US3557002A

US3557002A - Stabilized aqueous enzyme preparation

Info

Publication number: US3557002A
Application number: US683196A
Authority: US
Inventors: Charles Bruce Mccarty
Original assignee: Procter and Gamble Co
Current assignee: Procter and Gamble Co
Priority date: 1967-11-15
Filing date: 1967-11-15
Publication date: 1971-01-19
Anticipated expiration: 1988-01-19
Also published as: FI49179B; DE1808834C3; SE360675B; FI49179C; FR1599043A; DE1808834A1; DK132030B; BE723873A; NO128370B; NL162690B; DK132030C; AT314450B; NL162690C; GB1242596A; NL6816356A; DE1808834B2; CH534702A

Abstract

AQUEOUS ENZYME PREPARATION STABILIZED WITH A SHORT ALKYL CHAIN MONOHYDROXY ALCOHOL OR AN ALKOXY SHORT ALKYL CHAIN MONOHYDROXY ALCOHOL.

Description

3,55ZQQ2 .FM. 19, 1971 c. B. M CARTY STABILIZED AQUEOUS ENZYME PREPARATION Filed Nov. 15, 1967 O 1 is M n G h M M J 0 2 M e g A .m M .m n H O b D. 0 o S W '0 0 .5 n o w n m H A Du E 5 .0 O O O O O O O Q 0 8 7 6 5 4 3 2 .l

:09 8 2w .3 m :13 53:04 252m o oEwm 33 ATTORNEYS United States Patent STABILIZED AQUEOUS ENZYME PREPARATION Charles Bruce McCarty, Cincinnati, Ohio, assignor to The Procter & Gamble Company, Cincinnati, Ohio, a corporation of Ohio Filed Nov. 15, 1967, Ser. No. 683,196 Int. Cl. C11d 7/50 U.S. Cl. 252--89 7 Claims ABSTRACT OF THE DISCLOSURE Aqueous enzyme preparation stabilized with a short alkyl chain monohydroxy alcohol or an alkoxy short alkyl chain monohydroxy alcohol.

FIELD OF THE INVENTION The present invention relates to an aqueous enzyme preparation containing water, specific enzymes, short alkyl chain monohydroxy alcohols and/or alkoxy short alkyl chain monohydroxy alcohols and, as optional ingredients, nonionic or zwitterionic detergents. More particularly, the alcohols and alkoxy alcohols are added as stabilizing agents to aqueous solutions of enzymes. Addition of a nonionic or zwitterionic detergent enhances the stabilizing effect of the alcohols and the alkoxy alcohols and adds desirable surface active properties to the preparation.

PRIOR ART Enzymes are generally sold commercially in a dry powdered form. The powdered enzyme preparations must be protected from high temperatures and high relative humidities or the enzymes will quickly be degraded and/or deactivated. See, for example, Alcalase, an industrial bulletin published by Novo Industri A/ S, Copenhagen, Denmark. Aqueous enzyme preparations are not generally available because the enzymes are degraded and/or deactivated quickly in an aqueous environment.

lSome attempts have been made to stabilize enzymes in aqueous solutions. Cayle, U.S. Pat. 3,296,094, issued Jan. 3, 1967, utilizes partially hydrolyzed and solubilized collagen and glycerol to stabilize aqueous solutions of proteolytic enzymes. This method of stabilizing the enzymes, however, requires large quantities of glycerol, e.g., 35% to 60% by weight of the total solution, and, therefore, adds significantly to the cost of the enzyme solution.

Meister, U.S. Pat. 3,095,358, issued June 25, 1963, utilizes sorbitol to stabilize aqueous solutions containing enzymes such as papain and mixtures of protease and amylase obtained from Bacillus Subtilis. This method, too, requires large amounts (65% to 90% by weight of an 80% sorbitol solution) of the stabilizing agent, sorbitol. The large volume of sorbitol required to stabilize the enzymes in aqueous solutions acts as a diluent and adds significantly to the cost of the preparation.

These two prior art attempts are illustrative of the major problems confronted in attempts to stabilize aqueous solutions of enzymes: (1) expense; (2) vast dilution of the aqueous product with a stabilizing agent.

SUMMARY OF THE INVENTION The stabilized aqueous enzyme preparation of this invention masters the problems presented by the prior art. Less than 30% of the enzyme preparation is comprised of the stabilizing agent, i.e., short alkyl chain monohydroxy alcohols, and/or alkoxy short alkyl chain monohydroxy alcohols and the stabilizing agents are readily available at reasonable prices. The alcohols and ice alkoxy alcohols of this invention possess useful solvent properties and are, therefore, especially desirable herein when this preparation is used in detergent applications. The alcohols, e.g., ethanol, are also excellent astringents and thus serve a valuable function when the preparation of this invention is utilized as a mouthwash. Addition of the optional nonionic or zwitterionic detergent components enhances the stability of the enzymes in the aqueous preparation and enhances the detergent capabilities of this preparation.

The stabilized aqueous enzyme preparation of this invention comprises:

(1) From 65% to 97% water;

(2) From 0.001% to 1.0% enzymes selected from the group consisting of proteases and a-amylases;

(3) From 2% to 27% of a stabilizing agent selected from the group consisting of monohydroxy alcohols containing from 1 to 4 carbon atoms and alkoxy monohydroxy alcohols having the general formula wherein (R is an alkyl or alkoxy alkyl group which contains from 1 to 8 carbon atoms and from O to 1 ether linkages and [R is an alkylene group which contains from 1 to 4 carbon atoms; and

(4) From 0% to 15% of a detergent selected from the group consisting of nonionic detergents and zwitterionic detergents.

THE DRAWING FIG. 1 illustrates the stabilizing effects of various concentrations of the stabilizing agents of this invention in an aqueous solution containing 1% Alcalase (6% crystalline enzyme) and 5% ethoxylated tallow alcohol (1 mole of tallow alcohol ethoxylated with 30 moles of ethylene oxide) maintained at a pH of 7.0. -It is readily apparent that enzyme stability can be optimized by uitlizing the stabilizing agents of this invention in the preferred use ranges. It is also apparent that the preferred stabilizing agents, i.e., methanol, ethanol and isopropanol, can be utilized to maximize enzyme stability over long storage periods.

DETAILS AND DESCRIPTION OF THE INVENTION Granular detergent compositions containing enzymes have been used in Europe for several years and have recently been introduced into the United States. These enzyme-containing granular detergent compositions are particularly effective in removing stains from fabrics, household fixtures, floors and walls. These granular detergent compositions, however, are cumbersome to use in minor cleaning applications such as spot removing.

The above described disadvantages are overcome with the preparation of this invention. The aqueous enzyme preparation of this invention is convenient to use as a spot remover, detergent additive, detergent or mouthwash. In addition, the enzymes in the preparation of this invention are stable over long storage periods.

This preparation comprises three major essential components: water, enzymes and stabilizing agents. Nonionic or zwitterionic detergents can be added to this preparation as optional components. These components and the amounts in which they are utilized herein are discussed below.

Water comprises the major portion of the preparation of this invention and is generally utilized in amounts ranging from about 65 to about 97% by weight of the preparation and is preferably utilized in amounts ranging from 72% to Deionized water is preferred, although not mandatory for use herein.

Enzymes which are suitable for use herein and which are stabilized in aqueous solution by the stabilizing agents discussed hereinafter include the alkaline proteases, neutral proteases, acid proteases and the u-amylases. Proteases in these classifications are generally derived from fungal and bacterial sources. Enzymes derived from plant and animal sources can also be utilized herein but are not as readily classified in the above alkaline, neutral and acid subclasses. These enzymes are active in the pH range of from about 3 to about 11 and at temperatures ranging from about 40 F. to about 170 F. Optimum activity of these proteases is generally exhibited in the pH range of from about 5.0 to and preferably from 6.0 to 9.5. The proteases are particularly effective in degrading protein soil. The proteases catalyze the hydrolysis of the peptide linkage of proteins, polypeptides and related compounds. Free amino and carboxy groups are thus obtained and the long chain protein Structures are reduced to several shorter chains. These shorter chains can easily be removed from their environment with water or aqueous detergent compositions.

The alkaline proteases are particularly preferred en zymes for use herein. Alkaline proteases which are suitable for use in this invention include subtilisin, BPN, elastase, keratinase, carboxypeptidase, amino peptidase, aspergillopeptidase A and aspergillopeptidase B. Subtilisin and BPN' are especially preferred for use herein. The alkaline proteases are particularly preferred for use in this invention as they show optimum activity in the pH range of normal detergent components, i.e., 7.5 to 10.5, and the alkaline proteases show surprising stability in the preparation of this invention.

The neutral proteases which can also be utilized in the preparation of this invention include collagenase, chymotrypsin and trypsin and those proteolytic enzymes isolated from streptomyces species. Both chymotrypsin and trypsin show optimum activity in the neutral to alkaline range.

Examples of acid proteases suitable for use herein include pepsin, papain and bromelin. Both papain and bromelin show optimum activity in the acid to neutral range.

The a-amylases are also stabilized in the preparation of this invention. All of the a-amylases show optimum activity in the acid range. The a-amylases are particularly well suited for breaking down starch molecules as they attack the u -glycosidic linkages in starch. The remaining shorter chains are easily removed from their environment with water or aqueous solutions of detergents. The a-amylases may be obtained from animal sources, cereal grains, bacterial or fungal sources.

Commercial enzyme compositions containing the abovedescribed enzymes are suitable for use herein. These commercial enzyme compositions are generally sold in a dry, powdered form comprised of from about 2% to about 80% active enzymes in combination with an inert powdered vehicle such as sodium or calcium sulfate or sodium chloride as the remaining to 98%. The active enzyme content of commercial enzyme compositions is the result of manufacturing methods employed and is not critical herein so long as the finished preparation of this invention has the specified enzyme content. The insoluble inert materials are generally removed from the preparation of this invention to provide a preparation with good clarity which is free of precipitates.

Specific examples of commercial enzyme compositions suitable for use herein include: Alcalase; Maxatase. Protease B-4000 and Protease AP; CRD-Protease; Viokase; Pronase-P, Pronase-E, Pronase-AS and Pronase-AF; Bioprase; Rapidase P-2000; Takamine; Bromelain 1:10; HT proteolytic enzyme 200; Enzyme L-W; Miles a-amylase'; Rhozyme P-11 concentrate; Pectinol; Rhozyme PF; Rhozyme J-; (Rhozyme PF and .T-25 have salt and cornstarch vehicles and are' proteases having diastase activity); Amprozyme 200; and Wallerstein 627-P.

CRD-Protease (also known as Monsanto DA-10) is a 4 useful powdered enzyme composition. CRD-Protease is reported to be obtained by mutation of a Bacillus subtilis organism. It is comprised of neutral and alkaline proteases and a-amylases. The neutral protease has a molecular weight of about 44,000 and contains from 1 to 2 atoms of zinc per molecule. The CRD-Protease can be used in aqueous systems such as the present invention. The active enzyme content of CRD-Protease on a weight percent basis generally ranges from about 20% to about 75%.

Pronase-P, Pronase-E, Pronase-AS and Pronase-AF are powdered enzyme compositions which can also be used to advantage in this invention. These enzymes are produced from the culture broth of Streptomyces griseus used for streptomycin manufacture. They are isolated by a successive resin column treatment. The major component of pronase is a neutral protease, Streptomyces Griseus protease. This enzyme composition contains a calcium stabilizer salt and is fairly stable over a wide pH range, e.g., 4 to 10, and is fairly stable over a temperature range of 50 F. to 150 F.

Another enzyme composition preferred for use in the preparation of this invention is Alcalase which is a proteolytic enzyme preparation manufactured by submerged fermentation of. a special strain of Bacillus subtilis. The primary enzyme component of Alcalase is subtilisin. In addition to proteases, Alcalase contains small amounts of a-amylase. Alcalase is a fine grayish free-flowing powder having a crystalline active enzyme content of about 6%. The remainder of the powder is comprised primarily of sodium sulfate, calcium sulfate and various inert organic vehicle materials. Alcalase has unusually stable properties in the aqueous preparation of this invention.

Biophase is a powdered enzyme composition which contains alkaline proteases (BPN) and a-amylases. This enzyme composition can be obtained with or without the presence of diluents such as sodium and calcium sulfate.

Large variations in the amount of enzymes in the prep aration of this invention are contemplated. The preparation can contain from about 0.001% to about 1% enzymes by weight of the preparation. For best results, the preparation preferably contains from 0.01% to about 0.5% enzymes by weight of the preparation. When one of the preferred enzyme compositions is utilized herein, the preparation of this invention preferably contains from about 0.1% to about 4.0% of the enzyme composition as it is sold in commercial form, e.g., from about 2% to about active enzymes. The active enzyme content of the aqueous enzyme composition of this invention should, in any event, range between 0.001% and 1% as above delineated.

The stabilizing agents which stabilize the enzymes described above are selected from the group consisting of monohydroxy alcohols containing from 1 to 4 carbon atoms and alkoxy monohydroxy alcohols having the general formula wherein R contains from 1 to about 8 carbon atoms and from 0 to 1 ether linkages and R contains from 1 to 4 carbon atoms. Specific examples of these stabilizing agents include methanol, ethanol, propanol, isopropanol, butanol, isobutanol, methoxy methanol, Z-methoxy ethanol, 3- methoxy propanol, Z-methoxy propanol, ethoxy methanol, Z-ethoxy ethanol, 3-ethoxy propanol, 2-ethoxy propanol, propoxy methanol, 2-propoxy ethanol, 3-propoxy propanol, 2-propoxy propanol, butoxy methanol, Z-butoxy ethanol, 3-butoxy propanol, 2-butoxy propanol and diethylene glycol monobutyl ether. Mixtures of these stabilizing agents can be utilized to advantage in this preparation. Preferred stabilizing agents for use herein are methanol, ethanol, isopropanol, propanol, 3-propoxy propanol and diethylene glycol monobutyl ether. Methanol, ethanol and isopropanol are particularly preferred for use herein. Methanol and the methanol derived stabilizing agents, because of their poisonous nature, should not be utilized in aqueous enzyme preparations which may be ingested into the body.

The alcohols and alkoxy alcohols described above can be utilized in the preparation of this invention in effective amounts ranging from about 2% to about 27% by weight of the preparation. However, to obtain optimum stabilizing effects for long storage periods at high temperatures, the stabilizing agents should be utilized in the use ranges described below. The preferred use ranges set forth below delineate amounts of methanol, ethanol and isopropanol which must be present in this aqueous enzyme preparation to maintain about 50% enzyme activity after storage at 100 F. for five weeks.

TABLE I Use range in Preferred use weight percent range in of the weight percent enzyme of the enzyme preparation preparation Stabilizing agent Water-soluble nonionic and zwitterionic detergents can be utilized, as optional ingredients, in the preparation of this invention. These detergents enhance the storage stability of the enzymes utilized herein and significantly improve the detergent characteristics of the preparation. Because of these useful characteristics, it is preferred to include nonionic and zwitterionic detergents in the aqueous enzyme preparation of this invention. The nonionics and zwitterionics can be utilized herein in amounts ranging from about to about 15%, preferably from 4% to 10%, by weight of the enzyme preparation.

Examples of suitable nonionics for use herein include:

(1) The polyethylene oxide condensates of alkyl phenols, e.g., the condensation products of alkyl phenols having an alkyl group containing from about 6 to 12 carbon atoms in either a straight chain or branched chain configuration with ethylene oxide, the said ethylene oxide being present in amounts equal to to 25 moles of ethylene oxide per mole of alkyl phenol. The alkyl substituent in such compounds may be derived from polymerized propylene, diisobutylene, octene or nonene, for example.

(2) Those nonionic synthetic detergents derived from the condensation of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylene diamine. For example, compounds containing from about 40% to about 80% polyoxyethylene by weight and having a molecular weight of from about 5,000 to about 11,000 resulting from the reaction of ethylene oxide groups with a hydrophobic base constituted of the reaction product of ethylene diamine and excess propylene oxide, said base having a molecular weight of the order of 2,500 to 3,000 are satisfactory.

(3) The condensation product of 1 mole of aliphatic alcohols having from 8 to 22 carbon atoms, in either straight chain or branched chain configuration, with from 5 to 40 moles of ethylene oxide, e.g., a coconut alcoholethylene oxide condensate having from 5 to 40 moles of ethylene oxide per mole of coconut alcohol, the coconut alcohol fraction having from 10 to 14 carbon atoms.

(4) The unsubstituted amides and the monoethanol (5) Long chain tertiary amine oxides corresponding to the following general formula wherein R is an alkyl radical of from about 8 to about 22 carbon atoms, R and R are each methyl, ethyl or hydroxyethyl radicals, R is ethylene, and n equals from 0 to about 10. The arrow in the formula is a conventional representation of a semipolar bond. Specific examples of amine oxide detergents include: dimethyldodecylamine oxide and bis-(2-hydroxyethyl)-dodecylamine oxide.

(6) Long chain tertiary phosphine oxides corresponding to the following general formula RRRP O wherein R is an alkyl, alkenyl or monohydroxyalkyl radical ranging from 10 to 22 carbon atoms in chain length and R' and R" are each alkyl or monohydroxyalkyl groups containing from 1 to 3 carbon atoms. The arrow in the formula is a conventional representation of a semipolar bond. Examples of suitable phosphine oxides are found in US. Pat. 3,304,263, which issued Feb. 14, 1967, and include: dimethyldodecylphosphine oxide and bis-(2- hydroxyethyl) -dodecylphosphine oxide.

(7) Long chain sulfoxides having the formula wherein R is an alkyl radical containing from about 10 to about 22 carbon atoms, from 0 to about 5 ether linkages and from 0 to about 2 hydroxyl substituents, at least one moiety of R being an alkyl radical containing 0 ether linkages and containing from about 10 to about 18 carbon atoms, and wherein R is an alkyl radical containing from 1 to 3 carbon atoms and from one to two hydroxyl groups. Specific examples of these sulfoxides are: dodecyl methyl sulfoxide and 3-hydroxy tridecyl methyl sulfoxide.

The zwitterionic synthetic detergents suitable for use herein can be broadly described as derivatives of aliphatic quaternary ammonium, phosphonium and sulfonium compounds, in which the aliphatic radical may be straight chain or branched, and wherein one of the aliphatic substituents contains from about 8 to 22 carbon atoms and one contains an anionic water solubilizing group, e.g., carboxy, sulfo, sulfato, phosphato or phosphono. Examples of compounds falling within this definition are 3-(N,N-dimethyl-N-hexadecylammonio) propane-l-sulfomate and 3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy prapane-l-sulfonate. For more examples of zwitterionic synthetic detergents, see Diehl and Smith, Laundering Fabrics in Cold Water Containing a Synthetic Detergent Composition, Canadian Patent 708,147, issued Apr. 20, 1965 at page 6, lines 1-2. This disclosure is specifically incorporated herein by reference.

Mixtures of various nonionic detergents or mixtures of nonionic detergents and zwitterionic detergents can be utilized to advantage herein.

The various components of the enzyme preparation of this invention can be mixed together in any order. However, it is preferred that the alcohol-water mixture be prepared first and the enzymes added thereto to prevent any degradation or deactivation in solutions predominately consisting of either water or alcohol. The optional detergent components can be added at any time.

The pH of the stabilized aqueous enzyme preparation of this invention generally ranges from about 5.0 to 10.0 and preferably ranges from about 6.5 to about 8.5. Maximum stabilizing effects are obtained in the preferred pH range. The pH can be raised with a base, e.g., sodium or potassium hydroxide, or lowered with an acid, e.g., hydrochloric acid.

It is also preferred, although not mandatory, that a preservative be added to this preparation to prevent bacterial and fungal growth. One particularly good preservative is phenyl mercuric acetate which is generally utilized herein in amounts ranging from about 10 to about 40 parts per million of the preparation. Any preservative compatible with the components of this preparation can be utilized herein.

8 can be utilized as the sole detergent in a washing operation.

This preparation is also suitable for use as a mouthwash. The alcohol stabilizing agent, preferably ethanol, acts as an astringent while the enzymes are effective in 5 removing dental plaque, removing food particles from the mo 'n mu h UTILITY OF THE ENZYME PREPARATION Igliglglitylal crevice and re V1 g cous coatings fr m t e EXAMPLES The enzyme preparatlon of this invention contaimng Water, stabilizing agents, enzymes and, optionally non- 10 The following examples merely serve to illustrate the ionic and/or zwitterionic detergents, can be utilized as a invention in specific detail and when read in conjunction spot remover, detergent additive or as a detergent comwith the foregoing description will aid in determining the position, This preparation can be packaged in a sprayfull scope of the present invention. The examples are type bottle and conveniently used to remove relatively merely illustrative and are not intended to restrict this small spots from fabrics or it can also be utilized in larger 0 invention. All parts, percentages and ratios set forth herequantities as a detergent additive. This preparation can in are by weight unless otherwise indicated. be substituted for hypochlorite bleaches as it removes The following preparations (Examples l29) were premany of the stains which these bleaches remove and, as pared and stored in closed glass bottles for the indicated an added advantage, does not attack or degrade fluo- 20 lengths of time. Examples 1, 2 and 24 are not examples rescers and whiteners. With the addition of the optional of this invention but are inserted herein only for comnonionic and/or zwitterionic detergents, this preparation parative purposes.

Percent by weight 1 Percent remaining activity 1 50 F. storage,

weeks 80 F. storage, weeks 100 F. storage, weeks Example Stabilizing agents 'IAEa Alcalase Water 3 8 3 6 8 16 2 3 8 30% methanoL. 5% isopropanol.-. 10% isopropanol isopropanol 4% propanol 7% propanol 2% normal butanol 4% normal bntanol UiOcnHmimvvbrmcncnmmmmmmumu HHHHHl-H-Hl-HHHHHHHHHHHHHH See footnotes at bottom of following table.

Percent by weight Percent remaining activity, storage 80 F., weeks Alcal- 100 F.,

Example Stabilizing agents HAPS 8 ase Water 3 8 12 4 weeks i 3. 1 12% diethylene glycol monobutyl ether- 3. 25 1 12% 3-propoxy propanol 3. 25 1 3% ethylene glycol monobntyl ether; 3% diethylenc glycol monobutyl ether 3. 3g 1 29 10% 3-propoxy propai 3.25 1

1 30 parts per million of phenyl mercuric acetate was added to each preparation to retard bacterial and fungal growth. The pH of each of the preparations in Examples 1 to 29 is maintained at 7.0.

9 Percent remaining activity was determined by the Azocoll method. The Azoeoll method is based on the release of a water-soluble dye from a water-insoluble proteimdye substrate (Azocoll) by a proteolytic enzyme. The amount of dye released under carefully controlled conditions is measured spectrophotometrieally. Enzymatic activity is calculated from the amount of dye released. Initial activity equals 3 1 mole of tallow alcohol cthoxylated with 30 moles of ethylene oxide. 4 A commercial proteolytic enzyme preparation having a crystalline enzyme content of about 6%.

5 22 Week data. 0 12 week data. 1 4 week data.

a 3-(N,N-dimethyl-N-alkylannnonio)-2-hydroxy propane-l-sulfonate wherein the alkyl group is derived from middle-cut coconut alcohol: 2% C10, 66% C12, 23% C14 and 9% On.

As is readily apparent from Example 2, proteases (Alcalase) degrade and/or deactivate rapidly in aqueous solution when no stabilizing agent is present. After three weeks storage at 100 F., nearly 90% of the enzyme activity was lost. Less enzyme activity was lost at the milder 10 during high temperature (100 F.) storage and during lower'temperature (50 F.) storage. All perrorm well as spot removers, detergent additives and as detergents per se. The nonionic (TAEg and the zwitterionic (HAPS) additives enhanced storage stability in each case.

PERCENT BY WEIGHT Stabilizing agent Nonionic or Enzyme Zwitterionic Isopro- B-propoxy Example Ethanol panol Propanol 'IAE '3 1 HAPS 2 Alcalase 3 Amylase 4 Water 5 1 Tallow alcohol ethoxylated with moles of ethylene oxide. 2 3-(N,N-dimethyl-N-alkylammonio-Z-hydroxy propane-l-sulfonate. (See footnote 5, column 8.)

3 See Examples 1 through 29.

4 Wallerstein bacterial a-arnylase, lot number 4546A. 5 Each preparation in Examples 30-77 contains 30 parts per million of phenyl mercuric acetate. The pH of these preparations was maintained at 7.0.

conditions, i.e., F. and 80 F., but the loss was very substantial even at these mild conditions.

The addition of a nonionic detergent, TAE to the aqueous enzyme solution (Example 1) impedes degradation and/or deactivation of the enzymes at low storage temperatures; but, at high storage temperatures, the nonionic detergent appears to increase the rate of degradation and/or deactivation of the enzymes.

The addition of the stabilizing agents of this invention in Examples 3 through 23 and 25 through 29 universally results in increased enzyme stability at storage conditions of 50 F. However, to obtain significantly increased enzyme stability at higher temperatures, the preferred stabilizing agents, i.e., methanol, ethanol, propanol, isopropanol, 3-propoxy propanol or diethylene glycol monobutyl ether must be used in the hereinbefore described use ranges. Examples 4, 5, 9, 11, 12 and 13 illustrate the preferred stabilizing effect of the particularly preferred stabilizing agents, methanol, ethanol and isopropanol, at high temperatures (eight weeks at 100 F. storage).

The following stabilized aqueous enzyme preparations (Examples 30-77) were prepared. In each example, the water and stabilizing agent were thoroughly mixed, the nonionic or zwitterionic was added and the enzymes were added last. The enzymes were stabilized in each example EXAMPLE 78 A stabilized aqueous enzyme preparation was formulated according to this invention from the following com- The pH of this preparation was adjusted to 7.0 with so dium hydroxide.

This preparation was utilized in the series of stain removal and washing tests set forth below. Similarly soiled swatches were washed in aqueous solutions containing the equivalent of 1% cups of a commercial detergent composition (Tide) in 16 gallons of 7 grain per gallon hardness water at F. for a 10 minute period.

The preparation of this invention was used as is, i.e., no dilution, in the 5 minute, 30 minute and 3 hour soaks. From about one-half ml. to about one ml. of this preparation was sprayed directly onto each stained or soiled area of approximately to square centimeters. The soiled swatches were retained for the above designated times and then washed as described above in the commercial detergent composition.

This preparation was also utilized as an additive to amino peptidase, aspergillopeptidase A, aspergillopeptidase B, collagenase, chymotrypsin, trypsin, pepsin, papain, bromelin, Maxatase, Protease B-4000, Protease AP, Alcalase, CRD Protease, Viokase, Pronase-P, Pronase-E, Pronase-AS, Pronase-AF, Bioprase Rapidase P-2000,

the commercial detergent composition. About 1.2 ml. of 5 Takamine, HT Proteolytic Enzyme 2000, Enzyme L-W, the preparation of this invention was utilized per gallon Rhozyme P-11 concentrate, Pectinol, Rhozyme PF, of wash water in these washes. The results are summarized Rhozyme J-25 and Amprozyme 200. below. Results substantially similar to those in the previous EXAMPLE 7:;

Tidc

plus 5 min. 30 min. 3 hr. Pospreparasoaksoaksoaksible Tide tion Tide Tide Tide Stain score wash wash wash wash wash Protein 80 30 49 54 61 68 Pigment 35 12 19 21 21 T-shirt plus pillowcase 4 8 10 10 14 Synthetic soil 10 2 1 5 5 7 EMPA blood 112 10 5 e s 10 9 French dressing 5 3 4 4 4 4 Other stains 12 16 17 19 22 Cotton tracer 5 1 2 2 2 3 Shirt collars u 5 1 3 4 2 Total scores 195 70 101 122 136 150 l Naturally soiled T-shirt and pillowcase swatches. 2 Special synthetically soiled swatches.

3 Blood soaked swatches obtained from the Swiss Testing House in St Gallen, Switzerland (Eidgenossiche Material Prufungsund, Mersuchsanstant Fur Iudustrie, Banevcscn und The possible score for protein is derived as follows. Sixteen separate and ditferent protein stains are applied to each protein test swatch. Each stain, after the wash cycle, is graded visually on a 0 to 5 scale. A grade of 5 indicates complete removal (the possible score) while 0 indicates no removal. Grades between 0 and 5 indicate intermediate partial removal. The possible protein score of 80 is derived by multiplying the 16 stains by 5 possible points per stain. The possible scores for the other stains were calculated in the same manner. The total scores are indicative of broad spectrum cleaning capabilities.

The addition of the preparation of this invention to the wash water containing the commercial detergent increase the score (Tide+Preparation Wash) over the detergent wash (Tide Wash) by about 40% which in dicates a significant cleaning advantage. By utilizing a 5 minute soak, the total score (5 Min. Soak-Tide Wash) is increased by 70% over the Tide wash score while a minute or 3 hour soak increases the total score by nearly 100% over the Tide wash score.

EXAMPLE 79 A mouthwash preparation is formulated from the following components:

Components: Weight percent Ethanol 16.50

Water 79. 10 Monsanto 0RD Protease (DA-10) (34% enzyme) 2.00 Flavor, dyes and sweeteners 2.15 Sodium hydroxide to adjust pH to 7.0 0.25

The enzymes are stable over long storage periods at relatively high temperatures. The composition is useful in removing dental plaque from the teeth, food particles from the gingival crevice and mucous coatings in the mouth.

EXAMPLE 80 examples are obtained when the following stabilizing agents are substituted for those utilized in the previous examples in that the enzymes are stabilized in aqueous solution: methanol, ethanol, propanol, isopropanol, butanol, isobutanol, methoxy methanol, Z-methoxy ethanol, 3-methoxy propanol, 2-methoxy propanol, ethoxy methanol, 2-ethoxy ethanol, 3-ethoxy propanol, 2-ethoxy propanol, propoxy methanol, 2-propoxy ethanol, 3-propoxy propanol, 2-propoxy propanol, butoxy methanol, 2-butoxy ethanol, 3+butoxy propanol, 2-butoxy propanol, ethylene glycol monobutyl ether and diethylene glycol monobutyl ether.

Results substantially similar to those in Examples 1 through 78 are obtained when the following nonionic and zwitterionic detergents are substituted for tallow alcohol ethoxylated with 30 moles of ethylene oxide per mole of tallow alcohol and 3-(N,N-dimethyl-N-middlecut-coconut alkylammonio) 2 hydroxypropane-l-sulfonate in that the stabilizing effects of the alcohols and alkoxy alcohols are enhanced: decyl phenol ethoxylated with 20 moles of ethylene oxide per mole of decyl phenol, hexadecanoic amide, hexadecanoic diethanol amide, dimethyldodecylamine oxide, dimethyldodecylphosphine oxide and dodecyl methyl sulfoxide, the condensation product of ethylene oxide with the condensation product of propylene oxide with propylene glycol, the ethylene oxide portion of the compound being 50% of the total weight of the compound and the total molecular weight of the compound being about 1700; the condensation product of ethylene oxide with the condensation product of propylene oxide and ethylene diamine wherein the product contains about 65% polyethylene oxide by weight and the total molecular weight of the compound is 6000.

In Examples 3 through 24 and 26 through 78, the enzymes are stabilized in the aqueous preparation when no nonionic or zwitterionic is present in the aqueous solution.

Results substantially similar to those in Examples 1 through 79 are obtained when the pH of the preparation of this invention is maintained at 6.5, 8.0, and 8.5, in that the enzymes are stabilized for long periods of time. Enzyme stabilization is also obtained in Examples 1 through 79 when the pH of the preparation is maintained at 5.0, 9.0 and 10.0.

The foregoing description of the invention has been 13 presented describing certain operable and preferred embodiments. It is not intended that the invention should be so limited since variations and modifications thereof will be obvious to those skilled in the art, all of which are within the spirit and scope of this invention.

What is claimed is:

1. A stabilized aqueous enzyme preparation consisting essentially of by weight of the preparation:

(1) from 65% to 97% water;

(3) from 2% to 27% of stabilizing agents selected from the group consisting of monohydroxy alcohols containing from 1 to 4 carbon atoms and alkoxy monohydroxy alcohols having the general formula wherein R contains from 1 to 8 carbon atoms and from to 1 ether linkages and R contains from 1 to 4 carbon atoms; and

(4) from 0% to 15% of a detergent selected from the group consistin of nonionic detergents and zwitterionic detergents.

2. The preparation of claim 1 which consisting essentially of:

(1) from 72% to 95% water;

(2) from 0.01% to about 0.5% enzymes;

(3) stabilizing agents selected from the group consisting of methanol, ethanol, isopropanol, propanol, 3- propoxy propanol and diethylene glycol monobutyl ether, wherein the methanol is utilized in amounts ranging from about 16% to about 27%; the ethanol is utilized in amounts ranging from about 3% to about 19%; the propanol is utilized in amounts ranging from about 2% to about 8%; the isopropanol is utilized in amounts ranging from about 4% to about 22%; the propoxy propanol is utilized in amounts ranging from about 8% to 17%, and the diethylene glycol monobutyl ether is utilized in amounts ranging from 8% to 17%; and

(4) from 4% to of a detergent selected from the group consisting of nonionic detergents and zwitterionic detergents.

3. The preparation of claim 1 wherein the pH of the preparation is adjusted to between about 5.0 and 10.0.

4. The preparation of claim 1 wherein the pH of the preparation is adjusted to between 6.5 and 8.5.

5. The preparation of claim 1 wherein the enzymes are alkaline proteases and are selected from the group consisting of subtilisin, BPN', elastase, keratinase, carboxypeptidase, amino peptidase, aspergillopeptidase A and aspergillopeptidase B.

6. The preparation of claim 1 wherein the enzymes are alkaline proteases and are selected from the group consisting of subtilisin and BPN.

7. The preparation of claim I wherein the stabilizing agent is selected from the group consisting of methanol, ethanol and isopropanol; wherein the methanol is utilized in amounts ranging from about 17% to about 25% by weight of the preparation; the ethanol is utilized in amounts ranging from about 6% to about 18% by weight of the preparation; and the isopropanol is utilized in amounts ranging from about 9% to about 20% by weight of the preparation.

References Cited UNITED STATES PATENTS 2,922,749 1/ 1960 Snyder et al. 195-68 3,272,717 9/ 1966 Fukumoto et al. 195-68 3,451,935 6/1969 Roald et al. 252

OTHER REFERENCES Rose: The Condensed Chem. Dict., 6th ed. (1961), Reinhold Publ. Co., p. 882.

Extra Pharmacopoeia, The Pharmaceutical Press, London, 25th ed., pp. 7045, 881.

Microbiology Abstracts, 3A (1967-12) 3, p. 124.

LEON D. ROSDOL, Primary Examiner W. E. SCHULZ, Assistant Examiner U.S. Cl. X.R.

22 3 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. ,002 Dated January 19, 1971 I Charles Bruce McCarty It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Columns 7 and 8, the table at the bottom of the page, Example 27, under the headings of "HAPS and "Water" please change the amounts 3 .35" and "83.75" to read 3.25

and 89.75 --a Column 14, lines 25 and 27, please insert the following references cited in applicant's Statement of Prior Art, dated March 3, 1969: 412,836 10/15/1889 Carnrick 3,098,015 7/16/63 Ayra'pai Column 14, line 35, 36 and '37, please insert the followi references cited in applicant's Statement of Prior Art, dated March 3, 1969: Takemori et a1. Nature, Vol. 215, July 22 J. F. Brandts, Thermobiology, Academic Pres New York (1965) pages 57-62.

Signed and sealed this 2nd day of November 1 971 (SEAL) Attest:

EDWARD M.FLETCI ER,JR. ROBERT GOTTSCHALK Attesting Officer Acting Commissioner of Patents