AU686653B2

AU686653B2 - Stabilized enzyme solution and detergent prepared therefrom

Info

Publication number: AU686653B2
Application number: AU74883/94A
Authority: AU
Inventors: Josef K Zehetmair
Original assignee: DIVERSEY CORP
Current assignee: DIVERSEY Corp
Priority date: 1993-08-26
Filing date: 1994-08-25
Publication date: 1998-02-12
Anticipated expiration: 2014-08-25
Also published as: ZA946535B; NZ271293A; WO1995006101A1; EP0672100A1; CA2153066C; AU7488394A; CA2153066A1

Description

WO 95/06101 PCT/CA94/00463 1 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.

Prior Art: Numerous efforts have been made to formulate both stabilized enzyme-containing compositions and stabilized enzyme-containing detergent composition.

For example, Berry, U.S. Patent No. 3, 19,528, discloses a stabilized aqueous enzyme composi 'ion containing water, an amylase, a calcium io and an organic co-stabilizing compound selected rom aliphatic glycols and 1,3-propanediol. It was found that the addition of the calcium ion helped stabilize the enzyme in aqueous solution.

Also, Letton et al, Patent No. 4,318,818 discloses another stabilized aqueous enzyme composition which contains a stabiliz' g system comprising calcium ions and a low molecula weight carboxylic acid or salt, preferably, a format and, optionally, containing a low molecular weight a cohol. A detergent composition containing a st ilized enzyme composition is, also, disclosed in he Letton reference.

her efforts have been made to stabilize enzymes n aqueous solutions by using a compound of boron in an queous enzyme-containing solution, U.S.

Pat nt 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 Sproteolytic enzyme. Manganese as a metal ion is also S provided in the composition for stabilizing the enzyme, WO 95/06101 PCT/CA94/00463 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 compositi which contains calcium ions as the stabilizing met ion for the enzyme. Kaminsky et al also teach t t zinc and magnesium ions can replace the calci ion for purposes of stabilizing the enzyme. Howev the substitution of calcium with zinc or manganese ppears to be somewhat speculative in view of the ck of examples demonstrating comparable stabilizatio when magnesium or zinc is used in place of calcium.

Magn eum has also been used in high pH aqueous liqui detergent compositions to stabilize an enzyme as escribed in de Buzzaccarini et al, U.S. Patent 5,275, 3. The only stabilizing metal ion described is ma sium, because it was surprisingly found that at high magnesium provided a stabilizing effect for the enzyme when present in an amount ranging from 10 ppm to 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 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 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, yttrium, ytterbium, lanthanum, and mixtures thereof have proven to be helpful in inhibiting degradation of enzymes.

w Y In addition, polycarboxylic acids and short-chained alcohols have been shown to be helpful in inhibiting enzyme degradation in aqueous solutions.

Dicarboxylic acids are particularly preferred.

proteases, amylases and cellulases and mixtures thereof.

According to one aspect of the present invention, there is provided a stabilized aqueous enzyme composition which includes: water; an enzyme selected from the group consisting of amylase, protease, cellulase and mixtures thereof; and an ionic compound which comprises a cation other than calcium, boron, manganese, magnesium, strontium, or zinc, the cation having an effective nuclear charge greater than 2.6.

According to a further aspect of the invention, a stabilized composition includes: water; an enzyme which is a protease; an additive which is a source of oxygen-anion in aqueous solution; and a salt of a metallic element selected from the group consisting of cerium, yttrium, ytterbium, lanthanum, and mixtures thereof.

According to another aspect of the invention, a liquid detergent concentrate includes: from about 10 to about 70 percent by weight, based on the total concentrate weight, of water; from about 0.01 to about 2 percent by weight, based on the total concentrate weight, of a salt of a metallic element selected from the group consisting of cerium, yttrium, ytterbium, lanthanum, and mixtures thereof; from about 0.1 to about 4 percent by weight, based on the total concentrate weight, of an I O l\WINWORDUENNIFERWNSNODELETEP74883

DOC

*i l C C)

C

C.

0e additive which serves as a source of an oxygen-anion in aqueous solution; from about 8 to about 40 percent by weight, based on the total concentrate weight, of a surfactant component which comprises at least one nonionic surfactant; and an enzyme which is a protease, the enzyme being present in at least 10 ppm by weight, based on the total concentrate weight.

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 1.0 to about percent by weight, ha3ed on the total concentrate weight, of the additive which is a polycarboxylic acid salt or short chain alcohol, from about 8 to about 40 percent by weight, based on the 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 pH range of the detergent composition in accordance herewith is from about 6.0 to about 9.5. A particularly preferred pH range is from about 6.0 to about For a more complete understanding of the present invention, reference is made to the following detailed description and accompanying examples.

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 the practice of the present invention, that the effective nuclear charge of a cationic enzyme stabilizing agent is important. The present invention provides a stabilized aqueous enzyme composition, which, includes: CC C*

C

0S S S .C water; an enzyme selected from the group consisting of amylases, proteases, cellulases and mixtures thereof; and, an ionic compound which comprises a cation other than calcium, boron, manganese, magnesium, strontium or zinc, 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 percent to about 70 percent by weight, based on the total composition weight.

00 0° 0* *0 9 9 9 WO 95/06101 PCT/CA94/00463 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, mixtures of proteases or 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 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.OL, or in a slurry called 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 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 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.

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 fall within the above-identified range and which are suitable in the practice of the present invention are strontium, cerium, yttrium, ytterbium, lanthanum, and WO 95/06101 PCT/CA94/00463 6 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 chlorides), oxides, acetates, malates, 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 (Slater) Atomic Symbol Number Test Charge Results Element 2.20 Cesium Potassium Rubidium Sodium 2.55 Carbon 2.58 Sulfur 2.60 Boron 2.85 Barium Calcium Cerium Dysprosium Erbium Europium Gedoliniuni Lanthanum Magnesium Neodymium Praseodymium Promethium Samarium Strontium Terbium Thulium Ytterbium 3.00 Lutetium Scandium Yttrium 3.15 Hafnium Titanium Zirconium 3.30 Niobium Tantalium Vanadium 3.45 Chromium 3.50 Aluminium 3.60 Manganese 3.75 Iron 3.90 Cobalt 4.05 Nickel

N-

0+

N+

0+ 0- ~Rarna~ars~a~ -41 C1 I- WO 95/06101 PCT/CA94/00463 7 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 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 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 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 1 to 5 carbon atoms are preferred. Representative carboxylic acids are the dicarboxylic acids, including maleic acid, malonic acid, succinic acid, glutaric acid, 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 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 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 containing compounds in accordance with the present invention.

a C~ a ~aa~PP-~cll~Pl~a~Ywrrs V/O 95/06101 PCT/CA94/00463 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 bmployed in the practice of the present invention. The anionic surfactant chosen is selected for its ability to solubilize with the chosen salt in an aque,-? medium.

The anionic surfactant employed in the .ice'anttte and composition made therefrom is selected fra ron'a group consisting of alpha-olefin sulfonates, sultonates of ethoxylated linear alcohols, alkyl aryl sulfonates, naphthalene sulfonates and mixtures thereof. The sulfonates of ethoxylated linear alcohols which can be employed in the present invention have the general formula: R- (OCH 2

CH

2 )x S03M wherein x is an integer between about 1 and about 5 and M is an anion selected from the group 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

CH

3

(CH

2 )x 0o H S0 3

M

wherein x is an integer between about 6 and 12 and M is an anion selected from the group consisting of alkali metals, amine cations, ammonium cations and mixtures thereof.

WO 95/06101 PCT/CA94/00463 9 The naphthalene sulfonates which can be employed in the present invention have the general formula: R- SO 3

M

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 are, preferably, selected from the group consisting of C 1 to

C

1 8 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-CH

2

-SO

3

M

wherein M is either an alkali metal, amine cation or ammonium cation as well as mixtures thereof and 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 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 surfactants.

The nonionic surfactants which are advantageously employed in the compositions of the WO 95/06101 PCITCA94/00463 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 with hydrophobic bases to provide a polyoxyalkylene portion having an oxygen/carbon atcm 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 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 being prepared. The exact amount of ethylene oxide condensed with the hydrophobic base will depend upon the chemical characteristics of the base emplo ed and is readily apparent to those of ordinary skill in the art relating to the synthesis of oxyalkylene surfactant 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 1 to 6 carbon atoms and at least one 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 alky? portion and from about 5 to 15 ethenoxy groups in the polyoxyethylene radical. The alkyl substituent on 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

I

VO 95106101 PCT/CA94/00463 11 polyoxyethylene condensates of monoalkyl phenols impart good properties to the compositions of the invention. A typical product corresponds to the formula: CiH 7

(OCFICH

2 ),OCHz 2

C

6

H

Higher polyalkyloxyethylated phenols corresponding to the formula: R' 1 00 0--(CH 2

CH

2 0) -H

R"

wherein R is hydrogen or an alkyl radical having from about 1 to 12 carbon atoms, R' and R" are 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.

Other suitable nonionic surface active agents are 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 hydrophilic oxyalkylene chain in which the oxygen/carbon atom ratio is greater than 0.40.

Polymers of oxyalkylene groups obtained from propylene oxide, butylene oxide, amylene oxide, styrene oxide, mixtures of such oxyalkylene groups with each 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 ethylene oxide, butadiene dioxide, glycidol, mixtures of such oxyalkylene groups with each other and with minor amounts of oxyalkylene groups obtained from propylene WO 95/06101 PCT/CA94/00463 12 oxide, butylene oxide, amylene oxide, and styrene oxide are illustrative of hydrophilic oxyalkylene chains having an oxygen/carbon atom ratio greater than 0.40.

Further suitable nonionic surface active agents 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 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.

Illustrative products 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 aliphatic alcohol and thioalcohol having from about 8 to 22 carbon atoms in the aliphatic portion and about 3 to carbon atoms in the oxyalkylene portion. Typical products are the synthetic fatty alcohol, such as ndecyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, nhexadecyl, n-octadecyl and mixtures thereof condensed with 3 to 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 sequentially with 2 to 20 moles 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 than 0.40 followed by a mixture of propylene and ethylene 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 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 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 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 invention is preferably in a pH range from about 6.0 to about 9.5 and, more preferably, from about 6.0 to about For a more complete understanding of the present invention, the reader is referred to the following examples. In the examples, which are to be construed as illustrative, rather than limitative of the invention, all parts are by weight, absent indications to the contrary.

EXAMPLES A series of five enzyme containing solutions were prepared according to the following formula: Deionized water 9.2 Sodium Formate Propylene Glycol 76.7 Enzyme 10.0 ESPERASE 8.OL sold by Navo Industries.

Into each of these solutions, then, was added 0.1 part by weight of one component selected, in sequential order, from the following list: s^^lWU WO 95/06101 PCICA94/00463 14 Strontium Chloride Zn Chloride Yttrium Oxide Yttrium Chloride 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 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.

I

TABLE I Solution 1 Solution 2 Solution 3 Solution 4 Solution 5 Initial T, 486 516 insoluble 565 391 610 713 x 575 582 Af ter 2 days 49 cC 1435 x x 4709 3787 After 4 days 49 0

C

3408 x x x x After 6 days 49 0

C

5648 x x x 7107 Af ter 1 Month 32-C (RT) 572 x x 567 567 WO 95/06101 PCT/CA9,4/00463 16 EXAMPLES 6-12 A standard stabilized enzyme solution was prepared, as a base line for purposes of comparison, which included calcium ions and formate ions. This 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 under two different conditions; one batch was stored at room temperature and one batch was stored in a "hot box" at 40 0 C. Periodically, the enzyme activity was measured and compared to a previously measured initial value.

The following table, Table II, lists the components used to formulate the experimental solutions, and the observed enzyme activity after 4 days, 8 days and 12 days at 40 0 C and, also, after 11 days at room temperature (approx. 23 0 It will be noted that not all ingredients are used in each example.

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.

~~Ii~W~DII~ o TABLE II Examples 6 7 8 9 10 11 12 Di Water 25.9 85.9 85.9 85.9 85.9 85.9 85.9 Na Formate Na Malonate 4.0 Na Maleate 4.0 Tamol 6-1588 4.0 Ca Chloride 0.1 Ce Chloride 0.1 0.1 0.1 Y Acetate 0.1 0.1 0.1 Prep. Glycol 60.0 Esperase 8.SL 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Initial Activity 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% Act. 3 days 24 0 C 99.6 90.1 87.7 81.5 93.8 85.2 86.4 Act. 10 days 400C 98.2 50.3 48.7 35.2 48.9 44.2 39.4 Act. 21 days 40 0 C 93.0 33.3 30.5 20.3 31.4 24.7 23.6 Act. 30 days 40 0 C 94.7 29.2 28.8 19.6 25.9 23.6 20.7 Act. 31 days 24 0 C 78.9 62.4 60.6 48.9 62.7 56.4 52.9 After 30 days of Std. 40°C 100 62.4 60.6 48.9 62.7 56.4 52.9 After 30 days of Std. at 24 0 C 100 79.1 762.0 79.5 71.5 67.2 \s

J.

b 1 FI -p IIIP Ps e- i WO 95/06101 PCT/CA94/00463 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 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 was stored in a "hot box" at 38 0 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 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 0 C and, also, the observed activity after 25 days at room temperature. It will be noted that not all of the listed ingredients are 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 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: o TABLE III Examples 14 15 16 17 18 19 20 21 22 23 24 Water 30.0 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 Glycol 60.0 11.7 0.0 46.7 0.0 11.7 23.3 35.0 0.0 35.0 70.0 Surfactant A' 0.0 10.6 10.6 10.6 10.6 10.6 10.6 10.6 10.6 10.6 10.6 Surfactant B 2 0.0 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4 Enzyme 3 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Initial Activity 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 38 0 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 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 380C 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 380C 0.75 0.66 0.32 0.68 0.28 0.63 0.76 0.79 0.26 0.62 0.10 After 22 days 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 6 RT 4 0.62 0.75 0.57 0.78 0.53 0.72 0.69 0.73 0.78 0.73 0.31 tu,

%J

I-

TABLE III (Continued) Vxamples 14 15 16 17~ 1& 19 20 21 22 23 24 Remaining (RT) 68.78 82.42 S9.38 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- 0 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 100M0 108.51 70.82 110.74 70.48 108.07 115.82 111.63 109.93 122.26 43.63 A nonionic surfactant sold by Shell as Neodol 91-2.5 A nonionic surfactant sold by Shell as Neodol 23-6.5 A liquid proteolytic enzyme solution which is an alkalophilic variant RT =room temperature approximately 23 0

C

IF

Claims

1. A stabilized aqueous enzyme composition, including: water; an enzyme selected from the group consisting of amylase, protease, cellulase and mixtures thereof; and an ionic compound which comprises a cation other than calcium, boron, manganese, \xr.w% strontium, magnesium or zinc, the cation having an effective nuclear charge greater than 2.6. e.

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

3. The composition of Claim 1, wherein the ionic compound is a salt of a metallic element selected from the group consisting of cerium, yttrium, ytterbium, lanthanum, and mixtures thereof. 00 0

4. The composition of Claim 2, wherein the anion source is selected from the group consisting of a polycarboxylic acid, a short chain alcohol, a S,0 polycarboxylic acid salt, and mixtures thereof. 9 0 9

5. The composition of any one of Claims 1 to 4, further including a nonionic surfactant.

6. A stabilized aqueous enzyme composition including: water; an enzyme which is a protease; an additive which is a source of oxygen-anion in aqueous solution; and a salt of a metallic element selected from the group consisting of strontium, cerium, yttrium, ytterbium, lanthanum, and mixtures thereof.

7. A liquid detergent concentrate including: from about 10 to about 70 percent by weight, based on the total concentrate weight, of water; from about 0.01 to about 2 percent by weight, based on the total concentrate weight, of a salt of a metallic element selected from the group consisting of cerium, yttrium, ytterbium, lanthanum, and mixtures thereof; S* from about 0.1 to about 4 percent by weight, based on the total concentrate weight, of an additive which serves as a source of an oxygen-anion in aqueous solution; from about 8 to about 40 percent by weight, based on the total concentrate weight, of a *surfactant component which comprises at least c.'e nonionic surfactant; and an enzyme which is a protease, the enzyme being present in at least 10 ppm by weight, based on the total concentrate weight.

8. The composition of Claim wherein the anionic source is selected from the group consisting of a polycarboxylic acid, polycarboxylic acid salt, an alcohol having from one to fifteen carbon atoms and mixtures thereof. c I

9. The composition of Cla;c-,i 1, wherein the cation of the ionic compound has an effective nuclear charge in the range of 2.6 to 3.9. A stabilized aqueous enzyme compound substantially as described herein with reference to any one of the non-comparative examples. DATED: 23 April, 1997 PHILLIPS ORMONDE FITZPATRICK Attorneys for: DIVERSEY CORPORATION *R tO o 9 9 9* S 0* 0 *0 509* 04 00 V. S 4A *s J C*0 SDENFRWBNDEEEP483.C INTERNATIONAL SEARCH REPORT International application No. PCT/CA 94/00463 A. CLASSIFICATION OF SUB~JECT MATTERt IPC 6 CllD3/386 According to InternAtonal Patent Ca ifcadon or to both national elssification and [PC B. FIELDS SEARCHED Minimum documentation searched (datsficatinn syltem followed by clusification syraboli) IPC 6 C11D Documentation searched other than minimuim documnentation to the extent that such documents Ane included in the fields searched Electronic data base consulted during the intemornal search (name of data bas and, where practcal, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category' Ciso of documnt with idicationi, where appopriae, of the relevant passages J Relevant to claim No. X TENSIDE, 1,3 vol.8, no.4, 1971, MUNCHEN DEI pages 188 191 SABAS V. VAECK El AL. A 8 see the whole document P,X EP,A,0 588 413 (UNILEVER) 23 March 1994 1 A 8 see claims 6,8 A EP,A,0 378 262 (THE PROCTER GAMBLE 1,2,8 COMPANY) 18 July 1990 see claims 1,8,9 US,A,5 275 753 (BUZZACCARINI ET AL.) cited in the application [J Further documents are listed in the cooti-adon of box C.M Patent family memrbers are listed in sn= *Special categories of cited documents. t late docuament published afte the inenational filin; date A doumet deinig th geeralstae of~ ~wh~c r eort date and not in conflict with the apealication but cniered to be of particular relevance Inetiound adtepicpeo hoyudryn h W earlier document but published on or after the ineratonal W document of patcua e ;e the dcla d invention fiing date cannot be ooc r nVel or cannot be consdered to L document which may throw doubts on priority claim(s) or involve an inventive step when the document is taken aloae which is cited to establish the puhlication date of another document of particular relevance; the climed invention citchion or other special reason (as 5p~edc) cannot be conidered to involve an inveniAve step when the document referring to an oral disdocure, use. exhibition or document is combined with one or morv Other such docu. other nmn ments, such combhination being obMotta to a Person z&Jle PI document published pior to the international Mfing diate bu in the art. la ths the priority daue claimed W& document mnember of the samne patent family Date of the acuWa completion of the international search Date o f mailing of Wheinernational search report 13 December 1994 13, 01. Namc and mailing address of the ISA Authorized offcer Europecan Patent Office, P.B. 58 SPatentlinm 2 NI, 222 UH V Rijawjk Tel. +31.70) 340-2040, T2. 31 651 epo al, Vn Bl1ign L ~~Fax 31.70) 340-3016Va 0 ign I Farm PTIA20 ($*agi thaGl (July 19r" f ,t INrrmRNArJONAL SE~ARICH IUI0POWI frumadon on patent hfAmly zr~abcm international1 application No. PCT/CA 94/00463 Patent document I PbiaonPatent family I Publication cited in gearch report daemember(s) dat EP-A-0588413 23-03-94 CA-A- 2105703 16-03-94 EP-A-0378262 18-07-90 US-A-5275753 04-01-94 itV-B- 640358 AU-A- 4786790 DE-D- 69004809 DE-T- 69004809 JP-A- 3197597 TR-A- 25129 US-A- 5275753 640358 AU-A- 4786790 DE-D- 69004809 DE-T- 69004809 EP-A- 0378262 JP-A- 3197597 TR-A- 25129

26-08-93 19-07-90 13-01-94

30-06-94 28-08-91 01-11-92 04-01-94 26-08-93 19-07-90 13-01-94 30-06-94 18-07-90 28-08-91 01-11-92 w w- Fezm PCT/ASAIX 0 (pdot feUY anaz) (JUly IM9)