NZ271293A - Stabilised aqueous enzyme composition and detergent composition containing it - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £71 293 New Zealand No. 271293 International No. PCT/CA94/00463 Priority Datc<s): CcSpecification Filed: 0.«a: Pu&ication Dats: 2 -0-D.E*^-"^^- P.O. Journal No: '• IHHM4I % NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Stabilized enzyme solution and detergent prepared therefrom Name, address and nationality of applicant(s) as in international application form: DIVERSEY CORPORATION, a Canadian company of Suite 1600, 1 Robert Speck Parkway, Mississauga, Ontario L4Z 3S9, Canada WO 95/06101 PCT/CA94/00463 STABILIZED ENZYME SOLUTION AND DETERGENT PREPARED THEREFROM BACKGROUND OF THE INVENTION Field of the Invention: The present invention relates to an enzyme stabilizing composition. More particularly, the present invention relates to an enzyme stabilizing composition which contains a cation, other than calcium or boron, having an effective nuclear charge greater than 2.6. 10 prior Art; Numerous efforts have been made to formulate both stabilized enzyme-containing compositions and stabilized enzyme-containing detergent compositions.

For example. Berry, U.S. Patent No. 3,819,528, 15 discloses a stabilized aqueous enzyme composition containing water, an amylase, a calcium ion, and an organic co-stabilizing compound selected from aliphatic glycols and 1,3-propanediol. It was found that the addition of the calcium ion helped to stabilize the 20 enzyme in aqueous solution.

Also, Letton et al, U.S. Patent No. 4,318,81? discloses another stabilized aqueous enzyme composition which contains a stabilizing system comprising calcium ions and a low molecular weight carboxylic acid or salt, 25 preferably, a formate, and, optionally, containing a low molecular weight alcohol. A detergent composition containing a stabilized enzyme composition is, also, disclosed in the Letton reference. other efforts have been made to stabilize 30 enzymes in aqueous solutions by using a compound of boron in an aqueous enzyme-containing solution, e.g., U.S. Patent Nos. 4,261,868 and 4,537,707.

Oakes, in U.S. Patent 4,539,132, discloses a bleaching and cleaning composition which includes a 35 proteolytic enzyme. Manganese as a metal ion is also provided in the composition for stabilizing the enzyme, WO 95/06101 PCT/CA94/00463 2 where Oakes emphasizes that only manganese can be used to achieve stabilization of the proteolytic enzyme.

Kaminsky et al, U.S. Patent 4,305,837, describes a stabilized aqueous enzyme composition which 5 contains calcium ions as the stabilizing metal ion for the enzyme. Kaminsky et al also teach that zinc and magnesium ions can replace the calcium ion for purposes of stabilizing the enzyme. However, the substitution of calcium with zinc or manganese appears to be somewhat 10 speculative in view of the lack of examples demonstrating comparable stabilization when magnesium or zinc is used in place of calcium.

Magnesium has also been used in high pH aqueous liquid detergent compositions to stabilize an 15 enzyme as described in de Buzzaccarini et al, U.S. Patent 5,275,753. The only stabilizing metal ion described is magnesium, because it was surprisingly found that at high pH, magnesium provided a stabilizing effect for the enzyme when present in an amount ranging from 10 ppm to 20 10,000 ppm.

While various efforts have been made to prepare relatively stable aqueous enzyme-containing compositions, a need still exists in the art for additional compositions which can effectively stabilize enzymes in 25 aqueous solutions.

SUMMARY OF THE INVENTION It has been discovered, in accordance with the practice of the present invention, that the use of a cation having an effective nuclear charge greater than 30 2.6 assists in stabilizing enzymes in aqueous solution and such a cation appears to inhibit degradation of the molecular structure of the enzyme. In particular, a cation which is included in a salt of a metallic element selected from the group consisting of strontium, cerium, 35 yttrium, ytterbium, lanthanum, and mixtures thereof have proven to be helpful in inhibiting degradation of enzymes.

In addition, polycarboxylie acids and short-chained alcohols have been shown to be helpful in inhibiting enzyme degradation in aqueous solutions. Dicarboxylic acids are particularly preferred.

A stabilized aqueous enzyme composition in accordance with the present invention/ generally, comprises: (a) water; (b) em enzyme selected from the group consisting of amylases, proteases, cellulases and mixtures thereof; (c) an ionic compound which comprises a cation other them calcium, boron, manganese, magnesium or zinc, the cation having an effective nuclear charge greater than 2.6.

The present invention also contemplates a detergent composition utilizing the stabilized aqueous enzyme composition hereof. A liquid detergent concentrate in accordance with the present invention, generally, comprises: (a) water; (b) a salt of a metallic element which includes a cation having am effective nuclear charge greater than 2.6 and being selected from the group consisting of strontium, cerium, yttrium, ytterbium, lanthanum and mixtures thereof; (c) a polycarboxylic acid or salt thereof or a short chain alcohol; (d) a surfactant component which comprises at least one nonionic surfactant; and, (e) an enzyme selected from the group consisting of proteases, amylases and cellulases and mixtures thereof.

In a preferred embodiment of the liquid detergent concentrate hereof, the concentrate includes from about 10 to about 70 percent by weight, based on the total concentrate weights, of water, from about 0.01 to about 2 percent by weight, based on the total concentrate weight, of the metallic salt, from about l.o to about 6.0 percent by weight, based on the total concentrate weight, of the polycarboxylic acid salt or short chain alcohol, from about 8 to about 40 percent by weight, based on the 5 total concentrate weight, of the surfactant, and at least 10 ppm by weight, based on the total concentrate weight, of the pure enzyme.

The preferred pE range of the detergent composition in accordance herewith is from about 6.0 to 10 about 9.5. A particularly preferred pH range is from about 6.0 to about 8.0.

For a more complete understanding of the present invention, reference is made to the following detailed description and accompanying examples. 15 Throughout the following description and in the examples, any parts given are intended to be by weight, absent indications to the contrary.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It has now been discovered, in accordance with 20 the practice of the present invention, that the effective nuclear charge of a cationic enzyme stabilizing agent if important. The present invention provides a stabilized aqueous enzyme composition, which, generally, comprises: (a) water; (b) an enzyme selected from the group consisting of amylases, proteases, cellulases and mixtures thereof; and, (c) an ionic compound which comprises a cation other than calcium, boron, manganese, magnesium or zinc, 30 the cation having an effective nuclear charge greater than 2.6.

Preferably, the water used is de-ionized water in order to be free from contaminants. Generally, the water is present in an amount ranging from about 10 35 percent to about 70 percent by weight, based on the total composition weight.

Enzymes which are useful in the practice of the present invention include proteases, amylases, cellulases and mixtures thereof. Mixtures of one class of enzymes such as, e.g., mixtures of proteases or 5 mixtures of different amylases, as well as mixtures of different classes of enzymes are contemplated for use in the present invention. Proteases are preferred enzymes in the practice of the present invention. A particularly preferred protease is a protease derived from Bacillus 10 Lichenformis. A commercially available product which is useful in the practice of the present invention is a product sold by Navo Laboratories under the name "ESPERASE". This is available in a liquid form as a product called ESPERASE 8.0L, or in a slurry called 15 ESPERASE 8.0 SL, which includes a proteolytic enzyme suspended in a surfactant (TERGITOL 15-S-9, a polyethoxylated secondary fatty alcohol from Union Carbide).

It has now been discovered in accordance with 20 the practice of the present invention, that a cationic enzyme stabilizing additive is generally most effective when the cation thereof has an effective nuclear charge greater than 2.6. Preferred cationic enzyme stabilizing additives are those having an effective nuclear charge 25 ranging from 2.6 to 3.9. The effective nuclear charge may be calculated by Slater's method. This method is outlined in the article entitled "Atomic Shielding Constants", Physical Review. Volume 36, (1930) page 57, which is hereby incorporated by reference. 30 In particular, it has been found that an ionic compound, particularly a metallic salt, which comprises a cation having an effective nuclear charge greater than 2.6 is effective, in aqueous solutions, to help stabilize the enzyme. Particularly preferred metallic salts which 35 fall within the above-identified range and which are suitable in the practice of the present invention are strontium, cerium, yttrium, ytterbium, lanthanum, and mixtures thereof. It is understood that these metals are stable in their salt form, where the salt of the metal may be any of the well known types; for example, such as halogens (e.g. chlorides), oxides, acetates, malates, 5 formates to name only a few. A listing of some cations which are relevant to the present invention, along with their effective nuclear charges can be found below.

Effective Nuclear Atomic Test Charcre (Slater) Element Svmbol Number 2.20 Cesium Cs 55 0 Potassium K 19 (-) Rubidium Kb 37 0 Sodium Na 11 (-) 2.55 Carbon C 6 0 2.58 Sulfur s 16 0 2.60 Boron B (+) 2.85 Barium Ba 56 (+) Calcium Ca (+) Cerium Ce 58 (+) Dysprosium Dy 66 (+) Erbium Er 68 (+) Europium Eu 63 () Gedoliniuni Gd 64 (+) Lanthanum La 57 (+) Magnesium Mg 21 (+) Neodymium Nd 60 (+) Praseodymium Pr 59 0 Promethium P 61 () Samarium Sm 62 (+) Strontium Sr (+) Terbium Tb 65 0 Thulium Tm 69 () Ytterbium Yb 70 (+) 3.00 Lutetium Lu 71 0 Scandium SC 21 (+) Yttrium Y 39 (+) 40 3.15 Hafnium Hf 72 0 Titanium Ti 22 0 Zirconium Zr 40 (+) 3.30 Niobium Nb 41 (+) Tantalium Ta 73 0 45 Vanadium V 23 0 3,45 Chromium Cr 24 (-) 3.50 Aluminium Al 13 (+) 3.60 Manganese Mn (+) 3.75 Iron Fe 26 (+) 50 3.90 Cobalt Co 27 (+) 4.05 Nickel Nc 28 (-) Although boron and calcium fall within this preferred range for effective nuclear charges, it is known that calcium and boron have previously been used as enzyme stabilizers and, thus, are known compounds for 5 this purpose. Similarly, manganese and magnesium are also included in the above list, but are not preferred compounds and were not tested. Also, Zn is not included in the group of effective metal ion enzyme stabilizing compounds because, as the following test results 10 indicate, Zn did not maintain enzyme activity.

It has additionally been discovered that the stabilizing influence of the cation selected from the above group is further enhanced by the presence of an oxygen-anion containing compound such as a deprotonated 15 or partially deprotonated carboxylic acid or short chain alcohol or a salt thereof, and particularly, by a dicarboxylic acid salt. By short chain alcohol, an alcohol having 1 to 15 carbon atoms is meant. Alcohols having l to 5 carbon atoms are preferred. Representative 20 carboxylic acids are the dicarboxylic acids, including maleic acid, malonic acid, succinic acid, glutaric aci.c. adipic acid, pimelic acid and the like, as well as the water-soluble salts thereof, as well as mixtures thereof.

Particularly preferred dicarboxylic acids for 25 use in the practice of the present invention are those which are water-soluble, and in particular, pimelic acid and adipic acid, as well as the sodium salts thereof. Disodium malonate and disodium maleate have been shown to be particularly effective when used in combination with a 30 cation having an effective nuclear charge in the described range, in stabilizing enzymes in aqueous solutions. Disodium adipate, disodium malonate, disodium maleate, disodium succinate, and disodium glutarate are, also, suitable for use as sources of oxygen-anion 35 containing compounds in accordance with the present invention. 8 The composition of the present invention may, also, further include a surfactant. This surfactant may be anionic or nonionic, and is preferably a nonionic surfactant.

Various anionic surfactants may be employed in the practice of the present invention. The anionic surfactant chosen is selected for its ability to solubilize with the chosen salt in an aqueous medium. The anionic surfactant employed in the concentrate and 10 composition made therefrom is selected from the group consisting of alpha-olefin sulfonates, sulfonates of ethoxylated linear alcohols, alkyl aryl sulfonates, naphthalene sulfonates and mijctures thereof. The sulfonates of ethoxylated llm&r alcohols which can be 15 employed in the present invention have the general formula: R- (OCHjCH^ SOjH wherein x is an integer between about 1 and about 5 and H is an anion selected from the group 20 consisting of alkali metals, an amine cation, an ammonium cation and mixtures thereof, and R is a hydrocarbon radical having between 12 and 15 carbon atoms. Useful alkali metal cations include sodium, potassium, and mixtures thereof.

The alkyl aryl sulfonates which can be employed in the present invention include those having the general formula: OH <2*3 (CH2), wherein x is an integer between about 6 and 12 and M is an anion selected from the group consisting of 35 alkali metals, amine cations, ammonium cations and mixtures thereof.

The naphthalene sulfonates which can be employed in the present invention have the general formula: R ~ (sXs)—SOsM wherein R is a hydrocarbon radical having between 8 and 10 carbon atoms and M is an anion selected from the group consisting of alkali metals, amine cations, ammonium cations and mixtures thereof.

Alpha-olefin sulfonates are the preferred anionic surfactant in the present invention. Where alpha-olefin sulfonates are employed, they sure, preferably, selected from the group consisting of C12 to C1( alpha-olefin sulfonates and mixtures thereof.

Alpha-olefin sulfonates are well known and commercially available products. Generally, they are available as alkali metal salts. The alpha-olefin sulfonates contemplated for use herein can be represented by the following formula: RCH=CH-CH2-S03M wherein M is either an alkali metal, amine cation or ammonium cation as well as mixtures thereof and 25 R is an unsubstituted hydrocarbon radical ranging from C9 to C15 and mixtures thereof. Representative of the useful alkali metal cations are sodium and potassium, as well as mixtures thereof.

As noted herein above, nonionic surfactants are 30 also useful in the practice of the present invention. Generally, nonionic surfactants, where employed, are present in an amount ranging from about 8 percent to about 40 percent by weight, based on the total concentrate weight. The surfactant may be a mixture of 35 surfactants.

The nonionic surfactants which are advantageously employed in the compositions of the PCT7CA94/00463 present invention are basically the polyoxyalkylene adducts of hydrophobic bases wherein the oxygen/carbon atom ratio in the oxyalkylene portion of the molecule is greater than 0.40. Those compositions which are condensed 5 with hydrophobic bases to provide a polyoxyalkylene portion having an oxygen/carbon atom ratio greater than 0.40 include ethylene oxide, butadiene dioxide and glycidol, mixtures of these alkylene oxides with each other and with minor amounts of propylene oxide, butylene 10 oxide, amylene oxide, styrene oxide, and other higher molecular weight alkylene oxides. Ethylene oxide, for example, is condensed with the hydrophobic base in an amount sufficient to impart water dispersibility or solubility and surface active properties to the molecule 15 being prepared. The exact amount of ethylene oxide condensed with the hydrophobic base will depend upon the chemical characteristics of the base employed and. is readily apparent to those of ordinary skill in the art relating to the synthesis of oxyalkylene surfactant 20 condensates. Typical hydrophobic bases which can be condensed with ethylene oxide in order to prepare nonionic surface active agents include mono and polyalkylphenols, polyoxypropylene condensed with a base having from about l to 6 carbon atoms and at least one 25 reactive hydrogen atom, fatty acids, fatty amines, fatty amides and fatty alcohols. The hydrocarbon ethers such as the benzyl or lower alkyl ether of the polyoxyethylene surfactant condensates are also advantageously employed in the compositions of the invention.

Among the suitable nonionic surface active agents are the polyoxyethylene condensates of alkylphenols having from about 6 to 20 carbon atoms in the alkyl portion and from about 5 to 15 ethenoxy groups in the polyoxyethylene radical. The alkyl substituent on 35 the aromatic nucleus may be octyl, diamyl, n-dodecyl, polymerized propylene such as propylene tetramer and trimer, isoctyl, nonyl, etc. The benzyl ethers of the 11 polyoxyethylene condensates of monoalkyl phenols impart good properties to the compositions of the invention. A typical product corresponds to the formula: Higher polyalkyloxyethylated phenols corresponding to the formula: R wherein R is hydrogen or an alkyl radical having from 15 about 1 to 12 carbon atoms, R' and R" sore alkyl radicals having from about 6 to 16 carbon atoms and n has a value from about 10 to 40, are also suitable as nonionic surfactants, a typical oxyethylated polyalkylphenol is dinonylphenol condensed with 14 moles of ethylene oxide. 20 other suitable nonionic surface active agents axe cogeneric mixtures of conjugated polyoxyalkylene compounds containing in their structure at least one hydrophobic oxyalkylene chain in which the oxygen/carbon atom ratio does not exceed 0.40 and at least one 25 hydrophilic oxyalkylene chain in which the oxygen/carbon atom ratio is greater than 0.40. propylene oxide, butylene oxide, amylene oxide, styrene oxide, mixtures of such oxyalkylene groups with each 30 other and with minor amounts of polyoxyalkylene groups obtained from ethylene oxide, butadiene dioxide, and glycidol are illustrative of hydrophobic oxyalkylene chains having an oxygen/carbon atom ratio not exceeding 0.40. Polymers of oxyalkylene groups obtained from 35 ethylene oxide, butadiene dioxide, glycidol, mixtures of such oxyalkylene groups with each other and with minor amounts of oxyalkylene groups obtained from propylene R" Polymers of oxyalkylene groups obtained from PCT/CA94/0G463 12 oxide, butylene oxide, aaylene oxide, and styrene oxide aire illustrative of hydrophilic oxyalkylene chains having an oxygen/carbon atom ratio greater than 0.40.

Further suitable nonionic surface active agents 5 are the polyoxyethylene esters of higher fatty acids having from about 8 to 22 carbon atoms in the alkyl group and from 8 to 15 ethanoxy units in the oxyethylene portion. Typical products are the polyoxyethylene adducts of tall oil, rosin acids, lauric, stearic and 10 oleic acids and the like. Additional, nonionic surface active agents are the polyoxyethylene condensates of higher fatty acid amines and amides having from about 8 to 22 carbon atoms in the fatty alkyl or alkyl group and about 10 to 15 ethanoxy units in the oxyethylene portion. 15 Illustrative product* are coconut oil, fatty acid amines and amides condensed with about 10 to 15 moles of ethylene oxide.

Other suitable polyoxyalkylene nonionic surface active agents are the alkylene oxide adducts of higher 20 aliphatic alcohol and thioalcohol having from about 8 to 22 carbon atoms in the aliphatic portion and! about 3 to 15 carbon atoms in the oxyalkylene portion. Typical products (ore the synthetic fatty alcohol, such as n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n~ 25 hexadecyl, n-octadecyl and mixtures thereof condensed with 3 -o 15 moles of ethylene oxide, a mixture of normal fatty alcohol condensed with 10 to 30 moles of a mixture of ethylene and propylene oxides, a mixture of several fatty alcohol condensed Requentially with 2 to 20 moles 30 of ethylene oxide and 3 to 10 moles of propylene oxide, in either order; or a mixture of normal fatty alcohol condensed with a mixture of propylene and ethylene oxides, in which the oxygen/carbon atom ratio is less them 0.40 followed by a mixture of propylene and ethylene )5 oxides in which the oxygen/carbon atom ratio is greater than 0.40 or a linear secondary alcohol condensed with 3 to 10 moles of ethylene oxides, or a linear secondary 13 alcohol condensed with a mixture of propylene and ethylene oxides, or a linear secondary alcohol condensed with a mixture of ethylene, propylene and higher alkylene oxides. Linear ethoxylated alcohol nonionic surfactants 5 are particularly preferred surfactant for use in the practice of the present invention, and have been found to be most compatible with the total composition hereof, although other surfactants are acceptable.

A stabilized enzyme composition according to 10 the present invention may further contain an alcohol or glycol as an additional enzyme stabilizing agent. The alcohol or glycol is, however, optional. Preferred additional stabilizers are ethylene glycol and glycol.

The composition in accordance with the present :.5 invention is in a pH range from about 6.0 to about 9.5 and, preferably, from about 6.0 to about 8.0.

For a more complete understanding of the present invention, the reader is referred to the following examples. In the examples, which are to be 20 construed as illustrative, rather than limitative of the invention, all parts are by weight, absent indications tc the contrary.

EXAMPLES 1-5 A series of five enzyme containing solutions were prepared according to the following formula: Deionized water 9.2 Sodium Formate 4.0 Propylene Glycol 76.7 3 0 Enzyme (A) 10.0 (A) ESPERASE 8.0L sold by Navo Industries.

Into each of these solutions, then, was added 0.1 part by weight of one component selected, in sequential 35 order, from the following list: (1) Strontium Chloride (2) Zn Chloride (3) Yttrium Oxide (4) Yttrium Chloride (5) Ytterbium Chloride Each of the solutions was checked for stability according to the following table, Table I, in which the numbers refer to the number of seconds required to clear the silver halide emulsion off of an exposed and 10 developed piece of black-and-white film, and an x indicates that the enzyme is inactivated.

A review of the data from Table I shows that solutions 1, 4, and 5 were relatively stable over time, with solution 1 being the most stable of the series.

• • • • TABLE I Initial T, It After 2 days § 4?#C After 4 days § 49°C After 6 days 6 4?°C After 1 Month 6 33°C fRT) Solution 1 486 610 1435 3408 5648 572 Solution 2 516 713 X X X X Solution 3 insoluble X X X X X Solution 4 565 575 4709 X X 567 Solution 391 582 3787 X 7107 567 16 6-1? A standard stabilized enzyme solution was prepared, as a base line for purposes of comparison, which included calcium ions and formate ions. This 5 standard solution is set out as Example 6. A series of additional comparative solutions were prepared using various proportions of disodium malonate, disodium maleate, cerium chloride, and yttrium acetate, as examples 6-12. Each of the solutions was then stored 10 under two different conditions; one batch was stored at room temperature and one batch was stored in a **hot box" at 40°C. Periodically, the enzyme activity was measured and compared to a previously measured initial value.

The following table, Table II, lists the 15 components used to formulate the experimental solutions, and the observed enzyme activity after 4 days, 8 days and 12 days at 40°C and, also, after 11 days at room temperature (approx. 23°C). It will be noted that not all ingredients are used in each example. 20 A review of the data from Table II shows that several of the experimental solutions exhibited an enzyme activity which was relatively high at ambient temperatures (as a percent of the standard) , even in the complete absence of propylene glycol as an additive.

TABL] Examples £ 2 8 Di Water .9 85.9 85.9 Na Formate 4.0 Na Malonate 4.0 Na Maleate 4.0 Tamol 6-1588 Ca Chloride H • o Ce Chloride 0.1 0.1 Y Acetate Prep. Glycol 60.0 .0 Esperase 8.SL .0 .0 Initial Activity 100.0% 100.0% 100.0% Act. 3 days ® 24°C 99.6 90.1 87.7 Act. 10 days § 40"C 98.2 50.3 48.7 Act. 21 days § 40°C 93.0 33.3 .5 Act. 30 days § 40°C 94.7 29.2 28.8 Act. 31 days § 24°C 78.9 62.4 60.6 After 30 days % of Std. 6 40°C 100 62.4 60.6 After 30 days % of std. at 24°C 100 79.1 76.8 II 2 85.9 4.0 0.1 .0 85.9 4.0 0.1 10.0 IX 85.9 4.0 0.1 10.0 11 85.9 4.0 0.1 10.0 £ d vo 1,1 H 100.0% 81.5 .2 .3 19.6 48.9 48.9 62.0 100.0% 93.8 48.9 31.4 25.9 62.7 62.7 79.5 100.0% 85.2 44.2 24.7 23.6 56.4 56.4 71.5 100.0% 86.4 39.4 23.6 .7 52.9 52.9 67.2 n H n > <. 5 Cs 18 EXAMPLES 14-24 A standard stabilized enzyme solution was prepared, as a base line for purposes of comparison/ which included calcium ions and formate ions. This 5 standard solution is Example 14. A series of additional comparative solutions were prepared using cerium acetate and disodium malonate as Examples 15-24. Each of the solutions was then stored under two different conditions; one batch was stored at room temperature and one batch 10 was stored in a "hot box" at 38°C. Periodically, the enzyme activity was measured and compared to a previously measured initial value.

The following table, Table III, lists the components used to formulate the experimental stabilized 15 enzyme solutions, and the observed enzyme activity after four days, eight days, ten days, sixteen days, and twenty-two days in the "hot box" at 38°C and, also, the observed activity after 25 days at room temperature. It will be noted that not all of the listed ingredients are 20 included in each example. This series of tests was done primarily to optimize the relative proportions of cerium acetate and disodium malonate in the test solution, as well as the other components.

A review of the data from Table III shows that 25 several of the experimental solutions exhibited an enzyme activity which was superior to that of the standard.

Having, thus, described the invention, what is claimed is: TABLE III Example? 11 16 11 13. 12 23 21 22. 21 ZA Hater .0 56.9 66.7 22.2 64.0 54.2 44.5 34.7 69.4 32.0 0.0 Ca Chloride 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Na Formate 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Ce Acetate 0.0 0.4 0.3 0.1 0.0 0.1 0.2 0.3 0.6 0.0 0.0 Na Malonate 0.0 1.0 3.0 1.0 6.0 4.0 2.0 0.0 0.0 3.0 0.0 Propylene .0 Glycol 60.0 11.7 0.0 46.7 0.0 11.7 23.3 .0 0.0 70.0 Surfactant A1 0.0 .6 .6 .6 .6 .6 .6 .6 .6 .6 .6 Surfactant B' 0.0 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4 Enzyme ' .0 .0 .0 .0 .0 .0 .0 .0 .0 .0 .0 Initial Actirity 0.90 0.91 0.96 0.95 0.87 0.86 0.86 0.90 0.82 0.82 0.71 After 4 days 0 38°C 0.78 0.87 0.63 0.92 0.51 0.82 0.92 0.90 0.98 0.93 0.26 After 8 days e 38°C 0.71 0.67 0.42 0.75 0.35 0.60 0.78 0.74 0.45 0.70 0.13 After 10 days § 38°C 0.84 0.80 0.40 0.88 0.40 0.74 0.86 0.84 0.42 0.84 0.12 After 15 days § 38°C 0.75 0.66 0.32 0.68 0.28 o.63 0.76 0.79 0.26 0.62 0.10 After 22 days e 38°C? 0.86 0.60 0.27 0.67 0.26 0.57 0.70 0.76 0.39 0.52 0.10 After 25 days % RT4 0.62 0.75 0.57 0.78 0.53 0.72 0.69 0.73 0.78 0.73 0.31

Claims (12)

TABLE III (Continued) Examolea 11 11 1£ 11 IS 12 22 21 21 21 21 % Remaining (RT) 68.78 82.42 59.38 82.11 60.92 83.72 80.23 81.11 95.12 89.02 43.66 % Remaining (38*C) 95.40 65.93 28.13 70.53 29.89 66.28 81.40 84.44 47.56 63.41 14.08 Ave. % Remaining (38*C) 86.16 85.71 50.35 89.47 48.28 83.72 99.22 91.85 75.20 100.41 23.94 Overall Rating 154.94 168.13 109.72 171.58 109.20 167.44 179.46 172.96 170.33 189.43 67.61 % of Standard 100.00 10S.S1 70.82 110.74 70.48 108.07 115.82 111.63 109.93 122.26 43.63 (1) A nonionic surfactant sold by Shell as Neodol 91-2.5 (2) A nonionic surfactant sold by Shell as Neodol 23-6.5 (3) A liquid proteolytic enzyme solution which is an alkalophilic variant (4) RT = room temperature - approximately 23°C 271293 21 CLAIMS

1. A stabilized aqueous enzyme composition, comprising: 5 (a) water; (b) an enzyme selected from the group consisting of amylase, proteaser cellulase and mixtures thereof; and (c) an ionic compound which comprises a cation of a metallic salt other than calcium, boron, 10 manganese, magnesium or zinc, the cation having an effective nuclear charge greater than 2.6.

2. The composition of Claim 1, further comprising an additive which serves as a source of oxygen-anion 15 in aqueous solution.

3. The composition of Claim l, wherein the ionic compound is a salt of a metallic element selected from the group consisting of strontium, cerium, 20 yttrium, ytterbium, lanthanum, and mixtures thereof.

4. The composition of Claim 2, wherein the oxygen-anion source is selected from the group consisting of a polycarboxylie acid, a short chain C, 25 - C15 alcohol, a polycarboxylie acid salt, and mixtures thereof.

5. The composition of Claim l, further comprising: a nonionic surfactant. 30

6. A stabilized aqueous enzyme composition comprising: (a) water; (b) an enzyme which is a protease; 27 ) 29 J 22 (c) an additive which is a source of oxygen-anion in aqueous solution; and (d) a salt of a metallic element other than calcium, zinc, magnesium, manganese or boron 5 and which metallic element is selected from the group consisting of strontium, cerium, yttrium, ytterbium, lanthanum, and mixtures thereof.

7. The composition of Claim 4, further comprising 10 a nonionic surfactant.

8. A liquid detergent concentrate comprising: (a) from about 10 to about 70 percent by weight, based on the total concentrate weight, of 15 water; (b) from about 0.01 to about 2 percent by weight, based on the total concentrate weight, of a salt of a metallic element other than calcium, zinc, magnesium, manganese or boron and which 20 metallic element is selected from the group consisting of strontium, cerium, yttrium, ytterbium, lanthanum, and mixtures thereof; (c) from about 0.1 to about 4 percent by weight, based on the total concentrate weight, of an 25 additive which serves as a source of an oxygen-anion in aqueous solution; (d) from about 8 to about 40 percent by weight, based on the total concentrate weight, of a surfactant component which comprises at least one 30 nonionic surfactant; and (e) an enzyme which is a protease, the enzyme being present in an amount of at least 10 ppm by weight, based on the total concentrate weight. N.Z. PATENT OFFICE ] t 4 NOV 1996 j 27 J 29 J 23

9. The composition of Claim 8, wherein the oxygen-anion source is selected from the group consisting of a polycarboxylic acid, polycarboxylic acid salt, an alcohol having from one to fifteen 5 carbon atoms and mixtures thereof.

10. The composition of Claim 1, wherein the -* cation of the ionic compound has an effective nuclear charge in the range of 2.6 to 3.9. 0

11. A stabilized aqueous enzyme composition as claimed in Claim 1 substantially as herein described with reference to any example thereof.

12. A liquid detergent concentrate as claimed in Claim 8 substantially as herein described with reference to any example thereof. authorised aflenta N.Z. PATENT OFFICE 1 4 NOV 1996