US3557002A - Stabilized aqueous enzyme preparation - Google Patents

Stabilized aqueous enzyme preparation Download PDF

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US3557002A
US3557002A US683196A US3557002DA US3557002A US 3557002 A US3557002 A US 3557002A US 683196 A US683196 A US 683196A US 3557002D A US3557002D A US 3557002DA US 3557002 A US3557002 A US 3557002A
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preparation
enzyme
enzymes
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propanol
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Charles Bruce Mccarty
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Procter and Gamble Co
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38663—Stabilised liquid enzyme compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S516/00—Colloid systems and wetting agents; subcombinations thereof; processes of
    • Y10S516/01—Wetting, emulsifying, dispersing, or stabilizing agents

Definitions

  • 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
  • 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.
  • 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.
  • This method 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.
  • 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:
  • enzymes selected from the group consisting of proteases and a-amylases
  • 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
  • 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.
  • 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.
  • ethoxylated tallow alcohol 1 mole of tallow alcohol ethoxylated with 30 moles of ethylene oxide
  • the preferred stabilizing agents i.e., methanol, ethanol and isopropanol, can be utilized to maximize enzyme stability over long storage periods.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Alcalase is a proteolytic enzyme preparation manufactured by submerged fermentation of. a special strain of Bacillus subtilis.
  • the primary enzyme component of Alcalase is subtilisin.
  • 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.
  • BPN alkaline proteases
  • the preparation can contain from about 0.001% to about 1% enzymes by weight of the preparation.
  • the preparation preferably contains from 0.01% to about 0.5% enzymes by weight of the preparation.
  • 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.
  • 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.
  • 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.
  • Suitable nonionics for use herein include:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the Azocoll 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%.
  • proteases 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.
  • Examples 3 through 23 and 25 through 29 universally results in increased enzyme stability at storage conditions of 50 F.
  • 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).
  • Examples 30-77 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.
  • 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,
  • Rhozyme J-25 and Amprozyme 200 were summarized. Rhozyme J-25 and Amprozyme 200. below. Results substantially similar to those in the previous EXAMPLE 7:;
  • a mouthwash preparation is formulated from the following components:
  • 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.
  • 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.
  • a stabilized aqueous enzyme preparation consisting essentially of by weight of the preparation:
  • enzymes selected from the group consisting of proteases and a-amylases
  • 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
  • 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
  • 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.
  • 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.

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Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3717550A (en) * 1970-09-25 1973-02-20 Pabst Brewing Co Liquid compositions of bacterial protease and/or amylase and preparation thereof
US3839214A (en) * 1969-06-27 1974-10-01 United States Borax Chem Tetraborate composition
US3855142A (en) * 1971-07-15 1974-12-17 Lever Brothers Ltd Enzymatic denture cleanser
US3860536A (en) * 1970-01-02 1975-01-14 Cpc International Inc Enzyme-detergent combination
US3876563A (en) * 1972-09-22 1975-04-08 Procter & Gamble Liquid detergent compositions
USB458819I5 (de) * 1971-12-23 1976-04-13
DE2740957A1 (de) * 1976-09-13 1978-03-16 Ivan Endre Modrovich Stabilisierte fluessige enzym- und koenzymmassen und verfahren zu ihrer herstellung
US4250254A (en) * 1978-09-11 1981-02-10 Modrovich Ivan Endre Stabilized liquid enzyme and coenzyme compositions
US4271264A (en) * 1976-09-13 1981-06-02 Modrovich Ivan Endre Stabilized liquid enzyme and coenzyme compositions
US4277562A (en) * 1976-09-13 1981-07-07 Modrovich Ivan Endre Stabilized liquid enzyme and coenzyme compositions
US4285276A (en) * 1978-11-15 1981-08-25 Howard A. Fromson Method for printing employing lithographic fountain dampening solution
US4287082A (en) * 1980-02-22 1981-09-01 The Procter & Gamble Company Homogeneous enzyme-containing liquid detergent compositions containing saturated acids
US4305837A (en) * 1980-10-30 1981-12-15 The Procter & Gamble Company Stabilized aqueous enzyme composition
US4310625A (en) * 1976-03-17 1982-01-12 Modrovich Ivan Endre Stabilized liquid enzyme compositions for diagnostic determinations
US4318818A (en) * 1979-11-09 1982-03-09 The Procter & Gamble Company Stabilized aqueous enzyme composition
US4372874A (en) * 1976-09-13 1983-02-08 Modrovich Ivan Endre Stabilization of hydrolysis prone labile organic reagents in liquid media
US4391745A (en) * 1979-03-09 1983-07-05 Diamalt Aktiengesellschaft Desizing agent and process for preparation thereof
EP0130756A1 (de) * 1983-06-24 1985-01-09 Genencor International, Inc. Prokaryotische Carbonyl-Hydrolasen, Verfahren, DNA, Vektoren und transformierte Wirte zu ihrer Herstellung und diese Hydrolasen enthaltende Detergenszusammensetzungen
US4548727A (en) * 1983-10-06 1985-10-22 The Drackett Company Aqueous compositions containing stabilized enzymes
EP0171007A2 (de) * 1984-08-04 1986-02-12 Henkel Kommanditgesellschaft auf Aktien Geschirreinigungsmittel
US4711739A (en) * 1986-12-18 1987-12-08 S. C. Johnson & Son, Inc. Enzyme prespotter composition stabilized with water insoluble polyester or polyether polyol
WO1989003888A1 (en) * 1987-10-28 1989-05-05 Gds Technology, Inc. Test composition and method for the determination of anilides
US4927558A (en) * 1986-11-25 1990-05-22 Novo Industri A/S Proteolytic detergent additive and compositions containing the same
AU604640B2 (en) * 1985-08-03 1991-01-03 Henkel Kommanditgesellschaft Auf Aktien An alkaline protease, a process for the preparation of hybrid vectors and genetically transformed microorganisms
US5030378A (en) * 1990-01-02 1991-07-09 The Procter & Gamble Company Liquid detergents containing anionic surfactant, builder and proteolytic enzyme
US5102422A (en) * 1987-02-13 1992-04-07 Rohm Gmbh Methods for leather processing including liquid enzyme formulation
US5156773A (en) * 1989-12-12 1992-10-20 Novo Nordisk A/S Stabilized enzymatic liquid detergent composition
US5198353A (en) * 1988-02-11 1993-03-30 Novo Nordisk A/S Method for preparing stabilized enzyme dispersion
US5447649A (en) * 1990-03-01 1995-09-05 Novo Nordisk A/S Lipase containing liquid pre-spotter and use of such pre-spotter
US5858117A (en) * 1994-08-31 1999-01-12 Ecolab Inc. Proteolytic enzyme cleaner
US20080124783A1 (en) * 2002-01-16 2008-05-29 Poulose Ayrookaran J Multiply-substituted protease variants
AU2006288018B2 (en) * 2005-09-11 2010-04-01 Peter Alan Royle Hidden fitting locator

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1091174A (en) * 1976-03-17 1980-12-09 Ivan E. Modrovich Stabilized liquid enzyme
FR2520753B1 (fr) * 1982-02-01 1986-01-31 Transgene Sa Nouveaux vecteurs d'expression de la catechol 2,3-oxygenase, enzymes obtenues et leurs applications
MX161813A (es) * 1982-12-13 1990-12-28 Colgate Palmolive Co Mejoras a composicion detergente liquida
DK563986A (da) * 1986-11-25 1988-06-17 Novo Industri As Fremstilling af en lav-temperatur-aktiv protease
DE3927286C2 (de) * 1989-08-18 1997-07-24 Roehm Gmbh Wäßrige Enzym-Flüssigformulierungen
DE4226162A1 (de) * 1991-08-16 1993-02-18 Sandoz Ag Stabiles enzympraeparat
IL117948A0 (en) * 1995-04-18 1996-08-04 Horiuchi Co Ltd Reusable cleaning solutions containing stabilized enzymes

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3839214A (en) * 1969-06-27 1974-10-01 United States Borax Chem Tetraborate composition
US3860536A (en) * 1970-01-02 1975-01-14 Cpc International Inc Enzyme-detergent combination
US3717550A (en) * 1970-09-25 1973-02-20 Pabst Brewing Co Liquid compositions of bacterial protease and/or amylase and preparation thereof
US3855142A (en) * 1971-07-15 1974-12-17 Lever Brothers Ltd Enzymatic denture cleanser
USB458819I5 (de) * 1971-12-23 1976-04-13
US4169817A (en) * 1971-12-23 1979-10-02 Midwest Biochemical Corporation Liquid cleaning composition containing stabilized enzymes
US3876563A (en) * 1972-09-22 1975-04-08 Procter & Gamble Liquid detergent compositions
US4310625A (en) * 1976-03-17 1982-01-12 Modrovich Ivan Endre Stabilized liquid enzyme compositions for diagnostic determinations
US4277562A (en) * 1976-09-13 1981-07-07 Modrovich Ivan Endre Stabilized liquid enzyme and coenzyme compositions
US4271264A (en) * 1976-09-13 1981-06-02 Modrovich Ivan Endre Stabilized liquid enzyme and coenzyme compositions
US4372874A (en) * 1976-09-13 1983-02-08 Modrovich Ivan Endre Stabilization of hydrolysis prone labile organic reagents in liquid media
DE2740957A1 (de) * 1976-09-13 1978-03-16 Ivan Endre Modrovich Stabilisierte fluessige enzym- und koenzymmassen und verfahren zu ihrer herstellung
US4250254A (en) * 1978-09-11 1981-02-10 Modrovich Ivan Endre Stabilized liquid enzyme and coenzyme compositions
US4285276A (en) * 1978-11-15 1981-08-25 Howard A. Fromson Method for printing employing lithographic fountain dampening solution
US4391745A (en) * 1979-03-09 1983-07-05 Diamalt Aktiengesellschaft Desizing agent and process for preparation thereof
US4318818A (en) * 1979-11-09 1982-03-09 The Procter & Gamble Company Stabilized aqueous enzyme composition
US4287082A (en) * 1980-02-22 1981-09-01 The Procter & Gamble Company Homogeneous enzyme-containing liquid detergent compositions containing saturated acids
US4305837A (en) * 1980-10-30 1981-12-15 The Procter & Gamble Company Stabilized aqueous enzyme composition
EP0357157A2 (de) 1983-06-24 1990-03-07 Genencor International, Inc. Prokaryontische Carbonylhydrolasen und Mutanten von diesen sowie Verfahren zu deren Herstellung und deren Verwendung
EP0130756A1 (de) * 1983-06-24 1985-01-09 Genencor International, Inc. Prokaryotische Carbonyl-Hydrolasen, Verfahren, DNA, Vektoren und transformierte Wirte zu ihrer Herstellung und diese Hydrolasen enthaltende Detergenszusammensetzungen
US4548727A (en) * 1983-10-06 1985-10-22 The Drackett Company Aqueous compositions containing stabilized enzymes
EP0171007A2 (de) * 1984-08-04 1986-02-12 Henkel Kommanditgesellschaft auf Aktien Geschirreinigungsmittel
EP0171007A3 (en) * 1984-08-04 1989-05-03 Henkel Kommanditgesellschaft Auf Aktien Dish cleaning agent
AU604640B2 (en) * 1985-08-03 1991-01-03 Henkel Kommanditgesellschaft Auf Aktien An alkaline protease, a process for the preparation of hybrid vectors and genetically transformed microorganisms
US4927558A (en) * 1986-11-25 1990-05-22 Novo Industri A/S Proteolytic detergent additive and compositions containing the same
US4711739A (en) * 1986-12-18 1987-12-08 S. C. Johnson & Son, Inc. Enzyme prespotter composition stabilized with water insoluble polyester or polyether polyol
US5102422A (en) * 1987-02-13 1992-04-07 Rohm Gmbh Methods for leather processing including liquid enzyme formulation
WO1989003888A1 (en) * 1987-10-28 1989-05-05 Gds Technology, Inc. Test composition and method for the determination of anilides
EP0395680A1 (de) * 1987-10-28 1990-11-07 Gds Technology Inc Testsatz und verfahren zur bestimmung von aniliden.
EP0395680A4 (en) * 1987-10-28 1991-01-09 Gds Technology, Inc. Test composition and method for the determination of anilides
US4999288A (en) * 1987-10-28 1991-03-12 Gds Technology, Inc. Test composition and method for the determination of anilides
US5198353A (en) * 1988-02-11 1993-03-30 Novo Nordisk A/S Method for preparing stabilized enzyme dispersion
US5156773A (en) * 1989-12-12 1992-10-20 Novo Nordisk A/S Stabilized enzymatic liquid detergent composition
US5030378A (en) * 1990-01-02 1991-07-09 The Procter & Gamble Company Liquid detergents containing anionic surfactant, builder and proteolytic enzyme
US5447649A (en) * 1990-03-01 1995-09-05 Novo Nordisk A/S Lipase containing liquid pre-spotter and use of such pre-spotter
US5858117A (en) * 1994-08-31 1999-01-12 Ecolab Inc. Proteolytic enzyme cleaner
US6197739B1 (en) 1994-08-31 2001-03-06 Ecolab Inc. Proteolytic enzyme cleaner
US20080124783A1 (en) * 2002-01-16 2008-05-29 Poulose Ayrookaran J Multiply-substituted protease variants
US20080176313A1 (en) * 2002-01-16 2008-07-24 Poulose Ayrookaran J Multiply-substituted protease variants
US20110086412A1 (en) * 2002-01-16 2011-04-14 Danisco Us Inc. Multiply-Substituted Protease Variants
US20110091959A1 (en) * 2002-01-16 2011-04-21 Danisco Us Inc. Multiply-Substituted Protease Variants
AU2006288018B2 (en) * 2005-09-11 2010-04-01 Peter Alan Royle Hidden fitting locator

Also Published As

Publication number Publication date
CH534702A (de) 1973-03-15
NL6816356A (de) 1969-05-19
DE1808834A1 (de) 1969-07-17
BE723873A (de) 1969-05-14
FI49179B (de) 1974-12-31
DK132030C (da) 1976-03-01
FR1599043A (de) 1970-07-15
AT314450B (de) 1974-04-10
DE1808834C3 (de) 1980-04-24
DK132030B (da) 1975-10-13
GB1242596A (en) 1971-08-11
SE360675B (de) 1973-10-01
DE1808834B2 (de) 1979-08-09
NL162690B (nl) 1980-01-15
FI49179C (fi) 1975-04-10
NL162690C (nl) 1980-06-16
NO128370B (de) 1973-11-05

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