WO2022106400A1 - Combination of immunochemically different proteases - Google Patents

Combination of immunochemically different proteases Download PDF

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Publication number
WO2022106400A1
WO2022106400A1 PCT/EP2021/081815 EP2021081815W WO2022106400A1 WO 2022106400 A1 WO2022106400 A1 WO 2022106400A1 EP 2021081815 W EP2021081815 W EP 2021081815W WO 2022106400 A1 WO2022106400 A1 WO 2022106400A1
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seq
proteases
detergent composition
protease
detergent
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PCT/EP2021/081815
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French (fr)
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Jürgen Carsten Franz KNÖTZEL
Maria Norman Hockauf
Esben Peter Friis
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Novozymes A/S
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Publication of WO2022106400A1 publication Critical patent/WO2022106400A1/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
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • 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
    • C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
    • C11D17/04—Detergent materials or soaps characterised by their shape or physical properties combined with or containing other objects
    • C11D17/041—Compositions releasably affixed on a substrate or incorporated into a dispensing means
    • C11D17/042—Water soluble or water disintegrable containers or substrates containing cleaning compositions or additives for cleaning compositions

Definitions

  • the present invention relates to detergent compositions comprising high concentration of at least two immunochemically different proteases said combination providing the same or better wash performance compared to the individual proteases.
  • US 2009/0186795 A1 describes a dry dishwashing detergent comprising, inter alia, from about 0.1% to about 3% of an enzyme system including (i) less than about 0.2 of the enzyme product Esperase 6.0T and (ii) an alkaline stable protease product comprising the balance.
  • the present invention discloses a detergent composition comprising at least two immunochemically different proteases, wherein the total amount of protease is more than 0.1 wt% of the detergent composition, and the amount of each protease in the detergent composition is below 0.1 wt%, and the total amount of two or more immunochemically identical proteases is below 0.1 wt%.
  • Fig. 1 provides matrix identifying which of the proteases of SEQ ID NO 1 to SEQ ID NO 17 are immunochemically different.
  • AEP active enzyme protein: Enzyme protein which has a catalytic activity. There is various way to determine AEP. For example, AEP can be calculated by dividing total activities by the enzyme’s specific activity. The amount of enzyme (e.g. protease) is throughout the application considered to be active enzyme protein. In commercially available protease products, the amount of AEP typically constitutes 1-10 wt% of the enzyme product (see, e.g., Pettitt et al., Biofouling, vol. 20(6), 2004, pp. 299-311).
  • Bacterial in relation to polypeptide (such as an enzyme, e.g. a protease) 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.
  • polypeptide such as an enzyme, e.g. a protease
  • bacterial protease or “polypeptide having protease activity obtained from a bacterial source” or “polypeptide is of bacterial origin” thus refers to a protease 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 protease.
  • a sequence encoding a bacterial polypeptide having protease activity may also be referred to as a wildtype protease (or parent protease).
  • Bacterial polypeptide having protease activity includes recombinant produced wild types.
  • the invention provides polypeptides having protease activity, wherein said polypeptides are substantially homologous to a bacterial protease.
  • substantially homologous denotes a polypeptide having protease 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 protease.
  • Biosurfactants are active compounds that are produced at the microbial cell surface or excreted and reduce surface and interfacial tension. Microbial surfactants offer several advantages over synthetic ones, such as low toxicity and high biodegradability, and often remain active at extreme pH and salinity. Biosurfactants are produced by bacteria, yeasts, and filamentous fungi and are generally classified into low molecular-mass molecules (lipopeptides, glycolipids) and high molecular-mass polymers (polymeric and particulate surfactants). Biosurfactants include but are not limited to rhamnolipids and sophorolipids.
  • the detergent adjunct ingredient is different to the proteases of this invention.
  • Suitable adjunct materials include, but are not limited to the components described below such as surfactants, builders, flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, s, s, 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, solvents, and/or pigments.
  • Detergent composition refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles.
  • the detergent composition may be used to e.g. clean textiles 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., liguid, gel, powder, granulate, paste, bar, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liguid and/or solid laundry detergents and fine fabric detergents; fabric fresheners; fabric softeners; laundry boosters; and textile and laundry pre-spotters/pre-treatment).
  • detergent compositions e.g., liguid and/or solid laundry detergents and fine fabric detergents; fabric fresheners; fabric softeners; laundry boosters; and textile and laundry pre-spotters/pre-treatment.
  • the detergent formulation may contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglu- canases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or detergent adjunct ingredients such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers (as set forth herein), 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, bluing agents and fluorescent dyes, antioxidants, and solubilizers.
  • additional enzymes such as proteases, amylases, lipases, cutina
  • Detergent load is the amount of detergent used in a wash cycle.
  • 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-redepo- sition), 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). Also included is the maintenance of whiteness, e.g., the prevention of greying or dullness.
  • 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.
  • textile care benefits are prevention or reduction of dye transfer from one fabric to another fabric or another part of the same fabric (an effect that is also termed dye transfer inhibition or anti-backstaining), removal of protruding or broken fibers from a fabric surface to decrease pilling tendencies or remove already existing pills or fuzz (an effect that also is termed anti-pilling), improvement of the fabric-softness, colour clarification of the fabric and removal of particulate soils which are trapped in the fibers of the fabric or garment.
  • Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.
  • fragment means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has protease activity.
  • fungal in relation to polypeptide (such as an enzyme, e.g. a protease) 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 seguence in the genome by recombinant DNA technology.
  • the term “fungal protease” or “polypeptide having protease activity obtained from a fungal source” thus refers to a protease 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 protease.
  • the nucleotide seguence encoding the fungal polypeptide having protease activity is a seguence naturally in the genetic background of a fungal species.
  • the fungal polypeptide having protease activity encoding by such seguence may also be referred to a wildtype protease (or parent protease).
  • the invention provides polypeptides having protease activity, wherein said polypeptides are substantially homologous to a fungal protease.
  • the term “substantially homologous” denotes a polypeptide having protease 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 protease.
  • the polypeptides being substantially homologous to a fungal protease may be included in the detergent of the present invention and/or be used in the methods of the present invention.
  • 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.
  • Immunochemically different If two enzyme proteins (proteases) cross-react with the same antibody, they are immunochemically different. Immunochemical difference can be tested e.g. by use of the Ouchterlony test.
  • Improved wash performance is defined herein as an enzyme displaying an increased wash performance in a detergent composition relative to the wash performance of same detergent composition without the enzyme e.g. by increased stain removal or improved bleaching.
  • improved wash performance includes wash performance in laundry.
  • 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.
  • Laundering relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a detergent composition and optionally one or more enzymes.
  • the laundering process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand.
  • 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.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
  • sequence identity is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), pref-erably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled “longest identity” is used as the percent identity and is calculated as follows:
  • the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix.
  • the output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • Textile means any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles).
  • the textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and toweling.
  • the textile may be cellulose based such as natural cellulosics, includ-ing 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, cash- mere, 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, cash- mere, 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.
  • Fabric may be conventional washable laundry, for example stained household laundry.
  • fabric or garment it is intended to include the broader term textiles as well.
  • textile also covers fabrics.
  • the term “textile” is used interchangeably with fabric and cloth.
  • variant means a polypeptide having same activity as the parent enzyme comprising an alteration, /.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 protease has the enzymatic activity of the parent.
  • the protease activity of the variant is increased with reference to the parent protease, e.g. the mature polypeptide of SEQ ID NO:1.
  • wash cycle is defined herein as a washing operation wherein textiles are immersed in the wash liquor, mechanical action of some kind is applied to the textile in order to release stains and to facilitate flow of wash liquor in and out of the textile and finally the superfluous wash liquor is removed. After one or more wash cycles, the textile is generally rinsed and dried.
  • Wash liquor is defined herein as the solution or mixture of water and detergent components optionally including one or more enzymes.
  • Wash performance is used as detergent composition’s, enzyme’s or polymer’s capability to remove stains present on the object to be cleaned or maintain color and whiteness of textile during wash.
  • the improvement in the wash performance may be quantified by calculating the so-called delta REM as described in Experimental section.
  • Weight percentage is abbreviated w/w%, wt% or w%. The abbreviations are used interchangeably.
  • Whiteness is defined herein as a broad term with different meanings in different regions and for different consumers. Whiteness can be on white textiles or be used interchangely as brightness for colored textiles. Loss of whiteness or brightness can e.g. be due to greying, yellowing, or removal of optical brighteners/hueing agents. Greying and yellowing can be due to soil redeposition, stain redeposition, dirt/mud redeposition, pollution particles, body soils, colouring from e.g. iron and copper ions or dye transfer. Loss of whiteness might include one or several issues from the list below: colourant or dye effects; incomplete stain removal (e.g.
  • SEQ ID NO:1 is a protease from Bacillus circulans
  • SEQ ID NO:2 is a protease from Bacillus lentus
  • SEQ ID NO:3 is a protease from Bacillus lentus
  • SEQ ID NO:4 is a protease from Bacillus lentus
  • SEQ ID NO:5 is a protease from Bacillus lentus
  • SEQ ID NO:6 is a protease from Bacillus lentus
  • SEQ ID NO:7 is a protease from Bacillus subtilis
  • SEQ ID NO:8 is a protease from Bacillus subtilis
  • SEQ ID NO:9 is a protease from Bacillus subtilis
  • SEQ ID NO: 10 is a protease from Bacillus gibsonii
  • SEQ ID NO: 11 is a protease from Bacillus gibsonii
  • SEQ ID NO: 12 is a protease from Bacillus amyloliquefaciens
  • SEQ ID NO: 13 is a protease from Bacillus amyloliquefaciens
  • SEQ ID NO: 14 is a protease from Bacillus amyloliquefaciens
  • SEQ ID NO: 15 is a protease from Bacillus amyloliquefaciens
  • SEQ ID NO: 16 is a protease from Bacillus amyloliquefaciens
  • SEQ ID NO: 17 is a protease from Bacillus lentus
  • the inventors of the present invention have found a solution to the problem, namely the addition of two or more immunochemically different proteases to reach the requested higher protease concentration without increasing the risk of hazardous reactions, such as allergic reactions, in particular respiratory allergic reactions.
  • the immunochemical difference between two proteases may for the purpose of this invention be determined by the in silico method disclosed in the experimental section below. The settings in the in silico method was chosen so the results match the experimental Ouchterlony test results for immunochemical identity as close as possible.
  • the proteases of the present invention may comprise one or more conservative amino acid substitutions that do not alter their immunochemical properties.
  • conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
  • Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L.
  • amino acid changes are of such a nature that the physico-chemical properties, but not the immunochemical properties of the polypeptides are altered.
  • amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
  • Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for enzyme activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.
  • the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a/., 1988, DNA 7: 127).
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • the polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
  • the polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention.
  • a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention.
  • Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoters) and terminator.
  • Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper eta/., 1993, EMBO J. 12:2575- 2583; Dawson et al., 1994, Sc/ence 266:776-779).
  • a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides.
  • cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol.
  • the total concentration of the proteases in the wash liquor is typically from about 0.2 ppm to 20 ppm in solution.
  • the water temperatures typically range from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C and about 90°C.
  • the water to fabric ratio is typically from about 1 :1 to about 30:1.
  • the proteases 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 polypeptide of the present invention may also be incorporated in the detergent formulations disclosed in W097/07202, which is hereby incorporated by reference.
  • Liquid enzyme formulations are disclosed in W097/07202, which is hereby incorporated by reference.
  • the enzymes may be formulated as a liquid enzyme formulation, which is generally a pourable composition, though it may also have a high viscosity.
  • the physical appearance and properties of a liquid enzyme formulation may vary a lot - for example, they may have different viscosities (gel to water-like), be colored, not colored, clear, hazy, and even with solid particles like in slurries and suspensions.
  • the minimum ingredients are the enzymes (protease, lipase and other enzymes present) and a solvent system to make it a liquid.
  • the solvent system may comprise water, polyols (such as glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol, sugar alcohol (e.g. sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol or adonitol), polypropylene glycol, and/or polyethylene glycol), ethanol, sugars, and salts.
  • polyols such as glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol
  • sugar alcohol e.g. sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol or adonitol
  • polypropylene glycol e.g. sorbitol, mannitol, erythrito
  • a liquid enzyme formulation may be prepared by mixing a solvent system and an enzyme concentrate with a desired degree of purity (or enzyme particles to obtain a slurry/suspension).
  • liquid enzyme composition comprises:
  • the enzymes (proteases and optionally other enzymes present) in the liquid composition of the invention may be stabilized using conventional stabilizing agents.
  • stabilizing agents include, but are not limited to, sugars like glucose, fructose, sucrose, or trehalose; polyols like glycerol, propylene glycol; addition of salt to increase the ionic strength; divalent cations (e.g., Ca 2+ or Mg 2+ ); and enzyme inhibitors, enzyme substrates, or various polymers (e.g., PVP).
  • Selecting the optimal pH for the formulation may be very important for enzyme stability. The optimal pH depends on the specific enzyme but is typically in the range of pH 4-9.
  • surfactants like nonionic surfactant (e.g., alcohol ethoxylates) can improve the physical stability of the enzyme formulations.
  • compositions comprising a proteases, wherein the composition further comprises: (i) a polyol, preferably selected from glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol, polyethylene glycol, sugar alcohols, sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol and adonitol;
  • a polyol preferably selected from glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol, polyethylene glycol, sugar alcohols, sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol and adonitol;
  • an additional enzyme preferably selected from proteases, amylases, cellulases, lipases, DNases, mannanases, and/or xanthanases
  • a surfactant preferably selected from anionic and nonionic surfactants
  • Slurries or dispersions of enzymes are typically prepared by dispersing small particles of enzymes (e.g., spray-dried particles) in a liquid medium in which the enzyme is sparingly soluble, e.g., a liquid nonionic surfactant or a liquid polyethylene glycol. Powder can also be added to aqueous systems in an amount so not all go into solution (above the solubility limit).
  • Another format is crystal suspensions which can also be aqueous liquids (see for example WO2019/002356).
  • Another way to prepare such dispersion is by preparing water-in-oil emulsions, where the enzyme is in the water phase, and evaporate the water from the droplets.
  • Such slurries/suspension can be physically stabilized (to reduce or avoid sedimentation) by addition of rheology modifiers, such as fumed silica or xanthan gum, typically to get a shear thinning rheology.
  • the enzymes may also be formulated as a solid/granular enzyme formulation.
  • Non-dusting granulates may be produced, e.g. as disclosed in US 4,106,991 and US 4,661 ,452, and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • PEG poly(ethylene oxide) products
  • PEG polyethyleneglycol
  • the proteases may be formulated as a granule for example as a co-granule that combines one or more enzymes or benefit agents (such as MnTACN or other bleaching components).
  • additional enzymes include lipases, xyloglucanases, perhydrolases, peroxidases, proteases, laccases, hemicellulases, care cellulases, cellulases, cellobiose dehydrogenases, xylanases, phospho lipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pento- sanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, amylases, DNAse,
  • An embodiment of the invention relates to an enzyme granule/particle comprising proteases.
  • the granule is composed of a core, and optionally one or more coatings (outer layers) surrounding the core.
  • the granule/particle size, measured as equivalent spherical diameter (volume based average particle size), of the granule is 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibers), stabilizing agents, solubilising agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • the core may include binders, such as synthetic polymer, wax, fat, or carbohydrate.
  • the core may comprise a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
  • the core may consist of an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
  • the core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1 ; 1980; Elsevier. These methods are well-known in the art and have also been described in international patent application WO2015/028567, pages 3-5, which is incorporated by reference.
  • the core of the enzyme granule/particle may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule.
  • the optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are shown in WO 93/07263 and WO 97/23606.
  • the present invention provides a granule, which comprises:
  • Another aspect of the invention relates to a layered granule, comprising:
  • the enzymes may also be formulated as an encapsulated enzyme formulation (an ‘encapsulate’). This is particularly useful for separating the enzyme from other ingredients when the enzyme is added into, for example, a (liquid) cleaning composition, such as the detergent compositions described below.
  • Physical separation can be used to solve incompatibility between the enzyme(s) and other components. Incompatibility can arise if the other components are either reactive against the enzyme, or if the other components are substrates of the enzyme. Other enzymes can be substrates of proteases.
  • the enzyme may be encapsulated in a matrix, preferably a water-soluble or water dispersible matrix (e.g., water-soluble polymer particles), for example as described in WO 2016/023685.
  • a water-soluble polymeric matrix is a matrix composition comprising polyvinyl alcohol. Such compositions are also used for encapsulating detergent compositions in unit-dose formats.
  • the enzyme may also be encapsulated in core-shell microcapsules, for example as described in WO 2015/144784, or as described in the IP.com disclosure IPCOM000239419D.
  • Such core-shell capsules can be prepared using a number of technologies known in the art, e.g., by interfacial polymerization using either a water-in-oil or an oil-in-water emulsion, where polymers are crosslinked at the surface of the droplets in the emulsion (the interface between water and oil), thus forming a wall/membrane around each droplet/capsule.
  • the enzymes 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 wt% to 98 wt% moisture sink component and the composition additionally comprises from 20 wt% 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 a proteases and (a) one or more enzymes selected from the group consisting of lipases, xyloglucanases, perhydrolases, peroxidases, proteases, laccases and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, care cellulases, cellulases, cellobiose dehydrogenases, xylanases, phospho lipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, DNAse, and mixtures thereof
  • the enzymes (proteases and optionally other enzymes present) used in the above-mentioned enzyme formulations may be purified to any desired degree of purity. This includes high levels of purification, as achieved for example by using methods of crystallization - but also none or low levels of purification, as achieved for example by using crude fermentation broth, as described in WO 2001/025411 , or in WO 2009/152176.
  • the enzyme formulations may comprise one or more microorganisms or microbes.
  • any microorganism(s) may be used in the enzyme/detergent formulations in any suitable amount(s)/concentration(s).
  • Microorganisms may be used as the only biologically active ingredient, but they may also be used in conjunction with one or more of the enzymes described above.
  • the purpose of adding the microorganism(s) may, for example, be to reduce malodor as described in WO 2012/112718.
  • Other purposes could include in-situ production of desirable biological compounds, or inoculation/population of a locus with the microorganism(s) to competitively prevent other non-desirable microorganisms form populating the same locus (competitive exclusion).
  • microorganism generally means small organisms that are visible through a microscope. Microorganisms often exist as single cells or as colonies of cells. Some microorganisms may be multicellular. Microorganisms include prokaryotic (e.g., bacteria and archaea) and eukaryotic (e.g., some fungi, algae, protozoa) organisms. Examples of bacteria may be Grampositive bacteria or Gram-negative bacteria. Example forms of bacteria include vegetative cells and endospores. Examples of fungi may be yeasts, molds and mushrooms. Example forms of fungi include hyphae and spores. Herein, viruses may be considered microorganisms.
  • prokaryotic e.g., bacteria and archaea
  • eukaryotic e.g., some fungi, algae, protozoa
  • bacteria may be Grampositive bacteria or Gram-negative bacteria.
  • Example forms of bacteria include vegetative cells and endospores. Examples of fungi may be yeasts, molds and
  • Microorganisms may be recombinant or non-recombinant.
  • the microorganisms may produce various substances (e.g., enzymes) that are useful for inclusion in detergent compositions. Extracts from microorganisms or fractions from the extracts may be used in the detergents. Media in which microorganisms are cultivated or extracts or fractions from the media may also be used in detergents.
  • specific of the microorganisms, substances produced by the microorganisms, extracts, media, and fractions thereof, may be specifically excluded from the detergents.
  • the microorganisms, or substances produced by, or extracted from, the microorganisms may activate, enhance, preserve, prolong, and the like, detergent activity or components contained with detergents.
  • microorganisms may be cultivated using methods known in the art.
  • the microorganisms may then be processed or formulated in various ways.
  • the microorganisms may be desiccated (e.g., lyophilized).
  • the microorganisms may be encapsulated (e.g., spray drying).
  • Many other treatments or formulations are possible. These treatments or preparations may facilitate retention of microorganism viability over time and/or in the presence of detergent components.
  • microorganisms in detergents may not be viable.
  • the processed/formulated microorganisms may be added to detergents prior to, or at the time the detergents are used.
  • the microorganism is a species of Bacillus, for example, at least one species of Bacillus selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefa- ciens, Bacillus licheniformis, Bacillus atrophaeus, Bacillus pumilus, Bacillus megaterium, or a combination thereof.
  • Bacillus subtilis Bacillus subtilis
  • Bacillus amyloliquefa- ciens Bacillus amyloliquefa- ciens
  • Bacillus licheniformis Bacillus atrophaeus
  • Bacillus pumilus Bacillus megaterium
  • the aforementioned Bacillus species are on an endospore form, which significantly improves the storage stability.
  • Detergent compositions are on an endospore form, which significantly improves the storage stability.
  • the invention is directed to detergent compositions comprising at least two proteases in combination with one or more additional cleaning composition components.
  • the detergent composition comprises at least two polypeptides having protease activity with an amino acid sequence having at least 60% identity, such as 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% identity to the amino acid sequence set forth in SEQ ID NO:1 to SEQ ID NO:17.
  • the detergent composition is in solid form.
  • the detergent composition is in a liquid or gel form.
  • a bar form In one embodiment the detergent may be wrapped in water soluble PVOH film.
  • the liquid detergent composition may comprise a microcapsule, and thus form part of any detergent composition in any form, such as liquid and powder detergents, and soap and detergent bars.
  • the invention is directed to liquid detergent compositions comprising a microcapsule, as described above, in combination with one or more additional cleaning composition components.
  • the microcapsule may be added to the liquid detergent composition in an amount corresponding to from 0.1% to 5% (w/w) active enzyme protein; preferably from 0. 1% to 4%, such as from 0.1% to 3%, such as from 0.1% to 2.5%, such as from 0.1% to 2%, such as from 0.1% to 1.5%, such as from 0.1% to 1%, and most preferably from 0.01% to 1% (w/w) active enzyme protein.
  • the liquid detergent composition has a physical form, which is not solid (or gas). It may be a pourable liquid, a paste, a pourable gel or a non-pourable gel. It may be either isotropic or structured, preferably isotropic. It may be a formulation useful for washing in automatic washing machines or for hand washing. It may also be a personal care product, such as a shampoo, toothpaste, or a hand soap.
  • the liquid detergent composition may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to 70% water, up to 50% water, up to 40% water, up to 30% water, or up to 20% water.
  • Other types of liquids including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid detergent.
  • An aqueous liquid detergent may contain from 0-30% organic solvent.
  • a liquid detergent may even be non-aqueous, wherein the water content is below 10%, preferably below 5%.
  • Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
  • the detergent composition may take the form of a unit dose product.
  • a unit dose product is the packaging of a single dose in a non-reusable container. It is increasingly used in detergents for laundry.
  • a detergent unit dose product is the packaging (e.g., in a pouch made from a water-soluble film) of the amount of detergent used for a single wash.
  • Pouches can be of any form, shape and material which is suitable for holding the composition, e.g., without allowing the release of the composition from the pouch prior to 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, maltodextrin, polymethacrylates, 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 a blend composition comprising hydrolytically degradable and water-soluble polymer blends such as polyactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticizers like glycerol, ethylene glycerol, Propylene glycol, sorbitol and mixtures thereof.
  • the pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water-soluble film.
  • the compartment for liquid components can be different in composition than compartments containing solids (see e.g., US 2009/0011970).
  • detergent components may include, for textile care, the consideration of the type of textile to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product.
  • components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.
  • any detergent components known in the art for use in detergents may also be utilized.
  • Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/dis- integration agents, dyes, enzyme stabilizers (including boric acid, borates, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination.
  • Any ingredient known in the art for use in detergents may be utilized. The choice of such ingredients is well within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
  • the cleaning 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 and at least one non-ionic surfactant, the weight ratio of anionic to nonionic surfactant may be from 20:1 to 1 :20.
  • 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 (SLS); alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates
  • the detergent When included therein the detergent will usually contain from about 0,1% 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), dimethyl- distearylammonium 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 dimethyl- distearylammonium 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 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%.
  • nonionic surfactants 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 TWEEN, and combinations thereof.
  • PFA propoxylated fatty alcohols
  • 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-p-D-maltopyranoside, thiomaltopyranoside or a cyclic- maltopyranoside, such as described in EP2516606 B1.
  • Other biosurfactants may include rhamnolipids and sophorolipids.
  • a hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment).
  • hydrotropes typically 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 selfaggregation, see e.g. review by Hodgdon and Kaier (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming 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 detergent composition may contain about 0-65% by weight, such as about 5% to about 50% of a detergent builder or co-builder, or a mixture thereof.
  • 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"-nitrilotriethan-1-ol), and (carboxymethyl)inulin (CM
  • 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 or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). According to the present invention, these components can be included in lower levels than in currently available detergent compositions.
  • 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 1-hydroxyethane-1 ,1-diylbis(phosphonic acid
  • EDTMPA ethylenedia- minetetramethylenetetrakis(phosphonic acid)
  • DTMPA or DTPMPA N-(2-hydroxyethyl)iminodiacetic acid
  • ASMA aspartic acid-N-monoacetic acid
  • ASDA aspartic acid-N,N -di acetic acid
  • ASDA aspartic acid-N,N -di acetic acid (AS
  • 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.
  • 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: a) Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids such as peroxybenzoic acid and its ring-substituted derivatives, peroxy-a-naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, e-phthalimidoperoxycaproic acid [phthalimidoperoxyhexanoic acid (PAP)], and o-carboxybenzamidoperoxycaproic acid; aliphatic and aromatic diperoxydicarboxylic acids such as diperoxydodecanedio
  • 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-(deca- noyloxy)benzene-1 -sulfonate, 4-(decanoyloxy)benzoic acid (DOBA), sodium 4-(nona- noyloxy)benzene-1 -sulfonate (NOBS), and/or those disclosed in WO98/17767.
  • TAED tetraacetylethylenediamine
  • ISONOBS sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate
  • LOBS 4-(dodecanoyloxy)benzene-1 -sulfonate
  • DOBA 4-(decanoyloxy)
  • 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.
  • 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 bleaching systems are described, e.g. in W02007/087258, W02007/087244, W02007/087259, EP1867708 (Vitamin K) and W02007/087242.
  • Suitable photobleaches may for example be sulfonated zinc or aluminium phthalocyanines.
  • detergent compositions may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1 % of a polymer. Any polymer known in the art for use in detergents may be utilized.
  • the polymer may function as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties.
  • Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs.
  • Exemplary polymers include poly(vinyl alcohol) (P A), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of polyethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-/V-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI).
  • P A poly(vinyl alcohol)
  • PVP poly(vinylpyrrolidone)
  • PEG poly(ethylene oxide)
  • CMI carboxymethyl inulin
  • silicones copolymers of terephthalic acid and oli
  • polymers include polyethylene oxide and polypropylene oxide (PEO-PPO), diquaternium ethoxy sulfate, styrene/acrylic copolymer and perfume capsules
  • PEO-PPO polypropylene oxide
  • diquaternium ethoxy sulfate diquaternium ethoxy sulfate
  • styrene/acrylic copolymer and perfume capsules
  • Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.
  • 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.
  • certain of the above polymers namely, a polyacrylic acid, a modified polyacrylic acid polymer, a modified polyacrylic acid copolymer, a maleic acid-acrylic acid copolymer, carboxymethyl cellulose, cellulose gum, methyl cellulose, and/or combinations thereof, can be included in lower levels than in currently available detergent compositions, or even more preferably, excluded altogether.
  • the detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liguor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light.
  • fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum.
  • Suitable fabric hueing agents include dyes and dye-clay conjugates and may also include pigments.
  • Suitable dyes include small molecule dyes and polymeric dyes.
  • Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.l.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in W02005/03274, W02005/03275, W02005/03276 and EP1876226 (hereby incorporated by reference).
  • the detergent composition preferably comprises from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt% fabric hueing agent.
  • the composition may comprise from 0.0001 wt% to 0.2 wt% fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch.
  • Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and W02007/087243.
  • 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 /V-oxide polymers, copolymers of /V-viny I pyrrolidone and /V-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.
  • Fluorescent whitening agent include, but are not limited to, polyvinylpyrrolidone polymers, polyamine /V-oxide polymers, copolymers of /V-viny I pyrrolidone and /V-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles
  • 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 laundry detergent composition may be used in the composition of the present invention.
  • the most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulfonic acid derivatives, diaryl py- razoline derivatives and bisphenyl-distyryl derivatives.
  • diaminostilbene-sulfonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-dieth- anolamino-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-(/V-methyl-/ ⁇ /-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'-disul- fonate and sodium 5-(2/7-naphtho[1 ,2-d][1 ,2,3]triazol-2-yl)-2
  • 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.
  • Tinopal CBS-X is a 4.4'-bis-(sulfostyryl)-biphenyl disodium salt also known as Disodium Distyrylbiphenyl Disulfonate.
  • fluorescers suitable for use in the invention include the 1 -3-diaryl pyra- zolines 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).
  • the detergent compositions of the present invention may also include one or more antiredeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid.
  • CMC carboxymethylcellulose
  • PVA polyvinyl alcohol
  • PEG polyethyleneglycol
  • homopolymers of acrylic acid copolymers of acrylic acid and maleic acid.
  • the cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
  • certain of the above polymers namely, a polyacrylic acid, a modified polyacrylic acid polymer, a modified polyacrylic acid copolymer, a maleic acid-acrylic acid copolymer, carboxymethyl cellulose, cellulose gum, methyl cellulose, and/or combinations thereof, can by introduction of cellulases be included in lower levels than in currently available detergent compositions, or excluded altogether, thus improving the sustainability profile of the detergent composition.
  • the detergent compositions of the present invention may also include one or more rheology modifiers, structurants or thickeners, as distinct from viscosity reducing agents.
  • the rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, polymeric rheology modifiers which impart shear thinning characteristics to the ague- ous liguid matrix of a liguid detergent composition.
  • the rheology and viscosity of the detergent can be modified and adjusted by methods known in the art, for example as shown in EP 2169040.
  • adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liguid detergents and/or structure elasticizing agents.
  • the detergent additive as well as the detergent composition may comprise one or more [additional] enzymes such as lipases, cutinases, cellulases, amylases, carbohydrases, DNases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases, e.g., a laccase, and/or peroxidase.
  • additional enzymes such as lipases, cutinases, cellulases, amylases, carbohydrases, DNases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases, e.g., a laccase, and/or peroxidase.
  • the properties of the selected enzyme(s) should be compatible with the selected detergent, (/.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
  • cellulase means one or more (e.g., several) enzymes that hydrolyze a cellulosic material.
  • polypeptide having cellulase activity and cellulase are used interchangeably.
  • Cellulases may be selected from the group consisting of cellulases belonging to GH5, GH44, GH45, EC 3.2.1.4, EC 3.2.1.21 , EC 3.2.1.91 and EC 3.2.1.172.
  • Such enzymes include endoglu- canase(s) (e.g. EC 3.2.1.4), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
  • 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-glu- canase also referred to as endoglucanase.
  • DNase means a polypeptide with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA.
  • 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. licheni- formis, B. halodurans, B. clausii, or H. insolens.
  • Suitable mannanases are described in WO 1999/064619. A commercially available mannanase is Mannaway (Novozymes A/S).
  • Xanthan lyase (EC 4.2.2.12) is an enzyme that catalyzes the chemical reaction of cleaving the beta-D-mannosyl-beta-D-1 ,4-glucuronosyl bond on the polysaccharide xanthan.
  • This enzyme belongs to the family of lyases, specifically those carbon-oxygen lyases acting on polysaccharides.
  • 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 Sub- tilisin 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 tromBacillus.
  • Bacillus species from which subtilases have been derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amylolique- faciens, 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.
  • Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, DuralaseTM, DurazymTM, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, PrimaseTM, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coro- nase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Blaze Evity® 200T, Neutrase®, Everlase®, Esperase®, Progress® Uno, Progress® In and Progress® Excel (Novozymes A/S), those sold under the tradename MaxataseTM, MaxacaiTM, Maxapem®, Purafect® Ox, Purafect® OxP, Puramax®, FN2TM, FN3TM, FN4 ex TM, Excellase®, Ex
  • Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermo- myces, 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.
  • Thermo- myces e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216
  • cutinase from Humicola e.g
  • strain SD705 (W095/06720 & W096/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyces lipases (W010/065455), cutinase from Magnaporthe grisea (WO10/107560), cutinase from Pseudomonas mendocina (US5,389,536), lipase from Thermobifida fusca (W011/084412), Geobacillus stearothermophilus lipase (W011/084417), lipase from Bacillus subtilis (W011/084599), and lipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis (WO12/137147).
  • lipase variants such as those described in EP407225, WO92/05249, WO94/01541 , WO94/25578, WO95/14783, WO95/30744, WO95/35381 , WO95/22615,
  • Preferred commercial lipase products include include Lipolase 100T/L, Lipex 100T/L, Li- pex 105T, Lipex Evity 100L, Lipex Evity 200L (all Novozymes A/S), Preferenz® L 100 (DuPont).
  • 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 include an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus li- cheniformis, 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 N0:6 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.
  • amylase variants such as those described in WQ2011/098531 , WQ2013/001078 and WQ2013/001087.
  • amylases are DuramylTM, TermamylTM, FungamylTM, Stainzyme TM , Stainzyme PlusTM, NatalaseTM, Liquozyme X and BANTM Amplify; Amplify Prime; (from Novo- zymes A/S), and RapidaseTM , PurastarTM/EffectenzTM, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc./DuPont).
  • 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 GuardzymeTM (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 Nomenclature Committee of the 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, /.e., a van- adate-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 ver- ruculosa 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 Genic- ulosporium 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).
  • 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, Pomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. ci- nerea, 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, Pol- yporus, 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 proteases 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.
  • 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 NH4 + and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.
  • 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, proteases, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared, and the mixture is then plodded.
  • the proteases 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 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. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition 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 laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water-soluble film.
  • the compartment for liquid components can be different in composition than compartments containing solids: 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 invention is further summarized in the following embodiments.
  • the embodiments are indicated as E1 , E2 and so forth.
  • a detergent composition comprising at least two immunochemically different proteases, wherein a. the total amount of protease is at least 0.1 wt% of the detergent composition, b. the amount of each protease in the detergent composition is below 0.1 wt%, and c. the total amount of two or more immunochemically identical proteases is below 0.1 wt% d.
  • the detergent composition optionally comprises at least one additional enzyme which is not a protease.
  • the detergent composition according to E1 wherein the at least two immunochemically different proteases are selected from the group consisting of proteases having at least 60% identity to any of the proteases of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, and SEQ ID N0:17.
  • proteases having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as
  • the detergent composition according to any of E1 to E3 comprising a protease that has at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to the protease of SEQ ID NO:1 .
  • the detergent composition according to E4 further comprising a protease selected from the group of polypeptides that has at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to any of the proteases of SEQ ID NO:3, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
  • the detergent composition according to E5 comprising the protease of SEQ ID NO 1 in and at least one other protease selected from the group consisting of SEQ ID NO:3, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
  • the detergent composition according to E1 comprising any of the following combinations of proteases:SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11 , SEQ ID NO:1 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:17, SEQ ID NO:2 + SEQ ID NO:5,
  • the detergent composition according to E7 comprising any of the following combinations of proteases:SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11 , SEQ ID NO:1 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:2
  • E10 The detergent composition according to any of E1 to E8, wherein the detergent is supplied as a unit dose product, such as a pouch or tablet.
  • E11 The detergent composition according to any of E1 to E8, wherein the detergent is supplied as a liquid detergent or as a powder product.
  • E12 The detergent composition according to any of E1 to E11 comprising at least one additional enzyme selected from the group consisting of cellulases, mannanases, amylases, DNases, lipases, xanthanases, pectate lyases and oxidoreductases.
  • E13 Use of the detergent composition of any of E1 to E12 for laundering, hand dishwashing, automatic dishwashing or hard surface cleaning.
  • Composition 1 Liquid detergent
  • Composition 2 Unit Dose
  • composition 3 Powder detergent
  • the invention is directed to an ADW (Automatic Dish Wash) compositions comprising an enzyme of the present invention in combination with one or more additional ADW composition components.
  • ADW Automatic Dish Wash
  • Surfactant ingredients can be obtained from BASF, Ludwigshafen, Germany (Lutensol®); Shell Chemicals, London, UK; Stepan, Northfield, III, USA; Huntsman, Huntsman, Salt Lake City, Utah, USA; Clariant, Sulzbach, Germany (Praepagen®).
  • Sodium tripolyphosphate can be obtained from Rhodia, Paris, France.
  • Zeolite can be obtained from Industrial Zeolite (UK) Ltd, Grays, Essex, UK.
  • Citric acid and sodium citrate can be obtained from Jungbunzlauer, Basel, Switzerland.
  • NOBS is sodium nonanoyloxybenzenesulfonate, supplied by Eastman, Batesville, Ark., USA.
  • TAED is tetraacetylethylenediamine, supplied under the Peractive® brand name by Clariant GmbH, Sulzbach, Germany.
  • Sodium carbonate and sodium bicarbonate can be obtained from Solvay, Brussels, Belgium.
  • Polyacrylate, polyacrylate/maleate copolymers can be obtained from BASF, Ludwigshafen, Germany.
  • Repel-O-Tex® can be obtained from Rhodia, Paris, France.
  • Texcare® can be obtained from Clariant, Sulzbach, Germany.
  • Sodium percarbonate and sodium carbonate can be obtained from Solvay, Houston, Tex., USA.
  • HEDP Hydroxy ethane di phosphonate
  • Enzymes Savinase®, Savinase® Ultra, Stainzyme® Plus, Lipex®, Lipolex®, Lipoclean®, Celluclean®, Carezyme®, Natalase®, Stainzyme®, Stainzyme® Plus, Termamyl®, Termamyl® ultra, and Mannaway® can be obtained from Novozymes, Bagsvaerd, Denmark. Enzymes Purafect®, FN3, FN4 and Optisize can be obtained from Genencor International Inc., Palo Alto, California, US.
  • Direct violet 9 and 99 can be obtained from BASF DE, Ludwigshafen, Germany.
  • Solvent violet 13 can be obtained from Ningbo Lixing Chemical Co., Ltd. Ningbo, Zhejiang, China.
  • Brighteners can be obtained from Ciba Specialty Chemicals, Basel, Switzerland.
  • the performance of the enzyme variants and their blends was measured as the brightness of the colour of the textile samples washed with that specific protease/protease-blend. Brightness can also be expressed as the intensity of the light reflected from the textile sample when illuminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a clean textile. Therefore, the intensity of the reflected light can be used to measure wash performance of the proteases and the protease blends.
  • ⁇ Novozymes Color Vector Analyzer ⁇ Novozymes Color Vector Analyzer
  • the program retrieves the values from the image and converts them into values for red, green and blue (RGB).
  • the intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
  • the detergent for the wash performance tests is a liquid laundry model detergent (see below).
  • Standard textile pieces are obtained from Center for Testmaterials BV, P.O. Box 120, 3133 KT Vlaardingen, The Netherlands, and from EMPA Testmaterials AG, Mdvenstrasse 12, CH-9015 St. Gallen, Switzerland.
  • type PC-03 polyyester/cotton textile stained with chocolate milk with carbon black
  • PC-10 polyyester/cotton textile stained with pigment, oil and milk
  • EMPA 117 EH extra heated
  • This method compares two protein sequences by
  • the fragments were compared using the Fasta software, version 3.4, and filtering for 100% identity over window length amino acids.
  • a set of three or more protein sequences are considered immunochemically different if all possible pairs within the set are immunochemically different according to the pairwise method above.
  • window length and “match threshold” are chosen so the results match the experimental Ouchterlony test results as close as possible. In this case a window length of 19 and a match threshold of 10 was used.
  • the zone of equivalence lines may give a full identity (i.e. a continuous line), partial identity (i.e. a continuous line with a spur at one end), or a non-identity (i.e. the two lines cross completely).
  • SEQ ID NO:2 was immunochemically different from SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:12
  • SEQ ID NO:3 was immunochemically different from SEQ ID NO:5 and SEQ ID NO:6
  • SEQ ID NO:5 was immunochemically different from SEQ ID NO:6 and SEQ ID NO: 12
  • SEQ ID NO:6 was immunochemically different from SEQ ID NO: 12
  • SEQ ID NO:2 was not immunochemically different from SEQ ID NO:3 and SEQ ID NO:4
  • SEQ ID NO:1 was immunochemically different from SEQ ID NO:2
  • SEQ ID NO:12 was immunochemically different from SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6
  • the delta intensity values for each of the four enzyme concentrations were added together for each treatment (single enzyme or 1 :1 mixture) to obtain a sum of delta intensity for each treatment for each of the three stains wo stains PC-10, PC-03, and EMPA117EH.
  • the listed protease combinations perform better than SEQ ID NO:1 alone on at least one of the three tested stains, see Table 1 below.

Abstract

The present invention relates to detergent compositions comprising immunochemically different proteases.

Description

COMBINATION OF IMMUNOCHEMICALLY DIFFERENT PROTEASES
Reference to sequence listing
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to detergent compositions comprising high concentration of at least two immunochemically different proteases said combination providing the same or better wash performance compared to the individual proteases.
BACKGROUND OF THE INVENTION
The ability of a detergent to remove stains from the surface of textiles is an obvious care- about for the consumer and various surfactant ingredients play a role in that process. However, there is a desire to reduce the amount of detergents used in household care for several reasons. One reason is that some of the ingredients in detergents are derived from petrochemical resources and face scrutiny due to environmental concerns, most of all for not being sustainable because they are from a non-renewable source and are poorly biodegradable or even persistent in the environment. Another reason is compaction of detergent compositions: making detergent compositions reduces production cost, eases transportation, and ultimately means less burden on the environment, but reduction of surfactants also calls for the development of sustainable solutions that ensures continued performance of the detergent compositions.
US 2009/0186795 A1 describes a dry dishwashing detergent comprising, inter alia, from about 0.1% to about 3% of an enzyme system including (i) less than about 0.2 of the enzyme product Esperase 6.0T and (ii) an alkaline stable protease product comprising the balance.
SUMMARY OF THE INVENTION
Compaction of the detergents and the desire to replace surfactants with active ingredients that are more sustainable causes higher enzyme concentrations, including higher concentrations of proteases. The drive towards higher protease concentrations is nevertheless hampered by the fact that the allergenic potential of proteases potentially can cause respiratory allergic reactions when immunochemically identical proteases are present in high concentration, both when handled in the production of detergents and at the end-user during laundering, dish washing or hard surface cleaning. The inventors of the present invention have found a solution to the problem, namely the addition of two or more immunochemically different proteases to reach the requested higher protease concentration without increasing the risk of causing allergic reactions. Accordingly, the present invention discloses a detergent composition comprising at least two immunochemically different proteases, wherein the total amount of protease is more than 0.1 wt% of the detergent composition, and the amount of each protease in the detergent composition is below 0.1 wt%, and the total amount of two or more immunochemically identical proteases is below 0.1 wt%.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 provides matrix identifying which of the proteases of SEQ ID NO 1 to SEQ ID NO 17 are immunochemically different.
DEFINITIONS
As used herein, the articles "a" and "an" are understood to mean one or more of what is claimed or described
AEP (active enzyme protein): Enzyme protein which has a catalytic activity. There is various way to determine AEP. For example, AEP can be calculated by dividing total activities by the enzyme’s specific activity. The amount of enzyme (e.g. protease) is throughout the application considered to be active enzyme protein. In commercially available protease products, the amount of AEP typically constitutes 1-10 wt% of the enzyme product (see, e.g., Pettitt et al., Biofouling, vol. 20(6), 2004, pp. 299-311).
Bacterial: The term “bacterial” in relation to polypeptide (such as an enzyme, e.g. a protease) 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 protease” or “polypeptide having protease activity obtained from a bacterial source” or “polypeptide is of bacterial origin” thus refers to a protease 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 protease. A sequence encoding a bacterial polypeptide having protease activity may also be referred to as a wildtype protease (or parent protease). Bacterial polypeptide having protease activity includes recombinant produced wild types. In a further aspect, the invention provides polypeptides having protease activity, wherein said polypeptides are substantially homologous to a bacterial protease. In the context of the present invention, the term “substantially homologous” denotes a polypeptide having protease 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 protease.
Biosurfactants: Biosurfactants are active compounds that are produced at the microbial cell surface or excreted and reduce surface and interfacial tension. Microbial surfactants offer several advantages over synthetic ones, such as low toxicity and high biodegradability, and often remain active at extreme pH and salinity. Biosurfactants are produced by bacteria, yeasts, and filamentous fungi and are generally classified into low molecular-mass molecules (lipopeptides, glycolipids) and high molecular-mass polymers (polymeric and particulate surfactants). Biosurfactants include but are not limited to rhamnolipids and sophorolipids.
Detergent adjunct ingredient: The detergent adjunct ingredient is different to the proteases of this invention. The precise nature of these additional adjunct components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the operation for which it is to be used. Suitable adjunct materials include, but are not limited to the components described below such as surfactants, builders, flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, s, s, 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, solvents, and/or pigments.
Detergent composition: The term “detergent composition” refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles. The detergent composition may be used to e.g. clean textiles 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., liguid, gel, powder, granulate, paste, bar, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liguid and/or solid laundry detergents and fine fabric detergents; fabric fresheners; fabric softeners; laundry boosters; and textile and laundry pre-spotters/pre-treatment). In addition to containing the enzyme of the invention, the detergent formulation may contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglu- canases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or detergent adjunct ingredients such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers (as set forth herein), 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, bluing agents and fluorescent dyes, antioxidants, and solubilizers. The terms “detergent” and “detergent composition” may be used interchangeably.
Detergent load is the amount of detergent used in a wash cycle.
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-redepo- sition), 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). Also included is the maintenance of whiteness, e.g., the prevention of greying or dullness. 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 fabric to another fabric or another part of the same fabric (an effect that is also termed dye transfer inhibition or anti-backstaining), removal of protruding or broken fibers from a fabric surface to decrease pilling tendencies or remove already existing pills or fuzz (an effect that also is termed anti-pilling), improvement of the fabric-softness, colour clarification of the fabric and removal of particulate soils which are trapped in the fibers of the fabric or garment. Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.
Fragment: The term “fragment” means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has protease activity.
Fungal: In the context of the present invention the term “fungal” in relation to polypeptide (such as an enzyme, e.g. a protease) 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 seguence in the genome by recombinant DNA technology. In the context of the present invention, the term “fungal protease” or “polypeptide having protease activity obtained from a fungal source” thus refers to a protease 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 protease. Thus, the nucleotide seguence encoding the fungal polypeptide having protease activity is a seguence naturally in the genetic background of a fungal species. The fungal polypeptide having protease activity encoding by such seguence may also be referred to a wildtype protease (or parent protease). In a further aspect, the invention provides polypeptides having protease activity, wherein said polypeptides are substantially homologous to a fungal protease. In the context of the present invention, the term “substantially homologous” denotes a polypeptide having protease 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 protease. The polypeptides being substantially homologous to a fungal protease may be included in the detergent of the present invention and/or be used in the methods of the present invention.
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.
Immunochemically different: If two enzyme proteins (proteases) cross-react with the same antibody, they are immunochemically different. Immunochemical difference can be tested e.g. by use of the Ouchterlony test.
Improved wash performance: The term “improved wash performance” is defined herein as an enzyme displaying an increased wash performance in a detergent composition relative to the wash performance of same detergent composition without the enzyme e.g. by increased stain removal or improved bleaching. The term “improved wash performance” includes wash performance in laundry.
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.
Laundering: The term “laundering” relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a detergent composition and optionally one or more enzymes. The laundering process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand.
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.
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined 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), pref-erably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment).
Textile: The term “textile” means any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and toweling. The textile may be cellulose based such as natural cellulosics, includ-ing 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, cash- mere, 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. In the context of the present invention, the term “textile” is used interchangeably with fabric and cloth.
Variant: The term “variant” means a polypeptide having same activity as the parent enzyme comprising an alteration, /.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 protease has the enzymatic activity of the parent. In one embodiment, the protease activity of the variant is increased with reference to the parent protease, e.g. the mature polypeptide of SEQ ID NO:1.
Wash cycle: The term “wash cycle” is defined herein as a washing operation wherein textiles are immersed in the wash liquor, mechanical action of some kind is applied to the textile in order to release stains and to facilitate flow of wash liquor in and out of the textile and finally the superfluous wash liquor is removed. After one or more wash cycles, the textile is generally rinsed and dried.
Wash liquor: The term “wash liquor” is defined herein as the solution or mixture of water and detergent components optionally including one or more enzymes.
Wash performance: The term “wash performance” is used as detergent composition’s, enzyme’s or polymer’s capability to remove stains present on the object to be cleaned or maintain color and whiteness of textile during wash. The improvement in the wash performance may be quantified by calculating the so-called delta REM as described in Experimental section.
Weight percentage is abbreviated w/w%, wt% or w%. The abbreviations are used interchangeably.
Whiteness: The term “Whiteness” is defined herein as a broad term with different meanings in different regions and for different consumers. Whiteness can be on white textiles or be used interchangely as brightness for colored textiles. Loss of whiteness or brightness can e.g. be due to greying, yellowing, or removal of optical brighteners/hueing agents. Greying and yellowing can be due to soil redeposition, stain redeposition, dirt/mud redeposition, pollution particles, body soils, colouring from e.g. iron and copper ions or dye transfer. Loss of whiteness might include one or several issues from the list below: colourant or dye effects; incomplete stain removal (e.g. body soils, sebum etc.); redeposition (greying, yellowing or other discolourations of the object) (removed soils reassociate with other parts of textile, soiled or unsoiled); chemical changes in textile during application; and clarification or brightening of colours.
SEQUENCE OVERVIEW
SEQ ID NO:1 is a protease from Bacillus circulans
SEQ ID NO:2 is a protease from Bacillus lentus
SEQ ID NO:3 is a protease from Bacillus lentus
SEQ ID NO:4 is a protease from Bacillus lentus
SEQ ID NO:5 is a protease from Bacillus lentus
SEQ ID NO:6 is a protease from Bacillus lentus
SEQ ID NO:7 is a protease from Bacillus subtilis
SEQ ID NO:8 is a protease from Bacillus subtilis
SEQ ID NO:9 is a protease from Bacillus subtilis
SEQ ID NO: 10 is a protease from Bacillus gibsonii
SEQ ID NO: 11 is a protease from Bacillus gibsonii
SEQ ID NO: 12 is a protease from Bacillus amyloliquefaciens
SEQ ID NO: 13 is a protease from Bacillus amyloliquefaciens
SEQ ID NO: 14 is a protease from Bacillus amyloliquefaciens
SEQ ID NO: 15 is a protease from Bacillus amyloliquefaciens
SEQ ID NO: 16 is a protease from Bacillus amyloliquefaciens
SEQ ID NO: 17 is a protease from Bacillus lentus
DETAILED DESCRIPTION OF THE INVENTION
Classification of an enzyme mixture is based on Section 3.4.3.3.1. in the ECHA guidance. This hazard class stipulates that enzymes with the same catalytic activity (same IUB name and number) should be viewed additively unless they are known to be immunochemically different. As discussed above, there is a drive towards higher protease concentrations (polymer replacement, compaction of detergents compositions) in detergents.
The inventors of the present invention have found a solution to the problem, namely the addition of two or more immunochemically different proteases to reach the requested higher protease concentration without increasing the risk of hazardous reactions, such as allergic reactions, in particular respiratory allergic reactions. The immunochemical difference between two proteases may for the purpose of this invention be determined by the in silico method disclosed in the experimental section below. The settings in the in silico method was chosen so the results match the experimental Ouchterlony test results for immunochemical identity as close as possible.
The Ouchterlony test for immunochemical identity is well known in the literature (e.g. in Ouchterlony, O. (1958). "Diffusion-in-gel methods for immunological analysis". Progress in allergy. 5: 1-78) and the actual test conditions as well as results for some of the proteases of SEQ ID NO:1 to SEQ ID NO: 17 is disclosed in the experimental section below.
Based on in silico method for identifying immunochemically different proteases the following combinations of immunochemically different proteases are comprised by the present inven- tion:SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11 , SEQ ID NO:1 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:13, SEQ ID
NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:16, SEQ ID
NO:1 + SEQ ID NO:17, SEQ ID NO:2 + SEQ ID NO:5, SEQ ID NO:2 + SEQ ID NO:6, SEQ ID NO:2 + SEQ ID NO:7, SEQ ID NO:2 + SEQ ID NO:8, SEQ ID NO:2 + SEQ ID NO:9, SEQ ID
NO:2 + SEQ ID NQ:10, SEQ ID NO:2 + SEQ ID NO:11 , SEQ ID NO:2 + SEQ ID NO:12, SEQ ID
NO:2 + SEQ ID NO:13, SEQ ID NO:2 + SEQ ID NO:14, SEQ ID NO:2 + SEQ ID NO:15, SEQ ID
NO:2 + SEQ ID NO:16, SEQ ID NO:3 + SEQ ID NO:5, SEQ ID NO:3 + SEQ ID NO:6, SEQ ID NO:3 + SEQ ID NO:7, SEQ ID NO:3 + SEQ ID NO:8, SEQ ID NO:3 + SEQ ID NO:9, SEQ ID NO:3 + SEQ ID NQ:10, SEQ ID NO:3 + SEQ ID NO:11 , SEQ ID NO:3 + SEQ ID NO:12, SEQ ID NO:3 + SEQ ID NO:13, SEQ ID NO:3 + SEQ ID NO:14, SEQ ID NO:3 + SEQ ID NO:15, SEQ ID NO:3 + SEQ ID NO:16, SEQ ID NO:4 + SEQ ID NO:5, SEQ ID NO:4 + SEQ ID NO:6, SEQ ID NO:4 + SEQ ID NO:7, SEQ ID NO:4 + SEQ ID NO:8, SEQ ID NO:4 + SEQ ID NO:9, SEQ ID NO:4 + SEQ ID NQ:10, SEQ ID NO:4 + SEQ ID NO:11 , SEQ ID NO:4 + SEQ ID NO:12, SEQ ID NO:4 + SEQ ID NO:13, SEQ ID NO:4 + SEQ ID NO:14, SEQ ID NO:4 + SEQ ID NO:15, SEQ ID NO:4 + SEQ ID NO:16, SEQ ID NO:5 + SEQ ID NO:6, SEQ ID NO:5 + SEQ ID NO:7, SEQ ID NO:5 + SEQ ID NO:8, SEQ ID NO:5 + SEQ ID NO:9, SEQ ID NO:5 + SEQ ID NQ:10, SEQ ID NO:5 + SEQ ID NO:11 , SEQ ID NO:5 + SEQ ID NO:12, SEQ ID NO:5 + SEQ ID NO:13, SEQ ID NO:5 + SEQ ID NO:14, SEQ ID NO:5 + SEQ ID NO:15, SEQ ID NO:5 + SEQ ID NO:16, SEQ ID NO:5 + SEQ ID NO:17, SEQ ID NO:6 + SEQ ID NO:9, SEQ ID NO:6 + SEQ ID NQ:10, SEQ ID NO:6 + SEQ ID NO:11 , SEQ ID NO:6 + SEQ ID NO:12, SEQ ID NO:6 + SEQ ID NO:13, SEQ ID NO:6 + SEQ ID NO:14, SEQ ID NO:6 + SEQ ID NO:15, SEQ ID NO:6 + SEQ ID NO:16, SEQ ID NO:6 + SEQ ID NO:17, SEQ ID NO:7 + SEQ ID NO:9, SEQ ID NO:7 + SEQ ID NQ:10, SEQ ID NO:7 + SEQ ID NO:11 , SEQ ID NO:7 + SEQ ID NO:12, SEQ ID NO:7 + SEQ ID NO:13, SEQ ID NO:7 + SEQ ID NO:14, SEQ ID NO:7 + SEQ ID NO:15, SEQ ID N0:7 + SEQ ID N0:16, SEQ ID
N0:7 + SEQ ID N0:17, SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:8 + SEQ ID NQ:10, SEQ ID
N0:8 + SEQ ID N0:11, SEQ ID N0:8 + SEQ ID N0:12, SEQ ID N0:8 + SEQ ID N0:13, SEQ ID
N0:8 + SEQ ID N0:14, SEQ ID N0:8 + SEQ ID N0:15, SEQ ID N0:8 + SEQ ID N0:16, SEQ ID
N0:8 + SEQ ID N0:17, SEQ ID N0:9 + SEQ ID N0:12, SEQ ID N0:9 + SEQ ID N0:13, SEQ ID
N0:9 + SEQ ID N0:14, SEQ ID N0:9 + SEQ ID N0:15, SEQ ID N0:9 + SEQ ID N0:16, SEQ ID
N0:9 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:12, SEQ ID NQ:10 + SEQ ID N0:13, SEQ ID NQ:10 + SEQ ID N0:14, SEQ ID NQ:10 + SEQ ID N0:15, SEQ ID NQ:10 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:12, SEQ ID N0:11 + SEQ ID N0:13, SEQ ID N0:11 + SEQ ID N0:14, SEQ ID N0:11 + SEQ ID N0:15, SEQ ID N0:11 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:17, SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:5, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID
N0:9, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:5, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID
N0:6, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID
N0:8, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID
N0:5, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID
N0:7, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID
N0:9, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID
N0:7, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID
N0:9, SEQ ID N0:1 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:7 + SEQ ID
N0:9, SEQ ID N0:1 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:12, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:13, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:14, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:15, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:12, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:13, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:14, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:15, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID NO:2 + SEQ ID NO:5 + SEQ ID NO:7, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:8,
SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:2 + SEQ ID N0:6 + SEQ ID N0:9,
SEQ ID N0:2 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:2 + SEQ ID N0:8 + SEQ ID N0:9,
SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:7,
SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:9,
SEQ ID N0:3 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:7 + SEQ ID N0:9,
SEQ ID N0:3 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:6,
SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:8,
SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:6 + SEQ ID N0:9,
SEQ ID N0:4 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:8 + SEQ ID N0:9,
SEQ ID N0:5 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:7 + SEQ ID N0:9,
SEQ ID N0:5 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID NQ:10 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:12 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:13 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:14 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:15 + SEQ ID N0:16 + SEQ ID N0:17.
In an embodiment, the proteases of the present invention may comprise one or more conservative amino acid substitutions that do not alter their immunochemical properties. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.
Alternatively, the amino acid changes are of such a nature that the physico-chemical properties, but not the immunochemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for enzyme activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a/., 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoters) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper eta/., 1993, EMBO J. 12:2575- 2583; Dawson et al., 1994, Sc/ence 266:776-779).
A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35- 48.
General methods of PCR, cloning, ligation nucleotides etc. are well-known to a person skilled in the art and may for example be found in in “Molecular cloning: A laboratory manual”, Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.); “Current protocols in Molecular Biology”, John Wiley and Sons, (1995); Harwood, C. R., and Cutting, S. M. (eds.); “DNA Cloning: A Practical Approach, Volumes I and II”, D.N. Glover ed. (1985); “Oligonucleotide Synthesis”, M.J. Gait ed. (1984); “Nucleic Acid Hybridization”, B.D. Hames & S.J. Higgins eds (1985); “A Practical Guide To Molecular Cloning”, B. Perbal, (1984).
The total concentration of the proteases in the wash liquor is typically from about 0.2 ppm to 20 ppm in solution. The water temperatures typically range from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C and about 90°C. The water to fabric ratio is typically from about 1 :1 to about 30:1.
The proteases 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 formulations disclosed in W097/07202, which is hereby incorporated by reference. Liquid enzyme formulations
The enzymes (protease, lipase and other enzymes present) may be formulated as a liquid enzyme formulation, which is generally a pourable composition, though it may also have a high viscosity. The physical appearance and properties of a liquid enzyme formulation may vary a lot - for example, they may have different viscosities (gel to water-like), be colored, not colored, clear, hazy, and even with solid particles like in slurries and suspensions. The minimum ingredients are the enzymes (protease, lipase and other enzymes present) and a solvent system to make it a liquid.
The solvent system may comprise water, polyols (such as glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol, sugar alcohol (e.g. sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol or adonitol), polypropylene glycol, and/or polyethylene glycol), ethanol, sugars, and salts. Usually the solvent system also includes a preservation agent and/or other stabilizing agents.
A liquid enzyme formulation may be prepared by mixing a solvent system and an enzyme concentrate with a desired degree of purity (or enzyme particles to obtain a slurry/suspension).
In an embodiment, the liquid enzyme composition comprises:
(a) at least 0.01 % w/w active enzyme protein,
(b) at least 0.5% w/w polyol,
(c) water, and
(d) optionally a preservation agent.
The enzymes (proteases and optionally other enzymes present) in the liquid composition of the invention may be stabilized using conventional stabilizing agents. Examples of stabilizing agents include, but are not limited to, sugars like glucose, fructose, sucrose, or trehalose; polyols like glycerol, propylene glycol; addition of salt to increase the ionic strength; divalent cations (e.g., Ca2+ or Mg2+); and enzyme inhibitors, enzyme substrates, or various polymers (e.g., PVP). Selecting the optimal pH for the formulation may be very important for enzyme stability. The optimal pH depends on the specific enzyme but is typically in the range of pH 4-9. In some cases, surfactants like nonionic surfactant (e.g., alcohol ethoxylates) can improve the physical stability of the enzyme formulations.
One embodiment of the invention relates to a composition comprising a proteases, wherein the composition further comprises: (i) a polyol, preferably selected from glycerol, (mono, di, or tri) propylene glycol, (mono, di, or tri) ethylene glycol, polyethylene glycol, sugar alcohols, sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol and adonitol;
(ii) optionally an additional enzyme, preferably selected from proteases, amylases, cellulases, lipases, DNases, mannanases, and/or xanthanases
(iii) optionally a surfactant, preferably selected from anionic and nonionic surfactants,
(iv) optionally a salt, divalent cation, polymer, or enzyme inhibitor;
(v) optionally having a pH in the range of pH 4-9; and
(vi) water.
Slurries or dispersions of enzymes are typically prepared by dispersing small particles of enzymes (e.g., spray-dried particles) in a liquid medium in which the enzyme is sparingly soluble, e.g., a liquid nonionic surfactant or a liquid polyethylene glycol. Powder can also be added to aqueous systems in an amount so not all go into solution (above the solubility limit). Another format is crystal suspensions which can also be aqueous liquids (see for example WO2019/002356). Another way to prepare such dispersion is by preparing water-in-oil emulsions, where the enzyme is in the water phase, and evaporate the water from the droplets. Such slurries/suspension can be physically stabilized (to reduce or avoid sedimentation) by addition of rheology modifiers, such as fumed silica or xanthan gum, typically to get a shear thinning rheology.
Granular enzyme formulations
The enzymes (proteases and optionally other enzymes present) may also be formulated as a solid/granular enzyme formulation. Non-dusting granulates may be produced, e.g. as disclosed in US 4,106,991 and US 4,661 ,452, and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
The proteases may be formulated as a granule for example as a co-granule that combines one or more enzymes or benefit agents (such as MnTACN or other bleaching components). Examples of such additional enzymes include lipases, xyloglucanases, perhydrolases, peroxidases, proteases, laccases, hemicellulases, care cellulases, cellulases, cellobiose dehydrogenases, xylanases, phospho lipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pento- sanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, amylases, DNAse, and mixtures thereof. 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-gran- ulate for the detergent industry are disclosed in the IP.com disclosure IPCOM000200739D.
An embodiment of the invention relates to an enzyme granule/particle comprising proteases. The granule is composed of a core, and optionally one or more coatings (outer layers) surrounding the core. Typically, the granule/particle size, measured as equivalent spherical diameter (volume based average particle size), of the granule is 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
The core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibers), stabilizing agents, solubilising agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances. The core may include binders, such as synthetic polymer, wax, fat, or carbohydrate. The core may comprise a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend. The core may consist of an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating. The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm. The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation. Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1 ; 1980; Elsevier. These methods are well-known in the art and have also been described in international patent application WO2015/028567, pages 3-5, which is incorporated by reference.
The core of the enzyme granule/particle may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are shown in WO 93/07263 and WO 97/23606.
Such coatings are well-known in the art, and have earlier been described in, for example, WO00/01793, W02001/025412, and WO2015/028567, which are incorporated by reference. In one aspect, the present invention provides a granule, which comprises:
(a) a core comprising a protease according to the invention; and
(b) optionally a (salt) coating consisting of one or more layer(s) surrounding the core.
Another aspect of the invention relates to a layered granule, comprising:
(a) a (non-enzymatic) core;
(b) a coating surrounding the core, wherein the coating comprises a protease; and
(c) optionally a (salt) coating consisting of one or more layer(s) surrounding the enzyme containing coating.
Encapsulated enzyme formulations
The enzymes (proteases and optionally other enzymes present) may also be formulated as an encapsulated enzyme formulation (an ‘encapsulate’). This is particularly useful for separating the enzyme from other ingredients when the enzyme is added into, for example, a (liquid) cleaning composition, such as the detergent compositions described below.
Physical separation can be used to solve incompatibility between the enzyme(s) and other components. Incompatibility can arise if the other components are either reactive against the enzyme, or if the other components are substrates of the enzyme. Other enzymes can be substrates of proteases.
The enzyme may be encapsulated in a matrix, preferably a water-soluble or water dispersible matrix (e.g., water-soluble polymer particles), for example as described in WO 2016/023685. An example of a water-soluble polymeric matrix is a matrix composition comprising polyvinyl alcohol. Such compositions are also used for encapsulating detergent compositions in unit-dose formats.
The enzyme may also be encapsulated in core-shell microcapsules, for example as described in WO 2015/144784, or as described in the IP.com disclosure IPCOM000239419D.
Such core-shell capsules can be prepared using a number of technologies known in the art, e.g., by interfacial polymerization using either a water-in-oil or an oil-in-water emulsion, where polymers are crosslinked at the surface of the droplets in the emulsion (the interface between water and oil), thus forming a wall/membrane around each droplet/capsule.
Formulation of enzyme in co-granule
The enzymes (proteases and optionally other enzymes present) 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 wt% to 98 wt% moisture sink component and the composition additionally comprises from 20 wt% 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 a proteases and (a) one or more enzymes selected from the group consisting of lipases, xyloglucanases, perhydrolases, peroxidases, proteases, laccases and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, care cellulases, 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, DNAse, and mixtures thereof.
Purity of enzyme in formulations
The enzymes (proteases and optionally other enzymes present) used in the above-mentioned enzyme formulations may be purified to any desired degree of purity. This includes high levels of purification, as achieved for example by using methods of crystallization - but also none or low levels of purification, as achieved for example by using crude fermentation broth, as described in WO 2001/025411 , or in WO 2009/152176.
Microorganisms
The enzyme formulations, as well as the detergent formulations described below, may comprise one or more microorganisms or microbes. Generally, any microorganism(s) may be used in the enzyme/detergent formulations in any suitable amount(s)/concentration(s). Microorganisms may be used as the only biologically active ingredient, but they may also be used in conjunction with one or more of the enzymes described above.
The purpose of adding the microorganism(s) may, for example, be to reduce malodor as described in WO 2012/112718. Other purposes could include in-situ production of desirable biological compounds, or inoculation/population of a locus with the microorganism(s) to competitively prevent other non-desirable microorganisms form populating the same locus (competitive exclusion).
The term “microorganism” generally means small organisms that are visible through a microscope. Microorganisms often exist as single cells or as colonies of cells. Some microorganisms may be multicellular. Microorganisms include prokaryotic (e.g., bacteria and archaea) and eukaryotic (e.g., some fungi, algae, protozoa) organisms. Examples of bacteria may be Grampositive bacteria or Gram-negative bacteria. Example forms of bacteria include vegetative cells and endospores. Examples of fungi may be yeasts, molds and mushrooms. Example forms of fungi include hyphae and spores. Herein, viruses may be considered microorganisms.
Microorganisms may be recombinant or non-recombinant. In some examples, the microorganisms may produce various substances (e.g., enzymes) that are useful for inclusion in detergent compositions. Extracts from microorganisms or fractions from the extracts may be used in the detergents. Media in which microorganisms are cultivated or extracts or fractions from the media may also be used in detergents. In some examples, specific of the microorganisms, substances produced by the microorganisms, extracts, media, and fractions thereof, may be specifically excluded from the detergents. In some examples, the microorganisms, or substances produced by, or extracted from, the microorganisms, may activate, enhance, preserve, prolong, and the like, detergent activity or components contained with detergents.
Generally, microorganisms may be cultivated using methods known in the art. The microorganisms may then be processed or formulated in various ways. In some examples, the microorganisms may be desiccated (e.g., lyophilized). In some examples, the microorganisms may be encapsulated (e.g., spray drying). Many other treatments or formulations are possible. These treatments or preparations may facilitate retention of microorganism viability over time and/or in the presence of detergent components. In some examples, however, microorganisms in detergents may not be viable. The processed/formulated microorganisms may be added to detergents prior to, or at the time the detergents are used.
In one embodiment, the microorganism is a species of Bacillus, for example, at least one species of Bacillus selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefa- ciens, Bacillus licheniformis, Bacillus atrophaeus, Bacillus pumilus, Bacillus megaterium, or a combination thereof. In a preferred embodiment, the aforementioned Bacillus species are on an endospore form, which significantly improves the storage stability. Detergent compositions
In one embodiment, the invention is directed to detergent compositions comprising at least two proteases in combination with one or more additional cleaning composition components. In one embodiment, the detergent composition comprises at least two polypeptides having protease activity with an amino acid sequence having at least 60% identity, such as 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% identity to the amino acid sequence set forth in SEQ ID NO:1 to SEQ ID NO:17. In one embodiment the detergent composition is in solid form. In another embodiment, the detergent composition is in a liquid or gel form. In another embodiment a bar form. In one embodiment the detergent may be wrapped in water soluble PVOH film. 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.
Figure imgf000021_0001
The liquid detergent composition may comprise a microcapsule, and thus form part of any detergent composition in any form, such as liquid and powder detergents, and soap and detergent bars.
In one embodiment, the invention is directed to liquid detergent compositions comprising a microcapsule, as described above, in combination with one or more additional cleaning composition components.
The microcapsule, as described above, may be added to the liquid detergent composition in an amount corresponding to from 0.1% to 5% (w/w) active enzyme protein; preferably from 0. 1% to 4%, such as from 0.1% to 3%, such as from 0.1% to 2.5%, such as from 0.1% to 2%, such as from 0.1% to 1.5%, such as from 0.1% to 1%, and most preferably from 0.01% to 1% (w/w) active enzyme protein.
The liquid detergent composition has a physical form, which is not solid (or gas). It may be a pourable liquid, a paste, a pourable gel or a non-pourable gel. It may be either isotropic or structured, preferably isotropic. It may be a formulation useful for washing in automatic washing machines or for hand washing. It may also be a personal care product, such as a shampoo, toothpaste, or a hand soap.
The liquid detergent composition may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to 70% water, up to 50% water, up to 40% water, up to 30% water, or up to 20% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid detergent. An aqueous liquid detergent may contain from 0-30% organic solvent. A liquid detergent may even be non-aqueous, wherein the water content is below 10%, preferably below 5%.
Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
The detergent composition may take the form of a unit dose product. A unit dose product is the packaging of a single dose in a non-reusable container. It is increasingly used in detergents for laundry. A detergent unit dose product is the packaging (e.g., in a pouch made from a water-soluble film) of the amount of detergent used for a single wash.
Pouches can be of any form, shape and material which is suitable for holding the composition, e.g., without allowing the release of the composition from the pouch prior to 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, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be a blend composition comprising hydrolytically degradable and water-soluble polymer blends such as polyactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticizers like glycerol, ethylene glycerol, Propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids (see e.g., US 2009/0011970).
Detergent ingredients
The choice of detergent components may include, for textile care, the consideration of the type of textile to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.
Any detergent components known in the art for use in detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/dis- integration agents, dyes, enzyme stabilizers (including boric acid, borates, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in detergents may be utilized. The choice of such ingredients is well within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
Surfactants
The cleaning 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 and at least one non-ionic surfactant, the weight ratio of anionic to nonionic surfactant may be from 20:1 to 1 :20. 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 (SLS); 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. Anionic surfactants may be added as acids, as salts or as ethanolamine derivatives.
When included therein the detergent will usually contain from about 0,1% 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), dimethyl- distearylammonium 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-p-D-maltopyranoside, thiomaltopyranoside or a cyclic- maltopyranoside, such as described in EP2516606 B1. Other biosurfactants may include rhamnolipids and sophorolipids.
Figure imgf000025_0001
A hydrotrope is a compound that solubilises 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 selfaggregation, see e.g. review by Hodgdon and Kaier (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming 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.
Builders and Co-Builders
The detergent composition may contain about 0-65% by weight, such as about 5% to about 50% of a detergent builder or co-builder, or a mixture thereof. 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 or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). According to the present invention, these components can be included in lower levels than in currently available detergent compositions. 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), ethylenedia- minetetramethylenetetrakis(phosphonic acid) (EDTMPA),diethylenetriaminepentamethylene- pentakis(phosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N -di acetic acid (ASDA), aspartic acid-N- monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N- (2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), a-alanine-N,N-diacetic acid (a-ALDA), serine-N,N- diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA) , taurine-N,N-di- acetic acid (TLIDA) 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 and US 5977053.
Figure imgf000026_0001
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-a-naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, e-phthalimidoperoxycaproic acid [phthalimidoperoxyhexanoic acid (PAP)], and o-carboxybenzamidoperoxycaproic acid; aliphatic and aromatic diperoxydicarboxylic acids such as diperoxydodecanedioic acid, diper- oxyazelaic 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., Oxone®) 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-(deca- noyloxy)benzene-1 -sulfonate, 4-(decanoyloxy)benzoic acid (DOBA), sodium 4-(nona- noyloxy)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, co- balt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1 ,4,7-trimethyl-1 ,4,7-triazacyclononane (Me3-TACN) or 1 ,2,4,7-tetramethyl-1 ,4,7-triazacyclononane (Me4-TACN), in particular Me3-TACN, such as the dinuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2, 2', 2"- nitrilotris(ethane-1 ,2-diylazanylylidene-KN-methanylylidene)triphenolato-K3O]manganese(lll). The bleach catalysts may also be other metal compounds, such as iron or cobalt complexes.
In some embodiments, 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 imgf000028_0001
(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 W02007/087258, W02007/087244, W02007/087259, EP1867708 (Vitamin K) and W02007/087242. Suitable photobleaches may for example be sulfonated zinc or aluminium phthalocyanines.
Polymers and dispersants
Generally, detergent compositions may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1 % of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include poly(vinyl alcohol) (P A), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of polyethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-/V-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include polyethylene oxide and polypropylene oxide (PEO-PPO), diquaternium ethoxy sulfate, styrene/acrylic copolymer and perfume capsules Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.
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.
According to the present invention, however, certain of the above polymers, namely, a polyacrylic acid, a modified polyacrylic acid polymer, a modified polyacrylic acid copolymer, a maleic acid-acrylic acid copolymer, carboxymethyl cellulose, cellulose gum, methyl cellulose, and/or combinations thereof, can be included in lower levels than in currently available detergent compositions, or even more preferably, excluded altogether.
Fabric hueing agents
The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liguor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.l.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in W02005/03274, W02005/03275, W02005/03276 and EP1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt% fabric hueing agent. The composition may comprise from 0.0001 wt% to 0.2 wt% fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and W02007/087243.
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 /V-oxide polymers, copolymers of /V-viny I pyrrolidone and /V-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 laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulfonic acid derivatives, diaryl py- razoline 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-dieth- anolamino-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-(/V-methyl-/\/-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'-disul- fonate and sodium 5-(2/7-naphtho[1 ,2-d][1 ,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesul- fonate. 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. Tinopal CBS-X is a 4.4'-bis-(sulfostyryl)-biphenyl disodium salt also known as Disodium Distyrylbiphenyl Disulfonate. Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyra- zolines 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).
Anti-redeposition agents
The detergent compositions of the present invention may also include one or more antiredeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
According to the present invention, however, certain of the above polymers, namely, a polyacrylic acid, a modified polyacrylic acid polymer, a modified polyacrylic acid copolymer, a maleic acid-acrylic acid copolymer, carboxymethyl cellulose, cellulose gum, methyl cellulose, and/or combinations thereof, can by introduction of cellulases be included in lower levels than in currently available detergent compositions, or excluded altogether, thus improving the sustainability profile of the detergent composition.
Rheology Modifiers
The detergent compositions of the present invention may also include one or more rheology modifiers, structurants or thickeners, as distinct from viscosity reducing agents. The rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, polymeric rheology modifiers which impart shear thinning characteristics to the ague- ous liguid matrix of a liguid detergent composition. The rheology and viscosity of the detergent can be modified and adjusted by methods known in the art, for example as shown in EP 2169040. Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liguid detergents and/or structure elasticizing agents.
Additional Enzymes
The detergent additive as well as the detergent composition may comprise one or more [additional] enzymes such as lipases, cutinases, cellulases, amylases, carbohydrases, DNases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases, e.g., a laccase, and/or peroxidase.
In general, the properties of the selected enzyme(s) should be compatible with the selected detergent, (/.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts. Cellulases
The term “cellulase” means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. The two terms polypeptide having cellulase activity and cellulase are used interchangeably. Cellulases may be selected from the group consisting of cellulases belonging to GH5, GH44, GH45, EC 3.2.1.4, EC 3.2.1.21 , EC 3.2.1.91 and EC 3.2.1.172. Such enzymes include endoglu- canase(s) (e.g. EC 3.2.1.4), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. 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-glu- canase also referred to as endoglucanase.
DNases (deoxyribonuclease)
The term “DNase” means a polypeptide with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA.
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. licheni- formis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 1999/064619. A commercially available mannanase is Mannaway (Novozymes A/S).
Xanthanases
Xanthan lyase (EC 4.2.2.12) is an enzyme that catalyzes the chemical reaction of cleaving the beta-D-mannosyl-beta-D-1 ,4-glucuronosyl bond on the polysaccharide xanthan. This enzyme belongs to the family of lyases, specifically those carbon-oxygen lyases acting on polysaccharides.
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 Sub- tilisin 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 tromBacillus. Examples of Bacillus species from which subtilases have been derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amylolique- faciens, 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.
Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase™, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coro- nase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Blaze Evity® 200T, Neutrase®, Everlase®, Esperase®, Progress® Uno, Progress® In and Progress® Excel (Novozymes A/S), those sold under the tradename Maxatase™, Maxacai™, Maxapem®, Purafect® Ox, Purafect® OxP, Puramax®, FN2™, FN3™, FN4ex™, Excellase®, Ex- cellenz™ P1000, Excellenz™ P1250, Eraser™, Preferenz® P100, Purafect Prime, Preferenz P110™, Effectenz P1000™, Purafect®, Effectenz P1050™, Purafect® Ox, Effectenz ™ P2000, Purafast™, Properase®, Opticlean™ and Optimase® (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 Thermo- myces, 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 (W095/06720 & W096/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyces lipases (W010/065455), cutinase from Magnaporthe grisea (WO10/107560), cutinase from Pseudomonas mendocina (US5,389,536), lipase from Thermobifida fusca (W011/084412), Geobacillus stearothermophilus lipase (W011/084417), lipase from Bacillus subtilis (W011/084599), 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,
W096/00292, W097/04079, W097/07202, WO00/34450, WO00/60063, W001/92502,
W007/87508 and WO09/109500.
Preferred commercial lipase products include include Lipolase 100T/L, Lipex 100T/L, Li- pex 105T, Lipex Evity 100L, Lipex Evity 200L (all Novozymes A/S), Preferenz® L 100 (DuPont).
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 include an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus li- cheniformis, 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 N0:6 in WO 02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having a deletion in positions 181 and 182 and a substitution in position 193.
Other amylases which are suitable are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO:4 of WO 2006/066594 or variants having 90% sequence identity thereof.
Other examples are amylase variants such as those described in WQ2011/098531 , WQ2013/001078 and WQ2013/001087.
Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme TM, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ Amplify; Amplify Prime; (from Novo- zymes A/S), and Rapidase™ , Purastar™/Effectenz™, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc./DuPont).
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™ (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, /.e., a van- adate-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 ver- ruculosa 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 Genic- ulosporium 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, Pomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. ci- nerea, 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, Pol- yporus, 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.
Laundry soap bars
The proteases 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 NH4+ and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.
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, proteases, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared, and the mixture is then plodded. The proteases 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.
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. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition 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 laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids: 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.
Embodiments of the invention
The invention is further summarized in the following embodiments. The embodiments are indicated as E1 , E2 and so forth.
E1. A detergent composition comprising at least two immunochemically different proteases, wherein a. the total amount of protease is at least 0.1 wt% of the detergent composition, b. the amount of each protease in the detergent composition is below 0.1 wt%, and c. the total amount of two or more immunochemically identical proteases is below 0.1 wt% d. the detergent composition optionally comprises at least one additional enzyme which is not a protease.
E2. The detergent composition according to E1 , wherein the at least two immunochemically different proteases are selected from the group consisting of proteases having at least 60% identity to any of the proteases of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, and SEQ ID N0:17.
E3. The detergent composition according to any of E1 or E2, wherein the at least two immunochemically different proteases are selected from the group consisting of proteases having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to any of the proteases of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
E4. The detergent composition according to any of E1 to E3 comprising a protease that has at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to the protease of SEQ ID NO:1 .
E5. The detergent composition according to E4 further comprising a protease selected from the group of polypeptides that has at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to any of the proteases of SEQ ID NO:3, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
E6. The detergent composition according to E5 comprising the protease of SEQ ID NO 1 in and at least one other protease selected from the group consisting of SEQ ID NO:3, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
E7. The detergent composition according to E1 comprising any of the following combinations of proteases:SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11 , SEQ ID NO:1 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:17, SEQ ID NO:2 + SEQ ID NO:5, SEQ ID NO:2 + SEQ ID NO:6, SEQ ID NO:2 + SEQ ID NO:7, SEQ ID NO:2 + SEQ ID NO:8, SEQ ID NO:2 + SEQ ID NO:9, SEQ ID NO:2 + SEQ ID NQ:10, SEQ ID NO:2 + SEQ ID NO:11 , SEQ ID NO:2 + SEQ ID NO:12, SEQ ID NO:2 + SEQ ID NO:13, SEQ ID NO:2 + SEQ ID NO:14, SEQ ID NO:2 + SEQ ID NO:15, SEQ ID NO:2 + SEQ ID NO:16, SEQ ID NO:3 + SEQ ID NO:5, SEQ ID N0:3 + SEQ ID N0:6, SEQ ID N0:3 + SEQ ID N0:7, SEQ ID N0:3 + SEQ ID N0:8, SEQ ID N0:3 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID NQ:10, SEQ ID N0:3 + SEQ ID N0:11 , SEQ ID N0:3 + SEQ ID N0:12, SEQ ID N0:3 + SEQ ID N0:13, SEQ ID N0:3 + SEQ ID N0:14, SEQ ID N0:3 + SEQ ID N0:15, SEQ ID N0:3 + SEQ ID N0:16, SEQ ID N0:4 + SEQ ID N0:5, SEQ ID N0:4 + SEQ ID N0:6, SEQ ID N0:4 + SEQ ID N0:7, SEQ ID N0:4 + SEQ ID N0:8, SEQ ID N0:4 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID NQ:10, SEQ ID N0:4 + SEQ ID N0:11, SEQ ID N0:4 + SEQ ID N0:12, SEQ ID N0:4 + SEQ ID N0:13, SEQ ID N0:4 + SEQ ID N0:14, SEQ ID N0:4 + SEQ ID N0:15, SEQ ID N0:4 + SEQ ID N0:16, SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID NQ:10, SEQ ID N0:5 + SEQ ID N0:11, SEQ ID N0:5 + SEQ ID N0:12, SEQ ID N0:5 + SEQ ID N0:13, SEQ ID N0:5 + SEQ ID N0:14, SEQ ID N0:5 + SEQ ID N0:15, SEQ ID N0:5 + SEQ ID N0:16, SEQ ID N0:5 + SEQ ID N0:17, SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:6 + SEQ ID NQ:10, SEQ ID N0:6 + SEQ ID N0:11, SEQ ID N0:6 + SEQ ID N0:12, SEQ ID N0:6 + SEQ ID N0:13, SEQ ID N0:6 + SEQ ID N0:14, SEQ ID N0:6 + SEQ ID N0:15, SEQ ID N0:6 + SEQ ID N0:16, SEQ ID N0:6 + SEQ ID N0:17, SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:7 + SEQ ID NQ:10, SEQ ID N0:7 + SEQ ID N0:11, SEQ ID N0:7 + SEQ ID N0:12, SEQ ID N0:7 + SEQ ID N0:13, SEQ ID N0:7 + SEQ ID N0:14, SEQ ID N0:7 + SEQ ID N0:15, SEQ ID N0:7 + SEQ ID N0:16, SEQ ID N0:7 + SEQ ID N0:17, SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:8 + SEQ ID NQ:10, SEQ ID N0:8 + SEQ ID N0:11 , SEQ ID N0:8 + SEQ ID N0:12, SEQ ID N0:8 + SEQ ID N0:13, SEQ ID N0:8 + SEQ ID N0:14, SEQ ID N0:8 + SEQ ID N0:15, SEQ ID N0:8 + SEQ ID N0:16, SEQ ID N0:8 + SEQ ID N0:17, SEQ ID N0:9 + SEQ ID N0:12, SEQ ID N0:9 + SEQ ID N0:13, SEQ ID N0:9 + SEQ ID N0:14, SEQ ID N0:9 + SEQ ID N0:15, SEQ ID N0:9 + SEQ ID N0:16, SEQ ID N0:9 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:12, SEQ ID NQ:10 + SEQ ID N0:13, SEQ ID NQ:10 + SEQ ID N0:14, SEQ ID NQ:10 + SEQ ID N0:15, SEQ ID NQ:10 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:12, SEQ ID N0:11 + SEQ ID N0:13, SEQ ID N0:11 + SEQ ID N0:14, SEQ ID N0:11 + SEQ ID N0:15, SEQ ID N0:11 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:17, SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:5, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:6, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:5, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:12, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:13, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:14, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:15, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:12, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:13, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:14, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:15, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:2 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:2 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:2 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID NQ:10 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID NO:14 + SEQ ID NO:17, SEQ ID NO:10 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID NO:10 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:12 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:13 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:14 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:15 + SEQ ID N0:16 + SEQ ID N0:17.
E8. The detergent composition according to E7 comprising any of the following combinations of proteases:SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11 , SEQ ID NO:1 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:6 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:7 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:8 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:12 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:12 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:16 + SEQ ID N0:17.
E9. The detergent composition according to any of E1 to E8, wherein the detergent composition is supplied in a liquid form.
E10. The detergent composition according to any of E1 to E8, wherein the detergent is supplied as a unit dose product, such as a pouch or tablet. E11. The detergent composition according to any of E1 to E8, wherein the detergent is supplied as a liquid detergent or as a powder product.
E12. The detergent composition according to any of E1 to E11 comprising at least one additional enzyme selected from the group consisting of cellulases, mannanases, amylases, DNases, lipases, xanthanases, pectate lyases and oxidoreductases. E13. Use of the detergent composition of any of E1 to E12 for laundering, hand dishwashing, automatic dishwashing or hard surface cleaning.
Detergent compositions
The below mentioned ranges of detergent components are generally useful.
Composition 1 : Liquid detergent
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Composition 2: Unit Dose
Figure imgf000045_0002
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Composition 3: Powder detergent
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
In one embodiment, the invention is directed to an ADW (Automatic Dish Wash) compositions comprising an enzyme of the present invention in combination with one or more additional ADW composition components.
Surfactant ingredients can be obtained from BASF, Ludwigshafen, Germany (Lutensol®); Shell Chemicals, London, UK; Stepan, Northfield, III, USA; Huntsman, Huntsman, Salt Lake City, Utah, USA; Clariant, Sulzbach, Germany (Praepagen®).
Sodium tripolyphosphate can be obtained from Rhodia, Paris, France.
Zeolite can be obtained from Industrial Zeolite (UK) Ltd, Grays, Essex, UK.
Citric acid and sodium citrate can be obtained from Jungbunzlauer, Basel, Switzerland.
NOBS is sodium nonanoyloxybenzenesulfonate, supplied by Eastman, Batesville, Ark., USA.
TAED is tetraacetylethylenediamine, supplied under the Peractive® brand name by Clariant GmbH, Sulzbach, Germany.
Sodium carbonate and sodium bicarbonate can be obtained from Solvay, Brussels, Belgium.
Polyacrylate, polyacrylate/maleate copolymers can be obtained from BASF, Ludwigshafen, Germany.
Repel-O-Tex® can be obtained from Rhodia, Paris, France.
Texcare® can be obtained from Clariant, Sulzbach, Germany. Sodium percarbonate and sodium carbonate can be obtained from Solvay, Houston, Tex., USA.
Na salt of Ethylenediamine-N,N'-disuccinic acid, (S,S) isomer (EDDS) was supplied by Octel, Ellesmere Port, UK.
Hydroxy ethane di phosphonate (HEDP) was supplied by Dow Chemical, Midland, Mich., USA.
Enzymes Savinase®, Savinase® Ultra, Stainzyme® Plus, Lipex®, Lipolex®, Lipoclean®, Celluclean®, Carezyme®, Natalase®, Stainzyme®, Stainzyme® Plus, Termamyl®, Termamyl® ultra, and Mannaway® can be obtained from Novozymes, Bagsvaerd, Denmark. Enzymes Purafect®, FN3, FN4 and Optisize can be obtained from Genencor International Inc., Palo Alto, California, US.
Direct violet 9 and 99 can be obtained from BASF DE, Ludwigshafen, Germany.
Solvent violet 13 can be obtained from Ningbo Lixing Chemical Co., Ltd. Ningbo, Zhejiang, China.
Brighteners can be obtained from Ciba Specialty Chemicals, Basel, Switzerland.
All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on active concentration of the total composition unless otherwise indicated.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
EXAMPLES
Materials and Methods
AMSA-test method
Washing experiments were performed in order to assess the wash performance of selected protease variants in laundry or dish wash detergent compositions. The proteases of the present application were tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA, the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the dish wash sample, the melamine tile to be washed against all the slot openings. During the washing time, the plate, test solutions, melamine tile and lid are vigorously shaken to bring the test solution in contact with the soiled melamine tile and apply mechanical stress in a regular, periodic oscillating manner. For further description see WO 02/42740 especially the paragraph "Special method embodiments" at page 23-24.
The performance of the enzyme variants and their blends was measured as the brightness of the colour of the textile samples washed with that specific protease/protease-blend. Brightness can also be expressed as the intensity of the light reflected from the textile sample when illuminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a clean textile. Therefore, the intensity of the reflected light can be used to measure wash performance of the proteases and the protease blends.
Colour measurements are made with a professional flatbed scanner (Epson Expression 10000XL), which is used to capture an image of the washed textile samples.
To extract a value for the light intensity from the scanned images, a special designed software application is used {Novozymes Color Vector Analyzer) . The program retrieves the values from the image and converts them into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
Figure imgf000053_0001
Detergent
The detergent for the wash performance tests is a liquid laundry model detergent (see below).
Textiles
Standard textile pieces are obtained from Center for Testmaterials BV, P.O. Box 120, 3133 KT Vlaardingen, The Netherlands, and from EMPA Testmaterials AG, Mdvenstrasse 12, CH-9015 St. Gallen, Switzerland. Especially type PC-03 (polyester/cotton textile stained with chocolate milk with carbon black), PC-10 (polyester/cotton textile stained with pigment, oil and milk), and EMPA 117 EH (extra heated) (polyester/cotton textile stained with blood/milk/ink). In general, PC-03 is considered a cocoa stain, PC-10 is considered a milk stain, and EMPA 117 EH is considered a blood stain.
Experimental conditions
The experiments were conducted under the experimental conditions specified below:
Figure imgf000054_0001
Figure imgf000055_0001
The following proteases were tested:
First protease
• SEQ ID NO:1
Second protease
• SEQ ID NO:2
• SEQ ID NO:3
• SEQ ID NO:4
• SEQ ID NO:5
• SEQ ID NO:6
• SEQ ID NO:7
• SEQ ID NO:8
• SEQ ID NO:9
• SEQ ID NQ:10
• SEQ ID NO:11
• SEQ ID NO:12
• SEQ ID NO:13
• SEQ ID NO:14
• SEQ ID NO:15
• SEQ ID NO:16
• SEQ ID NO:17
In silico method for identifying immunochemically different proteases
This method compares two protein sequences by
• Splitting each protein sequence into fragments of a specified window length
• Comparing all fragments of protein 1 to all fragments of protein 2 and counting the number of exact matches. • If the number of exact matched does not exceed a specified match threshold, the two proteins are considered immunochemically different.
The fragments were compared using the Fasta software, version 3.4, and filtering for 100% identity over window length amino acids.
A set of three or more protein sequences are considered immunochemically different if all possible pairs within the set are immunochemically different according to the pairwise method above.
The parameters "window length" and "match threshold" are chosen so the results match the experimental Ouchterlony test results as close as possible. In this case a window length of 19 and a match threshold of 10 was used.
The result of analyzing immunochemical difference between the proteases of SEQ ID NO 1 to SEQ ID NO 17 is shown in figure 1.
Ouchterlony test for identifying immunochemmically different proteases
The ’’Ouchterlony” technique is a classic, fast and simple tool for comparing the immunochemical properties of enzymes and is described e.g. in Ouchterlony, O. (1958). "Diffusion-in-gel methods for immunological analysis". Progress in allergy. 5: 1-78.
When a few micrograms of the enzymes to be compared are applied in 10 pL wells next to each other in an agarose gel and left over-night, they will diffuse radially from the wells and, if recognized, be precipitated by antibodies applied in another well. After drying and protein staining of the gel with Coomassie Blue the precipitation pattern of the antigen-antibody complexes will reveal if the enzyme surfaces are different, partially or totally identical: the zone of equivalence lines may give a full identity (i.e. a continuous line), partial identity (i.e. a continuous line with a spur at one end), or a non-identity (i.e. the two lines cross completely).
Ouchterlony test for immunochemical difference between SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:12 was conducted with the following results:
• SEQ ID NO:2 was immunochemically different from SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:12
• SEQ ID NO:3 was immunochemically different from SEQ ID NO:5 and SEQ ID NO:6
• SEQ ID NO:5 was immunochemically different from SEQ ID NO:6 and SEQ ID NO: 12
• SEQ ID NO:6 was immunochemically different from SEQ ID NO: 12
• SEQ ID NO:2 was not immunochemically different from SEQ ID NO:3 and SEQ ID NO:4
• SEQ ID NO:3 was not immunochemically different from SEQ ID NO:4
• SEQ ID NO:1 was immunochemically different from SEQ ID NO:2 • SEQ ID NO:12 was immunochemically different from SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6
These results are consistent with in silico method for identifying immunochemically different proteases.
Example 1
Wash performance of the protease with SEQ ID NO:1 was tested in AMSA as described above either alone or together with a protease with SEQ ID NO:2 to SEQ ID NO.16. For each enzyme concentration (10-20-40-80 nmol/L enzyme as described above) a delta intensity value was calculated for the single enzymes and the 1 :1 mixtures as the intensity value (I nt) of a test material washed with the detergent containing a single enzyme or mixture minus the intensity value of a test material washed with the detergent alone, i.e. without any enzyme. The delta intensity values for each of the four enzyme concentrations were added together for each treatment (single enzyme or 1 :1 mixture) to obtain a sum of delta intensity for each treatment for each of the three stains wo stains PC-10, PC-03, and EMPA117EH.
The sums of delta intensity for the 1 :1 mixtures were then compared to the sums of delta intensity for the single enzymes in the mixture, and the relative performance of the mixture compared to a single enzyme was determined for the PC-10, PC-03, and EMPA117EH stains.
The relative performance values in the Table X below for the individual stains PC-10, PC- 03, and EMPA117EH expressed as percent relative performance against SEQ ID NO:1 , were determined by dividing the sum of delta intensity for a mixture by the sum of delta intensity for the single enzyme SEQ ID NO:1.
The listed protease combinations perform better than SEQ ID NO:1 alone on at least one of the three tested stains, see Table 1 below.
Figure imgf000057_0001
Figure imgf000058_0001
TABLE 1 : Relative performance of mixtures of immunochemically different proteases with SEQ ID NO:1 against SEQ ID NO:1 alone.

Claims

1. A detergent composition comprising at least two immunochemically different proteases, wherein a. the total amount of protease is at least 0.1 wt% of the detergent composition, b. the amount of each protease in the detergent composition is below 0.1 wt%, and c. the total amount of two or more immunochemically identical proteases is below 0.1 wt% d. the detergent composition optionally comprises at least one additional enzyme which is not a protease.
2. The detergent composition according to claim 1 , wherein the at least two immunochemically different proteases are selected from the group consisting of proteases having at least 60% identity to any of the proteases of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
3. The detergent composition according to any of claims 1 or 2, wherein the at least two immunochemically different proteases are selected from the group consisting of proteases having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to any of the proteases of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
4. The detergent composition according to any of claim 1 to 3 comprising a protease that has at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to the protease of SEQ ID NO:1 .
5. The detergent composition according to claim 4 further comprising a protease selected from the group of polypeptides that has at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to any of the proteases of SEQ ID NO:3, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
6. The detergent composition according to claim 5 comprising the protease of SEQ ID NO 1 in and at least one other protease selected from the group consisting of SEQ ID NO:3, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
58
7. The detergent composition according to claim 1 comprising any of the following combinations of proteases: SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11, SEQ ID NO:1 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:17, SEQ ID NO:2 + SEQ ID NO:5, SEQ ID NO:2 + SEQ ID NO:6, SEQ ID NO:2 + SEQ ID NO:7, SEQ ID NO:2 + SEQ ID NO:8, SEQ ID NO:2 + SEQ ID NO:9, SEQ ID NO:2 + SEQ ID NQ:10, SEQ ID NO:2 + SEQ ID NO:11, SEQ ID NO:2 + SEQ ID NO:12, SEQ ID NO:2 + SEQ ID NO:13, SEQ ID NO:2 + SEQ ID NO:14, SEQ ID NO:2 + SEQ ID NO:15, SEQ ID NO:2 + SEQ ID NO:16, SEQ ID NO:3 + SEQ ID NO:5, SEQ ID NO:3 + SEQ ID NO:6, SEQ ID NO:3 + SEQ ID NO:7, SEQ ID NO:3 + SEQ ID NO:8, SEQ ID NO:3 + SEQ ID NO:9, SEQ ID NO:3 + SEQ ID NQ:10, SEQ ID NO:3 + SEQ ID NO:11, SEQ ID NO:3 + SEQ ID NO:12, SEQ ID NO:3 + SEQ ID NO:13, SEQ ID NO:3 + SEQ ID NO:14, SEQ ID NO:3 + SEQ ID NO:15, SEQ ID NO:3 + SEQ ID NO:16, SEQ ID NO:4 + SEQ ID NO:5, SEQ ID NO:4 + SEQ ID NO:6, SEQ ID NO:4 + SEQ ID NO:7, SEQ ID NO:4 + SEQ ID NO:8, SEQ ID NO:4 + SEQ ID NO:9, SEQ ID NO:4 + SEQ ID NQ:10, SEQ ID NO:4 + SEQ ID NO:11, SEQ ID NO:4 + SEQ ID NO:12, SEQ ID NO:4 + SEQ ID NO:13, SEQ ID NO:4 + SEQ ID NO:14, SEQ ID NO:4 + SEQ ID NO:15, SEQ ID NO:4 + SEQ ID NO:16, SEQ ID NO:5 + SEQ ID NO:6, SEQ ID NO:5 + SEQ ID NO:7, SEQ ID NO:5 + SEQ ID NO:8, SEQ ID NO:5 + SEQ ID NO:9, SEQ ID NO:5 + SEQ ID NQ:10, SEQ ID NO:5 + SEQ ID NO:11, SEQ ID NO:5 + SEQ ID NO:12, SEQ ID
NO:5 + SEQ ID NO:13, SEQ ID NO:5 + SEQ ID NO:14, SEQ ID NO:5 + SEQ ID NO:15, SEQ ID
NO:5 + SEQ ID NO:16, SEQ ID NO:5 + SEQ ID NO:17, SEQ ID NO:6 + SEQ ID NO:9, SEQ ID NO:6 + SEQ ID NQ:10, SEQ ID NO:6 + SEQ ID NO:11, SEQ ID NO:6 + SEQ ID NO:12, SEQ ID
NO:6 + SEQ ID NO:13, SEQ ID NO:6 + SEQ ID NO:14, SEQ ID NO:6 + SEQ ID NO:15, SEQ ID
NO:6 + SEQ ID NO:16, SEQ ID NO:6 + SEQ ID NO:17, SEQ ID NO:7 + SEQ ID NO:9, SEQ ID NO:7 + SEQ ID NQ:10, SEQ ID NO:7 + SEQ ID NO:11, SEQ ID NO:7 + SEQ ID NO:12, SEQ ID
NO:7 + SEQ ID NO:13, SEQ ID NO:7 + SEQ ID NO:14, SEQ ID NO:7 + SEQ ID NO:15, SEQ ID
NO:7 + SEQ ID NO:16, SEQ ID NO:7 + SEQ ID NO:17, SEQ ID NO:8 + SEQ ID NO:9, SEQ ID NO:8 + SEQ ID NQ:10, SEQ ID NO:8 + SEQ ID NO:11, SEQ ID NO:8 + SEQ ID NO:12, SEQ ID
NO:8 + SEQ ID NO:13, SEQ ID NO:8 + SEQ ID NO:14, SEQ ID NO:8 + SEQ ID NO:15, SEQ ID
NO:8 + SEQ ID NO:16, SEQ ID NO:8 + SEQ ID NO:17, SEQ ID NO:9 + SEQ ID NO:12, SEQ ID
NO:9 + SEQ ID NO:13, SEQ ID NO:9 + SEQ ID NO:14, SEQ ID NO:9 + SEQ ID NO:15, SEQ ID
NO:9 + SEQ ID NO:16, SEQ ID NO:9 + SEQ ID NO:17, SEQ ID NQ:10 + SEQ ID NO:12, SEQ ID NQ:10 + SEQ ID NO:13, SEQ ID NQ:10 + SEQ ID NO:14, SEQ ID NQ:10 + SEQ ID NO:15, SEQ ID NQ:10 + SEQ ID NO:16, SEQ ID NQ:10 + SEQ ID NO:17, SEQ ID NO:11 + SEQ ID 59 NO:12, SEQ ID NO:11 + SEQ ID NO:13, SEQ ID NO:11 + SEQ ID N0:14, SEQ ID N0:11 + SEQ ID N0:15, SEQ ID N0:11 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:17, SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:5, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:8, SEQ ID
N0:1 + SEQ ID N0:2 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:5, SEQ ID
N0:1 + SEQ ID N0:3 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:7, SEQ ID
N0:1 + SEQ ID N0:3 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:9, SEQ ID
N0:1 + SEQ ID N0:4 + SEQ ID N0:5, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:6, SEQ ID
N0:1 + SEQ ID N0:4 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:8, SEQ ID
N0:1 + SEQ ID N0:4 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID
N0:1 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID
N0:1 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID
N0:1 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID
N0:1 + SEQ ID NQ:10 + SEQ ID N0:12, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:13, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:14, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:15, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:17,
SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:12, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:13,
SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:14, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:15,
SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:17,
SEQ ID N0:1 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:12 + SEQ ID N0:17,
SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:17,
SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:17,
SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:17,
SEQ ID N0:1 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:8,
SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:2 + SEQ ID N0:6 + SEQ ID N0:9,
SEQ ID N0:2 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:2 + SEQ ID N0:8 + SEQ ID N0:9,
SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:7,
SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:9,
SEQ ID N0:3 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:7 + SEQ ID N0:9,
SEQ ID N0:3 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:6,
SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:8,
SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:6 + SEQ ID N0:9,
60 SEQ ID NO:4 + SEQ ID NO:7 + SEQ ID NO:9, SEQ ID N0:4 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID NQ:10 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:12 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:13 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:14 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:15 + SEQ ID N0:16 + SEQ ID N0:17.
8. The detergent composition according to claim 7 comprising any of the following combinations of proteases: SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID
NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11 , SEQ ID NO:1 + SEQ ID NO:12, SEQ ID
NO:1 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID
NO:1 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID
NO:5, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID
NO:7, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID
NO:9, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID
NO:6, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID
NO:8, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID
NO:5, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID
NO:7, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID
NO:9, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID
NO:7, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID
NO:9, SEQ ID NO:1 + SEQ ID NO:6 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:7 + SEQ ID
NO:9, SEQ ID NO:1 + SEQ ID NO:8 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:11 61 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:12 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:12 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:13 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:13 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:14 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:14 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:15 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:15 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:16 + SEQ ID NO:17.
9. The detergent composition according to any of claims 1 to 8, wherein the detergent composition is supplied in a liquid form.
10. The detergent composition according to any of claims 1 to 8, wherein the detergent composition is supplied as a unit dose product, such as a pouch or tablet.
11. The detergent composition according to any of claims 1 to 8, wherein the detergent composition is supplied as a liquid detergent or as a powder product.
12. The detergent composition according to any of claims 1 to 11 comprising at least one additional enzyme selected from the group consisting of cellulases, mannanases, amylases, DNases, lipases, xanthanases, pectate lyases and oxidoreductases.
13. Use of the detergent composition of any of claims 1 to 12 for laundering, hand dishwashing, automatic dishwashing or hard surface cleaning.
14. Use or two or more immunochemically different proteases in a detergent composition.
15. The use according to claim 14, wherein a. the total amount of protease is at least 0.1 wt% of the detergent composition, b. the amount of each protease in the detergent composition is below 0.1 wt%, and c. the total amount of two or more immunochemically identical proteases is below 0.1 wt% d. the detergent composition optionally comprises at least one additional enzyme which is not a protease.
16. The use according to any of claims 14-15, wherein the at least two immunochemically different proteases are selected from the group consisting of proteases having at least 60% identity to any of the proteases of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
17. The use according to any of claims 14-16, wherein the at least two immunochemically different proteases are selected from the group consisting of proteases having at least 70%, such 62 as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to any of the proteases of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
18. The use according to any of claims 14-17, wherein the at least two immunochemically different proteases comprise protease that has at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to the protease of SEQ ID NO:1.
19. The use according to claim 18, wherein the at least two immunochemically different proteases further comprises a protease selected from the group of polypeptides that has at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to any of the proteases of SEQ ID NO:3, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
20. The use according to any of claims 14-19, wherein the at least two immunochemically different proteases comprises the protease of SEQ ID NO:1 and at least one other protease selected from the group consisting of SEQ ID NO:3, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
21. The use according to any of claims 14-20, wherein the at least two immunochemically different proteases comprise any of the following combinations of proteases: SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11 , SEQ ID NO:1 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:17, SEQ ID NO:2 + SEQ ID NO:5, SEQ ID NO:2 + SEQ ID NO:6, SEQ ID NO:2 + SEQ ID NO:7, SEQ ID NO:2 + SEQ ID NO:8, SEQ ID NO:2 + SEQ ID NO:9, SEQ ID NO:2 + SEQ ID NQ:10, SEQ ID NO:2 + SEQ ID NO:11 , SEQ ID NO:2 + SEQ ID NO:12, SEQ ID NO:2 + SEQ ID
NO:13, SEQ ID NO:2 + SEQ ID NO:14, SEQ ID NO:2 + SEQ ID NO:15, SEQ ID NO:2 + SEQ ID
NO:16, SEQ ID NO:3 + SEQ ID NO:5, SEQ ID NO:3 + SEQ ID NO:6, SEQ ID NO:3 + SEQ ID
NO:7, SEQ ID NO:3 + SEQ ID NO:8, SEQ ID NO:3 + SEQ ID NO:9, SEQ ID NO:3 + SEQ ID NQ:10, SEQ ID NO:3 + SEQ ID NO:11 , SEQ ID NO:3 + SEQ ID NO:12, SEQ ID NO:3 + SEQ ID
NO:13, SEQ ID NO:3 + SEQ ID NO:14, SEQ ID NO:3 + SEQ ID NO:15, SEQ ID NO:3 + SEQ ID NO:16, SEQ ID NO:4 + SEQ ID NO:5, SEQ ID NO:4 + SEQ ID N0:6, SEQ ID N0:4 + SEQ ID N0:7, SEQ ID N0:4 + SEQ ID N0:8, SEQ ID N0:4 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID NQ:10, SEQ ID N0:4 + SEQ ID N0:11 , SEQ ID N0:4 + SEQ ID N0:12, SEQ ID N0:4 + SEQ ID N0:13, SEQ ID N0:4 + SEQ ID N0:14, SEQ ID N0:4 + SEQ ID N0:15, SEQ ID N0:4 + SEQ ID N0:16, SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID NQ:10, SEQ ID N0:5 + SEQ ID N0:11, SEQ ID N0:5 + SEQ ID N0:12, SEQ ID N0:5 + SEQ ID N0:13, SEQ ID N0:5 + SEQ ID
N0:14, SEQ ID N0:5 + SEQ ID N0:15, SEQ ID N0:5 + SEQ ID N0:16, SEQ ID N0:5 + SEQ ID
N0:17, SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:6 + SEQ ID NQ:10, SEQ ID N0:6 + SEQ ID N0:11, SEQ ID N0:6 + SEQ ID N0:12, SEQ ID N0:6 + SEQ ID N0:13, SEQ ID N0:6 + SEQ ID
N0:14, SEQ ID N0:6 + SEQ ID N0:15, SEQ ID N0:6 + SEQ ID N0:16, SEQ ID N0:6 + SEQ ID
N0:17, SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:7 + SEQ ID NQ:10, SEQ ID N0:7 + SEQ ID N0:11, SEQ ID N0:7 + SEQ ID N0:12, SEQ ID N0:7 + SEQ ID N0:13, SEQ ID N0:7 + SEQ ID
N0:14, SEQ ID N0:7 + SEQ ID N0:15, SEQ ID N0:7 + SEQ ID N0:16, SEQ ID N0:7 + SEQ ID
N0:17, SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:8 + SEQ ID NQ:10, SEQ ID N0:8 + SEQ ID N0:11, SEQ ID N0:8 + SEQ ID N0:12, SEQ ID N0:8 + SEQ ID N0:13, SEQ ID N0:8 + SEQ ID
N0:14, SEQ ID N0:8 + SEQ ID N0:15, SEQ ID N0:8 + SEQ ID N0:16, SEQ ID N0:8 + SEQ ID
N0:17, SEQ ID N0:9 + SEQ ID N0:12, SEQ ID N0:9 + SEQ ID N0:13, SEQ ID N0:9 + SEQ ID
N0:14, SEQ ID N0:9 + SEQ ID N0:15, SEQ ID N0:9 + SEQ ID N0:16, SEQ ID N0:9 + SEQ ID
N0:17, SEQ ID NQ:10 + SEQ ID N0:12, SEQ ID NQ:10 + SEQ ID N0:13, SEQ ID NQ:10 + SEQ ID N0:14, SEQ ID NQ:10 + SEQ ID N0:15, SEQ ID NQ:10 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:12, SEQ ID N0:11 + SEQ ID N0:13, SEQ ID N0:11 + SEQ ID N0:14, SEQ ID N0:11 + SEQ ID N0:15, SEQ ID N0:11 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:17, SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:5, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:7, SEQ ID
N0:1 + SEQ ID N0:2 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:2 + SEQ ID N0:9, SEQ ID
N0:1 + SEQ ID N0:3 + SEQ ID N0:5, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:6, SEQ ID
N0:1 + SEQ ID N0:3 + SEQ ID N0:7, SEQ ID N0:1 + SEQ ID N0:3 + SEQ ID N0:8, SEQ ID
N0:1 + SEQ ID N0:3 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:5, SEQ ID
N0:1 + SEQ ID N0:4 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:7, SEQ ID
N0:1 + SEQ ID N0:4 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:4 + SEQ ID N0:9, SEQ ID
N0:1 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID
N0:1 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:1 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID NO:1 + SEQ ID NO:6 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:12, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:13, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:14, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:15, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:16, SEQ ID N0:1 + SEQ ID NQ:10 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:12,
SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:13, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:14,
SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:15, SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:16,
SEQ ID N0:1 + SEQ ID N0:11 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:12 + SEQ ID N0:16,
SEQ ID N0:1 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:16,
SEQ ID N0:1 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:16,
SEQ ID N0:1 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:16,
SEQ ID N0:1 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:1 + SEQ ID N0:16 + SEQ ID N0:17,
SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:7,
SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:2 + SEQ ID N0:5 + SEQ ID N0:9,
SEQ ID N0:2 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:2 + SEQ ID N0:7 + SEQ ID N0:9,
SEQ ID N0:2 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:6,
SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:7, SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:8,
SEQ ID N0:3 + SEQ ID N0:5 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:6 + SEQ ID N0:9,
SEQ ID N0:3 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:3 + SEQ ID N0:8 + SEQ ID N0:9,
SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:6, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:7,
SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:8, SEQ ID N0:4 + SEQ ID N0:5 + SEQ ID N0:9,
SEQ ID N0:4 + SEQ ID N0:6 + SEQ ID N0:9, SEQ ID N0:4 + SEQ ID N0:7 + SEQ ID N0:9,
SEQ ID N0:4 + SEQ ID N0:8 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:6 + SEQ ID N0:9,
SEQ ID N0:5 + SEQ ID N0:7 + SEQ ID N0:9, SEQ ID N0:5 + SEQ ID N0:8 + SEQ ID N0:9,
SEQ ID NQ:10 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID NQ:10 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID NQ:10 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:12 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:12 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:13 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:13 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:14 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:14 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:15 + SEQ ID N0:16, SEQ ID N0:11 + SEQ ID N0:15 + SEQ ID N0:17, SEQ ID N0:11 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:12 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:13 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:14 + SEQ ID N0:16 + SEQ ID N0:17, SEQ ID N0:15 + SEQ ID N0:16 + SEQ ID N0:17.
65
22. The use according to claim 21 comprising any of the following combinations of proteases:
SEQ ID NO:1 + SEQ ID NO:2, SEQ ID NO:1 + SEQ ID NO:3, SEQ ID NO:1 + SEQ ID NO:4, SEQ ID NO:1 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10, SEQ ID NO:1 + SEQ ID NO:11 , SEQ ID NO:1 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID
NO:2 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:2 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID
NO:3 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID
NO:3 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID NO:3 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID
NO:3 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:5, SEQ ID NO:1 + SEQ ID
NO:4 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID
NO:4 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:4 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID
NO:5 + SEQ ID NO:6, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID NO:7, SEQ ID NO:1 + SEQ ID
NO:5 + SEQ ID NO:8, SEQ ID NO:1 + SEQ ID NO:5 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID
NO:6 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NO:7 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID
NO:8 + SEQ ID NO:9, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NQ:10 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:12, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:13, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:14, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:15, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:11 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:12 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:12 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:13 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:13 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:14 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:14 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:15 + SEQ ID NO:16, SEQ ID NO:1 + SEQ ID NO:15 + SEQ ID NO:17, SEQ ID NO:1 + SEQ ID NO:16 + SEQ ID NO:17.
23. The use according to any of claims 14-22, wherein the detergent composition is supplied in a liquid form.
24. The use according to any of claims 14-23, wherein the detergent composition is supplied as a unit dose product, such as a pouch or tablet.
25. The use according to any of claims 14-24, wherein the detergent composition is supplied as a liquid detergent or as a powder product.
66
26. The use according to any of claims 14-25, wherein the detergent composition comprises at least one additional enzyme selected from the group consisting of cellulases, mannanases, amylases, DNases, lipases, xanthanases, pectate lyases, and oxidoreductases.
67
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