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
  • C11D3/38663—Stabilised liquid enzyme compositions

Definitions

• the present invention relates to a detergent composition comprising a protease and a second enzyme (which may be another protease or a non- proteolytic enzyme), to a method for stabilizing an enzyme in the presence of a protease and to an enzymatic detergent additive comprising a protease and a second enzyme.
• a second enzyme which may be another protease or a non- proteolytic enzyme
• proteases are widely used as ingredients in commercial detergents, including liquids. Two different proteases may be used together (US 4,511 ,490, WO 88/03946). Other enzyme types (i.e. non-proteolytic) may also be used in detergents, e.g. amylase, cellulase, lipase or peroxidase.
• the stability problem is aggravated as the protease is liable to digest and deactivate the other enzyme (i.e. the other protease or the non-proteolytic enzyme).
• WO 89/04361 discloses a detergent containing a protease and a lipase, where improved lipase stability is achieved by selecting a specified groups of lipases and a specified group of proteases.
• the stability of an enzyme in a detergent containing a protease can be improved by incorporation of a reversible protease inhibitor of the peptide or protein type.
• the invention provides a detergent composition comprising a protease and one or more other enzymes, characterized by further comprising a reversible protease inhibitor of the peptide or protein type.
• the invention provides a method for stabilizing an enzyme in the presence of a protease, characterized by incorporating a protease inhibitor.
• a further aspect of the invention provides an enzymatic detergent additive comprising a protease and one or more other enzymes in the form of a stabilized liquid or a non-dusting granulate, characterized by further comprising a reversible protease inhibitor of the peptide- or protein-type.
• JP-A 62-269689 discloses improvement of the stability of a protease in a liquid detergent by incorporation of a protease inhibitor, but no other enzymes were present.
• the protease used in the invention is preferably of microbial origin. It may be a serine protease, preferably an alkaline microbial protease or a trypsin- like protease.
• alkaline proteases are subtilisins, especially those derived from Bacillus, e.g. subtilisin Novo, subtilisi ⁇ Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (both described in WO 89/06279) and mutant subtilins such as those described in WO 89/06279.
• Examples of commercial Bacillus subtilisins are Alcalase ® , Savinase ® and Esperase ® , products of Novo Nordisk A/S.
• trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.
• the amount of protease in the detergent will typically be 0.2-40 ⁇ M, especially 1-20 ⁇ M (generally 5-1000 mg/l, especially 20-500 mg/l).
• Other enzymes generally be 0.2-40 ⁇ M, especially 1-20 ⁇ M (generally 5-1000 mg/l, especially 20-500 mg/l).
• the other enzyme(s) used in the invention may be another protease
• non-proteolytic enzyme e.g. an amylase, a cellulase, a lipase or an oxidoreductase, such as a peroxidase.
• the non- proteolytic enzyme is preferably of microbial origin, e.g. derived from a strain of
• the amount of the other enzyme(s) in the detergent will typically be 0.2-40 ⁇ M, especially 1-20 ⁇ M (generally 5-1000 mg/l, especially 20-500 mg/l).
• the inhibitor used in the invention may be any inhibitor of the peptide or protein type that reversibly inhibits the protease in question, e.g. those described in Lakowski, Jr. & Kato, Ann.Rev.Biochem. (1980) 49:593-626 and S. Murao et al., in Protein Protease Inhibitor - The Case of Streptomyces subtilisin Inhibitor (1985) at pp. 1-14. Examples are trypsin inhibitors of Family IV (described in the cited references) and subtilisin inhibitors of family III, VI and VII.
• Streptomyces subtilisin inhibitor SSI
• plasminostreptin from Streptomyces antifibrinolyticus barley subtilisin inhibitor Cl- 1 (e.g. described in Williamson et al., Plant Mol. Biol. 10, 1988, pp. 521 -535) and CI-2 (e.g. described in Williamson et al., Eur. J. Biochem. 165. 1987
• SSI Streptomyces subtilisin inhibitor
• Cl- 1 e.g. described in Williamson et al., Plant Mol. Biol. 10, 1988, pp. 521 -535) and CI-2 (e.g. described in Williamson
• eglin C from leech e.g. described in Seem ⁇ ller et al., Hoppe-Seylers Z. Phvsiol. Chem. 361 , 1980, pp. 1841-1846
• Vicia faba subtilisin inhibitor e.g. described in Svendsen et al., Carlsberq Res. Commun. 49, 1984, pp. 493-502
• leupeptin inhibitor e.g. described in S. Kondo et al., J. Antibiot. 22, 1969, pp. 558-568.
• the inhibitor may be a modified subtilisin inhibitor of Family VI with a weaker binding affinity for the protease.
• a modified inhibitor may have one -or more of the following amino acid substitutions at the indicated positions (numbered from the reactive site of the inhibitor, P1 , P2 etc. are in the direction of the N-terminal and P'1 , P'2 etc. are in the direction of the C-terminal of the inhibitor molecule):
• P3 Tyr, Glu, Ala, Arg, Pro, Ser, Lys or Trp
• P2 Ser, Lys, Arg, Pro, Glu, Val, Tyr, Trp or Ala
• P1 Arg, Tyr, Pro, Trp, Glu, Val, Ser, Lys or Ala
• P'1 Gin, Ser, Thr, He or Pro
• P'3 Glu, Gin, Asn, Val, Phe or Tyr.
• a preferred modified inhibitor is CI-2 substituted with Arg, Pro or Glu at position P3, Lys or Arg at P2, and/or Glu, Arg or Pro at P1.
• Modified inhibitors may be produced by known recombinant DNA techniques. Briefly, a DNA sequence (cDNA or a synthetic gene) encoding a known inhibitor is subjected to mutagenesis in order to replace the codon(s) for the amino acid(s) to be substituted with a new codon (codons) for the desired amino acid substitution (s). This may preferably be carried out by oligonucleotide- directed site-specific mutagenesis in bacteriophage M13 vectors (e.g. M.J. Zoller and M. Smith, Meth. Enzymol. 100 (1983) 468-500), in double-stranded DNA vectors (e.g. Y.
• M13 vectors e.g. M.J. Zoller and M. Smith, Meth. Enzymol. 100 (1983) 468-500
• double-stranded DNA vectors e.g. Y.
• the mutant gene is subsequently expressed in a suitable host strain.
• suitable hosts are bacteria (e.g. strains of Escherichia coli or Bacillus), fungi (e.g. strains of Saccharomyces cerevisiae or filamentous fungi like Aspergillus), plants such as tomato or potato or established human or animal cell lines.
• the mutant gene has to be inserted in an expression plasmid with promoter and terminator DNA elements for the formation of translatable mutant inhibitor mRNA in vivo.
• the plasmid is introduced into the host by genetic transformation. The choice of expression plasmid is dependent on the type of host strain used.
• the expression of the mutant inhibitor may be done intracellularly or extracellularly.
• the DNA sequence coding for the mutant inhibitor is fused in frame to a DNA sequence encoding a suitable peptide signalling secretion.
• the secretion signal should preferably be cleaved off in vivo, resulting in secretion of the mature mutant inhibitor into the growth medium.
• the amount of inhibitor preferably corresponds to a molar ratio of inhibitor reactive site to protease active site above 0.6, more preferably above 0.8 and most preferably above 1.
• the ratio is generally below 10, usually below 5.
• the type and amount of inhibitor is preferably chosen so as to provide at least 60% (e.g. at least 80%) inhibition in the detergent as such and below 10% inhibition when the detergent is diluted with water for use in washing, typically at a concentration of 0.3-10 g/l.
• the detergent of the invention may be in any convenient form, e.g. powder, granules or liquid.
• the invention is particularly applicable to the formulation of liquid detergents where enzyme stability problems are pronounced.
• a liquid detergent may be aqueous, typically containing 20-70% water and 0-20% organic solvent (hereinafter, percentages by weight).
• the detergent comprises surfactant which may be anionic, non- ionic, cationic, amphoteric or a mixture of these types.
• the detergent will usually contain 5-30% anionic surfactant such as linear alkyl benzene sulphonate (l-AS), alpha-olefin sulphonate (AOS), alcohol ethoxy sulphate (AES) or soap. It may also contain 3-20% anionic surfactant such as nonyl phenol ethoxylate or alcohol ethoxylate.
• the pH (measured in aqueous detergent solution) will usually be neutral or alkaline, e.g. 7-10.
• the detergent may contain 1-40% of a detergent builder such as zeolite, phosphate, phosphonate, citrate, NTA, EDTA or DTPA, or it may be unbuilt (i.e. essentially free of a detergent builder). It may also contain other conventional detergent ingredients, e.g. fabric conditioners, foam boosters, bactericides, optical brighteners and perfumes.
• a detergent builder such as zeolite, phosphate, phosphonate, citrate, NTA, EDTA or DTPA
• a detergent builder such as zeolite, phosphate, phosphonate, citrate, NTA, EDTA or DTPA
• other conventional detergent ingredients e.g. fabric conditioners, foam boosters, bactericides, optical brighteners and perfumes.
• the protease, other enzyme(s) and inhibitor may be included in the detergent of the invention by separate addition or by adding the combined additive provided by the invention.
• the additive will usually contain 0.2-8 mM protease (0.5-20%) and 0.2-8 mM (0.5-20%) of the second enzyme, and have an inhibitor/protease ratio as described above.
• the detergent additive may be in liquid form for incorporation in a liquid detergent.
• a liquid additive may contain 20-90% propylene glycol; 0.5-3% (as Ca) of a soluble calcium salt; 0-10% glycerol; minor amounts of short-chain fatty acids and carbohydrate; and water up to 100%.
• Fig. 1 is a graph showing the residual activity (in %) after 13 days at room temperature of lipase in a detergent composition containing lipase and protease alone compared to a composition containing lipase, protease and Streptomyces subtilisin inhibitor;
• Fig. 2 is a graph showing the residual activity (in %) after 13 days at room temperature of lipase in a detergent composition containing lipase and protease alone compared to a composition containing lipase, protease and barley subtilisin inhibitor CI-2;
• Fig. 3 is a graph showing the residual activity (in %) after 43 hours at room temperature of lipase in the presence of protease with or without added leupeptin inhibitor; and Fig. 4 is a graph showing the residual activity (in %) after 10 days at room temperature of cellulase in the presence of protease with or without added Streptomyces subtilisin inhibitor; and
• Fig. 5 is a graph showing the residual activity (in %) after 10 days at room temperature of cellulase in the presence of protease with or without added CI-2 inhibitor.
• a concentrated liquid detergent was formulated as follows (% by weight of active substance):
• a detergent according to the invention was prepared by addition of Streptomyces subtilisin inhibitor (SSI, 0.05 mg/ml, 4.5 ⁇ M) to a detergent of the composition: 52 (v/v) % of the above concentrated detergent in water containing 10 mg/ml (300 ⁇ M) Humicola lipase (LipolaseTM) and 0 1 mg mj (3 6 ⁇ M)
• Another detergent was prepared by addition of inhibitor CI-2 (0.03 mg/ml, 3.3 ⁇ M) to a detergent of the composition 55 (v/v) % concentrated detergent in water containing 10 mg/ml (300 ⁇ M) Humicola lipase (LipolaseTM) and 0.1 mg/ml (3.6 ⁇ M) Savinase ® .
• the lipase activity was measured at various times and expressed in % of initial lipase activity.
• the protection of lipase from proteolytic degradation in the presence of a protease inhibitor was determined by adding 0.67 g/l leupeptin inhibitor to a mixture of 0.5 g/l Pseudomonas cepacia lipase and 2 g/l Fusarium protease in 50 mM Tris-HCI, pH 8.0, at 20°C and measuring the residual lipase activity (in %) after 43 hours. From the results shown in Fig. 3 it appears that there is very little degradation of the lipase in the presence of the leupeptin inhibitor, whereas the lipase is almost completely degraded when no inhibitor is added. The protease activity may be restored by dilution.
• a concentrated liquid detergent was formulated as follows (% by weight of active substance):
• a detergent according to the invention was prepared by addition of Streptomyces subtilisin inhibitor (SSI, 0.09 mg/ml, 7.7 ⁇ M) to a detergent (90% (w/w) of the above concentrated detergent in water) containing 0.12 mg/ml (3.3 ⁇ M) Humicola cellulase and 0.18 mg/ml ( ⁇ .7 ⁇ M) Savinase ® .
• Another detergent was prepared by addition of inhibitor CI-2 (0.07 mg/ml, 7.8 ⁇ M) to a detergent (90% (w/w) of the above concentrated detergent in water) containing 0.12 mg/ml (3.3 ⁇ M) Humicola cellulase and 0.18 mg/ml (6.7 ⁇ M) Savinase ® .

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• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Wood Science & Technology (AREA)
• Organic Chemistry (AREA)
• Detergent Compositions (AREA)
• Enzymes And Modification Thereof (AREA)
• Cosmetics (AREA)

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Publications (1)

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Cited By (54)

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Citations (2)

• 1990
  • 1990-08-24 DK DK204290A patent/DK204290D0/da not_active Application Discontinuation
• 1991
  • 1991-08-23 EP EP91915580A patent/EP0544777B1/de not_active Revoked
  • 1991-08-23 DK DK91915580.4T patent/DK0544777T3/da active
  • 1991-08-23 ES ES91915580T patent/ES2062812T3/es not_active Expired - Lifetime
  • 1991-08-23 AT AT91915580T patent/ATE103632T1/de not_active IP Right Cessation
  • 1991-08-23 JP JP3514775A patent/JPH06500142A/ja active Pending
  • 1991-08-23 DE DE69101557T patent/DE69101557T2/de not_active Revoked
  • 1991-08-23 WO PCT/DK1991/000243 patent/WO1992003529A1/en not_active Application Discontinuation

Patent Citations (2)

Non-Patent Citations (1)

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