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
  • C11D3/38—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
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38618—Protease or amylase in liquid compositions only

Definitions

• the present invention relates to aqueous liquid or gel type detergent compositions comprising specific combinations of enzymes.
• the detergent compositions may further comprise a combination of boric acid or a boron compound capable of forming boric acid in the composition, a polyhydroxy compound, preferably propanediol, and relatively high level of calcium ion to stabilize a selected combination of a protease enzyme and other enzymes.
• the invention also relates to a process for enhancing stability of the non protease enzymes in combination of a protease enzyme with other enzymes in a liquid or gel detergent composition.
• the invention further relates to specific protease enzymes and their use in detergent compositions
• proteases have been used in detergent compositions for about 50 years and a number of such proteases have in the past 10 years been developed by protein engineering of a number of precursor proteases.
• subtilisin 309 - or Savinase® The most successful precursor protease on the market is subtilisin 309 - or Savinase®. Protein engineering of Savinase was first disclosed in 1989 in WO 89/06279. Subsequently a high number of patent applications relating to protein engineering of Savinase have been filed by the applicant and other companies, such as Genencor International, Inc., Procter & Gamble, Unilever NV, etc. Also, a number of Savinase variants have been marketed by Novozymes A/S and Genencor International, Inc.
• Aqueous liquid and gel detergent compositions containing enzymes, including proteases are well known in the art.
• the major problem encountered with such compositions is that of ensuring a sufficient storage stability of the enzymes in the compositions. It is particularly difficult to stabilize amylases in the presence of proteases, which can readily degrade amylases in aqueous liquid or gel detergent compositions but also other enzymes, such as lipases, cellulases, etc. are frequently degraded by the proteases.
• High-alkaline amylases such as alpha amylases are described in British Specification No. 1 ,296,839.
• the use of an enzyme stabilizing system comprising a mixture of boric acid or an alkali metal borate with calcium ion, and preferably with a polyol, is disclosed in U.S. Patent 4,537,706, Severson.
• Certain a-amylases that provide improved cleaning and stain removal are disclosed in W097/32961 , Baeck et al., and in WO 96/23873 and U.S. Patent 6,093,562.
• the present invention relates to detergent compositions comprising subtilisin KL and/or variants thereof in combination with at least one other enzyme, such as a protease, a lipase, a cutinase, an amylase, a carbohydrase; a cellulase; a pectinase; a pectate lyase; a hemicellulase, e.g. a mannanase, an arabinase, a galactanase, a xylanase; an oxidase, e.g., a laccase; and/or a peroxidase.
• a protease e.g., a lipase, a cutinase, an amylase, a carbohydrase
• a cellulase a pectinase
• a pectate lyase a hem
• amylases to be used in the detergent compositions of the invention are the amylase from B. licheniformis and other amylases, such as those disclosed in WO 2001/066712, WO 2006/002643, WO 2000/60060.
• the cellulases to be used in the detergent compositions of the invention are such as those disclosed in WO 1995/024471 , WO 91/17244, WO 2002/099091.
• the lipases to be used in the detergent compositions of the invention are such as those disclosed in WO 2000/060063.
• mannanases to be used in the detergent compositions of the invention are such as those disclosed in WO 99/64619, e.g. SEQ ID NO: 2.
• endoglucanase to be used in the detergent compositions of the invention are such as those disclosed in WO 91/17244
• subtilisin KL variants of the present invention are such as those indicated in WO 98/20115 and especially those indicated in Table 1 :
• subtilisin KL and variants thereof exhibit a remarkable compatibility to other enzymes used in liquid detergent compositions such as lipases, amylases, cellulases, peroxidases/oxidases and hemicellulases.
• This property results in a substantial increase in the residual activity of these enzymes in combination with subtilisin KL and variants thereof as compared to the residual activity in the presence of other proteases, even after long periods of storage.
• the result is an improved performance of the detergent composition or that similar results can be obtained with reduced amounts of enzyme NOMENCLATURE AND CONVENTIONS FOR DESIGNATION OF VARIANTS
• a frame of reference is first defined by aligning the parent enzyme with subtilisin BPN' (BASBPN).
• Another method is to use known recognized alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance.
• amino acid numbering correspond to that of the subtilase BPN' (BASBPN) sequence.
• BPN' sequence see Siezen et al., Protein Engng. 4 (1991 ) 719- 737.
• SAVINASE® Savinase® is marketed by Novozymes A/S. It is subtilisin 309 from B. Lentus.
• Modification(s) of a subtilisin KL variant is defined to include chemical modification as well as genetic manipulation of the DNA encoding subtilisin KL.
• the modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
• subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.
• homologous subtilase sequences The homology between two amino acid sequences is in this context described by the parameter "identity".
• identity In order to determine the degree of identity between two subtilases the GAP routine of the GCG package version 9.1 can be applied (infra) using the same settings. The output from the routine is besides the amino acid alignment the calculation of the "Percent Identity” between the two sequences. Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases, which can be modified according to the invention.
• Isolated polynucleotide when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
• isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
• Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
• the term "an isolated polynucleotide” may alternatively be termed "a cloned polynucleotide”.
• Isolated protein When applied to a protein, the term “isolated” indicates that the protein has been removed from its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e.
• An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE.
• the protein in a highly purified form, i.e., more than 40% pure, more than 60% pure, more than 80% pure, more preferably more than 95% pure, and most preferably more than 99% pure, as determined by SDS-PAGE.
• isolated protein may alternatively be termed "purified protein”.
• homologous impurities means any impurity (e.g. another polypeptide than the subtilase of the invention), which originate from the homologous cell where the subtilase of the invention is originally obtained from. Obtained from.
• the term Obtained from as used herein in connection with a specific microbial source, means that the polynucleotide and/or subtilase produced by the specific source, or by a cell in which a gene from the source has been inserted.
• substrate used in connection with a substrate for a protease should be interpreted in its broadest form as comprising a compound containing at least one peptide (amide) bond susceptible to hydrolysis by a subtilisin protease.
• wash performance is used as an enzyme's ability to remove proteinaceous or organic stains present on the object to be cleaned during e.g. wash or hard surface cleaning.
• the detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
• the invention provides a detergent additive comprising the enzyme of the invention.
• the detergent additive as well as the detergent composition comprises at least one other enzyme such as a protease, a lipase; a cutinase; an amylase; a carbohydrase; a cellulase; a pectinase; a pectate lyase; a hemicellulase, e.g. a mannanase, an arabinase, a galactanase, a xylanase; an oxidase, e.g., a laccase; and/or a peroxidase.
• the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
• Lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), or a Bacillus lipase as disclosed in WO 2000/060063. Other examples are lipase variants such as those described in WO 92/05249, WO
• Suitable amylases include those of bacterial or fungal origin.
• Amylases include, for example, ⁇ -amylases obtained from Bacillus.
• useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, WO 2000/60060, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181 , 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
• amylases are Duramyl®, Termamyl®, Stainzyme®, Stainzyme Plus®, Stainzyme ultra®, Fungamyl® and BAN® (Novozymes A/S), RapidaseTM, PurastarTM and Purastar OxAmTM (from Genencor International Inc.).
• Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care and whiteness maintenance benefits.
• cellulases examples include cellulases described in EP 0 531 372, WO 96/1 1262, WO 96/29397, WO 98/08940.
• Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
• Commercially used cellulases include Renozyme®, Celluzyme®, Celluclean®, Endolase® and Carezyme® (Novozymes A/S), ClazinaseTM, and Puradax HATM (Genencor Int.
• 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 used peroxidases include GuardzymeTM (Novozymes A/S). Hemicellulases: Suitable hemicellulases include those of bacterial or fungal origin.
• Suitable hemicellulases include mannanase, lichenase, xylanase, arabinase, galactanase, acetyl xylan esterase, glucorunidase, ferulic acid esterase, coumaric acid esterase and arabinofuranosidase as described in WO 95/35362. Suitable mannanases are described in WO 99/64619. Commercially used hemicellulases include Mannaway® (Novozymes A/S).
• the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
• a detergent additive of the invention i.e. a separate additive or a combined additive, can be formulated e.g. as a gel, a liquid, a slurry, etc.
• Preferred detergent additive formulations are liquids, in particular stabilized liquids, or slurries.
• Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
• Protected enzymes may be prepared according to the method disclosed in EP 238,216.
• the detergent composition of the invention may be in any convenient form, e.g. a paste, a gel or a liquid.
• a liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
• the detergent composition comprises one or more surfactants, which may be non- ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
• the surfactants are typically present at a level of from 0.1% to 60% by weight.
• the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha- sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
• an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, al
• glucamides N-acyl N-alkyl derivatives of glucosamine
• the detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
• a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
• the detergent may comprise one or more polymers.
• examples are carboxymethyl- cellulose, poly(vinylpyrrolidone), poly (ethylene glycol), polyvinyl alcohol), poly(vinylpyridine- N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
• the detergent may contain a bleaching system which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
• a bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
• the enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol, diethylene glycol, methylpropanediol, 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 or mono- or triethanolamine, and the composition may be formulated as described in e.g. WO 92/19709, WO 92/19708, US 5,972,873 or EP 0832174.
• a polyol such as propylene glycol, diethylene glycol, methylpropanediol, 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
• the detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
• fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
• any enzyme in particular the enzyme of the invention, may be added in an amount corresponding to 0.01- 100 mg of enzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzyme protein per litre of wash liquor, in particular 0.1 -1 mg of enzyme protein per litre of wash liquor.
• Detergent Examples 1 provide ranges for the composition of a liquid detergent. Materials and Methods
• protease designated subtilisin KL and variants thereof are used.
• Subtilisin KL is a Y167A+R170S+A194P variant of Savinase (using BPN' numbering)
• protease compatibility of the enzymes is determined by preparing the detergent compositions as indicated in each Example and measuring the residual activity of the other enzyme activities after the periods indicated in the Examples.
• Enzyme Activity Enzyme activities are measured using well known recognized standard methods.
• the detergent compositions used in the examples are either a model detergent according to the compositions provided below or commercial liquid laundry detergents e.g. Tide, Era, Gain, Cheer, Wisk, All, Purex, Arm & Hammer, Sun, Great Value, Ariel, Persil, Total, Skip, Dash, Dixan, Ava or any other brand extension or concentrated versions for the liquid detergent. If the commercial laundry detergent used comprises enzymes these are inactivated prior to use by heating the detergent in a microwave oven at 85°C for 5 minutes.
• a commercial liquid detergent for laundry was added commercial proteases, amylases, Lipase, and cellulases as listed below (if the detergent already contains enzymes then these can be inactivated by heating the detergent in a microwave oven up to 85°C for 5 minutes).
• Subtilisin KL was used in comparison with commercial protease, same amount of activity units was used.
• Subtilisin KL is selected as the protease instead of Alcalase 2.5L.
• the enzyme stability of Cellulase A 5000L, Lipase A 100L, Termamyl 300L and Amylase A 12L after 1 , 2, 3 and 4 weeks at 30 0 C is clearly improved if Subtilisin KL is the protease.
• the Subtilisin KL protease is just as stable as the reference protease, Alcalase 2.5L, used.
• Example 1 The commercial liquid detergent for laundry of Example 1 was added commercial proteases, amylases, Lipase, and cellulases as listed below (if the detergent already contains enzymes then these are inactivated by heating the detergent in a micro oven up to 85°C for 5 minutes). When Subtilisin KL was used in comparison with commercial protease, same amount of activity units was used.
• Subtilisin KL is selected as the protease instead of Alcalase 2.5L.
• the enzyme stability of Cellulase A 5000L, Lipase A 100L, Termamyl 300L and Amylase A 12L after 1 , 2, 3 and 4 weeks at 30 0 C is clearly improved if Subtilisin KL is selected as protease.
• the Subtilisin KL protease is just as stable as the reference protease, Alcalase 2.5L, used.
• Example 3 A commercial liquid detergent for laundry was added commercial proteases, amylases, and lipases as listed below (if the detergent already contains enzymes then these can be inactivated by heating the detergent in a micro oven up to 85 0 C for 5 minutes). When Subtilisin KL was used in comparison with commercial protease, same amount of activity units was used.
• a liquid detergent with the following formulation as shown in table 13 is prepared.
• Table 13 Detergent formulation
• Amylase Termamyl 300L
• protease are given in enzyme protein (active) per grammes [EP/g].
• the detergent formulations are stored in 2, and 4 weeks at 3O 0 C in closed glass vessels. After storage the residual protease and amylase activities are determined. Table 14 % Residual Protease activity
• Lipase Lipase A 100L (0,2%)
• Amylase Termamyl 300L (0,2%)
• Mannase Mannan A 4,OL (0,2%)
• the detergent formulations are stored in 2, and 4 weeks at 3O 0 C in closed glass vessels. After storage the residual protease, lipase (Lip.), mannase (Man.) and amylase (Ter.) activities are determined.
• Test set-up III Addition of enzymes: I) Savinase 16L (0,05mg EP/g det.)
• EP Enzyme Protein det ⁇ detergent
• Lipase Lipase A 100L (0,2%)
• Amylase Termamyl 300L (0,2%)
• the detergent formulations are stored in 1 , 2 and 3 weeks at 30 0 C in closed glass vessels. After storage the residual protease, lipase (Lip.), mannase (Man.) and amylase (Ter.) activities are determined.

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• 2007
  • 2007-10-08 JP JP2009530902A patent/JP5497440B2/ja active Active
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