CN1128543A - Synergistically stabilized liquid enzymatic compositions - Google Patents

Abstract

The present invention provides 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

Synergistically stabilized liquid enzyme composition

The present invention relates to novel formulations for stabilizing at least one enzyme contained in a liquid enzyme composition. The unique hydrodynamic properties of the components of these stabilized formulations allow the formulation to be synergistically well stabilized to other water-based enzyme dispersion mixtures even under conditions of mild to high heat and a wide pH range ability. Accordingly, the present invention also relates to stabilized liquid enzyme compositions. In addition, the present invention relates to novel methods of preparing stabilized liquid enzyme compositions, and methods of using said stabilized formulations in liquid enzyme compositions.

The industrial and commercial use of enzymes and liquid enzyme compositions has grown rapidly in recent years. It is well known that enzymes can be acidic, alkaline or neutral depending on the pH range in which they are active. All of these types of enzymes are contemplated for use in the present invention.

A number of enzymes and liquid enzyme combination properties have been combined with liquid detergents and have shown utility as solubilizing and cleaning formulations. In addition to their combination with liquid detergents, enzymes and liquid enzyme compositions have also shown utility in many commercial and industrial fields where multiple enzymes are currently used.

Proteases are a well-known class of enzymes that are frequently used for various industrial purposes. In these industrial uses, they work by hydrolyzing peptide bonds in proteins and proteinaceous substrates. Commercially, proteases find greatest use in the laundry industry (where proteases help remove protein-based stains such as blood or egg stains) and the cheesemaking industry (where proteases help curdle milk). Proteases are also used as meat tenderizers, for softening leather, improving food ingredients and flavor development. The liquid enzyme composition comprising alkaline protease can also be used as a dispersant for bacterial conjunctiva and algal and fungal clots in cooling tower water and metal fabrication liquid holding tanks.

Based on the pH range of their activity, proteases can be classified as acidic, neutral or alkaline proteases. Acid proteases include microbial rennet, chymosin, pepsin and fungal acid proteases. Neutral proteases include trypsin, papain, bromelain/ficin, and bacterial neutral protease. Alkaline proteases include subtilisin and related proteases. Commercially available liquid enzyme compositions containing proteases are available under the trade names of Rennilase ^R , "PTN" (Pancreatic Trypsin NOVO), "PEM" (Proteolytic Enzyme Mix), Neu-trase ^R , Alcalase ^R , Esperase ^R and Savinase ^R These compositions were provided by Novo Nordisk Bio-industials, Inc. of Danbury, CT. Another commercially available protease is available under the tradename HT-Proteolyti from Solvay Enzyme Products.

Another class of enzymes, amylases, have also been employed in many industrial and commercial processes where they are used to catalyze or accelerate the hydrolysis of starch. Amylases are used extensively in the corn steep liquor industry for the production of glucose syrup, malt syrup, and various other more refined starch hydrolysis end products such as high fructose syrup. As a class, they include alpha-amylases, beta-amylases, amyloglucosidases (glucoamylases), fungal amylases and pullulanases. Commercially available liquid enzyme compositions containing amylase are available under the trade names of BAN, Termamyl ^R , AMG, Fungamyl ^R and Promozyme ^™ (supplied by Novo Nordisk) and Dia-zyme L-200 (product of Solvay Enzyme Products).

Other enzymes of commercial interest are those affecting the hydrolysis of fibers. These enzymes include cellulases, hemicellulases, pectinases and beta-glucanases. Cellulases are enzymes that degrade cellulose, a linear glucose polymer found in plant cell walls. Hemicellulases are involved in the hydrolysis of hemicellulose, which, like cellulose, is a polysaccharide found in plants. Pectinases are enzymes involved in the degradation of pectin, a carbohydrate mainly composed of sugar acids. Beta-glucanases are enzymes involved in the hydrolysis of beta-glucans (which, like cellulose, are also linear polymers of glucose). In terms of commercial use, these enzymes have more or less utility in manufacturing processes based on their degradation of fibers.

It has been reported that cellulase can be used in the deinking process of old newsprint (ONP) without any surfactant and alkaline chemicals. The enzyme removes the ink from the fiber surface and disperses the ink particles to a certain size. See S. Say-Kyoun Ow, Biol-logical De-Inking Methods of Newsprint Wastepaper, Wordpulp and paper Technology, PP. 63, 64 (1992). In general, cellulases include endo-cellulases, exo-cellulases, exo-fiber-biohydrolases, mannanases and cellobiases. Commercially available liquid enzyme compositions comprising cellulase are available under the tradenames Cellu-clast ^R and Novozym ^R 188 (both supplied by Novo Nordisk).

Hemicellulases are also used in the deinking process to remove ink particles from the ONP fiber surface. See DYPrasad et al., Enzyme Deinking of Black and White Le-tterpress Printed Newsprint Waste, Progress in Paper Recycling, May 1992, PP.21, 22. Furthermore, hemicellulases such as xylanases are used in pulp bleaching processes. Pretreatment of kraft pulp with xylanase resulted in a substantial reduction in the use of bleaching chemicals (such as molecular chlorine) and also improved pulp quality, as reflected by higher brightness thresholds. See DJ Senior et al., Reduction in Chlorine Use During Bleaching of Kraft pulp Following Xylanase Treatment, Tappi Journal (in press, the contents of which were disclosed at the International Conference on Pulp Bleaching, Stockholm, 1991). The PULPZYM ^R product available from Novo Nordisk and the ECOPULP ^R product available from Alko Biotechnology are two examples of commercially available liquid enzyme compositions containing xylanase-based bleaching enzymes.

As a class, hemicellulases include hemicellulase mixtures and galactomannanases. Commercially available liquid enzyme compositions containing hemicellulase are available under the tradenames PULPZYMR (product of Novo), ECOPULP ^R (product of Alko Biotechnology) and Novozym ^R 280 and Gamanase ^™ (product of Novo Nordisk).

Pectinases are used commercially to weaken cell walls and enhance juice extraction, as well as to reduce viscosity and prevent gelation in these extracts. Pectinase consists of endogenous galacturonase, exogenous galacturonase, inner pectate lyase (transelimin-ase), outer pectate lyase (trans Elimination enzyme), and endopectin lyase (trans elimination enzyme). Commercially available liquid enzyme compositions containing pectinase are available under the tradenames Pectinex ^™ Ultra SP and Pectinex ^™* (both supplied by Novo Nordisk).

β-glucanases play an important role in the malting and brewing industry, where the treatment of barley cell walls containing β-glucans is necessary. β-glucanases consist of lichenases, laminarinases and exoglucanases. Commercially available liquid enzyme compositions containing β-glucanase are available under the tradenames Novozym ^R 234, Cereflo ^R , BAN, Finizym ^R and Ceremix ^R (all supplied by Novo Nordisk.

Two other classes of enzymes used industrially and commercially are lipases and phospholipases. Lipases and phospholipases are esterases that hydrolyze fats and oils by acting on the ester bonds in these compounds. Lipase acts on triglycerides while phospholipase acts on phospholipids. In the industrial sector, lipases and phospholipases represent commercially available esterases and both currently have many industrial and commercial uses.

In the pulp and paper industry, liquid enzyme preparations containing lipase have proven particularly useful in reducing resin deposition on drums and other equipment during production. For example, treatment of unbleached sulfite pulp with lipase prior to bleaching with chlorine has been reported to reduce the content of chlorinated triglycerides, which are believed to be responsible for resin deposition during paper manufacturing. See K. Fischer and K. Messner, Reducing Troublesome Pitch in Pu-lp Mills By Lipolytic Enzymes, Tappi Journal, Feb. 1995, p. 130. Novo Nordisk markets two liquid lipase preparations under the tradenames Resinase ^™ A and Resinase ^™ A 2X. Both formulations have been reported to significantly reduce resin deposition under certain conditions by breaking down resin in the pulp.

Another important use of lipase is in the degreasing of hides and hides during leather making. Alkaline lipase is used in combination with specific proteases and emulsification systems to help remove oil stains and improve soaking and liming clarification in leather making. See J. Christner, The Use of Lipases in the Beamhouse Processes, 87 J.A.L.C.A. 128 (1992).

Lipases have also been used to develop flavoring agents in cheese and to improve the palatability of beef tallow to dogs. In non-aqueous systems, lipases are used to synthesize esters from carboxylic acids and alcohols.

Commercial liquid enzyme compositions containing lipase are commercially available. For example, such compositions are commercially available under the tradenames Lipolase 100, Greasex 5OL, Palatase ^™ A, Palatase ^™ M and Lipozyme ^™ (all supplied by Novo Nordisk).

As far as phospholipases are used in industry, pancreatic phospholipase _A2 has been used to convert lecithin to lysolecithin. Lysolecithin has been reported to be a good emulsifier in the production of mayonnaise and baked bread. Phospholipase A ₂ is commercially available in the form of a liquid enzyme composition sold under the name LECITASE ^™ by Novo Nordisk.

Another class of commercially important enzymes are isomerases, which catalyze conversion reactions between isomers of organic compounds. Isomerases are of particular importance in the high fructose corn syrup industry. For example, the aldose-ketose isomerase reaction catalyzed by glucose isomerase involves the conversion of glucose to fructose and is one of the three key enzyme reactions in this industry. Sweetzyme ^R product is a liquid enzyme composition containing glucose isomerase provided by Novo Nordisk.

Oxidoreductases are enzymes that act as catalysts in chemical oxidation/reduction reactions and are accordingly involved in the decomposition and synthesis of many biochemical substances. Currently, many oxidoreductases have not gained a significant place in the industry since most oxidoreductases require the presence of cofactors. However, oxidoreductases are useful industrially, especially in food processing, when the cofactor is an integral part of the enzyme or is not necessarily present.

An oxidoreductase, glucose oxidase, is used to prevent the undesired browning reactions that affect food color and taste. Glucose oxidase is also used as an "oxygen scavenger" to prevent odor aggravation in fruit juices and to maintain the color and stability of certain sensitive food ingredients. Another oxidoreductase, catalase, has been used to break down residual hydrogen peroxide used as a sterilant. A third oxidoreductase, lipoxygenase (which occurs naturally in soy flour and is not usually purified for industrial use) is used in baking not only to obtain whiter bread but also to prevent dough softening effect. Other oxidoreductases have possible uses ranging from enzymatic synthesis of steroid derivatives to diagnostic assays. These oxidoreductases include peroxidase, superoxide dismutase, alcohol oxidase, polyphenol oxidase, xanthine oxidase, sulfhydryl oxidase, hydroxylase (hydroxylase), cholesterol oxidase, laccase, Alcohol dehydrogenase and steroid dehydrogenase.

When enzymes, such as those described above, are produced or sold for use in industrial processes, they are generally formulated as water-based or aqueous liquid enzyme compositions for the particular process. Water-based liquid enzyme compositions may contain additional solvents depending on the particular enzyme or use of the composition. However, these liquid enzyme compositions have historically suffered from difficult problems, such as chemical instability leading to loss of enzyme activity, especially upon storage. The critical problem of loss of enzyme activity upon storage particularly affects the liquid detergent industry.

All over the world, it is not uncommon for industrial products (eg, liquid enzyme compositions) to be stored in warehouses under various climatic conditions (where the product is subjected to temperatures ranging from freezing to above 50° C. for long periods of time). After many months of storage at extreme temperatures ranging from 0°C to 50°C, most liquid enzyme compositions lose 20% to 100% of their enzyme activity due to enzyme instability.

Various attempts have been made to stabilize enzymes contained in liquid enzyme compositions. Efforts to increase the stability of liquid enzyme compositions using formulations containing alcohols, glycerols, dialkylglycerol ethers and mixtures thereof with other compounds have only marginally succeeded, even in the mild storage temperature range.

In U.S. Patent No. 4,801,544, glycol and ethoxylated linear alcohol nonionic surfactants and hydrocarbon solvent systems are used as stabilizers and enzymes are described that will be present in the solvent/surfactant mixture Method of encapsulation in capsules. The water content of the composition was kept below 5% and the enzyme stability was checked at 35°, 70° and 100°F.

US Patent No. 4,548,727 describes the use of certain esters for stabilization of aqueous enzyme formulations. Esters useful as stabilizers have the general formula RcooR' wherein R is an alkyl group of from 1 to 3 carbon atoms or hydrogen and R' is an alkyl group of from 1 to 6 carbon atoms. The esters are present in the aqueous enzyme preparation in an amount from 0.1 to about 2.5% by weight.

US Patent No. 4,318,818 describes a stabilization system for aqueous enzyme compositions comprising calcium ions and a low molecular weight carboxylic acid or salt thereof. The pH of the stabilization system is from about 6.5 to 10.

US Patent No. 4,243,543 describes a process for the stabilization of liquid proteolytic enzyme-containing detergent compositions. The detergent composition is stabilized by incorporating therein an antioxidant and a hydrophilic polyol while stabilizing the pH of the composition.

US Patent No. 4,169,817 describes a liquid cleaner composition containing a stabilized enzyme. The composition is an aqueous solution containing from 10% to 50% by weight solid matter and contains a detergent builder, a surfactant, an enzyme system from Bacillus subtilis and an enzyme stabilizer. The stabilizer comprises a highly water-soluble sodium or potassium salt and/or a water-soluble hydroxy alcohol, and enables long-term storage of the solution without enzyme inactivation.

European Patent No. 0 352 244 A2 describes stabilized liquid detergent compositions using amphoteric surfactants.

The present invention provides a formulation capable of synergistically stabilizing one or more enzymes contained in a liquid enzyme composition.

Thus, the present invention also provides stabilized liquid enzyme compositions.

In addition, the invention also provides a method for preparing a stabilized liquid enzyme composition.

The objectives of the various aspects of the invention can be broadly accomplished using formulations for stabilizing liquid enzyme compositions comprising:

(a) at least one polymer selected from poly(cellulose) ethers, acrylic polymers, polyamides,

(b) a C ₂ -C ₆ polyhydric alcohol, and

(c) water,

Wherein components (a) and (b) are present in an amount effective to stabilize at least one enzyme contained in the liquid enzyme composition. Preferably, polymer (a) and _C2 - _C6 polyhydric alcohol (b) are present in a combined amount synergistically effective to stabilize at least one enzyme comprised in the liquid enzyme composition.

The stabilizing formulations of the present invention can be used with a variety of enzymes for use in liquid enzyme compositions with multiple functions. Enzymes and enzymes that can be used with this stabilizing formulation include, but are not limited to, those discussed above.

The present invention also relates to a stabilized liquid enzyme composition comprising:

(a) at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides,

(b) a C ₂ -C ₆ 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 contained in the liquid enzyme composition. Preferably, polymer (a) and _C2 - _C6 polyhydric alcohol (b) are present in a combined amount synergistically effective to stabilize at least one enzyme comprised in the liquid enzyme composition.

The present invention further relates to a method for preparing a stabilized liquid enzyme composition by combining the enzyme with the above stabilized formulation. In addition, the present invention also relates to a method for stabilizing a liquid enzyme composition using a stabilizing formulation, the method comprising the step of combining the stabilizing formulation with the liquid enzyme composition.

In a preferred embodiment, the invention provides a formulation for stabilizing a liquid enzyme composition, the formulation comprising:

(a) at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides,

(b) a C ₂ -C ₆ polyhydric alcohol, and

(c) water,

Wherein components (a) and (b) are present in a combined amount synergistically effective to stabilize at least one enzyme contained in the liquid enzyme composition.

Water-soluble, or at least partially water-soluble polymers are used in the formulations for the stabilized liquid enzyme compositions of the present invention or in the stabilized liquid enzyme compositions of the present invention. That is, the polymer must be sufficiently soluble so as to be readily miscible with water and form a single phase. With this solubility, the polymer does not segregate when combined with a _C2 - _C6 polyhydric alcohol and water in a stabilizing formulation or with a liquid enzyme composition. The formulation does not have to be a clear solution. Preferably, the formulation is an emulsion of substantially uniform composition.

The amount of the polymer present in the formulation for the stabilized liquid enzyme composition or in the stabilized liquid enzyme composition of the present invention depends on the molecular weight of the particular polymer used. The higher the molecular weight of the polymer used, generally the lower the amount of polymer required to stabilize the enzyme.

The polymer may preferably be used in amounts up to about 50% by weight of the stabilized formulation. More preferably, the polymer is present in an amount of 0.05 to 30% by weight, most preferably, 1% to 10% by weight. In a preferred embodiment, the polymer is of course present in an amount to achieve the desired synergistic stabilization of the liquid enzyme composition when combined with the polyhydric alcohol in an aqueous matrix or aqueous formulation.

The polymers used in the present invention are selected from poly(cellulose) ethers, acrylic polymers and polyamides. The polymer can be substituted or unsubstituted. The polymer may have a slight ionic charge, but is preferably non-ionic.

When the polymer used is poly(cellulose) ether, the polymer is preferably selected from the group consisting of: poly(carboxymethylcellulose) ether, poly(hydroxypropylmethylcellulose) ether, poly(hydroxyethylmethylcellulose) cellulose) ether, poly(hydroxybutylmethylcellulose) ether, poly(hydroxypropylcellulose) ether and poly(ethylhydroxyethylcellulose) ether. More preferably the polymer is poly(carboxymethylcellulose) ether. Certain poly(cellulose) ethers useful in the present invention, eg, poly(carboxymethylcellulose) ether, are sold as salts, sodium salts, and have a weak anionic charge. Preferred poly(cellulose) ethers have molecular weights ranging from 15,000 to 100,000, but more preferred molecular weights range from 20,000 to 75,000.

The acrylic polymer used in the present invention is preferably a polymer or copolymer of acrylic acid, methacrylic acid or derivatives thereof (eg acrylates or salts). Examples of preferred acrylic polymers are the Rohm and Haas polymers Acrysol GS product (a sodium polyacrylate polymer), Acrysol TI-935 product (an acrylic polymer) and Acrylin 22 product (an acrylic polymer). The molecular weight of the acrylic polymer can range from about 5,000 to greater than 4,000,000. Preferred acrylic polymers have molecular weights ranging from 100,000 to greater than 4,000,000, more preferred are those having molecular weights ranging from 750,000 to greater than 4,000,000. Particularly preferred are acrylic polymers having molecular weights of 1,250,000 and 4,000,000. Acrylic polymers of these various molecular weights are available from Aldrich Chemical Company. Acrylic polymers can have anionic charges. Preferred acrylic polymers are those polymers or derivatives thereof that have only weak or no charges, most preferably those polymers or derivatives that are uncharged.

When the polymer used is a polyamide, the most preferred polyamide is polyvinylpyrrolidone. Preferred polyvinylpyrrolidones are those having a molecular weight ranging from 5,000 to 400,000, but more preferred are those having a molecular weight ranging from 300,000 to 400,000. Preferred are those polymers of higher molecular weight.

The stabilizing formulation also contains a _C2 - _C6 polyhydric alcohol as a second component. The _C2 - _C6 polyhydric alcohol acts synergistically with the polymers described above to stabilize the enzyme in the liquid enzyme composition. The _C2 - _C6 polyhydric alcohol is preferably selected from diols and trihydric alcohols. More preferably, the _C2 - _C6 polyhydric alcohol is glycerol, sorbitol, propylene glycol, butylene glycol, hexylene glycol or ethylene glycol. The most preferred _C2 - _C6 polyol is glycerol.

The stabilizing formulation contains a sufficient amount of _C2 - _C6 polyol, together with a polymer, to stabilize at least one enzyme in the liquid enzyme composition. Preferably, the _C2 - _C6 polyol is used in an amount of about 0.50 to 60% by weight of the total stabilizing formulation, more preferably about 5 to 50% by weight, still more preferably 10 to 30% by weight between. Most preferably, the combined amount of _C2 - _C6 polyhydric alcohol and polymer in the stabilizing formulation or enzyme composition is such that it has a synergistic stabilizing effect.

The formulations of the present invention are water-based or contain water in an amount sufficient for the polymers to be miscible with the formulation without separation. In general, _C2 - _C6 polyhydric alcohols are water soluble, but sufficient water must be present so that the formulation forms a single phase. As noted above, the formulation need not be a clear solution, but is preferably an emulsion with a distinctly homogeneous character. Water-based formulations may contain solvents other than water.

Although the stabilizing formulations of the present invention can be prepared by mixing the components in any order, it is preferred to prepare the formulations by adding the desired amount of polymer to the _C2 - _C6 polyol/water mixture. C ₂ -C ₆ polyol/water mixtures can be prepared by methods known in the art. For example, the mixture can be prepared by simply mixing the desired polyol with an appropriate amount of water or by diluting a previously prepared mixture.

Preferred mixtures of _C2 - _C6 polyhydric alcohols and water may contain any percentage of _C2 - _C6 sufficient to stabilize (preferably co-stabilize) the at least one enzyme in the liquid enzyme composition together with the polymers described above alcohol. Preferably, the mixture contains 1-95% by weight of polyhydric alcohol or its water-soluble polymer; more preferably 10-50% by weight; most preferably 30-50% by weight. When the polyhydric alcohol is glycerol, the mixture is preferably a 50% by weight glycerin/water mixture. Applicants, without being bound by a particular theory, believe that the function of the mixture is to wet the polymers used in the invention and the _C2 - _C6 polyhydric alcohol in the mixture, thereby synergistically stabilizing the enzyme.

To sufficiently stabilize an enzyme in a liquid enzyme composition according to the invention, the enzyme should have at least 90% activity after 30 days at 25°C. The following examples illustrate the preferred synergistic stabilization of various enzymes after 30 days at 50°C.

The stabilization formulations described herein can be used with a variety of enzymes and industrial processes or commercial products. Enzymes, industrial processes or commercial products that can be used with this stabilizing formulation include, but are not limited to, those discussed above.

The use of the stabilizing formulation of the stabilized liquid enzyme composition forms a second embodiment of the present invention, a stabilized liquid enzyme composition. Thus the present invention also relates to a stabilized liquid enzyme composition comprising at least one polymer selected from the group consisting of poly(cellulose) ethers, acrylic acid polymers and polyamides; C ₂ -C ₆ polyhydric alcohols; water and at least one enzyme. The polymer and the _C2 - _C6 polyhydric alcohol are present in combined amounts effective to stabilize, preferably synergistically stabilize, at least one enzyme contained in the liquid enzyme composition. Preferred compositions according to the invention are capable of exhibiting a greater viscosity than a quantitatively comparable aqueous mixture containing the same polymer.

Desirable and preferred embodiments regarding the polymer, _C2 - _C6 polyhydric alcohol and water present in the stabilized liquid enzyme composition are the same as those discussed above in relation to the stabilizing formulation of the present invention.

As with the stabilizing formulations, the liquid enzyme compositions of the present invention can be applied to a variety of enzymes. These enzymes include, but are not limited to, the classes of enzymes and specific enzymes discussed hereinbefore. Enzymes which can be used are those obtained from animals, plants, fungi, bacteria and synthetically. Preferred water-dispersible enzymes for this system are proteases widely used in the laundry detergent and cheese making industries, including acidic, alkaline and neutral proteases; amylases used e.g. in the corn steep liquor industry, including acidic , alkaline and neutral amylases; lipases for cheese flavor development and for the pulp, paper and leather making industries; cellulase and xylanase (Xylase).

Depending on the intended use, enzymes are typically packaged and sold as concentrated liquid enzyme compositions that are diluted prior to use. Enzymes may also be provided in powder or dry form. Concentrated enzyme diluted enzyme content depends on the form of enzyme supply. In general, preferred enzyme levels may range from 0.05% to 40%, more preferably 0.5% to 25%, most preferably 10% to 20% by weight of the concentrated liquid enzyme composition. The stabilizing formulations of the present invention are particularly designed for use in concentrated liquid enzyme compositions and for use in already diluted compositions. The amount of stabilizing formulation required to stabilize or to co-stabilize the concentrated solution can, and likely will, differ from the amount used for the diluted composition. Suitable amounts of the stabilizing formulation or its components can be readily determined according to ordinary skill in the art using the methods set forth in the Examples below. However, it is well known that the enzyme content depends on the activity of the particular enzyme and the desired end use.

Depending on the enzymes involved and the intended use, the pH of the final stabilized liquid enzyme composition is preferably from 5.0 to 10.0, but more preferably around 7.0. Most preferably, the system should be allowed to adopt its own pH, generally around neutral. It is well known that small amounts of acidic or basic substances may be required to adjust the pH.

The stabilized liquid enzyme composition may be water-based or aqueous and may contain solvents or additives relevant to the use of the composition in a particular industrial process. For example, the stabilized liquid enzyme composition may contain some additives, such as surfactants, anti-foaming agents and the like known in the art. In terms of synergistic stabilization, these additives can be added in amounts that do not interfere with the synergistic stabilization of the liquid enzyme composition. This amount can be readily determined by one skilled in the art. Advantageously, when using the stabilized liquid enzyme composition of the present invention, the stabilized formulation can also be used as a dispersion aid for the enzyme in industrial process waters.

The present invention also relates to a method of preparing a stabilized liquid enzyme composition comprising the step of combining at least one enzyme with the stabilizing formulation of the present invention. The present invention further relates to a method of stabilizing a liquid enzyme composition using a stabilizing formulation, the method comprising the step of combining the liquid enzyme composition with the stabilizing formulation. Illustrative and preferred components for use in the method and the amounts of such components are the same as discussed above.

Those of ordinary skill will appreciate that the components for the formulation of the stabilized liquid enzyme composition or the components of the stabilized liquid enzyme composition may be combined in any order or even simultaneously. However, it is best to use the following order:

(a) mixing C ₂ -C ₆ polyhydric alcohols with water,

(b) adding at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides to the mixture prepared in step (a); and

(c) adding a liquid enzyme composition comprising at least one enzyme to the mixture formed in steps (a) and (b). The polymer and the _C2 - _C6 polyhydric alcohol are preferably present in combined amounts synergistically effective to stabilize at least one enzyme in the resulting liquid enzyme composition.

Another method of preparing a stabilized liquid enzyme composition comprises the steps of:

(a) mixing C ₂ -C ₆ polyhydric alcohols with water,

(b) adding at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides to the mixture prepared in step (a); and

(c) combining the formulation formed in steps (a) and (b) with a liquid enzyme composition comprising at least one enzyme. The polymer and the _C2 - _C6 polyhydric alcohol are preferably present in a combined amount synergistically effective to stabilize the enzyme in the at least one liquid enzyme composition.

In the process according to the invention, the polymer added in step (b) can be added alone, or as an aqueous dispersion or a solution (polymer dissolved in water or a suitable organic solvent). When other additives are included in the stabilized liquid enzyme composition, these additives may be added at any time, but are preferably added before step (c) or in a separate step after the addition of the enzyme.

The following examples are given for the purpose of illustrating the invention. It is to be understood, however, that the invention is not to be limited to the specific conditions or details set forth in these examples.

Example 1

By testing glycerol (referred to as compound A), poly(carboxymethylcellulose) ether (CMC) with a molecular weight of 250,000 (referred to as compound B), or polyvinylpyrrolidone (PVP) with a molecular weight of 360,000 (referred to as compound B') Demonstrate synergy. As shown in Tables 1 and 2, experiments were carried out by varying the ratio of compound A to compound B or B' over a range of concentrations and testing various types of enzymes for stabilization of increased enzyme activity at 50°C for 30 days. The concentration of each compound required to detect 90% of the original enzyme activity was taken as the endpoint. Concentrations are expressed as weight percent of compound in the final composition including added enzyme and water therein. Water was added to Compound A, glycerin to form a glycerol-water mixture. The endpoints for the composition containing Compound A and Compound B or B' are then compared to the endpoints for Compound A alone and Compound B or B' alone.

Synergy was determined using the method described by Kull, FC, Eisman, PC, Sylwestrowicz, HO, and Mayer, RL, Applied Microbiology 9:538-541 (1961) using the ratio determined by: $\frac{\mathrm{QA}}{Q}+\frac{\mathrm{QB}}{\mathrm{Qb}}$ where Qa = the percentage of the aqueous mixture of compound A acting alone to produce the endpoint (weight

quantity).

Qb = Percentage of the aqueous mixture of compound B acting alone to produce the endpoint (weight

quantity).

QA = Percentage of the aqueous mixture of compound A to compound B that produced the endpoint (weight

quantity).

QB = Percentage of compound B to compound A aqueous mixture that produced the endpoint (weight

quantity). Antagonism was indicated when the sum of QA/Qa and QB/Qb was greater than 1. If the sum is equal to 1, it indicates additive effect. If the sum is less than 1, synergy is indicated.

The method used to illustrate the synergistic effect of the compounds of the invention is a widely used and accepted method. More details are given in Kull et al. Further details of this method are contained in US Patent No. 3,231,509, the contents of which are incorporated herein by reference.

The results obtained shown in Tables 1 and 2 illustrate the improved stabilization of the HT-Proteolytic L-175 enzyme, an alkaline protease sold by Solvay Enzymes, Inc. The sum of QA/Qa+QB/Qb was calculated for all compositions containing glycerol (Compound A) and CMC (Compound B) using the method described by Kull et al. An example calculation is given below for some endpoints where there is a clear synergy. As shown in Table 1 and the calculation example, these endpoint values were 0.58, 0.67, 0.67 and 0.67, respectively, indicating the presence of synergy. Similarly, the sum of QA/Qa+QB'/Qb' was calculated for all compositions containing glycerol (Compound A) and PVP (Compound B'). As shown in Table 1 and the example calculations, these endpoints were 0.85, 0.95, 0.65, 0.85, respectively, again indicating the presence of synergy.

Table 1

            Compound A (% by weight)

60 30 20 10 5 0 Compound B (% by weight)

6 + + + + + + ^*

3 + + + + ^* + ^* -

2 + + + ^* - - -

1 + + ^* - - - -

0 + ^* - - - - -

(1) (*) = endpoint of ≥90% enzyme activity after 30 days at 50°C

(2) (+) = ≥90% enzyme activity after 30 days at 50°C

(3) (-) = <90% enzyme activity was retained after 30 days at 50°C

(4) Compound A = Glycerol

(5) Compound B=CMC

calculate:

Qa Qb QA QB QA/Qa＋QB/Qb

(%)60 6 5 3 5/60+3/6=0.58

                                                                                        

20 2 20/60+2/6=0.67

30 1 30/60+1/6=0.67

Table 2

     Compound A (% by weight)

50 30 20 10 5 0Compound B' (% by weight) 40 + + + + + + ^* 30 + + + + ^* + ^* -20 + + + - - -10 + + ^* + ^* - - -5 + - - - - -0 + ^* - - - - -

(1) (*) = endpoint of ≥90% enzyme activity after 30 days at 50°C

(2) (+) = ≥90% enzyme activity after 30 days at 50°C

(3) (-) = <90% enzyme activity was retained after 30 days at 50°C

(4) Compound A = Glycerin

(5) Compound B'=PVP

calculate:

  Qa Qb′ QA QB′ QA/Qa＋QB′/Qb′

(%)50 40 5 30 5/50+30/40=0.85

                                                                      

20 10 20/50+10/40=0.65

30 10 30/50+10/40=0.85

Example 2

The stabilized formulation of the invention of Example 1 was tested with another enzyme, Diazyme L-200 (a glucoamylase sold by Solvay Enzymes, Inc.). The results shown in Tables 3 and 4 also show an increase in the stability of this enzyme. The sum of QA/Qa + QB/Qb was calculated for all compositions containing glycerol (Compound A) and CMC (Compound B). Calculation examples for certain endpoints where synergy is evident in this example are shown. As shown in Table 3 and the calculation example, these endpoint values were 0.59, 0.67, 0.67 and 0.84, respectively, indicating the presence of synergy. Similarly, the sum of QA/Qa+QB'/Qb' was calculated for all compositions comprising glycerol (Compound A) and PVP (Compound B'). As shown in Table 4 and the calculation example, these endpoint values were 0.77, 0.53, 0.73 and 0.77, respectively, indicating the presence of synergy.

table 3

                  Compound A (% by weight)

60 30 20 10 5 0 Compound B (% by weight)

6 + + + + + + + + +

3 + + + + ^* + ^* -

2 + + ^* + ^* - - -

1 + + - - - - - - -

0 + ^* - - - - -

(1) (*) = endpoint of ≥90% enzyme activity after 30 days at 50°C

(2) (+) = ≥90% enzyme activity after 30 days at 50°C

(3) (-) = <90% enzyme activity was retained after 30 days at 50°C

(4) Compound A = Glycerol

(5) Compound B=CMC

calculate:

Qa Qb QA QB QA/Qa＋QB/Qb

(%) 60 6 5 3 5/60+3/6=0.59

                                                                                               

20 2 20/60+2/6=0.67

30 2 30/60+2/6=0.84

Table 4

                  Compound A (% by weight)

50 30 20 10 5 0Compound B'(% by weight)

30 + + + + + + ^*

20 + + + + + ^* -

10 + + + ^* + ^* - -

5 + + ^* - - - -

0 + ^* - - - - -

(1) (*) = endpoint of ≥90% enzyme activity after 30 days at 50°C

(2) (+) = ≥90% enzyme activity after 30 days at 50°C

(3) (-) = <90% enzyme activity was retained after 30 days at 50°C

(4) Compound A = Glycerol

(5) Compound B'=PVP

calculate:

  Qa Qb′ QA QB′ QA/Qa＋QB′/Qb′

(%)50 30 5 20 5/50+20/30=0.77

                                                                                            

20 10 20/50+10/30=0.73

30 5 30/50+5/30=0.77

Example 3

Enzymes with esterase activity were detected with the stabilized formulation in Example 1 of the present invention. Tables 5 and 6 present the results obtained using the enzyme Lipolase, a lipase sold by Novo Nordisk Bioindustries, Inc., showing an improved stabilization of this enzyme. The sum of QA/Qa+QB/Qb was calculated for all compositions containing aqueous glycerol (Compound A) and CMC (Compound B). Calculation examples for certain endpoints where synergy is evident in this example are shown. As shown in Table 5 and the calculation example, these endpoint values were 0.43, 0.37, 0.57 and 0.77, respectively, indicating the presence of synergy. Similarly, the sum of QA/Qa+QB'/Qb' was calculated for all compositions containing glycerol (Compound A) and PVP (Compound B'). As shown in Table 6 and the calculation example, these endpoint values were 0.60, 0.70, 0.65 and 0.85, respectively, indicating the presence of synergy.

table 5

Compound A (% by weight)

50 30 20 10 5 0 Compound B (% by weight)

6 + + + + + + ^*

3 + + + + + + + -

2 + + + + + ^* -

1 + + ^* + ^* + ^* - -

0 + ^* - - - - -

(1) (*) = endpoint of ≥90% enzyme activity after 30 days at 50°C

(2) (+) = ≥90% enzyme activity after 30 days at 50°C

(3) (-) = <90% enzyme activity was retained after 30 days at 50°C

(4) Compound A = Glycerin

(5) Compound B=CMC

calculate:

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

Compound A (% by weight)

50 30 20 10 5 0Compound B'(% by weight)

30 + + + + + + ^*

20 + + + + + + + + +

10 + + + + ^* + ^* -

5 + + ^* + ^* - - -

0 + ^* - - - - -

(1) (*) = endpoint of ≥90% enzyme activity after 30 days at 50°C

(2) (+) = ≥90% enzyme activity after 30 days at 50°C

(3) (-) = <90% enzyme activity was retained after 30 days at 50°C

(4) Compound A = Glycerin

(5) Compound B'=PVP

calculate:

  Qa Qb′ QA QB′ QA/Qa＋QB′/Qb′

(%)50 20 5 10 5/50+10/20=0.60

                                                                                                                     

20 5 20/50+5/20=0.65

30 5 30/50+5/20=0.85

Example 4

The hemicellulase Pulpzyme HB (a xylanase sold by Novo Nordisk Bioindustrials, Inc.) was chosen to test the improved stability formulation as in Example 1. The results obtained for this example are shown in Tables 7 and 8 below. The sum of QA/Qa + QB/Qb was calculated for all compositions containing glycerol (Compound A) and CMC (Compound B). Calculation examples for those endpoints where synergy is evident in this example are shown. As shown in Table 7 and the calculation example, these endpoint values were 0.43, 0.53, 0.57 and 0.77, respectively, indicating the presence of synergy. Similarly, the sum of QA/Qa+QB/Qb was calculated for all compositions containing glycerol (Compound A) and PVP (Compound B'). Endpoint values were 0.35, 0.45, 0.65 and 0.85, respectively, indicating the presence of synergy.

Table 7

     Compound A (% by weight)

50 30 20 10 5 0 Compound B (% by weight)

6 + + + + + + ^*

3 + + + + + + + -

2 + + + + ^* + ^* -

1 + + ^* + ^* - - -

0 + ^* - - - - -

(1) (*) = endpoint of ≥90% enzyme activity after 30 days at 50°C

(2) (+) = ≥90% enzyme activity after 30 days at 50°C (3) (-) = <90% enzyme activity was retained after 30 days at 50°C (4) Compound A = glycerol (5) Compound B = CMC calculate:

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=0.57

30 1 30/50+1/6=0.77

Table 8

  Compound A (% by weight)

50 30 20 10 5 0Compound B'(% by weight)

40 + + + + + + ^*

30 + + + + + + + -

20 + + + + + + + -

10 + + ^* + ^* + ^* + ^* -

5 + + - - - - - - -

0 + ^* - - - - -

(1) (*) = endpoint of ≥90% enzyme activity after 30 days at 50°C

(2) (+) = ≥90% enzyme activity after 30 days at 50°C

(3) (-) = <90% enzyme activity was retained after 30 days at 50°C

(4) Compound A = Glycerin

(5) Compound B'=PVP calculation:

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 (20)

1. A formulation for stabilizing a liquid enzyme composition, comprising: (a) at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides, (b) a C ₂ -C ₆ polyhydric alcohol, and (c) water, wherein components (a) and (b) are present in a combined amount synergistically effective to stabilize at least one enzyme contained in the liquid enzyme composition. 2. The formulation of claim 1, wherein said polymer is selected from the group consisting of poly(carboxymethylcellulose) ether, poly(hydroxypropylmethylcellulose) ether, poly(hydroxyethylmethylcellulose) ether , poly(cellulose) ethers of poly(hydroxybutylmethylcellulose) ether, poly(hydroxypropylcellulose) ether, and poly(ethylhydroxyethylcellulose) ether. 3. The formulation of claim 1, wherein said _C2 - _C6 polyhydric alcohol is glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol or ethylene glycol, and said component (b) and (c) are contained in a mixture of 1-95% by weight of said _C2 - _C6 polyhydric alcohol and the remainder water. 4. The formulation of claim 1 wherein said polymer is a poly(cellulose) ether selected from poly(carboxymethylcellulose) ethers and said _C2 - _C6 polyhydric alcohol is glycerol. 5. The formulation of claim 1 wherein said polymer is a polyamide selected from polyvinylpyrrolidone and said _C2 - _C6 polyhydric alcohol is glycerol. 6. A stabilized liquid enzyme composition comprising: (a) at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides, (b) a mixture of C ₂ -C ₆ polyhydric alcohols, (c) water, and (d) at least one enzyme, wherein components (a) and (b) are present in a combined amount synergistically effective to stabilize the enzyme in said at least one liquid enzyme composition. 7. The composition of claim 6, wherein said polymer is selected from the group consisting of poly(carboxymethylcellulose) ether, poly(hydroxypropylmethylcellulose) ether, poly(hydroxyethylmethylcellulose) Ethers, poly(hydroxybutylmethylcellulose) ethers, poly(hydroxypropylcellulose) ethers, and poly(cellulose) ethers of poly(ethylhydroxyethylcellulose) ethers. 8. The composition of claim 6, wherein said _C2 - _C6 polyhydric alcohol is glycerol, sorbitol, propylene glycol, butylene glycol, hexylene glycol or ethylene glycol; said component (b) and (c) contained in a mixture of 1-95% by weight of said C ₂ -C ₆ polyhydric alcohol and the remainder water; said enzyme selected from the group consisting of protease, amylase, lipase, cellulase, mannan Enzyme and xylanase (Xylase). 9. The composition of claim 6, wherein said polymer is a poly(cellulose) ether selected from poly(carboxymethylcellulose) ethers and said _C2 - _C6 polyhydric alcohol is glycerol. 10. The composition of claim 6 wherein said polymer is a polyamide selected from polyvinylpyrrolidone and said _C2 - _C6 polyhydric alcohol is glycerol. 11. The composition of claim 6, wherein said composition is an opacifying agent. 12. A stabilized liquid enzyme composition comprising: (a) at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides, (b) a mixture of C ₂ -C ₆ polyhydric alcohols, (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 said liquid enzyme composition. 13. A method of preparing a liquid stabilized enzyme composition, the method comprising: Combining at least one enzyme with a formulation comprising: (a) at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides, (b) a C ₂ -C ₆ polyhydric alcohol, and (c) water, wherein components (a) and (b) are present in a combined amount synergistically effective to stabilize at least one enzyme in said composition. 14. The method of claim 13, further comprising, prior to said combining step, mixing said C ₂ -C ₆ polyhydric alcohol and said water, and mixing poly(cellulose) ethers, acrylic polymers A step in which a polymer of polyamide is added to said mixture of C ₂ -C ₆ polyhydric alcohol and water. 15. The method of claim 13, wherein said _C2 - _C6 polyhydric alcohol is glycerol, sorbitol, propylene glycol, butylene glycol, hexylene glycol or ethylene glycol; said mixture is 1-95% ( weight) of said C ₂ -C ₆ mixture of polyhydric alcohols and the rest of the water; said at least one enzyme selected from the group consisting of protease, amylase, lipase, cellulase, mannanase and xylanase (Xylase ). 16. The method of claim 13, wherein said polymer is a poly(cellulose) ether selected from poly(carboxymethylcellulose) ethers, and said _C2 - _C6 polyhydric alcohol is glycerol. 17. The method of claim 13 wherein said polymer is a polyamide selected from polyvinylpyrrolidone and said _C2 - _C6 polyhydric alcohol is glycerol. 18. A method of stabilizing a liquid enzyme composition using a formulation comprising: (a) at least one polymer selected from poly(cellulose) ethers, acrylic polymers and polyamides, (b) a C ₂ -C ₆ polyhydric alcohol, and (c) water, Wherein components (a) and (b) are present in a combined amount synergistically effective to stabilize at least one enzyme contained in the liquid enzyme composition. The method comprises the step of combining a liquid enzyme composition comprising at least one enzyme with said formulation. 19. The method of claim 18, wherein said polymer is a poly(cellulose) ether selected from poly(carboxymethylcellulose) ether, said _C2 - _C6 polyhydric alcohol is glycerol, and Said at least one enzyme is selected from protease, amylase, lipase, cellulase, mannanase and xylanase (Xylase). 20. The method of claim 18, wherein said polymer is a polyamide selected from polyvinylpyrrolidone, said _C2 - _C6 polyhydric alcohol is glycerol, and said at least one enzyme is selected from protease, Amylase, lipase, cellulase, mannanase and hydroglycanase (Xylase).