DE69033423T2 - Laundry process - Google Patents

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

The invention relates to a method for cleaning fabrics, and in particular the invention relates to a method for cleaning 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 that undergo oxidation in higher animal tissues must be in free form (i.e., non-esterified) before they can be activated and oxidized. Thus, the function of intracellular lipases is to hydrolyze triacylglycerols, producing free fatty acids and glycerol.

Bacterial lipases are classically classified as glycerol ester hydrolases (EC 3.1.1.3) because they are polypeptides capable of cleaving ester bonds. They have a high affinity for interfaces, a characteristic property that distinguishes them from other enzymes such as proteases and esterases. One interface to which lipases readily adsorb is the oil-water interface.

Cutinases are esterases that catalyze the hydrolysis of cutin. For example, cutinases allow fungi to penetrate the cutin layer into the host plant during the early stages of a fungal infection. The primary structures of several cutinases have been compared and they have been shown to be highly conserved. Ettinger, Biochemistry. 26, pp. 7883-7892 (1987), Sebastian et al., Arch. Biochem. Biophys., 263 (1), pp. 77-85 (1988) have recently found that the formation of cutinase is induced by cutin in a fluorescent P. putida strain. This cutinase catalyzes the hydrolysis of p-nitrophenyl esters of C4-C16 fatty acids.

Lipases have long been considered as possible ingredients in detergent compositions. An early lipase preparation in the form of pancreatin was described as an additive to detergent formulations by Rohm, Chem. Abs., Int., P2048 (1916). More recently, lipases obtained from certain Pseudomonas or Chromobacter microorganisms have been described 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.

It has long been known that lipases are generally inhibited by anionic detergents and by nonionic detergents. For example, Wills, Bioch., 560, pp. 529-534 (1955), and Andree et al., J. App. Biochem., 2, pp. 218-229 (1980), reported that lipase activity is increased by emulsifiers. Not contradicting these teachings, Attempts to use lipases in cleaning solutions containing anionic or nonionic surfactants have been largely unsuccessful, and the effective use of lipases to clean oily stains has been limited to soaking applications:

U.S. Patent 3,950,277, inventors Stewart et al., issued April 13, 1976, describes soak compositions employing a lipase enzyme and a lipase activator selected from the group consisting of naphthalene sulfonates, certain polyoxyalkylene derivatives of ethylenediamine, and certain acyl amino acid salts.

Lipases in aqueous solution without added surfactants have been found to be useful for pre-wash or soak applications for extended periods of time followed by conventional washing with fully formulated detergents. Under these conditions, lipases were effective in removing natural oil (triglyceride) stains. Despite the many attempts to use lipase in detergent formulations for cleaning solutions, the washing benefits demonstrated have been disappointing.

Recently, efforts have been made to find specific lipases that are less affected by detergents in washing solutions. European patent application 258 068, published March 2, 1988, shows a lipase from the genus Thermomyces that is said to be compatible with anionic surfactants and effective as a detergent additive. Japanese patent application 63039579, published February 20, 1988, states that a new lipase obtained from a Pseudomonas is only slightly inhibited by anionic surfactants and activated by nonionic surfactants.

In summary, there was no clear teaching regarding the compatibility or incompatibility of lipases in cleaning and washing formulations, although it was generally recognized that specific surfactants (when present in appropriate amounts in detergent formulations) inhibit the lipase activity of some lipases. As a result, there was a tendency for cleaning solutions containing lipases to be those requiring prolonged soaking.

The present invention provides a method for cleaning fabrics having triglyceride stains thereon in a solution containing a lipase or a cutinase and at least one surfactant, wherein to the solution is added an agent which is not a substrate for the enzyme and which is 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, alcohol, aldehydes, ketones and amides and mixtures thereof to activate the enzymatic hydrolysis.

Having defined the scope of the invention, it will now be described more generally.

The enzyme(s) used in the process of the invention is/are capable of hydrolyzing triglycerides on fabric and the additive will prevent inhibition of the enzymatic hydrolysis by the surfactant in the solution and thus allow hydrolysis of the triglyceride soil or stain on the fabric to begin.

It is another feature of the present process that surfactant systems for use in cleaning solutions including lipases and/or cutinases can be formulated without the need for prolonged soaking or high temperatures for triglyceride hydrolysis.

Thus, the onset of hydrolysis for such an enzyme depends on a critical ratio being exceeded. It has been found that such enzymes will only "turn on" and hydrolyze the oil stain when the molar ratio of oil to surfactant in the cleaning solution at the oil stain interface exceeds a certain value, referred to herein as the "critical value." The value of the critical ratio for each enzyme depends on the identity of the surfactant used.

The hydrolysis activator changes the ratio of oil to surfactant in a wash solution in which the composition is used such that the critical ratio is exceeded so that the enzyme is "turned on" and hydrolyzes the oil stain.

The enzymes used in the present process (in combination with the hydrolysis activating agent to alter either the oil to surfactant ratio or the critical surfactant ratio) have sufficient hydrolytic activity in a surfactant-containing cleaning solution to hydrolyze at least about 5% by weight of the total oil stain, i.e., triglyceride, in a cleaning solution within about 14 to 15 minutes at about 25°C. A particularly preferred enzyme for use in the present invention is isolable from Pseudomonasputida (hereinafter "P. putida") ATCC 53552.

Materials cleaned in cleaning solutions include clothing soiled by body oils (sebum) and laundry soiled by food and cooking oils. Mono-, di- and triglycerides are present in sebum soils and in cooking oils and can potentially be hydrolyzed by lipases.

Analysis of the amino acid sequence of a recently discovered enzyme described as having lipase activity and isolable from P. putida ATCC 53552 suggests that there are substantial homologies between the nucleotide sequence of this enzyme and the nucleotide sequence of the cutinase gene recently determined for C. capsici. (Compare European Patent Application 268 456 to inventors Wiersema et al., published May 25, 1988, with Ettinger et al., Biochemistry, 26 pp. 7883-7892 (1987)). On Because of the relationship between cutinases and lipases, the enzymes of interest in the present invention include both cutinases and lipases capable of hydrolyzing triglycerides on materials in aqueous solution, and which are sometimes referred to hereinafter as glycerol ester hydrolases. Such enzymes useful in the present invention are typically obtained from various Pseudomonas, Chromobacter, Fusarium or Aspergillus strains. For example, the enzymes listed in this invention include those expressed from genes present in (or obtainable from) P. putida ATCC 53552, in P. sp. (as Amano 68S), in P. fluorescens (as Amano P) and in Aspergillus oryzae (as Lipolase). Toyo Jozo Co., Japan, US Biochemical Co., USA and Diosynth Co., Netherlands, sell lipases from Chromobacter viscosum. European patent application No. 0 214 761, published on March 3, 1987, assigned to Novo Industri, describes a lipase from Fusarium oxysporum. Other strains are known or have been described to produce lipases. For example, PCT/NL86/00023, published on February 12, 1987, assigned to Gist-Brocades NV, describes strains including certain Acinetobacter. It is understood that the genes expressing such enzymes can be cloned into another organism, such as E. coli, to obtain higher levels of expression.

CRITICAL RELATIONSHIP

Using a critical ratio as described herein, the inventors have found that in a surfactant-containing wash solution, the hydrolysis of oils containing ester bonds by specific glycerol ester hydrolase in the presence of surfactant is dependent on the ratio of oil to surfactant in the solution of interest. The ratio of oil to surfactant in the solution of interest is sometimes referred to hereinafter as the "system ratio." For convenience, both this system ratio and the value of the ratio required for hydrolysis to begin (referred to as the "critical ratio") are calculated and expressed based on the molar ratio of oil to surfactant in the starting aqueous solution. The system ratio defines the ratio of substrate (oil) with respect to the specific surfactant on an absolute mole/mole basis. At the critical ratio and above, the molar ratio of substrate relative to a specific surfactant is such that the enzyme is activated or "turned on" so that hydrolysis begins. That is, the concentration of both components in an aqueous medium, such as a wash water solution, is not critical since the molar ratio between the two components remains constant despite dilution in the aqueous medium.

Without being limited to a single theory, it may be difficult for the enzyme to bind the substrate unless the substrate is altered in some way facilitated by the surfactant. That is, there appears to be a not to provide a flexible complex between substrate and surfactant which is formed so that the substrate can be hydrolysed by the enzyme.

In the examples that follow, various lipases and cutinases are shown to have related but different critical ratios, which applicants have determined empirically using the model described above. A key aspect of the critical ratio model is a focus on the amount of substrate present, relative to the amount of surfactant, rather than the amount of enzyme or surfactant. In contrast, the prior art has believed that either using large amounts of enzyme or surfactant, or repeatedly testing either enzyme or surfactant, or both, will result in increased cleaning performance. Following the teaching or assumptions of this prior art results in the use of uneconomically large amounts of enzyme, surfactant, or both.

The molecular weights of the various surfactants (and typical structures) and the oils discussed below are shown below.

Compound Nominal Molecular Weight*

SDS (sodium dodecyl sulfate) 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

* Used for molarity calculations and calculated as pure surfactant. Typical structures of common surfactants are: SDS C₁₂LAS

Neodol 25-9 Neodol 23-6.5

Surfonic JL80X Triton X-100 C₁₆DAPS Neodol 25-3S

The onset of substrate hydrolysis by glycerol ester hydrolases depends largely on the system ratio and not on the concentration of either substrate (triglyceride) or surfactant.

An example of the effect of system ratio on the hydrolysis of substrate by a glycerol ester hydrolase is shown by Table IA, in which the enzymatic activity was monitored for a number of different trioctanoin concentrations at two different surfactant concentrations. The surfactant used for the data in Table IA was a zwitterionic salt, sometimes abbreviated to C₁₆DAPS ("Zwittergent 3-16", available from Calbiochem). TABLE IA

¹ Trioctanoin

² Hermaphrodite 3-16

³ Initial hydrolysis rates, measured as released umol H⁺ · min⁻¹ · mgE⁻¹ enzyme from P. putida ATCC 53552 (2 ppm)

* Indicates the exceeded critical ratio

As can be seen from the data in Table IA, there is either no enzyme activity (i.e., the enzyme is "turned off") or observable hydrolyase activity, depending on the system ratio and independent of the surfactant concentration. These data demonstrate that neither the absolute concentration of triglyceride nor the absolute concentration of surfactant determines whether the enzyme is active or not. Instead, it is the ratio of oil to surfactant that best describes the kinetic profile of enzyme activity. For the enzyme tested in Table IA, the critical ratio value (i.e., the system ratio at which enzymatic activity begins) with respect to this particular zwitterionic surfactant is between 10 and 20. At the system ratio value of 10 and below, the enzyme is not active. At a system ratio value of 20 and above, the enzyme is active.

Another example of this phenomenon, using a different surfactant, sodium oleate, with which the P. putida ATCC 53552 enzyme shows a different critical ratio, is shown in Table IB. TABLE IB

The substrate used for the data in Table IB was triolein. The concentrations (not shown) were varied to produce the system ratios shown. The surfactant sodium oleate used in the experiment summarized in Table IB is interesting because oleic acid is a product of reaction hydrolysis.

The experiments to determine enzyme activity, such as those shown in Tables IA and IB, were carried out as follows:

(i) Sample preparation:

The desired amount of triglyceride was weighed in a suitable sized beaker on a Mettler balance (model no. AE163). The corresponding amount of surfactant was added from a previously prepared aqueous stock solution of the surfactant. surfactant was added to the triglyceride and the triglyceride and surfactant were mixed manually. The sample was then adjusted to the desired weight using double 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 approximately 2 minutes.

(ii) Enzyme activity measurement:

This was accomplished by monitoring the rate of acid release from the enzymatic hydrolysis of the triglycerides in the emulsion. The assay was initiated by adding approximately 2 ppm lipase to 10 ml of the prepared emulsion. Acid release was monitored by autotitration on a pH-stat radiometer (model no. ABU80) to an endpoint of pH 10. Initial rates were recorded during the first 5 minutes of the reaction and reaction rates were recorded as µmol H+-min-1 mgE-1 titrated. Occasionally, enzyme activity is expressed as % total oil hydrolyzed in 14 minutes. In these experiments, the reaction was allowed to proceed for 14 minutes and the amount of acid titrated was recorded. Percent total oil (triglyceride) hydrolyzed values were then calculated by dividing the recorded values by the theoretically calculated value, assuming that three equivalents of oleic acid were formed for each equivalent of triglyceride. All assays were performed at room temperature.

The dependence of the onset of hydrolysis on a critical ratio of oil to surfactant in aqueous solution is not specific to the particular glycerol ester hydrolase used for the data of IA and Table IB; rather, it was found to be a general principle for other glycerol ester hydrolases. This is demonstrated in 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 studied, shown in Tables II-V, were also studied at higher surfactant concentrations, and the dependence on system ratios was confirmed. TABLE II (Enzyme isolable from P. putida ATCC 53552)

¹SDS (sodium dodecyl sulfate)

2C₁₂-LAS (available from Pfaultz and Bauer Inc.)

³Neodol 25-3S (available from Shell)

&sup4;Neodol 25-9 (available from Shell)

&sup5;Surfonic JL-80X (available from Texaco)

&sup6;Triton X-100 (available from Röhm and Haas)

*As described in footnote 3 in Table IA. TABLE III (Amano P. enzyme, available from Amano Co., isolable from Pseudomonas fluroescens)

¹SDS

²C₁₂-LAS

³Neodol 25-3S

&sup4;Neodol 25-9

&sup5;Surfonic JL-80X

*As described in footnote 3 in Table IA. TABLE IV (Enzyme Amano 685, available from Amano Co., isolable from P. sp.)

¹SDS

²C₁₂-LAS

³Neodol 25-3S

&sup4;Neodol 25-9

&sup5;Surfonic JL-80X

*As described in footnote 3 in Table IA.

TABLE V (Lipolase enzyme, available from Novo Industri, isolatable from A. oryzae)

Surfactant type and concentration Critical ratio

Anionic², 0.5 mM 0.5-1

Anionic³, 0.5 mM 20-30

Non-ionic&sup4;, 0.5 mM 20-30

Non-ionic&sup5;, 0.5 mM 10-20

²C₁₂-LAS

³Neodol 25-3S

&sup4;Neodol 25-9

&sup5;Surfonic JL-80X

As can be seen from Tables I-V, the critical ratios for specific enzymes depend on the identity of the surfactant.

The following Tables VI-IX show that the hydrolysis also depends on the substrate type. The data in Tables VI-IX were obtained using triolein as an oil (instead of trioctanoin as in Tables IV). TABLE VI (Enzyme from P. putida ATCC 53552)

¹SDS

²C₁₂-LAS

³Neodol 25-3S

&sup4;Neodol 25-9

&sup5;Surfonic JL-80X

*As described in footnote 3 in Table IA. TABLE VII (Enzyme Amano P)

¹SDS

²C₁₂-LAS

³Neodol 25-3S

&sup4;Neodol 25-9

&sup5;Surfonic JL-80X

*As described in footnote 3 in Table IA. TABLE VIII (Enzyme Amano 68S)

¹SDS

²C₁₂-LAS

³Neodol 25-3S

&sup4;Neodol 25-9

&sup5;Surfonic JL-80X

*As described in footnote 3 in Table IA.

TABLE IX (Enzyme Lipolase)

Surfactant type and concentration Critical ratio

Anionic², 0.5 mM 30-40

²C₁₂-LAS

The data shown above can be summarized by the "Table Summary" shown 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. TABLE SUMMARY

¹SDS (sodium dodecyl sulfate)

²C₁₂-LAS (available from Pfaultz and Bauer Inc.)

³Neodol 25-3S (available from Shell)

&sup4;Neodol 25-9 (available from Shell)

&sup5;Surfonic JL-80X (available from Texaco)

&sup6;Triton X-100 (available from Röhm and Haas)

ND = Not Determined

¹SDS (sodium dodecyl sulfate)

²C₁₂-LAS (available from Pfaultz and Bauer Inc.)

³Neodol 25-3S (available from Shell)

&sup4;Neodol 25-9 (available from Shell)

&sup5;Surfonic JL-80X (available from Texaco)

USAGE RATIO

The four enzymes tested (Tables I-IX) all demonstrate a critical ratio for each of the surfactants tested. These surfactants represent some of the most commonly used surfactants in commercially available detergent compositions. Such detergent compositions are typically recommended for use in cleaning purposes in the United States in amounts that, when dissolved in a cleaning solution, result in a surfactant concentration of between about 0.2 mM and about 1.5 mM (assuming an average load of 2-3 kg in a 72 liter wash solution).

The average amount of oily soil on fabrics in domestic laundries is estimated to be 300 mg oil/100 g fabric (Andree et al., J. App. Biochem., 2 pp. 218-229 (1980)). This indicates that, based on the ratio dependence shown above, the inclusion of lipases in the detergents most commercially available would result in little or no washing benefit because the usage ratio (of the actual oil concentration to the actual moles of surfactant in the cleaning solution) is below the critical ratio at which enzyme activity is initiated. The situation is similar for Europe and Japan, as use ratios are typically less than about 0.6 for Japan and less than about 0.4 for Europe, although fabric loading, wash solution, and recommended detergent use differ from the United States.

That is, based on recommended detergent usage and taking into account a wide variety of detergent compositions and surfactant molecular weights, the system ratios for the most commonly used detergents are typically less than 1, more typically in the range of about 0.2-0.6. (In calculating usage ratios, the initial solution concentrations were assumed and any possible interfacial effects were ignored.) However, as can be seen from the data in Tables II-IX, the critical ratios for the common surfactants (with trioctanoin as an oil) studied are generally greater than about 1. Performance at usage ratios below the critical ratio generally rendered efforts to include lipases in cleaning solutions ineffective.

Typical detergent compositions for cleaning contain various additives, such as builder salts. The additives commonly used in detergents were found to have no significant effect on the critical ratio at use levels (data not shown).

Likewise, mixtures of surfactants can be used to manipulate the critical ratio, although this is not according to the present method. The use of combinations of surfactants with different critical ratios The mixing of the individual surfactants in the combination with the respective critical ratios can produce critical ratios which are different from those of the individual surfactants in the combination. In practicing the invention, surfactants can be mixed to obtain a critical ratio of the combined surfactants which is lower than or equal to the lower of the individual critical ratios. This will be explained further below.

A conventional detergent composition is a mixture of Neodol 25-3S and C₁₂LAS (at a molar ratio of 1:0.4). This conventional detergent composition exemplifies the difficulties encountered in previous attempts to include the lipases in cleaning solutions. By examining the appropriate data for the surfactant component of the detergent composition in Table VI, one might conclude that the critical ratios are much higher than the use ratios. This conclusion, as demonstrated by conducting a swatch test and a washing machine test in which a solution contained either the conventional detergent or the conventional detergent plus ATCC 53552 enzyme, proved to be correct as shown by the data in Table X. TABLE X

¹Detergent comparisons at 1.29 g/L; enzyme, if present, at a concentration of 2 ug/ml.

²Stain: Cotton fabric sample with sebum contamination (synthetic)

³% SR(E), as described below.

As shown by the data in Table X, the stain removal value of the detergent composition with enzyme was not statistically different from the stain removal value of the detergent composition without enzyme. Thus, the enzyme was substantially inactive. The calculation of the use ratio shows that the use ratio was below the specified critical ratio of 10-20 and thus the enzyme was inactive.

As previously noted, soil removal was measured on a stain removal scale labeled "%SR(E)". This is a scale in which the ratio of the change in appearance of a contaminated, treated test sample to its maximum possible change in appearance.

%SR(E) values are calculated as follows:

where Es and E0W 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 expressed by the following formulas

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 indices o, s and w refer to the original unstained and untreated test sample, the stained and untreated test sample and the stained and treated test sample, respectively.

The statistical test, referred to as "LSD," refers to the smallest difference between the means within groups that would be called statistically significant at the 95% confidence level using the two-sample t-test with the variance estimated from all groups in the analysis of variance.

In summary, it has been shown that enzymes in a cleaning solution capable of hydrolyzing natural oil stains on fabric are dependent on a critical value of the molar use ratio of oil to surfactant in the cleaning solution for hydrolysis to occur. This critical ratio depends on the type of surfactant in the cleaning solution (and also on the type of oil in the cleaning solution). However, since the use ratios for most commonly used detergents are typically less than 1 and the critical ratio for the commonly used surfactants that have been studied is generally greater than about 1, lipases are generally inactive.

However, the inventors have found ways to "turn on" the hydrolysis by the enzyme using hydrolysis activating agents to change the ratio of oil to surfactant or to change the critical ratio of surfactants. Ways to modify the critical ratio are now described in more detail below. In addition, more than one lipase or cutinase can advantageously be used in the present process.

INCREASING THE SYSTEM RATIO BY 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 cleaning solution so that the enzyme hydrolyses oil stains. This added oil (which is in addition to the triglyceride present on stained fabrics being washed and which, together with the oily stains, forms the oil used as a numerator in calculating the critical ratio) need not be a substrate for the enzyme. The use of the additional oil as a means of turning on the enzyme also enables the removal of lower levels of oily stains during cleaning than would otherwise be possible.

The added oils (which are not substrates) are hexadecane and octadecane. The addition of non-substrate oil is shown by the data in Table XI. TABLE XI

* As described in footnote 3 in Table 1.

As can be seen from the data in Table XI, there is no hydrolase activity when triolein is present at a concentration of 0.3 mM and the system ratio is 1. With 1.5 mM triolein, resulting in a system ratio of 5, there is hydrolase activity. When 1.2 mM hexadecane was added to 0.3 mM triolein, the hydrolase was found to be active in the presence of 0.3 hexadecane added to the 0.3 mM triolein, the hydrolase was found to be active in the presence of 0.3 mM Surfonic JL-80X surfactant, even when the substrate concentration was left at 0.3 mM.

Although not in accordance with the present method, mixtures of substrate oils may also be used to alter the critical ratio. Table XII shows an example where the added oil is a substrate and is used to increase the system ratio above the critical ratio to activate the enzyme. TABLE XII

*% total oil hydrolyzed in 14 min at pH 10 and room temperature.

As can be seen from the data in 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 not hydrolyzed. However, when both oils (0.25 mM triolein and 2.25 mM trioctanoin) are emulsified together in 0.5 mM Surfonic JL-80X, 56% of the total oil is hydrolyzed.

Other organic compounds that do not participate in the hydrolysis reaction (in addition to the earlier discussion of added oils, such as hexadecane) may be used to achieve the critical ratio. Suitable organic compounds are those that are relatively insoluble, as indicated above, and that preferably contain few to no polar groups, since polar groups can interfere with enzyme activity. However, if the polar groups of the organic compound are obstructed or obscured by suitable branched or long chain alkyl groups, then some degree of polarity can be tolerated. Charged substituents (for example, -COO⁻Na⁺) are not preferred. The relatively insoluble organic compounds (which do not act as substrates for the enzyme) can be selected, without limitation, from glycol (diol) derivatives (such as diethylene glycol monolaurate, ethylene glycol dimethyl ether), alcohols (such as lauryl alcohol), aldehydes, ketones (such as methyl butyl ketone, methyl nonyl ketone) and amides (for example N,N-diethyldodecanamide). These compounds have a solubility δ of between about 7-9.5 according to the formula

δ = ΣGd/M

where ΣG is the sum of all atoms and groups in the molecule, d is the density, and M is the molecular weight. Compounds with a solubility δ of between about 8.0-9.0 are preferred, such as those described by J. Brandrup and EH Immergut, eds., Polymer Handbook 2nd edition, John Wiley & Sons, 1975), pp. IV-337 to IV-353. It may be that these relatively insoluble organic compounds, preferably with a few to no polar groups, are sufficiently chemically analogous to the oils (substrate or no substrate) to increase the overall "effective" oil concentration. Thus, such relatively insoluble organic compounds represent another embodiment of the means for altering the oil to surfactant ratio.

Table XIII shows the use of a preferred, relatively insoluble, organic compound, N,N-diethyl dodecanamide, to achieve the desired critical ratio when trioctanoin was the substrate. TABLE XIII

* uEq of fatty acids produced in 14 min.

The data in Tables XI to XIII were determined using the enzyme from P. putida ATCC 53552; however, other enzymes can be activated in a similar manner, even in the presence of a surfactant for which the enzyme has a high critical ratio, by including an oil which is not a substrate for the enzyme in the detergent composition. This is demonstrated by the data in Table 14 where the enzyme was Amano P. TABLE XIV

*As in footnote 3 of Table IA.

The molar ratio of oil hydrolysis activating agent (whether substrate or non-substrate) to surfactant according to the present invention is preferably greater than 0.5. This is due to the desired critical ratio of no greater than about 1 when assuming an average of 0.34 mM oily stains on the substances to be cleaned and an average of 0.75 mM surfactant(s).

REDUCING THE CRITICAL RATIO BY ADDITION OF SURFACE-ACTIVE AGENTS

Although not in accordance with the present method, mixtures of surfactants can be used to manipulate the critical ratio as well.

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 to substrate is varied and the hydrolysis activity is monitored. For example, Table IA shows the type of data typically produced by varying the ratio.

Since a desired critical ratio is usually no greater than 1, one or more surfactants must be tested (and/or another hydrolase is tested) until a critical ratio of less than 1 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 cleaning composition can then be formulated by including the lipase or cutinase and the surfactant selected to have a critical ratio of less than 1 or about 1.

It has been found that mixing a surfactant having a high critical ratio for a particular enzyme with a surfactant having a low critical ratio for that enzyme can result in a reduced critical ratio of the enzyme for the admixed surfactant. This exemplifies a means of altering the critical ratio of the surfactants in such a way that the enzyme used in the present invention is "turned on" and hydrolysis occurs. This is demonstrated by Table XV in which trioctanoin was added as an oil or substrate and the hydrolase was that of Table II (2 µg/ml). Enzyme activity was measured via initial rates and the reaction was carried out at room temperature to an end point pH of 10.00.

TABLE XV

Molar 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 reduces the critical ratio to 1, this is nevertheless not low enough for the enzyme to be active. That is, as can be seen from the data in Table XV, a 0.1-0.2 mole fraction of SDS when mixed with a surfactant of high critical ratio was effective in lowering the critical ratio of the surfactant mixture to that of SDS alone, but this is not low enough for commercially useful detergent formulations.

Table XVI shows a mixture of surfactants effective in reducing the critical ratio. Again, trioctanoin was used as an oil at a concentration of 0.64 mM and the hydrolase was that of Table II. TABLE XVI

As can be seen from the data in Table XVI, when Neodol 25-9 was used as a surfactant at a concentration of 0.3 mM with 0.64 mM of the oil in a system ratio of 2, there was no hydrolysis. However, when a mixture in a 1:1 ratio of the surfactants Neodol 25-9 and Neodol 35-3S was used at the same molar system ratio, there was 60% total hydrolysis. This is a surfactant system that is commercially viable.

Table XVII shows another example of mixing high and low critical ratio surfactants to reduce the critical ratio of the mixture. A surfactant composition was prepared from Neodol 25-9/Neodol 25-3S at a constant molar ratio of 1:1. The substrate concentration (trioctanoin) was about three times the normal use level (0.64 mM) and the percent total hydrolysis of the substrate after 7 minutes was monitored as a function of the change in the total surfactant concentration in the solution. TABLE XVII

**% total oil hydrolyzed in 7 minutes.

As can be seen from the data in Table XVII, the resulting surfactant mixture exhibited enzyme activity at a system ratio of 0.5. Thus, the critical ratio was at least 0.5 or less. In contrast, the surfactant Neodol 25-9 itself had a system ratio of about 20 before the enzyme activity was measured. Therefore, the addition of Neodol 25-3S in a 1:1 molar ratio lowered the critical ratio of Neodol 25-9 from about 20 to about 0.5.

Table VIII shows another example in which a mixture of high and low critical ratio surfactants synergistically reduces the critical ratio of the mixture to a point below the surfactant points of the individual components. A surfactant composition mixture of C12LAS/Neodol 25-9 was prepared in a 2:1 molar ratio and tested for comparison with each of the individual surfactants.

TABLE XVIII

Composition and concentration of surfactant Critical ratio

C12 LAS (0.5 mM) 5-10

C₁₂LAS (0.5 mM) 1-5

Neodol 25-9 (0.5 mM) 10-20

C12 LAS/Neodol 25-9 (2mM/1mM) 0.05-0.1

In the tests represented by the data in Table XVIII, the surfactant mixture exhibited Pseudomonas putida enzyme activity at a critical ratio between 0.05-0.1. The oil was triolein. In contrast, the Neodol 25-9 surfactant itself (at a concentration of 0.5 mM) had a critical ratio of between 10-20 and the critical ratio of C12LAS surfactant itself was 5-10 (at a concentration of 0.5 mM). Thus, the combination of these two surfactants reduced the critical ratio of the combination to a value below the critical ratio of each surfactant alone.

For present purposes, a preferred detergent composition suitable in unit amounts to clean fabric having a triglyceride thereon comprises a surfactant formulation that provides a surfactant concentration of from about 0.2 mM up to about 1.5 mM when a unit amount of the total composition is dissolved in a cleaning solution. Particularly preferred compositions include an enzyme isolable from P. putida ATCC 53552 present in an amount sufficient to hydrolyze at least about 5% by weight of triglyceride on fabric when a unit amount of the total composition is dissolved in a cleaning solution.

For example, a composition suitable for use in the present invention (designated "Composition (a)") was prepared by mixing the nonionic surfactant Neodol 23-6.5 and the nonionic surfactant Surfonic JL-80X in a 1:0.2 molar ratio. Additional additives and ratios were as follows:

Component % by weight

Surfactants

(Neodol 23-6.5/ 3.7

Surfonic JL-80X 26.0

Deionized water 0.6

Sodium tripolyphosphate

Sodium carbonate 10.5

Sodium polysilicate¹ 1.5

Alkaline proteases2,3 0.8/0.6

Optical brightener&sup4; 0.9

Pigments 0.1

Fragrance 0.2

¹ Trademark Britesil, available from PQ Corporation

² Trademark Alcalase, available from Novo Industries

³ Trademark Savinase, available from Novo Industries

&sup4; Trademark Tinopal 5 BM-XC, available from Ciba-Geigy A. G.

The hydrolase which was part of this detergent composition was cultured and isolated from P. putida ATCC 53552 as described in Wiersema et al., European Patent Application 268 456, published on May 25, 1988, and as also described below for the convenience of the reader.

(A) Inoculation and fermentation

An inoculation medium was prepared with 0.6% nutrient medium (Difco) and 1% glucose (pH 6.5). 100 ml of this medium was sterilized in 500 ml Fernbach bottles. Each bottle was then filled with an inoculation loop filled with an overnight culture grown on nutrient agar. of P. putida ATCC 53552 and placed on a New Brunswick shaker for 12 hours at 37ºC at 250 rpm. The incubated 12 hour culture was then seeded in appropriate volumes (1-10 vol/vol%) into a 1 liter fermenter (250 ml working volume), a 15 liter Biolafitte fermenter (12 liter working volume) or a 100 liter Biolafitte fermenter equipped with temperature control, RPM, air flow and pressure control. The fermenter medium contained 0.6% nutrient medium (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 proceed in the fermenter for 12-15 hours.

(B) Enzyme extraction by microfiltration

The crude fermentation culture was first filtered with an Amicon unit equipped with two Romicon micropore membranes (0.22 u) to remove cells. The remaining enzyme in the retentate bound to the cutin particles was recovered by centrifugation.

The overall yield reached 90%.

(C) Concentration and dialysis of total cell filtrate

The recovered filtrate from the Amicon unit was concentrated to a volume of 3 liters in an Amicon ultrafiltration unit with two Romicon Pm 10 modules. The concentrated material was then dialyzed with 20 liters of 0.01 M phosphate buffer, pH 7.5, to remove salts and dyes. The yield at this stage averaged about 80%. The total activity for this crude preparation was 8.68 x 106 units. One unit of lipase activity is defined as the amount of enzyme that results in an increase in 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 can be completely separated from another enzyme also possessing 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 10 mM Tris(Cl), pH 8, 0.5M NaCl and washed to remove unbound protein. The following washes were then used: 10 mM Tris(Cl), pH 8, 7M urea; 10 mM sodium phosphate, pH 8, 10 mM phosphate, pH 8, 0.5M NaCl. After washing, the column was developed with a linear gradient up to 50% n-propanol. The column fractions were then tested for activity towards p-nitrophenylbutyrate (PNB) and p-nitrophenylcaprylate (PNC) to localize the lipase activities. Two enzymes were clearly separated, namely 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 REMOVAL OF OILY STAINS

Both the fabric sample tests and the washing machine tests were carried out using the method according to the invention, as will be described below.

In the fabric sample test (1), 2 ppm hydrolase was mixed with the detergent composition previously described as composition (a). In a washing machine test (1), 20 ppm hydrolase was mixed with this composition. In both tests, soiled fabrics were stained with synthetic sebum oil. The synthetic sebum oil was prepared as follows. Ten oils in the following proportions were mixed:

Oils wt%/wt

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 15 g of the above melted oils were added 0.6 g of oleic acid, 1.2 g of triethanolamine and 0.225 g of charcoal. Then 60 ml of water at 130ºF was added and the mixture was mixed for 1 minute.

Fabric sample examination (1)

Cotton fabric swatches were soiled with the synthetic sebum oil and then washed in test beakers by agitation for 14 minutes followed by a 5 minute rinse. The cleaning solution consisted of 0.205 g of composition (a) dissolved in 250 ml of water. A control composition without the hydrolase was also prepared and used to treat stained cotton fabric swatches using the same protocol. Table XIX shows the stain removal for composition (a) of the invention and for the control composition. TABLE XIX

¹Calculated from the concentrations of surfactant and the olive and coconut oils.

²LSD = 2.23 at a confidence level of 0.95.

As can be seen from the fabric sample test data in Table XIX, a statistical improvement in soil removal was observed for the composition.

Washing machine inspection (1)

Polyester fabric swatches were stained with sebum, vegetable oil or olive oil. These swatches were then washed in a 72 liter washing machine at 96°F for 12 minutes, rinsed in the normal rinse cycle. They were then allowed to air dry. One set of swatches was treated in the cleaning solution in which 59 g of the inventive composition (a) was dissolved, whereas another set of swatches was treated with a control composition which did not contain the hydrolase but was otherwise identical to the inventive composition (a). The stain removal data, expressed as %SR(E), are shown in Table XX. TABLE XX

¹ LSD = 1.52

² LSD = 6.08 (Wesson-Brand oil)

³ LSD = 5.60

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

Fabric sample and washing machine examination (2)

The polycotton sheet was cut into 2" x 2" swatches, each weighing approximately 0.39 g. The desired amount of triolein was dissolved in 2-methylpentane and pipetted onto each swatch (200 µl/swatch). The triolein stain was allowed to set/spread for 72 hours at room temperature. The reflectance of the stain was then determined using a Hunter spectrocolorimeter and a prewash value (which is proportional to the concentration of the absorbing species) was determined.

The soiled swatches were divided into groups of 4 and placed in 250 ml bottles, each containing 200 ml of the desired treatment. The bottles were then shaken for 12 minutes at room temperature and rinsed twice with 200 ml of double distilled H₂O. Finally, they were air dried and the post-wash value (which is proportional to the concentration of the absorbing species) was determined.

Comparative Treatment A: 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: As A, except that 5 ppm Lipase ATCC 53552 was added to the surfactant composition.

Comparative Treatment C: Swatches were washed in an alternative formulation containing the surfactant composition of 0.3 mM C₁₂LAS/Neodol 25-9 in a molar ratio of approximately 1:4. No lipase was added.

Treatment D: As Treatment C except that 5 ppm of Lipase ATCC 53552 was added to the surfactant composition.

The amount of oily stains removed in each treatment is summarized in Table XXI. TABLE XXI

*% Loaded Triolein reflects the amount of oil per 100 g of fabric.

**The mM concentrations were calculated based on the initial concentrations of Oil and surfactants.

***Percent removal was calculated by comparing pre- and post-wash values to a standard curve versus the oil on the fabric swatch.

# Outside the scope of the invention.

As can be seen from the data in Table XXI, the use of a composition containing a mixture of surfactants and lipase removed from 33% to 60% of the oil on the polycotton sheet, and this removal was clearly better for composition (a) (with the hydrolase) than without the hydrolase. The LSD values show that this removal was statistically significant.

In summary, the process of the present invention is useful for cleaning solutions and involves the use of an enzyme capable of removing natural oil stains on fabrics in a cleaning solution and hydrolysis activating agents to change the ratio of oil to surfactant or to change the critical ratio of surfactants. Several embodiments of hydrolysis activating agents for use in cleaning solutions have been exemplified such that the enzyme will be active in hydrolyzing the oil stains. Typically, hydrolysis can be observed to occur when the enzyme has an activity sufficient to hydrolyze at least about 5% by weight of the total triglyceride stains within about 14 or 15 minutes at about 25°C. Without the hydrolysis activating agents used in the present invention, the enzyme is normally inhibited from hydrolyzing natural oily soils or stains when the cleaning solution contains between about 0.1 mM to 5 mM surfactant. Another way to determine the effect of the hydrolysis activating agent of the present invention is that when a lipase or cutinase is mixed with a surfactant formulation according to the invention, the lipase or cutinase is then capable of hydrolyzing at least about 30 mg of triolein fatty acid formed when a unit amount of cleaning composition is dissolved in an aqueous solution at 25°C at a pH of 10 at an average rate of about 0.072 mmol/min for about 14 minutes. Thus, surfactant systems containing lipases and/or cutinases can be formulated for use in cleaning solutions without the need for extensive soaking or high temperatures.

Claims (4)

1. A method for cleaning fabric having triglyceride stains thereon in a solution containing a lipase or a cutinase and at least one surfactant, wherein to the solution is added an agent which is not a substrate for the enzyme and which is selected from a hydrocarbon which is hexadecane or octadecane, and a relatively insoluble organic compound which has a solubility 8 between 7 and 9.5 and which is selected from glycol derivatives, alcohols, aldehydes, ketones and amides and mixtures thereof, to activate the enzymatic hydrolysis. 2. Process according to claim 1, characterized in that the surfactant and the activating agent are added to the solution such that the molar ratio of activating agent and surfactant is above the critical ratio of the enzyme for said surfactant, and wherein the molar ratio of activating agent to surfactant is greater than 0.5. 3. Process according to claim 1 or 2, characterized in that the enzyme can be isolated from an organism which expresses a gene obtainable from a Pseudomonas, a Chromobacter, an Aspergillus, an Acinetobacter or a Fusarium and which preferably isolable from Pseudomonas putida ATCC 53552, mutants thereof or clones thereof. 4. Process according to one of claims 1 to 3, characterized in that the surfactant is selected from sodium dodecyl sulfate, C₁₂H₂₅-φ-SO₃⊃min;Na⁺.