluorescent brightener levels include lower levels of from about 0.01, from 0.05, from about 0.1 or even from about 0.2 wt %to upper levels of 0.5 or even 0.75 wt%.
Soil release polymers
The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Another type of soil release polymers are 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 co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference) . Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in EP 1867808 or WO 2003/040279 (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 detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC) , polyvinyl alcohol (PVA) , polyvinylpyrrolidone (PVP) , polyoxyethylene and/or polyethyleneglycol (PEG) , homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
Formulation of detergent products
The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
Pouches can be configured as single or multicompartments. They can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC) . Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blended compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticisers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid cleaning composition or part components and/or a liquid cleaning  composition or part components separated by the water soluble film. The compartment for liquid components can be different in composition than compartments containing solids: US2009/0011970 A1.
Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
A liquid or gel detergent , which is not unit dosed, may be aqueous, typically containing at least 20%by weight and up to 95%water, such as up to about 70%water, up to about 65%water, up to about 55%water, up to about 45%water, up to about 35%water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30%organic solvent.
A liquid or gel detergent may be non-aqueous.
Laundry soap bars
The enzyme of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps. The laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature. The term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in. The bar is a solid typically in bar form but can be in other solid shapes such as round or oval.
The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct) , boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na +, K + or NH 4 + and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.
The laundry soap bar may also contain complexing agents like EDTA and HEDP, perfumes and/or different type of fillers, surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching  activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
The laundry soap bar may be processed in conventional laundry soap bar making equipment such as but not limited to: mixers, plodders, e.g a two stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers. The invention is not limited to preparing the laundry soap bars by any single method. The premix of the invention may be added to the soap at different stages of the process. For example, the premix containing a soap, a DNase of the present invention, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and and the mixture is then plodded. The DNase of the present invention and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form. Besides the mixing step and the plodding step, the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.
Granular detergent compositions
A granular detergent may be formulated as described in WO09/092699, EP1705241, EP1382668, WO07/001262, US6472364, WO04/074419 or WO09/102854. Other useful detergent compositions are described in WO09/124162, WO09/124163, WO09/117340, WO09/117341, WO09/117342, WO09/072069, WO09/063355, WO09/132870, WO09/121757, WO09/112296, WO09/112298, WO09/103822, WO09/087033, WO09/050026, WO09/047125, WO09/047126, WO09/047127, WO09/047128, WO09/021784, WO09/010375, WO09/000605, WO09/122125, WO09/095645, WO09/040544, WO09/040545, WO09/024780, WO09/004295, WO09/004294, WO09/121725, WO09/115391, WO09/115392, WO09/074398, WO09/074403, WO09/068501, WO09/065770, WO09/021813, WO09/030632, and WO09/015951.
Formulation of enzyme in co-granule
The enzyme of the invention may be formulated as a granule for example as a co-granule that combines one or more enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of enzymes in the detergent. This also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates for the detergent industry are disclosed in the IP. com disclosure IPCOM000200739D.
Another example of formulation of enzymes by the use of co-granulates are disclosed in WO 2013/188331, which relates to a detergent composition comprising (a) a multi-enzyme co-granule; (b) less than 10 wt zeolite (anhydrous basis) ; and (c) less than 10 wt phosphate salt (anhydrous basis) , wherein said enzyme co-granule comprises from 10 to 98 wt%moisture sink component and the composition additionally comprises from 20 to 80 wt%detergent moisture sink component. WO  2013/188331 also relates to a method of treating and/or cleaning a surface, preferably a fabric surface comprising the steps of (i) contacting said surface with the detergent composition as claimed and described herein in an aqueous wash liquor, (ii) rinsing and/or drying the surface.
The multi-enzyme co-granule may comprise an enzyme of the invention and (a) one or more enzymes selected from the group consisting of first-wash lipases, cleaning cellulases, xyloglucanases, perhydrolases, peroxidases, lipoxygenases, laccases and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, proteases, care cellulases, cellobiose dehydrogenases, xylanases, phospho lipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, amylases, and mixtures thereof.
The present invention can be further summarized in following paragraphs:
1. Use of a polypeptide having deoxyribonuclease (DNase) activity for preventing, reducing and/or removing malodor from a porous item.
2. Use according to paragraph 1, wherein the polypeptide having DNase activity is capable of preventing, reducing or removing stickiness of the porous item.
3. Use according to paragraph 1 or 2, wherein the polypeptide having DNase activity is of bacterial or fungal origin.
4. Use according to any of paragraphs 1-3, wherein the polypeptide having DNase activity is selected from the group consisting of: a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
5. Use according to any of paragraphs 1-4, wherein the polypeptide having DNase activity is from Aspergillus or Bacillus.
6. Use according to any of paragraphs 1-5, wherein the polypeptide having DNase activity is from Aspergillus oryzae or Bacillus cibi.
7. Use according to any of paragraphs 1-6, wherein the porous item comprises a sponge.
8. Use according to any of paragraphs 1-7, wherein the porous item is a kitchen sponge.
9. A detergent composition comprising a polypeptide having deoxyribonuclease (DNase) activity and a surfactant, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from a porous item.
10. The detergent composition according to paragraph 9, wherein the polypeptide having DNase activity is capable of preventing, reducing or removing stickiness of the porous item.
11. The detergent composition according to paragraph 9 or 10, wherein the polypeptide having DNase activity is of bacterial or fungal origin.
12. The detergent composition according to any of paragraphs 9-11, wherein the polypeptide having DNase activity is selected from the group consisting of: a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
13. The detergent composition according to any of paragraphs 9-12, wherein the polypeptide having DNase activity is from Aspergillus or Bacillus.
14. The detergent composition according to any of paragraphs 9-13, wherein the polypeptide having DNase activity is from Aspergillus oryzae or Bacillus cibi.
15. The detergent composition according to any of paragraphs 9-14, wherein the porous item comprises a sponge.
16. The detergent composition according to any of paragraphs 9-15, wherein the detergent composition further comprises one or more components selected from the group consisting of flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric huing agents, anti-foaming agents, dispersants, processing aids, and/or pigments.
17. The detergent composition according to paragraph 16, wherein the enzyme is selected from the group consisting of proteases, amylases, lipases, phospholipases, esterases, cellulases, xylanases, mannanases, pullulanases, malanases, β-glucanases, arabinosidases, peroxidases, xanthanase and mixtures thereof.
18. The detergent composition according to paragraphs 16 or 17, wherein the enzyme is selected from proteases, amylase, mannanases and pectate lyases.
19. The detergent composition according to any of paragraphs 16-18, wherein the enzyme is a protease.
20. A method for cleaning a porous item soiled with a biofilm and/or a food stain, comprising steps of:
(1) Contacting the porous item with a wash liquor comprising the polypeptide having deoxyribonuclease (DNase) activity according to any of paragraphs of 1-8 or the detergent composition according to any of paragraphs 9-19; and
(2) Optionally rinsing the porous item,
wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from the porous item.
21. The method according to paragraph 20, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing stickiness from the porous item.
22. The method according to paragraph 20 or 21, wherein the method is carried out by hand or by machine.
23. The method according to any of paragraphs 20-22, wherein the porous item comprises a sponge.
Enzyme assays
Assay I
Testing of DNase activity
DNase activity was determined on DNase Test Agar with Methyl Green (BD, Franklin Lakes, NJ, USA) , which was prepared according to the manual from supplier. Briefly, 21 g of agar was dissolved in 500 ml water and then autoclaved for 15 min at 121℃. Autoclaved agar was temperated to 48℃ in water bath, and 20 ml of agar was poured into petri dishes and allowed to solidify by incubation o/n at room temperature. On solidified agar plates, 5 μl of enzyme solutions are added, and DNase activity are observed as colorless zones around the spotted enzyme solutions.
Examples
Material and Methods
Enzymes
Aspergillus oryzae DNase: mature form of the polypeptide of SEQ ID NO: 1 (i.e. SEQ ID NO: 1 in WO 2017/060518) .
Bacillus Protease: mature form of the polypeptide of SEQ ID NO: 5 with 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 of WO 2016/001449.
Detergent
Fairy Original (detergent 1) : a commercial hand dish wash detergent bought from P&G, containing 20.5%Sulphates, 3.0%Nonionics, 62.5%Water, 0.80%Ethanol, 0.10%Phenoxyethanol,  6.0%Amine Oxides, etc.
Natural Dish Liquid Free &Clear (detergent 2) : a commercial hand dish wash detergent purchased from Seventh Generation, containing 79.5%water, 16.2%sodium lauryl sulfate, 2.0%Glycerin, 2.11%auramine oxide, 0.07%caprylyl/myristyl glucoside, 0.08%magnesium chloride, 0.05%citric acid, 0.03%Benzisothiazolinone, 0.04%Methylisothiazolinone.
Preparation of food stains
Meat stain preparation:
20 g of defatted beef was ground together with 20 g of defatted pork in a meat grinder (BRAUN Power Plus 1300, using minimal filter pore) for two rounds. The defatted pork or beef was prepared by cutting off the fatty part with a kitchen knife. 3 g of the minced meat mixture was spread evenly on a dish plate by using a massage cushion (Inomata Round Shampoo Brush) . The plate soiled with the minced meat mixture was dried overnight at room temperature followed by heating at 140℃ for 2 hours in an oven (Thermo Electron LED GmbH, Thermo Scientific) . The soiled pate was cooled down to room temperature before the wash test.
Egg stain preparation:
50 g of egg was mixed with 50 g of tap water by using a spoon in a bowl. Then the mixture was cooked in a steamer (AUCMA AZF-12DY01) at 100℃for 20 min. Then roughly 90g of the steamed egg was taken out of the bowl slowly and 8-10g of steamed egg was left in the bowl as egg stain for the wash test.
Fish stain preparation:
220 g of fish skin was boiled with 450 of ml tap water for 10min. 175 g of the boiled fish skin solution was then blended with 25 g of fish meat by using an automatic blender (Media, MJ-PB80Easy218, vegetable mode) for 2.5 min. The resulting blended fish meat mixture was kept in a 50℃ water bath to avoid solidification. 6 g of the blended fish meat mixture was spread onto a plate evenly by using a massage cushion (Inomata Round Shampoo Brush) . The soiled plate was dried overnight at room temperature followed by heating at 140℃ for 2 hours in an oven (Thermo Electron LED GmbH, Thermo Scientific) . The soiled pate was cooled down to room temperature before the wash test.
Hand wash method
50 g of detergent 1 or detergent 2 was dissolved in 5 liter of water with a hardness of 16°dH  (Ca: Mg  = 4: 1) . In washes with DNase, Aspergillus oryzae DNase (final concentration: 1.7 ppm) was added. In washes with protease, the Bacillus protease (final concentration: 1 ppm) was added. In washes with both DNase and protease, 1.7 ppm of Aspergillus oryzae DNase and 1 ppm of Bacillus protease was added.
One soiled plate was soaked for 2 min in the wash liquor of detergent only, detergent with DNase, detergent with protease, or detergent with both DNase and protease, as prepared above. Then the soiled plate was scrubbed with the green or yellow side of the kitchen sponge (3M G6215, the green side is made with a felt and the yellow side is made with a sponge) until substantially all stain was removed away from the soiled plate. In the present invention, yellow side (i.e. the softer side) of the kitchen sponge was used to scrub egg stains. The green side (i.e. the rougher and stiffer side) of the kitchen sponge was used to scrub meat stains and fish stains, as these two stains are harder to remove. After finishing scrubbing one plate, the same sponge was used to scrub one more plate which was soiled with the same stain and soaked for 2 min in the same wash liquor. Then the sponge was rinsed under running tap water along with 5 times squeezing. 3 repetitions were carried out for each stain type.
After rinse, each kitchen sponge was photographed for subsequent visual evaluation of residual stain particles left on the sponge (more specifically on the side for scrubbing stains) . Afterwards, each kitchen sponge was put into a Ziplock plastic bag and sealed for the following tests.
Example 1
POLFA test on malodor removing benefit of DNase
The rinsed sponge was sealed in a Ziplock plastic bag and incubated overnight at room temperature. Microorganisms will grow during incubation and produce bad odor (malodor) . The malodor of each sponge after incubation was measured by POLFA, an odor sensor device bought from KALMOR company. For detecting malodor, the Ziplock bag was opened slightly to let the sensor of POLFA sense the sponge for about 400 seconds at which the malodor level reached plateau. The malodor value was recorded by POLFA device automatically and can be read from the display window of POLFA device. The malodor removal benefit of enzyme (s) was evaluated by malodor reduction percentage (%) , which is calculated by following formula.
Malodor reduction %= (V1 -V2) /V1 x 100%
V1: malodor value at 400 seconds of the sponge washed with detergent without enzyme (i.e. detergent only)
V2: malodor value at 400 seconds of the sponge washed with detergent plus enzyme
The POLFA malodor test results were summarized in below table 1. A greater malodor reduction %suggests a better malodor removal benefit.
Table 1: Malodor removal benefit evaluated by POLFA
Figure PCTCN2021124368-appb-000040
As can be seen from table 1, adding enzyme (s) to the detergent provided malodor removal benefit on sponges tested for all three stain types. Specifically, the DNase of the present invention showed overall a better malodor removal benefit than protease.
Further, by combining the DNase of the present invention with a protease showed a further improved malodor removal benefit. Particularly, a synergy effect of combining DNase with protease was observed for egg stain in detergent 1.
Example 2
Panel evaluation on malodor removing benefit of DNase
Six trained panelists were selected to form a panel group for malodor evaluation. Each panelist was presented with four sponge samples from the four test conditions (i.e., detergent only, detergent+protease, detergent+DNase, detergent+DNase+protease) in a random order and was asked to rank the four sponges according to the intensity of their malodor. Then the four sponge samples were placed in the order from the weakest to the strongest malodor and were scored by the  six panelists. Score range was from 1 to 4. A score of 1, 2, 3 and 4 indicates minor, small, medium and big difference in terms of malodor intensity, respectively.
The sensory odor value for the first sample (i.e. the one having the weakest malodor among the four samples) was preset as 1. The second sponge sample was scored against the first one and for example got a score of 2, then the sensory odor value assigned to the second sample would be 1+2=3. The third sample was scored against the second one and for example got a score of 1, then the sensory odor value assigned to the third sample would be 3+1=4. The fourth sponge samples were scored in the same way. The average sensory odor value for sponges washed for egg stain and meat stain are summarized in below table 2. A higher average sensory odor value represents a stronger malodor felt by the panel group.
Table 2: Malodor removal benefit evaluated by panel evaluation, detergent 1
Figure PCTCN2021124368-appb-000041
As can be seen from table 2, the malodor removal benefit of the DNase or DNase+protease of the present invention was confirmed by panel group. The trend of the enzyme benefit on malodor removal was the same as that determined by POLFA.
Example 3
BTB test on deep cleaning effect of DNase
LB liquid (10g/L Tryptone, 5 g/L yeast extract and 10g/L NaCl) was 1: 1 diluted with Mili Q water. 10g/L lactose and 0.17g/L Bromothymol Blue was added to the diluted LB liquid and stirred for 10 mins by using magnetic stirring apparatus (IKAMAG, rdeq-fs-0133) . The pH of the resulted solution was adjusted to 8.6 with 1 M NaOH to obtain a BTB solution having a blue color. Due to containing Bromothymol Blue, the BTB solution can change color with the change of acidity, i.e., the BTB solution shows a blue color when it is alkaline, a yellow color at an acidic pH, and a green color  at a neutral pH.
300ml of the BTB solution having a blue color was added to the Ziplock bag containing a rinsed sponge and incubated for 8 hours at 35℃. During incubation, microorganisms will grow and produce acidic substances which can decrease the pH value of the solution. As a result, the BTB solution will gradually change color from blue to green and to yellow depending on how many microorganisms have grown. As fewer microorganisms will grow on sponges having fewer residual stains (i.e. better deep cleaning effect achieved) , the color change of the BTB solution can be used to evaluate the cleaning effect. In other words, the cleaner the sponge is, the more blue hue/less yellow hue the BTB solution will appear. The color of the BTB solution after 8 hours incubation was observed and recorded.
For fish stains, the BTB solution containing sponges washed by detergent without enzyme changed from blue to green gradually and after 8 hours became a totally yellow-orange color. For the sponges washed by DNase, the BTB solution showed a bluish green color. For those washed by protease, a yellowish green color was observed.
Therefore, it can be concluded that the sponge washed in the presence of DNase showed overall better deep cleaning effect than without DNase, and by combining DNase with protease, a further improved deep cleaning effect was confirmed. It can also be expected that less microorganism growing will produce less malodor and/or less sticky substances on sponges washed in the presence of DNase.
Data are not shown here, but a similar benefit of DNase with/without protease was observed on other stains in both detergent 1 and detergent 2.
Example 4
Visual evaluation on residual stain particles left on the rinsed sponge
The visible residual stain particles left on the surface of each sponge (specifically, on the side of the sponge used for scrubbing stains) were evaluated by naked eyes on the photos taken after rinse.
Results showed that for all tested stain types, the sponges washed by the combination of DNase and protease had the least residues (very few residual stain particles or nearly no visible stain particles left) , indicating the best wash performance compared to other conditions. On the other hand, the sponges washed by detergent only were observed having the most residual stain particles.
Further, slightly fewer residual stain particles were observed for the sponges washed by DNase alone compared to by protease alone.
In addition, the size of the residual particles washed with enzyme (s) were in general smaller than those washed without enzyme (s) .
When taking the BTB test results together, one of the possible mechanisms for the observed malodor removal benefit of the DNase might be that the DNase of the present invention disrupted more stains, especially those associated with bad odors such as biofilms, into smaller particles which can be rinsed away easily from the porous/rough surface of the porous items. By further combining with other enzymes such as protease, more variety of stains were digested into more smaller pieces, resulting in a better deep cleaning effect, which further improved the malodor removal benefit.

Claims (23)

  1. Use of a polypeptide having deoxyribonuclease (DNase) activity for preventing, reducing and/or removing malodor from a porous item.
  2. Use according to claim 1, wherein the polypeptide having DNase activity is capable of preventing, reducing or removing stickiness of the porous item.
  3. Use according to claim 1 or 2, wherein the polypeptide having DNase activity is of bacterial or fungal origin.
  4. Use according to any of claims 1-3, wherein the polypeptide having DNase activity is selected from the group consisting of: a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  5. Use according to any of claims 1-4, wherein the polypeptide having DNase activity is from Aspergillus or Bacillus.
  6. Use according to any of claims 1-5, wherein the polypeptide having DNase activity is from Aspergillus oryzae or Bacillus cibi.
  7. Use according to any of claims 1-6, wherein the porous item comprises a sponge.
  8. Use according to any of claims 1-7, wherein the porous item is a kitchen sponge.
  9. A detergent composition comprising a polypeptide having deoxyribonuclease (DNase) activity and a surfactant, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from a porous item.
  10. The detergent composition according to claim 9, wherein the polypeptide having DNase activity is capable of preventing, reducing or removing stickiness of the porous item.
  11. The detergent composition according to claim 9 or 10, wherein the polypeptide having DNase activity is of bacterial or fungal origin.
  12. The detergent composition according to any of claims 9-11, wherein the polypeptide having DNase activity is selected from the group consisting of: a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  13. The detergent composition according to any of claims 9-12, wherein the polypeptide having DNase activity is from Aspergillus or Bacillus.
  14. The detergent composition according to any of claims 9-13, wherein the polypeptide having DNase activity is from Aspergillus oryzae or Bacillus cibi.
  15. The detergent composition according to any of claims 9-14, wherein the porous item comprises a sponge.
  16. The detergent composition according to any of claims 9-15, wherein the detergent composition further comprises one or more components selected from the group consisting of flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric huing agents, anti-foaming agents, dispersants, processing aids, and/or pigments.
  17. The detergent composition according to claim 16, wherein the enzyme is selected from the group consisting of proteases, amylases, lipases, phospholipases, esterases, cellulases, xylanases, mannanases, pullulanases, malanases, β-glucanases, arabinosidases, peroxidases, xanthanase and mixtures thereof.
  18. The detergent composition according to claim 16 or 17, wherein the enzyme is selected from proteases, amylases, mannanases and pectate lyases.
  19. The detergent composition according to any of claims 16-18, wherein the enzyme is a protease.
  20. A method for cleaning a porous item soiled with a biofilm and/or a food stain, comprising the steps of:
    (1) Contacting the porous item with a wash liquor comprising the polypeptide having deoxyribonuclease (DNase) activity according to any of claims 1-8 or the detergent composition according to any of claims 9-19; and
    (2) Optionally rinsing the porous item,
    wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing malodor from the porous item.
  21. The method according to claim 20, wherein the polypeptide having DNase activity is capable of preventing, reducing and/or removing stickiness from the porous item.
  22. The method according to claim 20 or 21, wherein the method is carried out by hand or by machine.
  23. The method according to any of claims 20-22, wherein the porous item comprises a sponge.
PCT/CN2021/124368 2020-10-19 2021-10-18 Use of polypeptide, detergent composition and cleaning method WO2022083538A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015155351A1 (en) * 2014-04-11 2015-10-15 Novozymes A/S Detergent composition
CN106164236A (en) * 2014-04-11 2016-11-23 诺维信公司 Composition of detergent
CN106232795A (en) * 2014-05-02 2016-12-14 诺维信公司 Composition of detergent
CN106459839A (en) * 2014-05-28 2017-02-22 诺维信公司 Use of polypeptide
WO2018108865A1 (en) * 2016-12-12 2018-06-21 Novozymes A/S Use of polypeptides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015155351A1 (en) * 2014-04-11 2015-10-15 Novozymes A/S Detergent composition
CN106164236A (en) * 2014-04-11 2016-11-23 诺维信公司 Composition of detergent
CN106232795A (en) * 2014-05-02 2016-12-14 诺维信公司 Composition of detergent
CN106459839A (en) * 2014-05-28 2017-02-22 诺维信公司 Use of polypeptide
WO2018108865A1 (en) * 2016-12-12 2018-06-21 Novozymes A/S Use of polypeptides

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