SK153695A3 - Stabilizing agent and synergistically by it stabilized liquid enzymatic composition - Google Patents

Abstract

A stabilizing formulation capable of enhancing the storage and shelf-life of liquid enzymatic compositions as well as acting as a dispersant aid for industrial process waters. Such stabilizing formulation contains at least one polymer selected from a poly(cellulosic)ether, an acrylic polymer, and a polyamide, a C2-C6 polyhydric alcohol and water. The polymer and mixture are present in an amount effective to stabilize, preferably in a combined amount effective to synergistically stabilize, one or more enzymes contained in a liquid enzymatic composition. A stabilized liquid enzymatic composition which contains one or more components of the stabilizing formulation and an enzyme. Methods for using the stabilizing formulation and preparing stabilized liquid enzymatic compositions are also described.

Description

Technique / ·

The present invention relates to novel compositions for stabilizing at least one enzyme present in a liquid enzymatic composition. The unique rheological properties of the components of these stabilized compositions preferably provide a synergistic stabilizing capacity over other aqueous mixtures of enzymatic dispersions even under medium to high temperature conditions and over a wide pH range. The invention therefore also relates to stabilized liquid enzymatic compositions. Furthermore, the invention relates to a novel method for preparing stabilized enzyme mixtures and methods using stabilized compositions based on liquid enzymatic mixtures.

BACKGROUND OF THE INVENTION

The use of enzymes and enzyme enzymatic mixtures has grown rapidly in industry and the industrial market over the past few years. As is well known, enzymes may be acidic, alkaline or neutral, depending on the pH range in which they are active. It is contemplated that all these types of enzymes be utilized in relation to the present invention.

Many enzymes and liquid enzymatic compositions are associated with liquid detergents and are used as solubilizers and cleaners. Furthermore, in conjunction with liquid detergents, enzymes and liquid enzymatic compositions are also used in a variety of commercial and industrial areas in which a wide range of enzyme classes is employed.

Proteases, a well known class of enzymes, are often used in a wide range of industrial applications where they hydrolyze peptide bonds in proteins and protein substrates. From a commercial point of view, proteases have the greatest use in the detergent industry where they help to remove protein stains, such as blood or egg stains, and in the cheese industry, where they help milk the milk. Proteases are also used as a means to soften meat, to soften the skin, to modify food ingredients and to release fragrances. It has been shown that liquid enzymatic compositions containing alkaline proteases can be used as dispersing agents for bacterial films and algae and fungal coatings in water cooling towers and liquid storage tanks in the metal industry.

Proteases can be characterized as acidic, neutral or alkaline proteases depending on the pH range in which they are active. Acid proteases include microbial renets, renin (chymotrypsin), pepsin, and fungal acid proteases. Neutral proteases include trypsin, papain, bromelain / ficin, and neutral bacterial proteases. Copper alkaline proteases include subtilisin and similar enzymes. Commercial liquid protease containing enzymatic compositions are available under the names Rennilase®, PTN (pancreatic trypsin NOVO), PEM (proteolytic enzyme mixture), Neutrase®, Alcalase®, Esperase®, and Savinase®, all of which are supplied by Novo Nordisk Bioindustrials, Inc. Danbury, CT. Another commercial protease is available under the name HT-Proteolytic and is available from Solvay Enzyme Products.

Amylases, another class of enzymes, are also used in many industrial and commercial processes in which they catalyze or accelerate the hydrolysis of starch. Amylases are widely used in the industrial processing of corn syrups in the production of glucose syrups, maitose syrups and a number of other refined end products of starch hydrolysis, which are high frutose syrups. Amylases as a class include α-amylase, β-amylase, amyloglucosidase (glucoamylase), fungal amylase and pulluianase. Commercial liquid amylase-containing enzymatic compositions are available under the names BAN, Termamyl®, AMG, Fungamy® and Promozyme® from Novo Nordisk, and Diazyme L / 200 manufactured by Solvay Enzyme Products.

Other commercially valuable classes of enzymes are those that cause fiber hydrolysis. These classes include cellulases, hemicellulases, pectinases and β-glucanases. Cellulases are enzymes that degrade cellulose, a linear glucose polymer found in plant cell walls. Hemicellulases are involved in the hydrolysis of hemicelluloses, which, like cellulose, are a plant polysaccharide. Pectinases are enzymes involved in the degradation of pectin, a carbohydrate whose main component is sugar acid. β-Glucanases are enzymes involved in the hydrolysis of β-glucans, which are also cellulose-like in that they are linear glucose polymers. From a commercial point of view, these enzymes have less or greater utility in fiber degradation manufacturing processes.

Cellulases are used in the process of removing black from old waste paper (ONP), so there is no need to use any surfactants and alkaline chemicals. Enzymes push the black from the fiber surface and disperse the black particles to the final form. See S. Say-Kyoun Ow, Methods for Biological Removal of Printer Black from Newsprint Waste, World Pulp and Paper Technology, pp. 63: 64 (1992). Collectively, cellulases include endocellulase, exocellulase, exocelo / biohydrolase, mannase and celobioase. Commercial liquid enzymatic compositions containing cellulases are available under the names Celluclast® and Novozyme® 188, both available from Novo Nordisk.

Hemicellulases are also used to de-ink to eject black particles from the surface of ONP fibers. See D. Y. Prasad et al., Enzymatic Removal of Printing Black from Black and White Printing Waste Newsprint, Progress in Paper Recycling, May 1992, pp 21, 22. Furthermore, hemicelllulases such as xylanases are used in the pulp bleaching process. Pretreatment of kraft pulp leads to a significant reduction in the consumption of bleaching chemicals such as molecular chlorine and also improves pulp quality, which is reflected in its higher gloss. See D. J. Senior et al., Reducing Chlorine Consumption in Bleaching Kraft Pulp after Xylanase Treatment, Tappi Journal (Preliminary Sharing, Publication Aspects Presented at the International Conference on Pulp Bleaching, Stockholm, 1991). PULPZYM®, a product supplied by Novo Nordisk, ECOPULP®, a product supplied by Alko Biotechnology, are two examples of commercially available liquid enzymatic compositions containing xylanase-based bleaching enzymes.

Hemicellulases as a class include a mixture of hemicellulases and galactomannanase. Commercial liquid enzymatic compositions containing hemicellulases are also available as PULPZYM® from Novo, ECOPULP® from Alko Biotechnology, Novozym® and Gamanase®, both of which are from Novo Nordisk.

Pectinases are used commercially to attenuate cell walls and to increase the extraction of fruit juices as well as to reduce viscosity and prevent gelation in these extracts. Pectinases consist of endopolygalacturonase, exopolygalacturonase, endopectate lyase (transeliminase), and endopectin lyase (transeliminase). Commercial liquid enzymatic compositions containing pectinases are available under the names Pectinex®, Ultra SP and Pectinex®, both manufactured by Novo Nordisk.

β-Glucanases play an important role in malting and brewing, where it is necessary to modify the cell walls of barley containing β-glucans. β-Glucanases include lichenase, laminarinase, and exoglucanase. Commercial liquid enzymatic preparations containing β-glucanases are Novozym® 234, Cereflo®, BAN, Finizym®, and Ceremix® · manufactured by Novo Nordisk. ^from

Two other classes of industrial and commercially important enzymes are lipases and phospholipases. Lipases and phospholipases are esterase enzymes that hydrolyze fats and oils by attacking the ester bond of these compounds. Lipases act on triglycerides, while phospholipases act on phospholipids. Lipases and phospholipases are commercially available esterases for industry and both of these classes have a number of industrial and commercial uses.

In the paper and pulp industry, liquid enzymatic preparations have proven to be particularly effective in reducing pitch deposits on rollers and other equipment used in the manufacturing process. For example, treating the unbleached pulp with lipases prior to bleaching the pulp with chlorine reduces the content of chlorinated triglycerides that are known to cause pitch deposits in the paper making process. See K. Fischer and K. Messner, Reduction of difficult deposits in pulp shredders by lipolytic enzymes, Tappi Journal, February 1992, p. 130. Novo Nordisk markets two liquid enzymatic preparations under the names Resinase ™ A and Resinase ™ A 2X, both of which, under certain conditions, reduce significantly pitch deposits by removing the wood resins present in the pulp.

Another important use of lipases is to degrease hides and boots in tanneries. Alkaline lipases are used for degreasing in conjunction with special proteases and emulsifying systems, which also improves the wetting and leaching effects of skin processing. See J. Christner, Use of Lipases in Wet Tanning Processes, 87 J. A. L. C. A. 128 (1992).

Lipases are also used to develop the taste of cheeses and to improve the palatable tallow for dogs. In non-aqueous systems, lipases are used to synthesize esters from carboxylic acids and alcohols.

Liquid lipase-containing enzymatic preparations are commercially available, for example under the tradenames Lipolase 100, Greasex 50L, Pallatase ™ A, Palatase ™ M and Lipozyme ™, available from Novo Nordisk.

In view of commercially used phospholipases, pancreatic phospholipase Az is used to convert lecithin to lysolecithin. Lysolecithin is an excellent emulsifier for mayonnaise and bread baking. Business is phospholipase

Az available in a liquid enzyme preparation supplied as LECITASE ™ by Novo Nordisk.

Another commercially valuable class of enzymes are isomerases. which catalyze conversion reactions between isomers of organic compounds. Isomerases are of significant use in the production of high fructose corn syrups. For example, glucose isomerase catalyzed aldose to ketose is also involved in the conversion of glucose to fructose and is thus one of the three key enzymatic reactions in the industry. Sweetzyme® is a liquid enzymatic preparation containing glucose isomerase and is available from Novo Nordisk.

Redox enzymes are enzymes that act as catalysts in chemical oxidation / reduction reactions and thus are useful in the degradation and synthesis of many biochemicals. At present, many redox enzymes have not found significant application in industry since most redox enzymes require the presence of cofactors. However, where cofactors are an integral part of the enzyme or do not need to be added, the redox enzymes are commercially used, especially in the food industry.

The redox enzyme, glucose oxidase, is used to prevent unwanted browning reactions that affect the color and taste of food. Glucose oxidase is also used as an oxygen scavenger to prevent the formation of flavors in juices and to protect the color and stability of some sensitive food ingredients.

The redox enzyme, catalase, is used to decompose residual hydrogen peroxide, which is used as a sterilizing agent. The third redox enzyme (lipoxygenase), naturally found in soybean meal and rarely purified for industrial use, is used in baking, not only to obtain whiter bread, but also to prevent dough softening caused by certain agents. Other redox enzymes can be used from enzymatic synthesis of steroid derivatives to diagnostic assays. These redox systems include peroxidases, superoxidizmutase, alcohol oxidase, polyphenol oxidase, xanthine oxidase, sufhydryloxidase, hydroxylase, cholesterol oxidase, laccase, alcohol dehydrooxygenase, and steroid dehydrogenases.

When the enzymes as described above are prepared or sold for industrial use, they are usually in the form of aqueous or predominantly aqueous liquid enzymatic preparations intended for a particular process. The predominantly aqueous liquid enzymatic compositions may contain additional solvents depending on the particular enzyme or use of the composition. These liquid enzymatic compositions have traditionally been associated with problems such as chemical instability, which can result in a loss of enzymatic activity, particularly when stored. In particular, the liquid detergent industry is affected by these considerable problems of enzyme storage loss. / ·

For industrial products, such as liquid enzymatic preparations, it is not uncommon for them to be stored in stores in various occurring climatic conditions where the products are exposed to temperatures from freezing to more than 50 ° C for a long time. After storage at extreme temperatures ranging from 0 ° C to 50 ° C for several months, many liquid enzyme preparations lose 20 to 100 percent of their enzymatic activity due to enzyme instability.

Many attempts have been made to stabilize the enzymes found in liquid enzymatic compositions. Attempts to increase the stability of liquid enzymatic formulations using formulations containing alcohols, glycerols, dialkyl glycol ethers, mixtures thereof, and other substances have only marginal success, even over moderate storage temperatures.

In U.S. Pat. No. 4,801,544, an ethylene glycol system and an ethoxylated linear nonionic surfactant with a hydrocarbon solvent are used as stabilizers, and the encapsulation of the enzyme in micelles formed in a solvent-surfactant mixture is described. The water content of the mixture was maintained at less than 5 percent and the enzyme stability was verified at 35 °, 70 ° and 100 ° F.

Stabilization of the aqueous enzyme preparation by other esters has been described in U.S. Patent 4,548,727. The ester used as stabilizer had the formula RCOOR ', wherein R is alkyl of one to three carbon atoms or hydrogen, and R' is alkyl of one to six carbon atoms. The ester is present in the aqueous enzyme preparation in an amount of from 0.1 to 2.5 weight percent.

U.S. Patent 4,318,818 discloses a stabilizing system for aqueous liquid enzymatic compositions wherein the stabilizing system comprises calcium ions and a low molecular weight carboxylic acid, or a salt thereof. The pH of the stabilizing system is in the pH range of about 6.5 to about 10.

U.S. Patent 4,243,543 relates to the stabilization of liquid detergent compositions containing proteolytic enzymes. The detergent composition is stabilized by adding an antioxidant and a hydrophilic polyol to the composition while stabilizing the pH of the composition.

U.S. Patent 4,169,817 relates to liquid cleaning compositions containing stabilized enzymes. The composition is an aqueous solution which contains from 10 to 50% by weight of a solid and contains detergent builders, surfactants, an enzyme system derived from Bacillus subtilis, and an enzyme stabilizing agent. The stabilizing agent contains water-soluble sodium or potassium salts and / or water-soluble hydroxy alcohols and allows the solution to be stored for extended periods without inactivating the enzyme.

European Patent 0 352 244 A2 discloses a stabilized liquid detergent composition using an amphoteric surfactant.

SUMMARY OF THE INVENTION

The present invention provides a composition capable of synergistically stabilizing one or more enzymes found in a liquid enzymatic composition.

Thus, the invention provides a stabilized liquid enzymatic composition.

Further, the invention provides processes for preparing stabilized liquid enzymatic compositions.

Various aspects of the invention can be broadly accomplished by the use of a means for stabilizing a liquid enzymatic composition comprising:

(a) at least one polymer selected from a cellulose polyether, an acrylic polymer, and a polyamide, (b) a C2-C8 polyhydric alcohol, and (c) water, wherein components (a) and (b) are present in an amount effective to stabilize at least one enzyme found in a liquid enzymatic composition. Preferably, the polymer (a) and the C2-C6 polyhydric alcohol (b) are present in an amount that, in combination, synergistically stabilizes at least one enzyme present in the liquid enzymatic composition.

The stabilizing agent of the invention can be used for a wide range of enzymes used in liquid enzymatic compositions while providing a wide range of functions. The enzymes and enzyme classes that can be used with this stabilizing agent include, but are not limited to, the above enzymes and enzyme classes.

The present invention also provides stabilized liquid compositions comprising:

(a) at least one polymer selected from a cellulose polyether, an acrylic polymer and a polyamide, (b) a C2-C8 polyhydric alcohol, (c) water, and (d) at least one enzyme wherein components (a) and (b) are present in an amount effective to stabilize at least one enzyme in the liquid enzymatic composition. Preferably, the polymer (a) and the C2-C5 polyhydric alcohol (b) are present in an amount that, in combination, synergistically stabilizes at least one enzyme present in the liquid enzymatic composition.

Furthermore, the invention relates to a method of preparing a stabilized liquid enzymatic composition by combining the enzyme with the above stabilizing agent. The invention also relates to a method of using a stabilized composition for stabilizing a liquid enzymatic composition, comprising the method of combining a stabilizing composition with a liquid enzymatic composition.

The invention provides in a preferred embodiment a composition for stabilizing a liquid enzymatic composition comprising:

(a) at least one polymer selected from a cellulose polyether, an acrylic polymer, and a polyamide; (b) a C2-C8 polyhydric alcohol; and (c) water, wherein components (a) and (b) are present in an amount that the combination synergistically effectively stabilizes at least one enzyme present in the liquid enzymatic composition.

The water-soluble or at least partially soluble polymer is used in the composition for stabilizing the liquid enzymatic compositions or in the stabilized liquid enzymatic compositions of the invention. Thus, the polymer must have sufficient solubility to achieve miscibility with water and to form a single phase. If the polymer has this solubility, the polymer must not be separated after being combined with the C2-C8 polyhydric alcohol and the water present in the stabilizer or the liquid enzymatic mixture. The composition need not be a clear solution. Preferably, the emulsion composition is of an apparently homogeneous structure.

In the composition for stabilizing a liquid enzymatic composition or in a stabilized liquid composition of the invention, the amount of polymer present also depends on the molecular weight of the particular polymer used. The higher the molecular weight of the polymer used, the lower the amount of polymer generally needed to stabilize the enzyme.

The polymer may preferably be used in amounts up to about 50% by weight of the stabilizing agent. Even more preferably, the polymer content is from 0.05 to 30% by weight, and most preferably from 1 to 10% by weight. Of course, in a preferred embodiment, the polymer is present in an amount that provides the desired synergistic stabilization of the liquid enzymatic composition when combined with a polyhydric alcohol in an aqueous or predominantly aqueous composition.

The polymer used in the present invention is selected from cellulose polyethers, acrylic polymers and polyamides. The polymer may be substituted or unsubstituted. The polymer may have a lower ionic charge, but is preferably non-ionic in nature.

When the selected polymer is a cellulose ether, the polymer is preferably selected from poly (carboxymethyl) cellulose ethers, poly (hydroxypropylmethyl) cellulose ethers, poly (hydroxyethylmethyl) cellulose ethers, poly (hydroxybutylmethyl) cellulose ethers, poly (hydroxypropyl) cellulose ethers, poly (ethylhydroxyethyl) ) of cellulose ethers.

Preferably, the polymer is a poly (carboxymethyl) cellulose ether. Certain cellulose polyethers used in the present invention, such as cellulose poly (carboxymethyl) ethers, are supplied as salts, i. j. sodium salts, and have a low anionic character. Preferred are cellulose polyethers having a molecular weight in the range of 15,000 to 100,000, but more preferably the polymers are in the range of 20,000 to 75,000.

Acrylic polymers used in the present invention; are preferably polymers or copolymers of acrylic acid, methacrylic acid or derivatives thereof, e.g. esters or salts of acrylic acid. Examples of preferred selected acrylic polymers are those of Rohm and Haas Acrylos GS (sodium polyacrylate), Acrysol TI-935 (acrylic polymer), and Acrylin 22 (acrylic polymer). The molecular weight of the acrylic polymers may range from about 5,000 to more than 4,000,000. Preferred acrylic polymers have molecular weights of from 100,000 to more than 4,000,000, but more preferably polymers with a molecular weight γ range from 750,000 to more than 4,000,000. Particularly preferred are acrylic polymers having a molecular weight of from 1.250.000 to 4.000.000. Acrylic polymers with these molecular weights are available from Aldrich Chemical Company. The acrylic polymers may have an anionic character. Preferred acrylic polymers are those polymers or derivatives thereof that are of a mild anionic nature or are uncharged, most preferably the ionic electroneutral polymers.

When a polyamide polymer is used, the most preferred polyamide is polyvinylpyrrolidone. Preferred are polyvinylpyrrolidones having a molecular weight in the range of 5,000 to 400,000, more preferably those with a molecular weight in the range of 300,000 to 400,000, with higher polymers being most preferred.

The stabilizing composition also comprises a C 2 -C 6 polyhydric alcohol as a second component, the C 2 -C 8 polyhydric alcohol has a synergistic effect with the above-described polymer to stabilize the enzyme in a liquid enzymatic composition. The C 2 -C 8 polyhydric alcohol is preferably a glycol or a trihydric alcohol. A more preferred C 2 -C 8 polyhydric alcohol is glycerol, sorbitol, propylene glycol, butylene glycol, hexylene glycol or ethylene glycol. The most preferred C2-C8 polyhydric alcohol is glycerol.

The stabilizing composition comprises a C2-C8 polyhydric alcohol in an amount sufficient, together with the polymer, to stabilize at least one enzyme in the liquid enzymatic composition. Preferably, the C 2 -C 8 polyhydric alcohol is present in an amount of 0.50 to 60% by weight of the stabilizing agent, more preferably in an amount of 5 to 50% by weight, and even more preferably between 10 to 30% by weight. Most preferably, the stabilizing composition or enzyme composition comprises a C 2 -C 8 polyhydric alcohol in an amount associated with the polymer to achieve a synergistic effect.

The compositions of the invention are compositions comprising water or aqueous compositions containing sufficient water to render the polymer miscible with the composition and not to separate. Generally, C2-C8 polyhydric alcohols are water soluble, but sufficient water is required to form the single phase. As mentioned above, the composition is not required to be a clear solution, preferably an emulsion with a seemingly homogeneous structure. The water-containing compositions may contain a solvent other than water.

Although the stabilizing agents of the invention can be prepared by mixing the ingredients in any order, the compositions are preferably prepared by adding the necessary amount of polymer to the C2-C8 polyhydric alcohol / water mixture. A mixture of C2-C8 polyhydric alcohol with water can be prepared by a conventional method. For example, a mixture may be prepared by simply mixing the selected polyhydric alcohol with an appropriate amount of water, or by diluting the previously prepared mixture.

The preferred mixture of C 2 -C 6 polyhydric alcohol and ^the - water can contain any number of C 2 -C 5 alcohol sufficient with the polymer discussed above to stabilize, preferably synergistically, at least one enzyme in the liquid enzymatic composition. Preferably, the composition comprises 1 to 95 weight percent of a polyhydric alcohol or water-soluble polymer: more preferably 10 to 50 weight percent; most preferably 30 to 50 weight percent. If the polyhydric alcohol is glycerol, the composition preferably contains 50% by weight glycerol in a mixture with water. Without wishing to be bound by theory, the Applicant believes that the composition causes wetting of the polymer used in the present invention that synergistically stabilizes the enzyme when mixed with a C 2 -C 8 polyhydric alcohol.

In order to adequately stabilize the enzyme in a liquid enzymatic composition in accordance with the present invention, the enzyme needs to exhibit at least 90% of its activity after 30 days at 25 ° C. The examples below demonstrate the advantageous synergistic stabilization of various enzymes at 50 ° C after 30 days.

The stabilizing agent described herein can be used for a wide range of enzymes, industrial processes and commercial products. The enzymes, industrial processes and commercial products with which this stabilizing agent can be used include, but are not limited to, the above.

The use of a stabilizing agent for stabilizing a liquid enzymatic composition results in a second object of the present invention, for a stabilized liquid enzymatic composition. The invention also relates to a stabilized liquid composition comprising at least one polymer selected from cellulose polyethers, acrylic polymers and polyamides; C 2 -C 8 polyhydric alcohol; water; and at least one enzyme. The polymer and the C 2 -C 8 polyhydric alcohol are present in a total amount effective for stabilization, in particular synergistic stabilization, of at least one enzyme present in the liquid enzymatic composition. Preferred compositions according to the invention are capable of achieving a higher viscosity than the quantitatively corresponding aqueous mixtures with the same polymer.

The observed and preferred embodiments with respect to the polymer, C2-C8 polyhydric alcohol and water present in this stabilized liquid enzymatic composition are the same as those discussed above for the stabilized formulation, which is also an object of the present invention.

As with the stabilizing formulation, the liquid enzymatic composition, which is also an object of the present invention, can be used for a wide range of enzymes. These enzymes include, but are not limited to, the enzymes and enzyme classes discussed herein. The enzymes that can be used can be derived from animal, plant, fungal, bacterial and synthetic sources. Preferably, the water-dispersible enzymes in this system are proteases, including acidic, alkaline and neutral proteases, which are widely used in the detergent and cheese industries; amylases, including acidic, alkaline and neutral amylases, which are used e.g. in the processing of corn syrup; lipases used to develop the taste of cheese, in the paper and pulp and leather industries; cellulases and xylases.

Depending on the intended use, the enzymes are often packaged and sold in concentrated liquid enzymatic compositions and diluted prior to use. The enzymes can also be supplied in powder or dried form. The amount of enzyme present after dilution of the concentrated enzyme depends on the form in which the enzyme was delivered. In general, the amount of enzyme is preferably in the range of 0.05 to 40% by weight of the concentrated liquid enzymatic composition, more preferably in the range of 0.5 to 25%, and most preferably in the range of 10 to 20%. The stabilizing agent of the present invention is specifically contemplated for use in a concentrated liquid composition as well as for a composition already diluted for use. The amount of stabilizing agent required to stabilize or synergistically stabilize the concentrated solution may and will likely be different from that for the diluted mixture. Determination of the appropriate amount of the stabilizing agent or its components can be readily determined by one of the conventional methods in the art given in the examples below. However, as is well known in the art, the amount of enzyme present is, however, dependent upon the activity of the particular enzyme and the desired end use.

Depending on the enzyme found and its intended use, the pH of the final stabilized enzyme mixture is preferably in the range of 5.0 to 10.0, but more preferably around 7.0. Most preferably, let the system adjust its own pH, generally roughly neutral. However, as is known in the art, it may be necessary to adjust the pH with a small amount of an acidic or alkaline substance.

The stabilized liquid enzymatic composition may contain water or be an aqueous solution and contain other solvents or additives aimed at using the composition in a particular industrial process. E.g. the stabilized liquid enzymatic composition may contain additives such as surfactants, antifoams and the like, as is commonly known in the art. These additives can be added in amounts not interfering with the synergistic stabilization of the liquid enzymatic composition. Those skilled in the art can readily determine these amounts. Advantageously, the stabilized liquid enzymatic composition of the present invention can be used and the stabilizing agent used as an enzyme dispersant in industrial process waters.

The present invention also provides a process for preparing a stabilized liquid enzymatic composition with a step comprising combining at least one enzyme with a stabilizing agent, which is also an object of the present invention. The invention further includes a method of using a stabilizing agent for stabilizing a liquid enzymatic composition comprising the step of combining the liquid enzymatic composition with a stabilizing agent. Illustrative and preferred components, as well as amounts thereof, used in the process are the same as in the foregoing discussion.

The skilled artisan will appreciate that the components of the composition for stabilizing a liquid enzymatic composition may be combined in any order or even simultaneously. Nevertheless, the following order is preferable:

(a) mixing the C2-C6 polyhydric alcohol with water, (b) adding at least one polymer selected from cellulose polyethers, acrylic polymers, and polyamides to the mixture prepared in step (a), and (c) adding at least one enzyme to the mixture formed in step (b).

The polymer and the C2-C8 polyhydric alcohol are preferably present in a combined amount synergistically effective to stabilize at least one enzyme in the resulting liquid enzymatic composition.

An alternative process for preparing a stabilized liquid composition is the following steps:

(a) mixing the C2-C8 polyhydric alcohol with water, (b) adding at least one polymer selected from cellulose polyethers, acrylic polymers and polyamides to the mixture prepared in (a), and (c) combining the composition formed in steps (a) and (b) a liquid enzymatic composition comprising at least one enzyme. The polymer and the C2-C8 polyhydric alcohol are preferably present in a combined amount synergistically effective to stabilize at least one enzyme in the liquid enzymatic composition.

In the processes corresponding to the invention, the polymer added in step (b) may be added alone, as an aqueous dispersion or solution, wherein the polymer is dissolved in water or in a suitable organic solvent. If additional additives are to be added to the stabilized enzyme mixture, these may be added at any time, but preferably before step (c) or in the next step after the enzyme has been added.

The following examples are given to illustrate the invention. However, it is to be understood that the invention is not limited to certain conditions or details set forth in these examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Synergism was demonstrated by testing glycerol, designated Component A, and a 250,000 molecular weight poly (carboxymethyl) cellulose ether (CMC) labeled Component B, or a 360,000 molecular weight polyvinylpyrrolidone (PVP) labeled Compound B '. As can be seen from Tables 1 and 2, experiments were performed with varying amounts of Compound A to Compound B or B 'over a range of concentrations, and various types of enzymes were verified to improve stabilization of enzymatic activity at 50 ° C for 30 days. The concentration of each component required to achieve 90% of the original enzymatic activity was taken as the endpoint. Concentrations are expressed as weight percent of the substance in the final blend, taking into account the added enzyme, the remainder being water. Water was added to Substance A, glycerol, to form a glycerol-water mixture. Endpoints for mixtures containing Compound A and Compound b or B 'were compared to endpoints for Compound A and Compound B or B'. substance A alone and substance B respectively; B '.

Synergism was determined by the method described by Kuli, F. C., Eisman, P. C., Sylwestrowicz, H. D., and Mayer, R. L., Applied Microbiology 9: 538-541 (1961) using the ratio determined by:

QA QB Q? Qb where Qa is the amount by weight of the aqueous mixture of Substance A acting alone, which provided the endpoint,

Qb - the quantity by weight of the aqueous mixture of Substance B acting alone which yielded a cation point,

QA - the quantity by weight of the aqueous mixture of Substance A to Substance B that provided the endpoint,

QB - amount by weight of the aqueous mixture of Substance B to Substance A that provided the endpoint.

If the sum of QA / Qa and QB / Qb is greater than one, it indicates antagonism. If this sum is equal to one, it indicates additivity. If this sum is less than one, it shows synergism.

This process is widely used and accepted to demonstrate the synergism of the composition of the invention. For more details on this process, see U.S. Patent 3,231,509, the disclosure of which is hereby incorporated by reference.

The results obtained from Tables 1 and 2 demonstrate an enhanced stabilization of the enzyme, HT-Proteolytic L-175, an alkaline protease supplied by Solvay Enzymes, Inc. Using the method described by Kuli aspol., QA / Qa + QB / Qb totals were calculated for all mixtures containing glycerol (Compound A) and CMC (Compound B). The calculation models are given for some endpoints where synergism is evident in these cases. As shown in Table 1 and the calculation model, the endpoint values are 0.58; 0.67; 0.67 and 0.67; respectively, demonstrating the existence of synergism. Similarly, totals for QA / Qa + QB '/ Qb' were calculated for all glycerol (Compound A) and PVP (Compound B ') formulations. As shown in Table 2 and the model calculation, the final points 0.85; 0.95; 0.65; 0.85 again demonstrate the existence of synergism.

Table 1

Substance B (% by weight) Substance A (% by weight) 60 30 20 10 5 0 6 + + + + + X + 3 + + + / X + + x X + 2 + + + x - - 1 + + x - - - - 0 + x - - - - -

(1) ( ^x ) (2) ω (3) (-) (4) Substance A (5) Substance B = endpoint> 90% of enzyme activity after 30 days at 50 ° C

-> 90% enzyme activity after 30 days at 50 ° C = <90% enzyme activity remains after 30 days at 50 ° C

- glycerol = CMC

calculations:

qa qb QA QB QA / Qa + QB / Qb 60 6 5 3 5/60 + 3/6 = 0.58 10 3 10/60 + 3/6 = 0.67 20 2 20/60 + 2/6 = 0.67 30 1 30/60 + 1/6 = 0.67

Table 2

Fabric B ' (% by weight) Substance A (% by weight) 50 30 20 10 5 0 40 + + + + + X + 30 + + + + χ ^z - + x - 20 + + + - - - 10 + + x + x - - - 5 + - - - - - 0 + x - - - - -

( ¹ ) ( ^x ) ~ endpoint> 90% enzyme activity after 30 days at 50 ° C (2) (+) => 90% enzyme activity after 30 days at 50 ° C (3) (-) = <90% enzyme activity remains after 30 days at 50 ° C (4) Substance A = glycerol (5) Substance B '= PVP

calculations:

qa qb ' QA QB ' QA / Qa + QB '/ Qb' 50 40 5 30 5/50 + 30/40 = 0.85 10 30 10/50 + 30/40 = 0.95 20 10 20/50 + 10/40 = 0.65 30 10 30/50 + 10/40 = 0.85

Example 2

The stabilizing composition of the invention, as in Example 1, was tested by another enzyme, Diazyme L-200, a glucoamylase supplied by Solvay Enzymes, Inc. The results from Tables 3 and 4 also show increased stabilization by this enzyme. The sum of QA / aa + QB / Qb was calculated for all mixtures containing glycerol (Compound A) and CMC (Compound B). Calculation patterns are given for some endpoints where there is clear synergism in this case. As shown in Table 3 and the calculation model, these endpoint values are 0.59; 0.67; 0.67 and 0.84, respectively. they show the existence of synergism. Similarly, the sums of QA / Qa + QB '/ Qb' were calculated for all mixtures containing glycerol (Compound A) and PVP (Compound B '). The endpoint values resulting from Table 4 and the calculation models were 0.77; 0.53; 0.53 and 0.77, respectively. and again suggesting the existence of synergism.

Table 3

Substance B (% by weight) Substance A (% by weight) 60 30 20 10 5 0 6 + + + + + + x 3 + + + + x + x 2 + X + + x - - - 1 + - - - - 0 X + - - - - -

(1) ( ^x ) (2) (+) (3) (-) (4) Substance A (5) Substance B = endpoint> 90% enzyme activity after 30 days at 50 ° C => 90% enzyme activity after 30 days at 50 ° C = <90% enzyme activity remains after 30 days at 50 ° C = glycerol = CMC

calculations:

qa qb ' QA QB ' QA / Qa + QB '/ Qb' 60 6 5 3 5/60 + 3/6 = 0.59 10 3 10/60 + 3/6 = 0.67 20 2 20/60 + 2/6 = 0.67 30 2 30/60 + 2/6 = 0.84

Table 4

Fabric B ' (% by weight) Substance A (% by weight) 50 30 20 10 5 0 30 + + + + + + x 20 + + + + x - 10 + + + x X + - 5 + X + - - - - 0 + x - - - -

(1) ( ^x ) (2) (+) (3) (-) (4) Substance A (5) Substance B = endpoint> 90% enzyme activity after 30 days at 50 ° C => 90% enzyme activity after 30 days at 50 ° C = <90% enzyme activity remains after 30 days at 50 ° C = glycerol = PVP

calculations:

qa qb ' QA QB ' QA / Qa + QB '/ Qb' 50 30 5 20 5/50 + 20/30 = 0.77 10 10 10/50 + 10/30 = 0.53 20 10 20/50 + 10/30 = 0.73 30 5 30/50 + 5/30 = 0.77

Example 3

Enzymes with esterase activity were tested with the stabilizing composition of the present invention, similar to Example 1. The results obtained with the enzyme, Lipolase, lipase supplied by Novo Nordisk Bioindustries, Inc. are shown in Tables 5 and 6 and show increased stabilization of this enzyme. Sums of QA / Qa + QB / Qb were calculated for all mixtures containing aqueous glycerol (Compound A) and CMC (Compound B). Calculation patterns are given for some endpoints where synergism is shown in this case. As shown in Table 5 and the calculation model, endpoint values 0.43; 0.37; 0.57 and 0.77, respectively, show the presence of synergism. Similarly, the sums of QA / Qa + QB / Qb 'were calculated for all mixtures containing glycerol (Compound A) and PVP (Compound B'). The endpoint values resulting from Table 6 and the calculation model were 0.60; 0.70; 0.65 and 0.85, respectively, show the presence of synergism.

Table 5

Substance B (% by weight) Substance A (w / w) __ 50 30 20 10 5 0 6 + + + / · + + + x 3 + + + + + - 2 + + + + + x - 1 + + x + x X + - - 0 + x - - - - -

( ¹ ) ( ^x ) = endpoint> 90% enzyme activity after 30 days at 50 ° C (2) (+) => 90% enzyme activity after 30 days at 50 ° C (3) (-) = <90% enzyme activity remains after 30 days at 50 ° C (4) Substance A = glycerol (5) Substance B = CMC

calculations:

qa qb ' QA QB ' QA / Qa + QB '/ Qb' 50 6 5 2 5/50 + 2/6 = 0.43 10 1 10/50 + 1/6 = 0.37 20 1 20/50 + 1/6 = 0.56 30 1 30/50 + 1/6 = 0.76

Table 6

Fabric B ' (% by weight) Substance A (% by weight) 50 30 20 10 5 0 30 + + + / - + + X + 20 + + + + + + 10 '+ + + + x + x - 5 + + x + x - - - 0 + x - - - - -

(1) ( ^x ) = endpoint> 90% enzyme activity after 30 days at 50 ° C (2) (+) => 90% enzyme activity after 30 days at 50 ° C (3) (-) = <90% enzyme activity remains after 30 days at 50 ° C (4) Substance A = glycerol (5) Substance B = PVP

calculations:

qa qb ' QA QB ' QA / Qa + QB '/ Qb' 50 20 5 10 5/50 + 10/20 = 0.60 10 10 10/50 + 10/20 = 0.70 20 5 20/50 + 5/20 = 0.65 30 5 30/50 + 5/20 = 0.85

Example 4

Hemicellulose, Pulpzyme HB, xylanase supplied by Novo Nordisk Bioindustrials, Inc. was selected to test the increased stability of the composition as in Example 1. For this example, Tables 7 and 8 show the following results. Sums of QA / Qa + QB / Qb were calculated for all mixtures containing glycerol (Compound A) and CMC (Compound B). Sample calculations are given for endpoints where synergism is evident in this case. Table 7 and the sample calculation show endpoint values of 0.43; 0.53; 0.57 and 0.77, respectively, which show the presence of synergism. Similarly, the sums of QA / Qa + QB '/ Qb' were calculated for all mixtures containing glycerol (Compound A) and PVP (Compound B '). Endpoint values 0.35; 0.45; 0.65 and 0.85, respectively, show the presence of synergism.

Table 7

Substance B (% by weight) Substance A (w / w) '/about) 50 30 20 10 5 0 6 + + + / · + + + x 3 + + + + + - 2 + + + X + + x - 1 + + x + x - - - 0 + x - - - - -

(1) ( ^x ) (2) (+) (3) (-) (4) Substance A (5) Substance B = endpoint> 90% enzyme activity after 30 days at 50 ° C => 90% enzyme activity after 30 days at 50 ° C = <90% enzyme activity remains after 30 days at 50 ° C = glycerol = CMC

calculations:

qa qb ' QA QB ' QA / Qa + QB '/ Qb' 50 6 5 2 5/50 + 2/6 = 0.43 10 2 10/50 + 2/6 = 0.53 20 1 20/50 + 1/6 = 0.56 30 1 30/50 + 1/6 = 0.76

Table 8

Fabric B ' (% by weight) Substance A (% by weight) 50 30 20 10 5 0 40 + + + / · + + X + 30 + + + + - 20 + + -h + + - 10 + + x + x + x X + - 5 + - - - - - 0 + x - - - - -

(1) ( ^x ) (2) (+) (3) (-) (4) Substance A (5) Substance B = endpoint> 90% enzyme activity after 30 days at 50 ° C => 90% enzyme activity after 30 days at 50 ° C = <90% enzyme activity remains after 30 days at 50 ° C = glycerol = PVP

calculations:

qa qb ' QA QB ' QA / Qa + QB '/ Qb' 50 40 5 10 5/50 + 10/40 = 0.35 10 10 10/50 + 10/40 = 0.45 20 10 20/50 + 10/40 = 0.65 30 10 30/50 + 10/40 = 0.85

Claims (10)

changed letter PATENT CLAIMS First • A composition for stabilizing liquid enzymatic compositions comprising; (a) at least one polymer selected from the group consisting of cellulose ether, acrylic polymer and polyamide (b) a C2-C8 polyhydric alcohol, and (c) water, wherein said components (a) and (b) are present in a combined amount synergistically * to stabilize at least one enzyme present in the liquid enzymatic composition. Second The composition of claim 1 wherein said polymer is a cellulose polyether selected from the group consisting of a poly (carboxymethyl) cellulose ether, poly (hydroxypropylmethyl) cellulose ether, poly (hydroxyethylmethyl) cellulose ether, poly (hydroxybutylmethyl) cellulose ether r a poly (hydroxypropyl) cellulose ether and a poly (ethylhydroxyethyl) cellulose ether. Third The composition of claim 1 wherein said C 2 -C 8 polyhydric alcohol is glycerol, sorbitol, propylene glycol, butylene glycol, hexylene glycol, or ethylene glycol, and said components (b) and (c) are present in a mixture containing 1 to 95% by weight of said C 2 -C 8 polyhydric alcohol and the rest is water. 4th A stabilized liquid enzymatic composition comprising: (a) at least one polymer selected from the group consisting of cellulose polyether, acrylic polymer and polyamide; (b) a mixture of C2-C8 polyhydric alcohols, (c) water; and altered sheet (d) at least one enzyme wherein components (a) and (b) are present in a combined amount synergistically effective to stabilize at least one enzyme in the liquid enzymatic composition. 5. The composition of claim 4 wherein said polymer is a cellulose polyether selected from the group consisting of a poly (carboxymethyl) cellulose ether, a poly (hydroxypropylmethyl) cellulose ether, a poly (hydroxyethylmethyl) cellulose ether, a poly (hydroxybutylmethyl) cellulose ether, a poly (hydroxypropyl) cellulose ether. cellulose ether and poly (ethylhydroxyethyl) cellulose ether. A composition according to claim 4, wherein said C 2 -C 6 polyhydric alcohol is glycerol, sorbitol, propylene glycol, and the like. butylene glycol, hexylene glycol or ethylene glycol; said components (b) and (c) being present in a mixture in an amount of 1 to 95% by weight of said C 2 -C 10 polyhydric alcohol and the remainder being water; and said enzyme is selected from protease, amylase, lipase, cellulase, mannase and xylase. 7. The composition of claim 4 wherein said composition is an emulsion. 8. A stabilized liquid enzymatic composition comprising: (a) at least one polymer selected from the group consisting of cellulose polyether, acrylic polymer and polyamide, (b) a mixture of -02-0θ polyhydric alcohols, (c) water, and (d) at least one enzyme, wherein components (a) and (b) are present in an amount effective to stabilize said at least one enzyme in the liquid enzymatic composition. 9. A process for the production of a liquid stabilized enzymatic composition comprising the step of mixing at least one enzyme and a composition comprising: the altered sheet (a) at least one polymer selected from the group consisting of cellulose ether, acrylic polymer and polyamide, (b) a C2-C6 polyhydric alcohol, and (c) water, wherein components (a) and (b) are present in a combined amount; which is synergistically effective to stabilize at least one enzyme in said mixture. 10. The method of claim 9, further comprising, prior to said mixing step, mixing steps of said C2-C8 polyhydric alcohol and water and adding a polymer selected from the group consisting of cellulose polyether, acrylic polymer and polyamide to said C2'Cg polyhydric alcohol mixture. and water. The method of claim 9, wherein said C 2 -C 6 polyhydric alcohol is glycerol, sorbitol, propylene glycol, butylene glycol, hexylene glycol or ethylene glycol; said mixture comprising 1 to 95% by weight of said C 2 -C 5 polyhydric alcohol and the remainder being water; said at least one enzyme is selected from protease, amylase, lipase, cellulase, mannase and xylase. 12. A method of using the composition, wherein the composition comprises: (a) at least one polymer selected from the group consisting of cellulose polyether, acrylic polymer and polyamide, (b) a C2-C6 polyhydric alcohol, and (c) water, wherein components (a) and (b) are present in a combined amount that is synergistically effective to stabilize at least one enzyme present in the liquid enzymatic composition, for stabilizing the liquid enzymatic composition comprising the step of mixing the liquid enzymatic composition comprising at least one enzyme with said composition. The method of claim 12, wherein said polymer is a cellulose polyether selected from a poly (carboxymethyl) cellulose ether and said C 2 -C 8 polyhydric alcohol is glycerol and said at least altered sheet one enzyme is selected from protease, amylase, lipase, cellulases, mannases and xylases. The method of claim 12, wherein said polymer is a polyamide selected from polyvinylpyrrolidone and said C 2 -C 6 polyhydric alcohol is glycerol and said at least one enzyme is selected from protease, amylase, lipase, cellulase, mannase, and xylase.