EP0399681B1 - Wäschewaschverfahren - Google Patents

Wäschewaschverfahren Download PDF

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Publication number
EP0399681B1
EP0399681B1 EP90304796A EP90304796A EP0399681B1 EP 0399681 B1 EP0399681 B1 EP 0399681B1 EP 90304796 A EP90304796 A EP 90304796A EP 90304796 A EP90304796 A EP 90304796A EP 0399681 B1 EP0399681 B1 EP 0399681B1
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Prior art keywords
surfactant
enzyme
ratio
neodol
oil
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French (fr)
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EP0399681A2 (de
EP0399681A3 (de
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Maha Y. El-Sayed
Sheldon N. Lewis
Susan A. Anderson
Richard J. Wiersema
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Clorox Co
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Clorox Co
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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
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • 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
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase

Definitions

  • This invention relates to a method of laundering fabrics; more particularly, it relates to a method of laundering fabrics having triglyceride stains thereon in a solution containing a lipase or a cutinase and at least one surfactant.
  • Lipases are enzymes naturally produced by a wide variety of living organisms from microbes to higher eukaryotes. Fatty acids undergoing oxidation in tissues of higher animals must be in free form (that is, non-esterified) before they can undergo activation and oxidation. Thus, intracellular lipases function to hydrolyze the triacylglycerols to yield free fatty acids and glycerol.
  • Bacterial lipases are classically defined as glycerolesterhydrolases (EC 3.1.1.3) since they are polypeptides capable of cleaving ester bonds. They have a high affinity for interfaces, a characteristic which separates them from other enzymes such as proteases and esterases. An interface onto which lipases readily absorb is that of oil/water.
  • Cutinases are esterases that catalyze the hydrolysis of cutin.
  • cutinase allows fungi to penetrate through the cutin barrier into the host plant during the initial stages of a fungal infection.
  • the primary structures of several cutinases have been compared and shown to be strongly conserved. Ettinger, Biochemistry, 26 , pp. 7883-7892 (1987).
  • Sebastian et al., Arch. Biochem. Biophys., 263 (1) , pp. 77-85 (1988) have recently found production of cutinase to be induced by cutin in a fluorescent P. putida strain. This cutinase catalyzed hydrolysis of p-nitrophenyl esters of C 4 -C 16 fatty acids.
  • Lipases have long been considered as potential components in detergent compositions. An early preparation of lipase in the form of pancreatin was taught for addition to detergent formulations by Rohm, Chem. Abs., Int. , P2048 (1916). More recently, lipases obtained from certain Pseudomonas or Chromobacter microorganisms have been disclosed as useful in detergent compositions: Thom et al., U.S. Patent No. 4,707,291, issued November 17, 1987 and Wiersema et al., European Patent Application 253,487, published January 20, 1988.
  • Lipases have long been known generally to be inhibited by anionic detergents and by nonionic detergents. Lipase activity has been reported as enhanced by emulsifiers such by Wills, Bioch., 60 , pp. 529-534 (1955) and Andree, et al., J. App. Biochem., 2 , pp. 218-229 (1980). Not withstanding such teachings, attempts to use lipases in laundry solutions which contain anionic or nonionic surfactants have been largely unsuccessful, and effective use of lipases for cleaning oily stains have been limited to presoak applications.
  • Lipases have been found useful in aqueous solution, without added surfactants, for prewash or presoak applications over extended periods of time, followed by a conventional washing with fully formulated detergents. Under these conditions, lipases have been effective in removing natural oil (triglyceride) stains. But despite the many attempts to use lipase in detergent formulations for laundering solutions, the demonstrated washing benefit has been disappointing.
  • the present invention provides a method of laundering fabrics having triglyceride stains thereon in a solution containing a lipase or a cutinase and at least one surfactant wherein there is added to the solution an agent, which is not a substrate for the enzyme, selected from a hydrocarbon, which is hexadecane or octadecane, and a relatively insoluble organic compound, which has a solubility ⁇ between 7 and 9.5 and which is selected from glycol derivatives, alcohols, aldehydes, ketones and amides, and mixtures thereof, so as to activate hydrolysis of the enzyme.
  • an agent which is not a substrate for the enzyme, selected from a hydrocarbon, which is hexadecane or octadecane, and a relatively insoluble organic compound, which has a solubility ⁇ between 7 and 9.5 and which is selected from glycol derivatives, alcohols, aldehydes, ketones and amides, and mixtures thereof, so
  • the enzyme(s) used in accordance with the present method is/are capable of hydrolyzing triglyceride on fabric and the additive agent will prevent inhibition of enzymatic hydrolysis by the surfactant in the solution, and will thus allow the onset of hydrolysis of the triglyceride fabric soil or stain.
  • surfactant systems may be formulated that include lipases and/or cutinases for use in laundering solutions, without requiring extended soaking or high temperatures for triglyceride hydrolysis.
  • the onset of hydrolysis for such an enzyme is dependent upon exceeding a critical ratio. It has been discovered that such enzymes will "turn on” and hydrolyze the oil stain only if the molar ratio of oil to surfactant in the laundry solution, at the oil stain interface, exceeds a certain value, referred to herein as the "critical ratio". The value of the critical ratio for each enzyme depends upon the identity of surfactant used.
  • the hydrolysis activating agent changes the ratio of oil to surfactant in a laundry solution in which the composition is employed to exceed the critical ratio, so that the enzyme will "turn on” and hydrolyze the oil stain.
  • the enzymes used in the present method has sufficient hydrolytic activity in a surfactant laundering solution to hydrolyze at least about 5 wt.% of the total oil stain, i.e. triglyceride, in a laundering solution within about 14 to 15 minutes at about 25°C.
  • a particularly preferred enzyme for use in the present invention is isolatable from Pseudomonas putida (hereinafter " P . putida ”) ATCC 53552.
  • Fabrics cleaned in laundering solutions include clothing soiled by body oils (sebum) and linens soiled by food and cooking oils.
  • body oils sebum
  • linens soiled by food and cooking oils Mono-, di- and triglycerides are present in sebum soils and cooking oils and potentially can be hydrolyzed by lipases.
  • enzymes of interest for the present invention include both cutinases and lipases which are capable of hydrolyzing triglyceride on fabric in aqueous solution and will sometimes hereinafter be described as glycerol ester hydrolases.
  • Such enzymes useful in the present invention are typically obtained from certain Pseudomonas , Chromobacter , Fusarium or Aspergillus strains.
  • P. putida ATCC 53552 from P. sp . (as Amano 68S), from P.
  • the enzyme has difficulty binding the substrate unless the substrate is changed in some way facilitated by surfactant. That is, there appears to be a necessary complex between substrate and surfactant formed in order for the substrate to be hydrolyzable by enzyme.
  • Typical structures of common surfactants are:
  • Initiation of substrate hydrolysis by the glycerol ester hydrolases depends strongly on the system ratio and not on the concentration of either substrate (triglyceride) or surfactant.
  • Table IA An example of the effect of the system ratio on hydrolysis of the substrate by a glycerol ester hydrolase is illustrated by Table IA where enzymatic activity was monitored for a number of different trioctanoin concentrations at two different surfactant concentrations.
  • the surfactant used for the Table IA data was a zwitterionic salt sometimes abbreviated C 16 DAPS ("Zwittergent 3-16" available from Calbiochem). Oil Conc. (mM) Surfactant Conc.
  • the substrate used for the data of Table IB was triolein.
  • the concentrations (not shown) were varied to produce the system ratios indicated.
  • the sodium oleate surfactant used in the experiment summarized by Table IB is interesting because oleic acid is a product of reaction hydrolysis.
  • the desired amount of triglyceride was weighed into an appropriate size beaker, on a Mettler balance (model number AE163).
  • the corresponding amount of surfactant was added to the triglyceride, from a previously prepared aqueous surfactant stock solution, and the triglyceride and surfactant mixed manually.
  • the sample was then adjusted to the desired weight using doubly distilled H 2 O. Emulsification of the sample was carried out, prior to assaying enzyme activity, with a probe sonicator (Braun-Sonic model 2000), on ice, for approx. 2 minutes.
  • % total oil (triglyceride) hydrolyzed was then calculated by dividing the recorded value with the theoretically calculated value assuming three equivalents of oleic acid was produced for each triglyceride equivalent. All assays were run at ambient temperatures.
  • Critical Ratio Enzyme Activity Anionic 1 mM 0.1 - 0.5 400 Anionic, 0.2 mM 5 - 10 175 Anionic, 0.5 mM 1 - 5 200 Nonionic, 0.5 mM ⁇ 0.1 750 Nonionic, 0.5 mM 0.5 - 1 700 (Enzyme Lipolase, available from Novo Industri, isolatable from A. oryzae ) Surfactant Type & Conc.
  • Critical Ratio Anionic 0.5 mM 0.5 - 1 Anionic, 0.5 mM 20 - 30 Nonionic, 0.5 mM 20 - 30 Nonionic, 0.5 mM 10 - 20
  • Tables VI-IX show that hydrolysis is also dependent upon substrate type.
  • the data of Tables VI-IX was collected using triolein as the oil (rather than trioctanoin as in Tables I-V).
  • (Enzyme from P. putida ATCC 53552) Surfactant Type & Conc.
  • Critical Ratio Enzyme Activity Anionic, 1 mM 5 - 10 60 Anionic, 0.5 mM 5 - 10 50 Anionic, 1 mM 1 - 5 125 Nonionic, 0.5 mM 10 - 20 60 Nonionic, 0.5 mM 0.5 - 1.0 150 (Enzyme Amano P) Surfactant Type & Conc.
  • the average amount of oily soil on fabrics in household laundries is an estimated 300 mg oil/100 g of fabric (Andree et al., J. App. Biochem, 2 , pp. 218-229 (1980).
  • the situation is similar for Europe and Japan because, although the fabric load, wash solution and recommended detergent usage differ from the United States, the use ratios are typically less than about 0.6 for Japan and less than about 0.4 for Europe.
  • the system ratios for most common detergents are typically less than 1, more usually on the order of about 0.2-0.6.
  • the critical ratio for the common surfactants studied are generally greater than about 1. The performance at use ratios below the critical ratio has made attempts to include lipases in laundering solutions generally ineffective.
  • Typical detergent compositions for laundering include various additives, such as builder salts. It has been discovered that at use levels the additives commonly utilized in detergents have no substantial effect on the critical ratio (data not shown).
  • mixtures of surfactants can be used to manipulate the critical ratio also.
  • One conventional detergent composition is a mixture of Neodol 25-3S and C 12 LAS (with a molar ratio of 1:0.4).
  • This conventional detergent composition exemplifies the difficulties encountered in prior attempts to include lipases in laundering solutions.
  • a solution included either the conventional detergent or the conventional detergent plus ATCC 53552 enzyme.
  • Treatment Use Ratio Stain Removal , 14 min. washes (Neodol 25-3S and C 12 LAS, 1:0.4 molar ratio), 5 min. rinse 0.05 49.61 14 min. washes (Neodol 25-3S and C 12 LAS, 1:0.4 molar ratio), and ATCC 53552 enzyme, 5 min. rinse 0.05 51.07
  • the stain removal value of the detergent composition with enzyme was not statistically different from the stain removal value with the detergent composition without enzyme.
  • the enzyme was substantially not active. Calculation of the use ratio shows the use ratio was below the determined critical ratio of 10-20, and thus the enzyme was inactive.
  • %SR(E) soil removal scale
  • %SR(E) ⁇ E s - ⁇ E ow ⁇ E s x 100 where E s and E ow are distances in the CIE L*a*b* color space [see, Hunter, The Measurement of Appearance (New York: John Wiley & Sons, 1975) pp.
  • E s (L* 0 - L* s ) 2 + (a* 0 - a* s ) 2 + (b* 0 - b* s ) 2
  • E ow (L*0 - L*W) 2 + (a*0 - a*W) 2 + (b*0 - b*W) 2 in which the subscripts o,s and w refer to the original unstained and untreated test sample, the stained and the untreated test sample, and the stained and treated test sample, respectively.
  • the statistical test denoted as the "LSD” refers to the smallest difference between within-group means that would be declared statistically significant at the 95% confidence level using the two-sample test t-test with the variance estimated from all groups in the analysis of variance.
  • the present method may beneficially use more than one lipase or cutinase.
  • Hydrolases can be "turned on” in the presence of a surfactant by the addition of an oil to increase the ratio of oil to surfactant in a laundry solution so that the enzyme will hydrolyze oil stains.
  • This added oil (that will be in addition to the triglyceride found on stained fabrics being washed and, together with the oily stain, constitutes the oil used as numerator in the critical ratio calculation) does not need to be a substrate for the enzyme.
  • Use of the additional oil as a means for turning on the enzyme also allows one to remove lower levels of oily stains during laundering than would otherwise be possible.
  • the added oils are hexadecane and octadecane.
  • the addition of non-substrate oil is illustrated by the data of Table XI.
  • triolein when triolein is at a concentration of 0.3 mM and the system ratio is 1, there is no hydrolase activity. With 1.5 mM triolein, which produces a system ratio of 5, there is hydrolase activity.
  • the hydrolase When 1.2 mM hexadecane was added to the 0.3 mM triolein, then the hydrolase was found to be active in the presence of 0.3 hexadecane was added to the 0.3 mM triolein, then the hydrolase was found to be active in the presence of 0.3 mM Surfonic JL-80X surfactant even though the substrate concentration remained at 0.3 mM.
  • mixtures of substrate oils can be used to manipulate the critical ratio also.
  • Table XII demonstrates an example where the oil added is a substrate and is used to increase the system ratio above the critical ratio to activate the enzyme.
  • Trioctanoin (mM) Triolein (mM) Surfonic JL-80X(mM) System Ratio Enzyme Activity 1.87 0 0.5 3.74 0 1.87 0.63 0.5 5 51 0 0.25 0.5 0.5 0 2.25 0 0.5 4.5 0 2.25 0.25 0.5 5 56
  • Suitable organic compounds are those that are relatively insoluble as indicated above and preferably contain few to no polar groups because polar groups may interfere with enzyme activity. However, if the organic compound's polar groups are hindered or obscured by suitable branched or long chain alkyl groups, then some polarity can be tolerated. Charged substituents (e.g., -COO - Na + ) are not preferred.
  • glycol (diol) derivatives such as diethylene glycol monolaureate, ethylene glycol dimethyl ether
  • alcohols such as lauryl alcohol
  • aldehydes such as methyl butyl ketone, methyl nony
  • Table XIII illustrates use of a preferred, relatively insoluble, organic compound, N,N-diethyl-dodecanamide, to achieve the desired critical ratio when trioctanoin was the substrate.
  • Tables XI through XIII were collected using the enzyme from P. putida ATCC 53552; however, other enzymes can similarly be activated even in the presence of a surfactant for which the enzyme has a high critical ratio by including an oil that is not a substrate for the enzyme in the detergent composition. This is illustrated by the data of Table XIV, where the enzyme was Amano P. Trioctanoin (mM) Hexadecane (mM) Neodol 25-3S System Ratio Enzyme Activity 5 - 0.5 10 0 15 - 0.5 30 181 5 10 0.5 40 45
  • the molar ratio of oil hydrolysis activating agent (whether substrate or non-substrate) to surfactant in accordance with the invention preferably is greater than 0.5. This is calculable from the desired critical ratio of not greater than about 1 when one assumes an average of 0.34 mM oily stains on the fabrics being laundered and an average of 0.75 mM surfactant(s).
  • mixtures of surfactants can be used to manipulate the critical ratio also.
  • the hydrolase is tested in aqueous solution for hydrolysis activity in aqueous solution with a surfactant and a hydrolyzable substrate.
  • the ratio of surfactant and substrate is varied while hydrolysis activity is monitored.
  • Table IA illustrates the type of data that will typically be generated by varying the ratio.
  • a desired critical ratio is normally not greater than 1, one or more surfactants may need to be tested (and/or another hydrolase tested) until a critical ratio of less than or about 1 is found.
  • the enzyme tested in Table IA had a critical ratio between 0.5 - 1 when the surfactant was Neodol 25-3S and the substrate was trioctanoin.
  • the laundering composition may then be formulated by including the lipase or cutinase and the surfactant selected to have a critical ratio of less than or about 1.
  • Table XVI illustrates a mixture of surfactants effective in reducing the critical ratio. Again trioctanoin was used as the oil at (0.64) mM and the hydrolase was as in Table II.
  • Surfactant (mM) System Ratio % Total Hydrolysis Neodol 25-9 0.3 2 0 Neodol 25-9 0.13 5 0 Neodol 25-9 0.064 10 0 Neodol 25-9 0.032 20 18 Neodol 25-9 0.016 40 47 Neodol 25-9 0.008 80 60 Neodol 25-9/ Neodol 25-3S (1:1) 5.0 0.06 26 Neodol 25-9/ Neodol 25-3S (1:1) 2.0 0.3 35 Neodol 25-9/ Neodol 25-3S (1:1) 1.0 0.6 50 Neodol 25-9/ Neodol 25-3S (1:1) 0.5 1 53 Neodol 25-9/ Neodol 25-3S (1:1) 0.3 2 60 Neodol 25-9/ Neodol 25
  • Neodol 25-9 surfactant at 0.3 mM was used with 0.64 mM of the oil for a system ratio of 2, there was no hydrolysis.
  • Neodol 25-9 and Neodol 35-3S surfactants for the same molar system ratio there is 60 percent total hydrolysis. This is a surfactant system which is commercially usable.
  • Table XVII illustrates another example of mixing high and low critical ratio surfactants in order to reduce the critical ratio for the admixture.
  • a surfactant composition was prepared of Neodol 25-9 - Neodol 25-3S at a constant molar ratio of 1:1.
  • the substrate concentration (trioctanoin) was at about three times normal use levels (0.64 mM) and the percent total hydrolysis of the substrate after seven minutes was monitored as a function of changing the total surfactant concentration in the solution.
  • Neodol 25-9 1.28 mM 0.5 0 Neodol 25-9 Neodol 25-3S(1:1) 1.28 mM 0.5 35 Neodol 25-9 0.64 mM 1.0 0 Neodol 25-9 Neodol 25-3S(1:1) 0.64 mM 1.0 55 Neodol 25-9 0.128 mM 5.0 0 Neodol 25-9 Neodol 25-3S(1:1) 0.128 mM 5.0 60 Neodol 25-9 0.064 mM 10.0 0 Neodol 25-9 Neodol 25-3S(1:1) 0.064 mM 10.0 60 Neodol 25-9 0.032 mM 20.0 30 Neodol 25-9 Neodol 25-3S(1:1) 0.032 mM 20.0 60
  • the resulting surfactant mixture showed enzyme activity at a system ratio of 0.5.
  • the critical ratio was at least 0.5 or lower.
  • the Neodol 25-9 surfactant by itself had a system ratio of about 20 before enzyme activity was measured. Therefore, the inclusion of Neodol 25-3S at a 1:1 molar ratio reduced the critical ratio for Neodol 25-9 from about 20 to about 0.5.
  • Table XVIII illustrates another example of where a mixture of high and low critical ratio surfactants synergistically reduces the critical ratio for the admixture to a point below that for either component surfactant.
  • a surfactant composition mixture of C 12 LAS/Neodol 25-9 was prepared at a molar ratio of 2:1 and tested for comparison against each of the individual surfactants.
  • the surfactant mixture showed the Pseudomonas putida enzyme activity at a critical ratio between 0.05-0.1.
  • the oil was triolein.
  • the Neodol 25-9 surfactant by itself had a critical ratio of between 10-20 and the critical ratio for C 12 LAS surfactant by itself was 5-10 (at 0.5mM).
  • the combination of these two surfactants reduced the critical ratio for the combination to a value below the critical ratio of either surfactant by itself.
  • a preferred detergent composition useful in unit amounts to launder fabric having a triglyceride thereon, comprises a surfactant formulation providing from about 0.2 mM to about 1.5 mM surfactant concentration when a unit amount of the total composition is dissolved in a laundry solution.
  • Particularly preferred compositions include an enzyme isolatable from P. putida ATCC 53552 and in an amount sufficient to hydrolyze at least about 5 wt.% triglyceride on fabric when a unit amount of the total composition is dissolved in a laundry solution.
  • composition(a) a composition suitable for use in accordance with the invention
  • composition(a) was prepared by admixing the nonionic surfactant Neodol 23-6.5 and the nonionic surfactant Surfonic JL-80X in a 1:0.2 mole ratio. Additional additives and proportions were: Component wt.% Surfactants (Neodol 23-6.5/ 3.7 Surfonic JL-80X) 26.0 deionized water 0.6 sodium tripolyphosphate sodium carbonate 10.5 sodium polysilicate 1.5 alkaline proteases , 0.8/0.6 brightener 0.9 pigment 0.1 fragrance 0.2
  • the hydrolase included in this detergent composition was grown and isolated from P. putida ATCC 53552 as is described in Wiersema et al., European Patent Application 268,456, published May 25, 1988, but also set out below for the reader's convenience.
  • a seed medium was prepared with 0.6% nutrient broth (Difco) and 1% glucose (pH 6.5). 100 ml of this medium was sterilized in 500 ml fernbach flasks. The flasks were each seeded with a loopful from an overnight culture of P. putida ATCC 53552 grown on nutrient agar, and placed on a New Brunswick shaker at 250 rpm, 37°C for 12 hours.
  • the incubated 12-hour culture was then seeded at appropriate volumes (1-10% v/v) into a 1 liter fermenter (250 ml working volume), a 15 liter Biolafitte fermenter (12 liters working volume), or a 100 liter Biolafitte fermerter provided with a temperature controller, RPM, airflow and pressure controller.
  • the fermenter medium contained 0.6% nutrient broth (Difco), 0.3% apple cutin, and 0.2% yeast extract (Difco), with an initial pH of 6.5. The medium was adjusted to pH 6.8 and sterilized for 40 minutes before seeding. Bacterial growth and enzyme production were allowed to continue in the fermenter for 12-15 hours.
  • the crude fermentation culture was first filtered in a Amicon unit outfitted with two Romicon microporous membranes (0.22 ⁇ ) to remove cells. Remaining enzyme in the retentate which was bound to the cutin particles was removed by centrifugation. Total recovery approached 90%.
  • the recovered filtrate from the Amicon unit was concentrated to a volume of 3 liters on an Amicon ultrafiltration unit with two Romicon Pm 10 modules.
  • the concentrated material was then dialised with 20 liters of 0.01M phosphate buffer, pH 7.5, to remove salts and color. Recovery at this stage averaged about 80%.
  • Total activity for this crude preparation was 8.68 x 10 6 units.
  • a unit of lipase activity is defined as the amount of enzyme which results in an increase of absorbance at 415 nm of 1.0/minute when incubated at 20°C with mM p-nitrophenylbutyrate in 0.1 M pH 8.0 Tris-HCl buffer containing 0.1 wt.% Triton X-100.
  • the desired enzyme may be separated completely from another enzyme also with lipase activity by chromatography on hydrophobic resins.
  • the enzyme solution of Example III(C) after ultrafiltration and difiltration was adjusted to 0.5M NaCl and applied to a 0.8 x 7 cm octyl Sepharose column equilibrated in 10mM Tris(Cl), pH 8, 0.5M NaCl and washed to removed unbound protein. The following washes were then employed: 10mM Tris(Cl), pH 8, 7M urea; 10mM Na phosphate, pH 8; 10mM phosphate, pH 8, 0.5M NaCl. After washing, the column was then developed with a linear gradient to 50% n-propanol.
  • fractions were then assayed for activity on p-nitrophenyl butyrate (PNB) and p-nitrophenyl caprylate (PNC) in order to locate the lipase activities.
  • PNB p-nitrophenyl butyrate
  • PNC p-nitrophenyl caprylate
  • composition (a) 2ppm hydrolase was admixed with the detergent composition previously described as composition (a).
  • 20ppm hydrolase was admixed with this composition. Both studies included staining fabrics with synthetic sebum soil.
  • the synthetic sebum soil was prepared as follows. Ten oils having the following proportions were admixed: Oils %w/w Stearic acid 5 Squalene 5 Cholesterol 5 Linoleic acid 5 Oleic acid 10 Paraffin oil 10 Palmitic acid 10 Coconut oil 15 Sperm wax 15 Olive oil 20
  • Cotton swatches were stained with the synthetic sebum soil and then washed in test beakers by agitating for 14 minutes followed by a 5 minute rinse. The laundering solution was 0.205g of composition(a) dissolved in 250ml water. A control composition without the hydrolase was also prepared and used to treat stained cotton swatches by the same protocol. Table XIX shows the stain removal for the inventive composition (a) and for the control composition.
  • Polyester swatches were stained with sebum, vegetable oil or olive oil. These swatches were then washed for 12 minutes at 96 F in a 72 liter washing machine, rinsed in the normal rinse cycle and then allowed to air dry. One set of swatches was treated in laundering solution having 59 g inventive composition(a) dissolved therein while another set of swatches was treated with a control composition identical to inventive composition(a) but without the hydrolase.
  • the stain removal data expressed as %SR(E) is shown by Table XX. Composition Sebum Vegetable Oil Olive Oil comp.(a) 89.69 51.82 60.79 Control 83.75 29.20 35.05
  • the polycotton fabric was cut into 2" x 2" swatches, each weighing about 0.39 g.
  • the desired amount of triolein was dissolved in 2-methyl pentane, and pipetted onto each swatch (200 ⁇ L/swatch).
  • the triolein stain was allowed to wick out for 72 hrs. at room temperature.
  • the reflection of the stain was then evaluated using a Hunter Spectracolorimeter, and a prewash value (proportional to the concentration of the absorbing species) was determined.
  • the soiled swatches were divided into groups of 4 and loaded into 250 ml bottles, each with 200 ml of the desired treatment. The bottles were then shaken for 12 minutes at room temperature, and rinsed twice with 200 ml of dd H 2 O. Finally they were air dried and the postwash value (proportional to the concentration of the absorbing species) determined.
  • the method of the invention is useful in laundering solutions and comprises use of an enzyme capable of hydrolyzing natural oil stains on fabric when in a laundry solution and hydrolysis activating means for changing the ratio of oil to surfactant or for changing the critical ratio of the surfactants.
  • hydrolysis activating agents have been exemplified for use in laundry solutions so that the enzyme will be active in hydrolyzing the oil stains.
  • the enzyme is normally inhibited from hydrolyzing natural oily soils or stains when the laundering solution contains between about 0.1 mM to 5 mM of surfactant.
  • Another way of stating the effect of the hydrolysis activating agent of the invention is that when a lipase or cutinase is admixed with a surfactant formulation in accordance with this invention, then the lipase or cutinase is capable of hydrolyzing at least about 30 mg triolein when a unit amount of laundering composition is dissolved in aqueous solution at 25°C at pH 10 with an average rate of about 0.0072 mmoles/min fatty acid being produced over about 14 minutes.
  • surfactant systems may be formulated that include lipases and/or cutinases for use in laundering solutions without requiring extended soaking or high temperatures.

Claims (4)

  1. Verfahren zum Reinigen von Stoff mit Triglyceridflecken darauf in einer Lösung, die eine Lipase oder eine Cutinase und wenigstens ein grenzflächenaktives Mittel enthält, wobei zu der Lösung ein Mittel zugefügt wird, das kein Substrat für das Enzym ist und das ausgewählt ist aus einem Kohlenwasserstoff, bei dem es sich um Hexadecan oder Octadecan handelt, und einer relativ unlöslichen organischen Verbindung, die eine Löslichkeit δ zwischen 7 und 9,5 besitzt und die ausgewählt ist aus Glykolderivaten, Alkoholen, Aldehyden, Ketonen und Amiden und Gemischen davon, um die enzymatische Hydrolyse zu aktivieren.
  2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß das oberflächenaktive Mittel und das Aktivierungsmittel der Lösung so zugesetzt werden, daß das Molverhältnis von Aktivierungsmittel und oberflächenaktivem Mittel oberhalb des kritischen Verhältnisses des Enzyms für das genannte oberflächenaktive Mittel liegt, und wobei das molare Verhältnis von Aktivierungsmittel zu oberflächenaktivem Mittel größer als 0,5 ist.
  3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß das Enzym aus einem Organismus isoliert werden kann, der ein Gen exprimiert, das aus einem Pseudomonas, einem Chromobacter, einem Aspergillus, einem Acinetobacter oder einem Fusarium erhältlich ist und das vorzugsweise aus Pseudomonas putida ATCC 53552, Mutanten davon oder Clonen davon isolierbar ist.
  4. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß das oberflächenaktive Mittel aus Natriumdodecylsulfat, C12H25--SO3 -Na+ ausgewählt ist.
EP90304796A 1989-05-15 1990-05-02 Wäschewaschverfahren Expired - Lifetime EP0399681B1 (de)

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US5512203A (en) * 1987-05-29 1996-04-30 Genencor International, Inc. Cutinase cleaning compositions
AU2014892A (en) * 1991-04-30 1992-12-21 Procter & Gamble Company, The Liquid detergents with an aryl boronic acid
GB9216387D0 (en) * 1992-07-31 1992-09-16 Unilever Plc Enzymatic detergent compositions
US5442100A (en) * 1992-08-14 1995-08-15 The Procter & Gamble Company β-aminoalkyl and β-N-peptidylaminoalkyl boronic acids
WO1994007989A1 (en) * 1992-09-25 1994-04-14 Unilever N.V. Cleaning process
BE1009312A3 (fr) * 1995-05-05 1997-02-04 Solvay Compositions detergentes.
EP1127423B1 (de) 1998-11-02 2005-11-23 Broadcom Corporation Messung der nichtlinearen verzerrung in sendern
US20070111914A1 (en) * 2005-11-16 2007-05-17 Conopco, Inc., D/B/A Unilever, A Corporation Of New York Environmentally friendly laundry method and kit
EP3556834B1 (de) 2016-05-09 2020-10-14 The Procter & Gamble Company Reinigungsmittelzusammensetzung umfassend eine fettsäure decarboxylase
ES2721224T3 (es) * 2016-05-09 2019-07-29 Procter & Gamble Composición detergente que comprende una enzima transformadora de ácido oleico
PL3540036T3 (pl) 2016-05-09 2021-04-19 The Procter & Gamble Company Kompozycja detergentowa zawierająca lipoksygenazę kwasu tłuszczowego
BR112021009789A2 (pt) * 2018-11-20 2021-08-17 Unilever Ip Holdings B.V. composição detergente, método de tratamento de um substrato de tecido e uso de uma enzima esterase
US20210199658A1 (en) * 2019-12-30 2021-07-01 The United States Of America, As Represented By The Secretary Of Agriculture Detection of lipase activity in honey bees

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US3974082A (en) * 1972-08-21 1976-08-10 Colgate-Palmolive Company Bleaching compositions
EP0268456A2 (de) * 1986-11-19 1988-05-25 The Clorox Company Enzymatisches Persäure-Bleichmittelsystem
WO1988009367A1 (en) * 1987-05-29 1988-12-01 Genencor, Inc. Cutinase cleaning composition
EP0375102A2 (de) * 1988-12-19 1990-06-27 The Clorox Company Modifiziertes Enzym enthaltendes, enzymatisches Persäure-Bleichmittelsystem

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US3950277A (en) * 1973-07-25 1976-04-13 The Procter & Gamble Company Laundry pre-soak compositions
GB8514707D0 (en) * 1985-06-11 1985-07-10 Unilever Plc Enzymatic detergent composition
GB8811045D0 (en) * 1988-05-10 1988-06-15 Unilever Plc Enzymatic detergent composition

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Publication number Priority date Publication date Assignee Title
US3974082A (en) * 1972-08-21 1976-08-10 Colgate-Palmolive Company Bleaching compositions
EP0268456A2 (de) * 1986-11-19 1988-05-25 The Clorox Company Enzymatisches Persäure-Bleichmittelsystem
WO1988009367A1 (en) * 1987-05-29 1988-12-01 Genencor, Inc. Cutinase cleaning composition
EP0375102A2 (de) * 1988-12-19 1990-06-27 The Clorox Company Modifiziertes Enzym enthaltendes, enzymatisches Persäure-Bleichmittelsystem

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EP0399681A2 (de) 1990-11-28
CA2016610A1 (en) 1990-11-12
DE69033423D1 (de) 2000-02-24
AU5488090A (en) 1990-11-15
JPH0388897A (ja) 1991-04-15
ATE188990T1 (de) 2000-02-15
JP3252962B2 (ja) 2002-02-04
DE69033423T2 (de) 2000-05-25
ES2141080T3 (es) 2000-03-16
EP0399681A3 (de) 1991-01-16

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