EP0399681B1

EP0399681B1 - Method of laundering fabrics

Info

Publication number: EP0399681B1
Application number: EP90304796A
Authority: EP
Inventors: Maha Y. El-Sayed; Sheldon N. Lewis; Susan A. Anderson; Richard J. Wiersema
Original assignee: Clorox Co
Current assignee: Clorox Co
Priority date: 1989-05-15
Filing date: 1990-05-02
Publication date: 2000-01-19
Anticipated expiration: 2010-05-02
Also published as: AU5488090A; JP3252962B2; EP0399681A3; EP0399681A2; CA2016610A1; JPH0388897A; DE69033423T2; DE69033423D1; ATE188990T1; ES2141080T3

Abstract

The activation of glycerol ester hydrolases, toward triglyceride oils, is correlated to the molar ratio of total triglyceride to surfactant concentrations in the test solution. This ratio dependency, when considered in laundry compositions, allows for predictable and improved utilization of these enzymes in the hydrolysis of triglyceride stains. A particularly preferred enzyme for this use is isolatable from Pseudomonas putida ATCC 53552. This enzyme, or hydrolase, has sufficient hydrolysis activity in a laundering solution to hydrolyze at least about 5 wt. % of total triglyceride stains in a laundering solution within about 15 minutes at about 25 DEG C.

Description

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. For example, 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 C₄-C₁₆ 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.
U.S. Patent 3,950,277, inventors Stewart et al., issued April 13, 1976 describes pre-soak compositions employing a lipase enzyme and a lipase activator selected from the group consisting of naphthalene sulfonates, certain polyoxyalkylene derivatives of ethylene diamine and certain acylamino acid salts.
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.
Attempts have recently been made to find particular lipases that are less affected by detergents in wash solutions. European Patent Application 258,068, published March 2, 1988 reports a lipase from the genus Thermomyces said to be compatible with anionic surfactants and effective as a detergent additive. Japanese Patent Application 63039579, published February 20, 1988 states that a novel lipase, obtained from a Pseudomonas, is only slightly inhibited by anionic surfactant and is activated by nonionic surfactant.
In summary, there have been no clear teachings on the compatibility or incompatibility of lipases in laundry and cleaning formulations, although it has been generally recognized that specific surfactants (when present in useful amounts in detergent formulations) will inhibit lipase activity for some lipases. As a consequence, laundering solutions including lipases have tended to be those that require extended soaking.
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 δ 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.
Having indicated the scope of the present invention, it will now be described more generally.
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.
It is another feature of the present method that 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.
Thus, 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 (in combination with hydrolysis activating agent for changing either the ratio of oil to surfactant or the critical ratio of surfactant) 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. Mono-, di- and triglycerides are present in sebum soils and cooking oils and potentially can be hydrolyzed by lipases.
Analysis of the amino acid sequence for a recently discovered enzyme described as having lipase activity and isolatable from P. putida ATCC 53552 suggests there are substantial homologies between the nucleotide sequence for this enzyme and the nucleotide sequence of the cutinase gene recently determined for C. capsici. (Compare European Patent Application 268,456, inventors Wiersema et al., published May 25, 1988 with Ettinger et al., Biochemistry, 26, pp. 7883-7892 (1987)). Because of the relationship between cutinases and 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. For example, among the enzymes with this invention has been listed are those expressed by genes present in (or obtainable from) P. putida ATCC 53552, from P. sp. (as Amano 68S), from P. fluorescens (as Amano P), and from Aspergillus oryzae (as Lipolase). Toyo Jozo Co. of Japan, U.S. Biochemical Co. of the U.S.A. and Diosynth Co. of The Netherlands sell lipases from Chromobacter viscosum. European Patent Application No. 0,214,761, published March 3, 1987, applicant Novo Industri, describes a lipase from Fusarium oxysporum. Yet other strains are known or described as producing lipases. For example, PCT/WL86/00023, published February 12, 1987, applicant Gist-Brocades N.V., describes strains including certain Acinetobacter. It should be understood that the genes expressing such enzymes can be cloned into another organism such as E. coli, for higher levels of expression.

CRITICAL RATIO

Using a critical ratio as described herein, we have discovered that when in a surfactant laundering solution, the hydrolysis of oils, having ester bonds, by specific glycerol ester hydrolases in the presence of surfactant is dependent upon the ratio of oil to surfactant in the solution of interest. The ratio of oil to surfactant in the solution of interest will sometimes hereinafter be called the "system ratio". For convenience, both this system ratio and the value of the ratio required for hydrolysis to begin (called the "critical ratio") will be calculated and expressed on the basis of the molar relationship of oil to surfactant in the bulk aqueous solution. The system ratio defines the relationship of substrate (oil) with respect to specific surfactant on an absolute mole/mole basis. At the critical ratio and above the molar relationship of substrate with respect to a specific surfactant is such that the enzyme is activated, or "turned on", such that hydrolysis begins. That is, concentration of either component in an aqueous medium, such as wash water solution, is not critical because the molar relationship between the two components remains constant in spite of dilution in the aqueous medium.
Without being restricted to a single theory, it may be that 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.
In the examples which follow, different lipases and cutinases are shown to have related, but different, critical ratios, which applicants have determined empirically through their above-described model. One key aspect of the critical ratio model is a focus upon the amount of substrate present relative to the amount of surfactant, rather than on the amount of enzyme or surfactant. By contrast, the prior art has assumed that either using large amounts of enzymes, or surfactants, or repeatedly testing with either or both, will result in enhanced cleaning performance. But following teachings or assumptions of the such prior art leads one to using wastefully large amounts of enzyme, surfactant, or both.

The molecular weights of various surfactants (and typical structures) and oils discussed hereinafter are set out below.

Compound	Nominal Molecular Weight
SDS (sodium dodecylsulfate) surfactant	288
C₁₂LAS surfactant	362
Neodol 25-9 surfactant	596
Neodol 25-3S surfactant	444
Surfonic JL80X surfactant	625
Triton X-100 surfactant	624
C₁₆DAPS surfactant	392
Trioctanoin substrate	470
Triolein substrate	884

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.

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 C₁₆DAPS ("Zwittergent 3-16" available from Calbiochem).

Oil Conc. (mM)	Surfactant Conc. (mM)	System Ratio	Enzyme Activity
0.5	0.5	1	0
10	10	1	0
2.5	0.5	5	0
50	10	5	0
5	0.5	10	0
10	0.5	20	334
200	10	20	398
15	0.5	30	405
300	10	30	370
20	0.5	40	370
400	10	40	417

As may be seen from the data of Table IA, there is either no enzyme activity (that is, the enzyme is "turned off") or observable hydrolyase activity, depending upon the system ratio, and independent of the surfactant concentration. This data shows that neither the absolute concentration of the triglyceride nor the absolute concentration of the surfactant determines whether or not the enzyme is active. Instead, it is the ratio of the oil to surfactant that best describes the kinetic profile of enzyme activity. For the enzyme tested in Table IA, the value of the critical ratio (i.e. system ratio at which enzymatic activity begins) with respect to this particular zwitterionic surfactant is between 10 and 20. At and below the system ratio value of 10, the enzyme is not active. At and above the system ratio value of 20, the enzyme is active.
Another example of this phenomenon, using another surfactant with which the P. putida ATCC 53552 enzyme displays a different critical ratio, sodium oleate, is presented in Table IB.

Na Oleate (mM) System Ratio Enzyme Activity

0.3 1 0

0.3 5 0

0.3 10 0

0.3 20 60

0.3 30 90
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 experiments determining enzyme activity, such as those set out in Tables IA and IB, were carried out as follows:

(i) Sample Preparations:

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 H₂O. 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.

(ii) Enzyme Activity Measurement:

This was achieved by monitoring the rate of acid liberation, from the enzymatic hydrolysis of the triglycerides in the emulsion. The assay was initiated by adding approx. 2ppm lipase to 10 ml of the prepared emulsion. The acid liberated was monitored by autotitration, on a Radiometer pH-stat (model number ABU80) to an endpoint of a pH of 10. Initial rates were recorded for the first 5 minutes of the reaction, and the reaction rates reported as µmole H⁺ titrated . min ^-1mgE^-1. Occasionally enzyme activity is reported as % total oil hydrolyzed in 14 min. In these examples, the reaction was allowed to run for 14 min. and the amount of acid titrated recorded. The % 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.

The dependency of the onset of hydrolysis upon a critical ratio of oil to surfactant in aqueous solution is not specific to the particular glycerol ester hydrolase used for the data of Table IA and Table IB; rather, the principle has been discovered to be general for other glycerol ester hydrolases. This is shown by Tables II-V, which show the critical ratio for a variety of different nonionic and anionic surfactants and several different enzymes (where the substrate was trioctanoin). The various enzymes examined as shown by Tables II-V were also examined at higher surfactant concentrations and the dependency upon the system ratios was confirmed.

(Enzyme isolatable from P. putida ATCC 53552)
Surfactant Type & Conc.	Critical Ratio	Enzyme Activity
Anionic, 2 mM	0.5 - 5.0	325
Anionic, 1 mM	5 - 10	250
Anionic, 2 mM	0.5 - 1	300
Nonionic, 0.5 mM	10 - 20	450
Nonionic, 0.5 mM	5 - 10	500
Nonionic, 2 mM	10 - 15	450

(Enzyme Amano P., available from Amano Co., isolatable from Pseudomonas fluroescens)
Surfactant Type & Conc.	Critical Ratio	Enzyme Activity
Anionic, 2 mM	1 - 5	100
Anionic, 1 mM	5 - 20	70
Anionic, 2 mM	10 - 20	200
Nonionic, 0.5 mM	1 - 5	400
Nonionic, 0.5 mM	0.5 - 1	550

(Enzyme Amano 68S, available from Amano Co., isolatable from P. sp.)
Surfactant Type & Conc.	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

As can be seen by Tables I-V, the critical ratios for particular enzymes are dependent upon surfactant identity.

The following 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.	Critical Ratio	Enzyme Activity
Anionic, 1 mM	5 - 10	160
Anionic, 0.5 mM	10 - 20	13
Anionic, 0.5 mM	5 - 10	20
Nonionic, 0.5 mM	5 - 10	20
Nonionic, 0.5 mM	5 - 10	40

(Enzyme Amano 68S)
Surfactant Type & Conc.	Critical Ratio	Enzyme Activity
Anionic, 1 mM	1 - 5	200
Anionic, 0.5 mM	10 - 20	30
Anionic, 0.5 mM	5 - 10	25
Nonionic, 0.5 mM	1 - 5	30
Nonionic, 2.5 mM	1 - 5	40

(Enzyme Lipolase)
Surfactant Type & Conc.	Critical Ratio
Anionic, 0.5 mM	30-40

The above data may be summarized by the "Table Summary" below where "++" means a critical ratio of 0.01 - 0.1, "+" means a critical ratio of 0.1-1.0, "0" means a critical ratio of 1.0-10, and "-" means a critical ratio of 10-100.

USE RATIO

The four enzymes tested (Tables I-IX) all demonstrated a critical ratio for each of the surfactants tested. These surfactants constitute some of the most commonly used surfactants in commercially available detergent compositions. Such detergent compositions are typically recommended for United States laundering use in amounts that, when dissolved in laundry solution, provide a surfactant concentration between about 0.2 mM and about 1.5 mM (assuming a 2-3 kg average load in a 72 liter wash solution).
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). This indicates that, based on the ratio dependency demonstrated above, inclusion of lipases in most commercially available detergents would provide little or no washing benefit because the use ratio (of actual oil concentration to actual moles of surfactant in the laundering solution) is below the critical ratio at which enzyme activity is initiated. 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.
That is, based on the recommended detergent use and considering a wide variety of detergent compositions and surfactant molecular weights, the system ratios for most common detergents are typically less than 1, more usually on the order of about 0.2-0.6. (In calculating the use ratios, the bulk concentrations in solution have been assumed and any possible interfacial effects ignored.) But as can be seen from the data of Tables II-IX, the critical ratio for the common surfactants studied (with trioctanoin as oil) 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).
Although not in accordance with the present method, mixtures of surfactants can be used to manipulate the critical ratio also.
Use of combinations of surfactants having different critical ratios can generate critical ratios that are different from the individual surfactants in the combination. By practicing the invention, one can admix surfactants to achieve a critical ratio of the combined surfactants that is at or below the lower of the individual critical ratios. This will be further explained hereinafter.

One conventional detergent composition is a mixture of Neodol 25-3S and C₁₂LAS (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. By examination of the appropriate data for the component surfactants of the detergent composition in Table VI, one could conclude that the critical ratios are much higher than the use ratio. This conclusion proves true when a swatch study and a washing machine study were conducted, as illustrated by the data of Table X, where 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 C₁₂LAS, 1:0.4 molar ratio), 5 min. rinse	0.05	49.61
14 min. washes (Neodol 25-3S and C₁₂LAS, 1:0.4 molar ratio), and ATCC 53552 enzyme, 5 min. rinse	0.05	51.07

As illustrated by the data of Table X, 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. Thus, 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.
As earlier noted, soil removal was measured on a stain removal scale designated "%SR(E)". This is a scale expressing the ratio of the change of appearance of a soiled, treated test sample to its maximum possible change of appearance.
Values of %SR(E) are calculated as follows: %SR(E) = ΔEs - ΔEow ΔEs 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. 302-303.] and are given by Es = (L*0 - L*s)2 + (a*0 - a*s)2 + (b*0 - b*s)2 Eow = (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.
As a brief summary then, enzymes capable of hydrolyzing natural oil stains on fabric when in a laundry solution have been shown to have a dependency for the onset of hydrolysis upon a critical value of the molar use ratio of oil to surfactant in the laundry solution. This critical ratio is dependent upon the type of surfactant in the laundering solution (and also upon the type of oil in the laundering solution). But because the use ratios for most common detergents are typically less than 1 and the critical ratio for the common surfactants studied are generally greater than about 1, lipases generally are inactive.
However, we have discovered ways of "turning on" hydrolysis by the enzyme through hydrolysis activating agents for changing the ratio of oil to surfactant or for changing the critical ratio of the surfactants. Ways to modify the critical ratio will now be more fully described. In addition, the present method may beneficially use more than one lipase or cutinase.

INCREASING THE SYSTEM RATIO WITH ADDITION OF OILS OR OTHER ORGANIC COMPOUNDS

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 (that are not substrates) are hexadecane and octadecane. The addition of non-substrate oil is illustrated by the data of Table XI.

Triolein(mM) Hexadecane(mM) Surfonic JL-80X(mM) System Ratio Enzyme Activity

0.3 - 0.3 1 0

1.5 - 0.3 5 15

0.3 1.2 0.3 5 8
As may be seen from the data of Table XI, 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. 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.
Although not in accordance with the present method, 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
As seen by the data of Table XII, 0.25 mM triolein, emulsified in 0.5 mM Surfonic JL-80X, is not hydrolyzed by this enzyme. Similarly, 2.25 mM trioctanoin emulsified in 0.5 mM surfonic JL-80X is also not hydrolyzed. However, when both these oils (0.25 mM triolein and 2.25 mM trioctanoin) are emulsified together in 0.5 mM surfonic JL-80X, then 56% of the total oil is hydrolyzed.
Other organic compounds which do not participate in the hydrolysis reaction (in addition to the earlier discussion of added oils such as hexadecane) can be used to reach the critical ratio. 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. The relatively insoluble organic compounds (which do not act as substrates for the enzyme) can be chosen, without limitation, from glycol (diol) derivatives (such as diethylene glycol monolaureate, ethylene glycol dimethyl ether), alcohols (such as lauryl alcohol), aldehydes, ketones (such as methyl butyl ketone, methyl nonyl ketone), and amides (e.g., N,N-diethyldodecanamide). These compounds have a solubility, δ, of between about 7-9.5, in accordance with the formula δ = ΣG dM where ΣG is the sum for all the atoms and groupings in the molecule, d is the density, and M, the molecular weight. Preferred are compounds with a solubility, δ, of between about 8.0-9.0 such as are illustrated and described by J. Brandrup and E.H. Immergut, Eds., Polymer Handbook, 2d Ed., John Wiley & Sons, 1975), pp. IV-337 to IV-353, It may be that these relatively insoluble organic compounds, preferably with few to no polar groups, are sufficiently chemically analogous to the oils (substrate or not) as to increase the total "effective" oil concentration. Thus, such relatively insoluble organic compounds represent another embodiment of the means for changing the ratio of oil to surfactant.
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.

Trioctanoin (mm) N,N-diethyldodecanamide (mm) Neodol 25-9 (mm) Enzyme Activity

2.5 -- 0.5 < 5

10.0 -- 0.5 35

-- 10.0 0.5 < 5

2.5 2.5 0.5 5

2.5 5.0 0.5 12

2.5 7.5 0.5 19
The data of 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).

REDUCING THE CRITICAL RATIO WITH ADDITION OF SURFACTANTS

Although not in accordance with the present method, mixtures of surfactants can be used to manipulate the critical ratio also.
In order to determine the critical ratio for a particular lipase or cutinase with different surfactants, 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. For example, Table IA illustrates the type of data that will typically be generated by varying the ratio.
Because 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. For example, 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.

It has been discovered that mixing a surfactant with a high critical ratio for a particular enzyme with one that has a low critical ratio for that enzyme can result in a lowered critical ratio of the enzyme for the admixed surfactant. This exemplifies a means for changing the critical ratio of the surfactants so that the enzyme used in accordance with the present invention will be "turned on" and hydrolysis will occur. This is illustrated by Table XV where trioctanoin was used as the oil, or substrate, and the hydrolase was as in Table II (2µg/ml). Enzyme activity was measured by initial rates and the reaction was carried out at ambient temperature to an end point pH of 10.00.

Mole ratio (SDS: Neodol 25-9)	Critical Ratio (0.5 mM total surfactant)
0:1	10 - 20
0.025:0.975	10 - 20
0.05:0.95	5 - 10
0.1:0.9	1 - 5
0.2:0.8	1 - 3
0.5:0.5	1 - 3
0.75:0.25	1 - 3
1:0	1 - 3

Although the combination shown reduced the critical ratio to 1, this is nevertheless not low enough for the enzyme to be active. That is, as may be seen by the data of Table XV, a 0.1-0.2 mole fraction of SDS, when admixed with a high critical ratio surfactant, was effective to reduce the critical ratio of the surfactant admixture to that of SDS alone, but this is not low enough for commercial detergent formulations.

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 25-3S (1:1)	0.1	6	60
Neodol 25-9/ Neodol 25-3S (1:1)	0.03	21	60

As can be seen from the data of Table XVI, when 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. However, with a mixture in a 1:1 ratio of 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.

Surfactant Composition & Molar Ratio	Total Surfactant Conc.	System Ratio	Enzyme Activity
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

As can be seen from the data of Table XVII, the resulting surfactant mixture showed enzyme activity at a system ratio of 0.5. Thus, the critical ratio was at least 0.5 or lower. By contrast, 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 C₁₂LAS/Neodol 25-9 was prepared at a molar ratio of 2:1 and tested for comparison against each of the individual surfactants.

Surfactant Composition And Concentration Critical Ratio

C₁₂LAS (0.5 mM) 5 - 10

C₁₂LAS (5.0 mM) 1 - 5

Neodol 25-9 (0.5 mM) 10 - 20

C₁₂LAS/Neodol 25-9 (2mM/1mM) 0.05 - 0.1
In the tests illustrated by the data of Table XVIII, the surfactant mixture showed the Pseudomonas putida enzyme activity at a critical ratio between 0.05-0.1. The oil was triolein. By contrast, the Neodol 25-9 surfactant by itself (at 0.5 mM) had a critical ratio of between 10-20 and the critical ratio for C₁₂LAS surfactant by itself was 5-10 (at 0.5mM). Thus, 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.
For present purposes, 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.

For example, a composition suitable for use in accordance with the invention (designated "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) Seeding and Fermenting

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.

(B) Enzyme Recovery by Microfiltration

The crude fermentation culture was first filtered in a Amicon unit outfitted with two Romicon microporous membranes (0.22µ) to remove cells. Remaining enzyme in the retentate which was bound to the cutin particles was removed by centrifugation. Total recovery approached 90%.

(C) Concentration and Dialysis of Whole Cell Filtrate

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 10⁶ 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.

(D) Complete Isolation of the Hydrolase

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. The column fractions were then assayed for activity on p-nitrophenyl butyrate (PNB) and p-nitrophenyl caprylate (PNC) in order to locate the lipase activities. Two enzymes were clearly resolved, fraction 32 with a PNB/PNC ratio of 4.6 (which is the desired enzyme) and fraction 51 with a PNB/PNC ratio of 1.40.

IMPROVED OILY STAIN REMOVAL

Both swatch studies and washing machine studies were conducted with the method of the invention as will now be described.
In the swatch study (1), 2ppm hydrolase was admixed with the detergent composition previously described as composition (a). In a washing machine study (1), 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
To 15g of the above melted oils was added 0.6g oleic acid, 1.2g triethanolamine and 0.225g charcoal. Then 60 ml water at 130°F was admixed, and the mixture blended for 1 minute.

Swatch Study (1)

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.

Composition System Ratio %SR(E)

comp.(a) 0.08 60.72

Control 0.08 57.37
As may be seen from the swatch study data of Table XIX, statistical enhancement of soil removal was seen for the composition.

Washing Machine Study (1)

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
As can be seen from the data of Table XX, statistically significant stain removal was achieved for all stains tested on polyester fabric.

Swatch and Washing Machine Study (2)

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 µL/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 H₂O. Finally they were air dried and the postwash value (proportional to the concentration of the absorbing species) determined.
Comparative Treatment A: The swatches were washed in a surfactant composition of 0.3 mM C₁₂LAS/Neodol 25-9 in a 2:1 molar ratio. No lipase was added.
Treatment B: Same as A:, except for the addition of a 5 ppm lipase ATCC 53552 to the surfactant composition.
Comparative Treatement C: The swatches were washed in an alternate formulae containing the surfactant composition of 0.3 mM C₁₂LAS/Neodol 25-9, in a molar ratio of about 1:4. No lipase was added.
Treatment D: Same as treatment C, except for the addition of 5 ppm lipase ATCC 53552 to the surfactant composition.

The amount of oily stain removed in each treatment is summarized by Table XXI.

Oil Loaded	System Ratio	Treatment outside the scope of the invention	% Triolein Removal	LSD
%	mM
1	0.072	0.24	A (comparative)	44	3.8
1	0.072	0.24	B (inventive)	55
3	0.22	0.73	A (comparative)	29	3.7
3	0.22	0.73	B (inventive)	34
1	0.072	0.25	C (comparative)	39	4.5
1	0.072	0.25	D (inventive)	60
3	0.22	0.76	C (comparative)	27	3.6
3	0.22	0.76	D (inventive)	33

As may be seen by the data of Table XXI, use of a composition comprising a surfactant mixture and lipase removed from 33% to 60% of the oil on the polycotton fabric, and this removal was distinctly better for the composition (a) (including the hydrolase) than without hydrolase. The LSD values show this removal was statistically significant.
In sum, 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. Several embodiments of hydrolysis activating agents have been exemplified for use in laundry solutions so that the enzyme will be active in hydrolyzing the oil stains. One can usually observe the onset of hydrolysis when the enzyme has an activity sufficient to hydrolyze at least about 5 wt.% of total triglyceride stains within about 14 or 15 minutes at about 25°C. Without the hydrolysis activating means used according to the invention, 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. Thus, surfactant systems may be formulated that include lipases and/or cutinases for use in laundering solutions without requiring extended soaking or high temperatures.

Claims

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 δ 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.
A method as claimed in claim 1 wherein surfactant and activator agent are added to the solution so that the molar ratio of activator agent and surfactant is above the critical ratio of the enzyme for the said surfactant, the molar ratio of activator agent to surfactant being greater than 0.5.
A method as claimed in claim 1 or claim 2 wherein the enzyme is isolatable from an organism expressing a gene obtainable from a Pseudomonas, a Chromobacter, an Aspergillus, an Acinetobacter, or a Fusarium, and is preferably isolatable from Pseudomonas putida ATCC 53552, mutants thereof or clones thereof.
A method as claimed in any of claims 1 to 3 wherein the surfactant is selected from sodium dodecylsulfate, C₁₂H₂₅--SO₃ ^-Na⁺