Classifications

• • 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
  • C11D3/26—Organic compounds containing nitrogen
  • C11D3/33—Amino carboxylic acids
• • 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
  • C11D7/00—Compositions of detergents based essentially on non-surface-active compounds
  • C11D7/22—Organic compounds
  • C11D7/32—Organic compounds containing nitrogen
  • C11D7/3272—Urea, guanidine or derivatives thereof

Definitions

• This invention relates to enzymatic stain removal of stains from fabrics.
• the invention relates to fabric stain removal composition for stain removal by direct application or pre-treatment of a stain on a stained fabric.
• Enzymes are used in detergent formulations to aid cleaning and stain removal.
• the objective of the invention is to improve low / ambient temperature enzyme stain removal of stains on stained fabrics.
• the present invention provides a fabric stain-removal composition comprising the combination of:
• said stains comprise biological material, preferably proteinaceous and/or starch material deposited on fabric.
• composition is ambient-active.
• composition comprises one or more surfactants.
• the invention provides a process for removing a stain from a stained fabric substrate, comprising the step of treating the stain with the composition of the first aspect of the invention.
• the method takes place in ambient conditions.
• the method of the second aspect comprises the step of applying said composition directly to the stained fabric, with or without the addition of water.
• the step of applying the composition may itself be a main washing process or it may be as a pre-step (as a pre-treatment) to a further, subsequent ('main') washing process steps.
• Such further, subsequent washing steps main comprise any manual washing process or any washing process in a fabric washing machine.
• the invention provides a fabric stain removal treatment device comprising a storage chamber for storing the composition of the first aspect of the invention and a treatment member for applying said composition directly to a substrate, preferably using the method of the second aspect.
• the invention provides use of an arginine compound in the enzymatic removal of fabric stains.
• the arginine compound enhances the enzymatic removal of stains present on fabric and is effective at lower temperatures. This offers improved laundry (i.e. fabric) cleaning of stained fabrics in regions where ambient water washing is prevalent. Improved washing performance at lower
• the invention provides enzymatic performance of proteinaceous and/or starch based soil and/or stains in an ambient temperature cleaning processes (with low temperature wash liquor) without serious
• the enzyme can therefore be selected more freely, on the basis of other
• the arginine compound improves the removal of based stains at ambient temperature and thus may significantly reduce the energy cost for each wash.
• substrate includes fabric, clothing etc.
• arginine compound is intended to include any suitable arginine compound including stereoisomeric and racemic forms, derivatives, and substituted derivatives, salts thereof, and any mixtures thereof.
• the arginine compound is present in any wash liquor in a concentration in the range 0.01 mg/ml - 10mg/ml and more preferably in the range 0.01 - 0.32 mg/ml, more preferably 0.08-0.16 mg/ml.
• the arginine compound is present in any composition of the invention in a concentration in the range 40 mg - 5000 mg per dose, preferable 320mg - 4000 mg per dose.
• the composition may be provided as a single dose format or as multiple dose, free flowing format (powder, liquid, gel, paste etc) which is measured out by the consumer using a dosing device.
• the dose may range from 10 ml to 100ml.
• concentration within the composition may be higher and the concentration per dose higher than in main wash formulations, so may in the range 300- 5000 mg per dose, preferably 500 mg - 2000 mg per dose.
• Pre-treatment device dose levels may vary from 0.1 - 10ml.
• ambient-active is intended to mean less that 25 degrees Celcius and preferably 22 degrees Celcius or less, more preferably 15 degrees or less but always greater than 1 degree Celcius and "active" means effective in achieving stain removal.
• treatment in the context of enzymatic fabric treatment composition preferably means cleaning and more preferably stain removal.
• stain removal is measured in terms of Remission units or a Remission index.
• Stain removal is preferably shown when there is a remission equal to or greater than 2 Remission units and more preferably greater or equal to 5 units. This is represents effective stain removal for a visible (by the human eye) effect.
• enzyme includes enzyme variants (produced, for example, by recombinant techniques) are included. Examples of such enzyme variants are disclosed, e.g., in EP 251 ,446 (Genencor), WO 91/00345 (Novo Nordisk), EP 525,610 (Solvay) and WO 94/02618 (Gist-Brocades NV).
• composition unless specified otherwise.
• abbreviation 'wt%' is to be understood as % by weight of the total composition.
• the pre-treatment composition is ambient-active. Accordingly, the temperature of the wash liquor step of aqueous washing process is therefore less than 40°C and preferably less than 30°C and more preferably less than 25°C and more preferably less than 22°C further more preferably 15°C or less at all times during the washing but excluding drying. Encouraging low temperature wash liquor is advantageous environmentally and financially.
• the enzymatic treatment composition is preferably packaged with instructions to treat a substrate at low temperatures using the composition for example in the method described herein, the low temperatures being preferably less than 40 °C, more preferably less than 30°C even more preferably less than 25°C preferably at 22°C or less most preferably at 15 degrees °C.
• the invention is especially advantageous for the particular situation where one requires enzymatic cleaning of stains in a ambient temperature cleaning
• the enzyme system preferably comprises a mesophilic or
• thermophilic enzyme system The enzyme system may even be a mesophilic and/or thermophilic enzyme system with the exclusion of pyschrophilic enzymes.
• Enzymes may be from animal, vegetable, bacterial origin (derived from bacteria) or fungal origin (derived from fungus) however enzymes from bacterial origin are preferred. Chemically modified or protein engineered mutants are included.
• Genes encoding such enzymes can be transferred from one host to a preferred expression production host which may or may not be the same as the original host.
• the one or more enzymes preferably comprises a protease.
• Preferred proteases are serine proteases or metallo proteases, preferably an alkaline microbial protease or a trypsin-like protease.
• protease enzymes include AlcalaseTM, SavinaseTM, PrimaseTM, DuralaseTM, DyrazymTM, EsperaseTM, EverlaseTM, PolarzymeTM, and KannaseTM, (Novozymes A S), MaxataseTM, MaxacalTM, MaxapemTM,
• the arginine compound is underivatised arginine and/or homo-arginine and more preferably underivatised arginine.
• the one or more enzymes preferably comprises an amylase.
• Suitable amylases include those 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 B. Iicheniformis, described in more detail in GB 1 ,296,839, or the Bacillus sp. strains disclosed in WO 95/026397 or WO 00/060060.
• amylases are DuramylTM, TermamylTM, Termamyl UltraTM, NatalaseTM, StainzymeTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (from Genencor International IncCommercially available amylases include StainzymeTM (Novozymes).
• the one or more enzymes may comprises a protease in combination with an amylase and the arginine compound is underivitised arginine.
• the enzymes are preferably present at 0.001 - 5%wt more preferably 0.01 - 3%.
• the composition preferably comprises further enzymes.
• the composition preferably comprises a lipase; the preferred lipases including so called ' first wash' lipases which comprise a polypeptide having an amino acid sequence which has at least 90 percent sequence identity with the wild-type lipase derived from Humicola lanuginosa strain DSM 4109 and compared to said wild-type lipase, comprises a substitution of an electrically neutral or negatively charged amino acid within 15 A of E1 or Q249 with a positively charged amino acid; and may further comprise:
• I. comprises a negatively charged amino acid in position E210 of said wild-type lipase
• III comprises a neutral or negatively charged amino acid at a position corresponding to N94 of said wild-type lipase;
• IV has a negative charge or neutral charge in the region corresponding to positions 90-101 of said wild-type lipase
• LipexTM Novozymes
• a similar enzyme from Novozymes but believed to fall outside of the above definition has been disclosed by Novozymes under the name LipocleanTM and this is also preferred.
• Other possible lipases include lipases from Humicola (synonym Thermomyces), e.g. from other H. lanuginosa (T. lanuginosus) strains or from H. insolens, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes, P.
• P. wisconsinensis a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1 131 , 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
• lipase enzymes include LipolaseTM and Lipolase UltraTM, and the Bacterial enzyme, Lipomax ® ex Genecor. This is a bacterially derived Lipase, of variant M21 L of the lipase of Pseudomonas alcaligenes as described in WO 94/25578 to Gist-Brocades (M. M.M.J. Cox, H.B.M. Lenting, L.J.S.M.
• the composition preferably comprises a phospholipase classified as EC 3.1 .1 .4 and/or EC 3.1 .1 .32.
• phospholipase is an enzyme which has activity towards phospholipids. Phospholipids, such as lecithin or
• phosphatidylcholine consist of glycerol esterified with two fatty acids in an outer (sn-1 ) and the middle (sn-2) positions and esterified with phosphoric acid in the third position; the phosphoric acid, in turn, may be esterified to an amino-alcohol.
• Phospholipases are enzymes which participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including
• phospholipases A1 and A2 which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group in
• Phospholipase C and phospholipase D release diacyl glycerol or phosphatidic acid respectively.
• the composition preferably comprises a cutinase. classified in EC 3.1 .1 .74.
• the cutinase used according to the invention may be of any origin.
• Preferably cutinases are of microbial origin, in particular of bacterial, of fungal or of yeast origin.
• the composition preferably comprises a cellulase include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Thielavia terrestris, Myceliophthora thermophila, and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757, WO 89/09259, WO 96/029397, and WO 98/012307.
• the composition preferably comprises peroxidases/oxidases, especially of bacterial origin. Chemically modified or protein engineered mutants are included.
• An example of an oxidative bacterium is, but not limited to, are Aeromonas sp wherefrom oxidases can be sourced.
• the composition preferably comprises a pectate lyase (also called
• polygalacturonate lyases include pectate lyases that have been cloned from different bacterial genera such as Erwinia, Pseudomonas, Klebsiella and
• the pectate lyase comprises the pectate lyase disclosed in Heffron et al., (1995) Mol. Plant-Microbe Interact. 8: 331 -334 and Henrissat et al., (1995) Plant Physiol. 107: 963-976.
• pectatel lyases are disclosed in WO 99/27083 and WO 99/27084.
• Other specifically contemplated pectate lyases derived from Bacillus
• pectate lyase variants are disclosed in WO 02/006442, especially the variants disclosed in the Examples in WO 02/006442 (which document is hereby incorporated by reference).
• alkaline pectate lyases include BIOPREPTM and
• the composition preferably comprises a mannanase:
• mannanases EC 3.2.1 .78
• mannanases include mannanases of bacterial and fungal origin.
• the mannanase is derived from a strain of the filamentous fungus genus Aspergillus, preferably Aspergillus niger or Aspergillus aculeatus (WO 94/25576).
• WO 93/24622 discloses a mannanase isolated from Trichoderma reseei. Mannanases have also been isolated from several bacteria, including Bacillus organisms. For example, Talbot et al., Appl. Environ.
• JP-A-63036775 relates to the Bacillus microorganism FERM P-8856 which produces beta-mannanase and beta-mannosidase.
• JP-A-08051975 discloses alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001 .
• a purified mannanase from Bacillus amyloliquefaciens is disclosed in WO 97/1 1 164.
• WO 91/18974 describes a hemicellulase such as a glucanase, xylanase or
• mannanase active Contemplated are the alkaline family 5 and 26 mannanases derived from Bacillus agaradhaerens, Bacillus licheniformis, Bacillus halodurans, Bacillus clausii, Bacillus sp., and Humicola insolens disclosed in WO 99/64619. Especially contemplated are the Bacillus sp. mannanases concerned in the Examples in WO 99/64619.
• mannanases examples include MannawayTM available from Novozymes A/S Denmark.
• the enzyme and any perfume/fragrance or pro-fragrance present may show some interaction and should be chosen such that this interaction is not negative. Some negative interactions may be avoided by encapsulation of one or other of enzyme and pro-fragrance and/or other segregation within the product.
• the composition preferably comprises a surfactant.
• the surfactant may be a synthetic surfactant or a biosurfactant which is mircrobially synthesized e.g. from bacteria, fungi or other microbe.
• biosurfactant preferably comprises a microbial ly-derived biosurfactant.
• a glycolipid biosurfactant which may be a rhamnolipid or sophorolipid or trehalolipid or a mannosylerythritol lipid (MEL).
• MEL mannosylerythritol lipid
• the biosurfactant may advantageously comprise a cellobiose, peptide based biosurfactants, lipoproteins and lipopeptides e.g. surfactin, fatty acids e.g. corynomucolic acids (preferably with hydrocarbon chain C12-C14) , phospholipids e.g.
• hexadecane to less than 1 mN m-1 and CMC of 30 mg L-1 (Kretschner et al., 1982) and Spiculisporic acid; polymeric biosurfactants including emulsan, liposan, mannoprotein and polysaccharide-protein complexes.
• polymeric biosurfactants including emulsan, liposan, mannoprotein and polysaccharide-protein complexes.
• biosurfactant comprises a rhamnolipid.
• the composition according to the invention comprises a surfactant, preferably a detersive surfactant.
• a detersive surfactant we mean that the surfactant, or at least one surfactant of any surfactant mixture, provides a detersive, i.e. cleaning effect to textile fabrics treated as part of a laundering process.
• Other surfactants which may or may not be detersive surfactants, can be used as part of the composition.
• the detersive surfactant is present by weight in the laundry detergent
• compositions at a level of from 3 to 85% by weight, preferably from 3 to 60% by weight, more preferably from 3 to 40% by weight, most preferably from 3 to 35% by weight.
• Additional surfactants can also be incorporated in the laundry compositions of the invention; these may be detersive or non-detersive
• the detersive surfactant comprises anionic surfactant, nonionic surfactant or a mixture of the two. More preferably the detersive surfactant mixture comprises anionic and nonionic surfactants. Cationic surfactant may optionally be present as part of the detersive surfactant.
• anionic surfactant is present at a level of from 0.1 to 95% by weight, preferably from 1 to 50% by weight, more preferably from 1 .5 to 25% by weight based on total weight of surfactants present.
• Nonionic surfactant if present, is incorporated at a level of from 0.1 to 95% by weight, preferably from 1 to 50% by weight, more preferably from 1 .5 to 25% by weight based on total weight of surfactants present. If a detersive surfactant mixture is used that incorporates both anionic and nonionic surfactants, then preferably the ratio of anionic surfactant to nonionic surfactant is from 10:1 to 1 :10.
• 'nonionic surfactant' shall be defined as amphiphilic molecules with a molecular weight of less than about 10,000, unless otherwise noted, which are substantially free of any functional groups that exhibit a net charge at the normal wash pH of 6-1 1 .
• nonionic surfactant may be used.
• fatty acid alkoxylates especially ethoxylates, having an alkyl chain of from C 8 -C 35 , preferably C8-C30, more preferably Cio-C2 4 , especially C10-C18 carbon atoms, and having preferably 3 to 25, more preferred 5 to 15 ethylene oxide groups, for example, Neodols from Shell (The Hague, The Netherlands); ethylene
• oxide/propylene oxide block polymers which may have molecular weight from 1 ,000 to 30,000, for example, Pluronic (trademark) from BASF (Ludwigshafen, Germany); and alkylphenol ethoxylates, for example Triton X-100, available from Dow Chemical (Midland, Mich., USA).
• nonionic surfactants considered within the scope of this invention include condensates of alkanolamines with fatty acids, such as cocamide DEA, polyol- fatty acid esters, such as the Span series available from Uniqema (Gouda, The Netherlands), ethoxylated polyol-fatty acid esters, such as the Tween series available from Uniqema (Gouda, The Netherlands), alkylpolyglucosides, such as the APG line available from Cognis (Dusseldorf, Germany) and n- alkylpyrrolidones, such as the Surfadone series of products marketed by ISP (Wayne, N.J ., USA).
• Anionic surfactant include condensates of alkanolamines with fatty acids, such as cocamide DEA, polyol- fatty acid esters, such as the Span series available from Uniqema (Gouda, The Netherlands), ethoxylated polyol-fatty acid
• 'Anionic surfactants' are defined herein as amphiphilic molecules comprising one or more functional groups that exhibit a net anionic charge when in aqueous solution at the normal wash pH of between 6 and 1 1 .
• Preferred anionic surfactants are the alkali metal salts of organic sulphur reaction products having in their molecular structure an alkyl radical containing from about 6 to 24 carbon atoms and a radical selected from the group consisting of sulphonic and sulphuric acid ester radicals.
• anionic surfactant hereinafter described can be used, such as alkyl ether sulphates, soaps, fatty acid ester sulphonates, alkyl benzene sulphonates, sulphosuccinate esters, primary alkyl sulphates, olefin sulphonates, paraffin sulphonates and organic phosphate; preferred anionic surfactants are the alkali and alkaline earth metal salts of fatty acid carboxylates, fatty alcohol sulphates, preferably primary alkyl sulfates, more preferably they are ethoxylated, for example alkyl ether sulfates; and alkylbenzene sulfonates or mixtures thereof.
• cationic, amphoteric surfactants and/or zwitterionic surfactants may be present in the compositions according to the invention.
• Preferred cationic surfactants are quaternary ammonium salts of the general formula R 1 R 2 RsR 4 N + X " , for example where Ri is a Ci 2 -Ci alkyl group, R 2 and R 3 are methyl groups, R is a 2-hydroxyethyl group, and X " is a chloride ion.
• This material is available commercially as Praepagen (Trade Mark) HY from Clariant GmbH, in the form of a 40% by weight aqueous solution.
• the composition according to the invention comprises an amphoteric or zwitterionic surfactant.
• Amphoteric surfactants are molecules that contain both acidic and basic groups and will exist as zwitterions at the normal wash pH of between 6 and 11.
• an amphoteric or zwitterionic surfactant is present at a level of from 0.1 to 20% by weight, more preferably from 0.25 to 15% by weight, even more preferably from 0.5 to 10% by weight.
• Suitable zwitterionic surfactants are exemplified as those which can be broadly described as derivatives of aliphatic quaternary ammonium, sulfonium and phosphonium compounds with one long chain group having about 8 to about 18 carbon atoms and at least one water solubilizing radical selected from the group consisting of sulfate, sulfonate, carboxylate, phosphate or phosphonate.
• a general formula for these compounds is:
• Ri contains an alkyl, alkenyl or hydroxyalkyi group with 8 to 18 carbon atoms, from 0 to 10 ethylene-oxy groups or from 0 to 2 glyceryl units;
• Y is a nitrogen, sulfur or phosphorous atom;
• R 2 is an alkyl or hydroxyalkyi group with 1 to 3 carbon atoms;
• x is 1 when Y is a sulfur atom and 2 when Y is a nitrogen or phosphorous atom;
• R 3 is an alkyl or hydroxyalkyi group with 1 to 5 carbon atoms and Z is a radical selected from the group consisting of sulfate, sulfonate, carboxylate, phosphate or phosphonate.
• Preferred amphoteric surfactants are amine oxides, for example coco dimethyl amine oxide.
• Preferred zwitterionic surfactants are betaines, and especially amidobetaines.
• Preferred betaines are C 8 to Ci 8 alkyl amidoalkyl betaines, for example coco amido betaine. These may be included as co-surfactants, preferably present in an amount of from 0 to 10 wt %, more preferably 1 to 5 wt %, based on the weight of the total composition.
• composition according to the present invention are betaine surfactants. Examples of these are mentioned in the following list.
• the sulfatobetaines such as 3-(dodecyldimethylammonium)-1 -propane sulfate; and 2-(cocodimethylammonium)-1 -ethane sulfate.
• the sulfobetaines such as: 3-(dodecyldimethyl-ammonium)-2-hydroxy-1 -propane sulfonate; 3-(tetradecyl-dimethylammonium)-1 -propane sulfonate; 3-(Ci2-Ci 4 alkyl- amidopropyldimethylammonium)-2-hydroxy-1 -propane sulfonate; and 3-
• carboxybetaines such as (dodecyldimethylammonium) acetate (also known as lauryl betaine); (tetradecyldimethylammonium) acetate (also known as myristyl betaine); (cocodimethylammonium) acetate (also known as coconut betaine);
• oleyldimethylammonium also known as oleyl betaine
• the sulfoniumbetaines such as: (dodecyldimethylsulfonium) acetate; and 3- (cocodimethyl-sulfonium)-l -propane sulfonate.
• the phosphoniumbetaines such as 4-(trimethylphosphonium)-1 -hexadecane sulfonate; 3-(dodecyldimethylphosphonium)-1 -propanesulfonate; and
• compositions according to the present invention preferably comprise carboxybetaines or sulphobetaines as amphoteric or zwitterionic surfactants, or mixtures thereof. Especially preferred is lauryl betaine.
• the treatment composition may comprise other ingredients commonly found in detergent liquids. Especially polyester substantive soil release polymers, hydrotropes, opacifiers, colorants, perfumes, other enzymes, other surfactants, microcapsules of ingredients such as perfume or care additives, softeners, polymers for anti redeposition of soil, bleach, bleach activators and bleach catalysts, antioxidants, pH control agents and buffers, thickeners, external structurants for rheology modification, visual cues, either with or without functional ingredients embedded therein and other ingredients known to those skilled in the art.
• Figure 1 is a bar chart displaying the average RFU readings from Example 1 for replicates 1 -4 from Table 1 vs arginine concentration. Error bars display standard deviation between the four replicates for each concentration;
• Figure 2 is a bar chart displaying the average RFU readings from Example 2 for replicates 1 -4 from Table 2 vs arginine concentration. Error bars display standard deviation between the four replicates for each concentration.
• Neodol 25-7 ex. C12-C15 alcohol 7-ethoxylate
• LAS acid Cio-Ci 4 alkyl benzene sulphonic acid
• Example 1 The effect of arginine on Savinase activity
• Tris.HCI (2-Amino-2-hydroxymethyl-1 ,3-propanediol hydrochloride Trizma® hydrochloride ex. Sigma Aldrich)(pH 8.0)
• BODIPY TR-X dye conjugated casein substrate solution prepared as follows: 1 mg of BODIPY TR-X dye labelled casein substrate (Invitrogen, Cat No. E6639) was thoroughly dissolved in 200 ⁇ of 0.1 M sodium bicarbonate buffer (pH 8.5). This 200 ⁇ was then added to 19.8 ml of 50 mM T s.HCI (pH 8.0) to make a working solution of 10 Mg/ml. Fresh substrate was prepared shortly before each assay and stored in the dark at room temperature until required. - DL-Arginine (Sigma Cat No. 1 1020, EC Number: 230-571 -3)
• reaction mixtures were prepared in a 300 ⁇ well / 96 well microtitre (microwell) plate:
• fluorescence is proportional to protease activity. Fluorescence intensity was measured in a fluorescence microplate reader set for excitation at 485 nm and emission detection at 530 nm. Example 1 Results
• Table 1 Relative fluorescent units (RFU) measured for 1 .3 ⁇ / ⁇ Savinase and 4 ⁇ / ⁇ BODIPY TR-X dye labelled casein substrate with a range arginine
• Arginine increases relative Savinase activity up to a maximum of 51.4 % at 0.25 M.
• Example 2 The effect of arginine on Stainzyme activity
• BODIPY FL dye conjugated DQ starch substrate solution 1 mg of BODIPY FL dye conjugated DQ starch substrate (Invitrogen, Cat No. E33651 ) was thoroughly dissolved in 100 ul of 50 mM sodium acetate buffer (pH 4.0). This 100 ul was added to 900 ul of 50 mM MOPS (pH 6.9) to make a working solution of 10 ug/ml. Fresh substrate was prepared shortly before each assay and stored in the dark at room temperature until required.
• BODIPY FL dye conjugated DQ starch substrate (10 ⁇ g/ml) 100 ⁇
• Table 2 Relative fluorescent units (RFU) measured for Stainzyme and BODIPY FL dye conjugated DQ starch substrate with a range DL-arginine concentrations. Four replicates were carried out for each concentration in parallel.
• REU Relative fluorescent units
• Amylase sensitive stain CS27: potato starch, coloured (Testfabrics Inc.) - Laundry Enzymes (Novozymes):
• Example composition A 5 mg/L 100 ⁇
• Example c A 5 mg/L 100 ⁇
• deltaE [ ( ⁇ _) 2 + (Aa) 2 + (Ab) 2 ] 1 2 wherein ⁇ _ is a measure of the difference in darkness between the washed and white cloth; Aa and Ab are measures for the difference in redness and yellowness respectively between both cloths. From this equation, it is clear that the lower the value of deltaE, the whiter the cloth will be. With regard to this colour
• Table 3.1 The results of Table 3.1 are shown in Figure 3.1 .
• Table 3.2 End-point stain removal assays using CS27 stained cloth treated with Stainzyme and a range of DL-arginine concentrations in composition A. Four replicates were performed in parallel on the same 96 well plate. The plates were scanned and the deltaE and SRI values calculated. Rep 1 Rep 2 Rep 3 Rep 4 Average

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