Classifications

• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38663—Stabilised liquid enzyme compositions

Definitions

• the present invention relates to novel formulations for stabilizing at least one enzyme contained in a liquid enzymatic composition.
• the unique rheological properties of the components of these stabilizing formulations preferably afford synergistic stabilizing capacity over other water- based mixtures of enzymatic dispersions, even under conditions of moderate to high heat and wide pH ranges.
• the invention therefore, also relates to stabilized liquid enzymatic compositions. Additionally, the invention relates to novel methods for the preparation of stabilized liquid enzymatic compositions, and methods using the stabilizing formulations with liquid enzymatic compositions.
• enzymes and liquid enzymatic compositions in industry and in the commercial marketplace has grown rapidly over the last several years.
• enzymes can be acid, alkaline or neutral, depending upon the pH range in which they are active. All of these types of enzymes are contemplated to be useful in connection with the invention disclosed herein.
• enzymes and liquid enzymatic compositions have been associated with liquid detergents and have shown utility as solubilizing and cleaning formulations. In addition to their association with liquid detergents, enzymes and liquid enzy ⁇ matic compositions have also shown utility in a number of different commercial and industrial areas in which a wide variety of enzyme classes are now used.
• proteases are a well-known class of enzymes frequently utilized in a wide variety of industrial applications where they act to hydrolyze peptide bonds in proteins and proteinaceous substrates.
• Commercially, the greatest uses of proteases are made in the laundry detergent industry, where they help to remove protein-based stains such as blood or egg stains, and in the cheese-making industry, where they aid in curdling milk.
• Proteases are also used as meat tenderizers, for softening leather, for modifying food ingredients, and for flavor development.
• Liquid enzymatic compositions con ⁇ taining alkaline proteases have also shown to be useful as dispersants of bacterial films and algal and fungal mats in cooling tower waters and metalworking fluid containment bays.
• Proteases can be characterized as acid, neutral, or alkaline proteases depending upon the pH range in which they are active.
• the acid proteases include the microbial ren ⁇ nets, rennin (chymosin), pepsin, and fungal acid proteases.
• the neutral proteases include trypsin, papain, bromelain/ ficin, and bacterial neutral protease.
• the alkaline proteases include subtilisin and related proteases.
• Amylases another class of enzymes, have also been utilized in many industrial and commercial processes in which they act to catalyze or accelerate the hydrolysis of starch.
• Amylases are used largely in the corn syrup industry for the production of glucose syrups, maltose syrups, and a variety of other more refined end products of starch hydrolysis such as high fructose syrups.
• they include ⁇ -amylase, / 3-amylase, amyloglucosidase (glucoamylase) , fungal amylase, and pullulanase.
• liquid enzymatic compositions containing amylases are available under the names BAN, Termamyl®, AMG, Fungamyl®, and PromozymeTM, which are supplied by Novo Nordisk, and Diazyme L-200, a product of Solvay Enzyme Products.
• cellulases are enzymes that degrade cellulose, a linear glucose polymer occurring in the cell walls of plants.
• Hemicellulases are involved in the hydrolysis of hemicellulose which, like cellulose, is a polysaccharide found in plants.
• the pectinases are enzymes involved in the degradation of pectin, a carbohydrate whose main component is a sugar acid.
• -glucanases are enzymes involved in the hydrolysis of j ⁇ -glucans which are also similar to cellulose in that they are linear polymers of glucose. In a commercial context, these enzymes have utility to a greater or lesser degree in manufacturing processes dependent on fiber degra ⁇ dation.
• Cellulases have reported utility in the de-inking process of old newsprint (ONP) wastepaper, eliminating the need for any surfactants and alkaline chemicals.
• the enzymes dislodge inks from fiber surfaces and disperse ink particles to a finite size. See S. Say-Kyoun Ow, Biological De-Inl ing Methods of Newsprint Wastepaper, World Pulp and Paper Technology, pp. 63, 64 (1992).
• cellulases include endocellulase, exocellulase, exocello-biohydrolase, mannase, and cellobiase.
• Commercial liquid enzymatic compositions containing cellulases are available under the names Celluclast® and Novozym®188 which are both supplied by Novo Nordisk.
• Hemicellulases are also used in the de-inking process to dislodge ink particles from the fiber surface of ONP. See D. Y. Prasad et al.. Enzyme Deinking of Black and White Letterpress Printed Newsprint Waste, Progress in Paper Recy ⁇ cling, May 1992, pp. 21, 22. Additionally, hemicellulases, such as the xylanases, are employed in the pulp bleaching process. Xylanase pretreat ent of kraft pulps has resulted in major reductions in bleaching chemical requirements, such as molecular chlorine, and has also improved pulp quality as reflected by higher brightness ceilings. See D. J. Senior et al..
• hemicellulases include hemicellulase mixture and galactomannanase.
• Commercial liquid enzymatic composi ⁇ tions containing hemicellulases are available as PULPZYM® from Novo, ECOPULP® from Alko Biotechnology and Novozym®280 and GamanaseTM, which are both products of Novo Nordisk.
• pectinases are used commercially to weaken cell walls and enhance extraction of fruit juice, as well as to aid in decreasing viscosity and preventing gelation in these extracts.
• Pectinases consist of endopolygalacturonase, exopoly-galacturonase, endopectate lyase (transeliminase), exopectate lyase (transeliminase), and endopectin lyase (transeliminase).
• Commercial liquid enzymatic compositions containing pectinases are available under the names PectinexTM Ultra SP and PectinexTM*, both supplied by Novo Nordisk.
• y ⁇ -glucanases are comprised of lichenase, laminarinase, and exoglucanase.
• Commercial liquid enzymatic compositions containing ⁇ - glucanases are available under the names Novozym®234, Cereflo®, BAN, Finizym®, and Ceremix®, all of which are supplied by Novo Nordisk.
• Lipases and phospholipases are esterase enzymes which hydrolyze fats and oils by attacking the ester bonds in these compounds. Li ⁇ pases act on triglycerides, while phospholipases act on phospholipids. In the industrial sector, lipases and phospholipases represent the commercially available esterases, and both currently have a number of industrial and commercial applications.
• liquid enzyme preparations containing lipases have proven to be particularly useful in reducing pitch deposits on rolls and other equipment during the production process.
• the treatment of unbleached sulfite pulp with lipases prior to bleaching with chlorine to reduce the content of chlorinated triglycerides, which are reportedly the cause of pitch deposition during the paper manufacturing process has been reported.
• K. Fischer and K. Messner Reducing Troublesome Pi tch in Pulp Mills By Lipolytic Enzymes, Tappi Journal, Feb. 1992, p. 130.
• Novo Nordisk markets two liquid lipase preparations under the names ResinaseTM A and ResinaseTM A 2X, both of which, under certain conditions, reportedly reduce pitch deposits significantly by breaking down wood resins in pulp.
• lipases Another important use of lipases is to degrease hides and pelts in the leather-making process.
• Alkaline lipases are used in conjunction with special proteases and emulsify ⁇ ing systems to aid degreasing, as well as to improve the soaking and liming effect 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 for the development of fla ⁇ vors in cheese and to improve the palatability of beef tallow to dogs. In nonaqueous systems, lipases have been employed to synthesize esters from carboxylic acids and alcohols.
• liquid enzymatic compositions containing li ⁇ pases are available.
• such compositions are available under the trade names Lipolase 100, Greasex 50L, PalataseTMA, Palatase m M, and LipozymeTM which are all supplied by Novo Nordisk.
• pancreatic phospholipase A 2 has been used to convert lecithin into lysolecithin.
• Lysolecithin reportedly is an excellent emulsifier in the production of mayonnaise and the baking of bread.
• phospholipase A « is available in a liquid enzymatic composition sold as LECITASETM by Novo Nordisk.
• the isomerases are particu ⁇ larly important in the high fructose corn syrup industry.
• the aldose-ketose isomerase reaction catalyzed by glucose isomerase, involves the conversion of glucose to fructose and is just one of three key enzyme reactions in the industry.
• Sweetzyme® product is a liquid enzymatic composition containing glucose isomerase which is supplied by Novo Nordisk.
• Redox enzymes are enzymes that act as catalysts in chemical oxidation/reduction reactions and, consequently, are involved in the breakdown and synthesis of many biochemicals.
• redox enzymes have not gained a prominent place in industry since most redox enzymes require the presence of a cofactor.
• cofactors are an integral part of an enzyme or do not have to be supplied, redox enzymes are commercially useful, particularly in the food processing industry.
• the redox enzyme glucose oxidase
• Glucose oxidase is also used as an "oxygen scavenger" to prevent the development of off-flavors in juices and to preserve color and stability in certain sensitive food ingredients.
• the redox enzyme, catalase has been utilized to decompose residual hydrogen peroxide used as a sterilizing agent.
• a third redox enzyme, lipoxidase (lipoxygenase) found naturally in soya flour and not usually purified for industrial use, is used in baking not only to obtain whiter bread, but also to reverse the dough softening effects caused by certain agents.
• redox enzymes have possible applications ranging from the enzymatic synthesis of steroid derivatives to use in diagnostic tests.
• These redox enzymes include peroxidase, superoxide dismutase, alcohol oxidase, polyphenol oxidase, xanthine oxidase, sulfhydryl oxidase, hy- droxylases, cholesterol oxidase, laccase, alcohol dehydrogenase , and steroid dehydrogenases.
• enzymes When enzymes, such as those described above, are prepared or sold for use in industrial processes, they generally are formulated as water-based or aqueous liquid enzymatic compositions designed for a particular process. Water-based liquid enzymatic compositions may contain additional solvents depending upon the particular enzyme or use of the composition. These liquid enzymatic compositions, however, have historically been plagued with problems such as chemical instability which can result in the loss of enzymatic activity, particularly upon storage. This critical problem of loss of enzymatic activity upon storage has particularly affected the liquid detergent industry.
• the stabilization of an aqueous enzyme preparation using certain esters has been described in U.S. Patent No. 4,548,727.
• the ester used as a stabilizer has the formula, RCOOR', where R is an alkyl of from one to three carbons or hydrogen, and R' is an alkyl of from one to six carbons.
• the ester is present in the aqueous enzyme preparation in an amount from 0.1 to about 2.5% by weight.
• U.S. Patent No. 4,318,818 describes a stabilizing system for aqueous enzyme compositions where the stabilizing system comprises calcium ions and a low molecular weight carboxylic acid or its salt.
• the pH of the stabilizing system is from about 6.5 to about 10.
• U.S. Patent No. 4,243,543 teaches the stabilization of liquid proteolytic enzyme-containing detergent compositions.
• the detergent compositions are stabilized by adding an antioxidant and a hydrophilic polyol to the composition while stabilizing the pH of the composition.
• U.S. Patent No. 4,169,817 teaches a liquid cleaning composition containing stabilized enzymes.
• the composition is an aqueous solution containing from 10% to 50% by weight of solids and including detergent builders, surface active agents, an enzyme system derived from Bacillus subtilis and an enzyme stabilizing agent.
• the stabilizing agents comprise highly water soluble sodium or potassium salts and/or water soluble hydroxy alcohols and enable the solution to be stored for extended periods without deactivation of the enzymes.
• European Patent No. 0 352 244 A2 describes stabilized liquid detergent compositions using an amphoteric surfactant.
• the present invention provides a formulation capable of synergistically stabilizing one or more enzymes contained in a liquid enzymatic composition.
• the invention thus, also provides stabilized liquid enzymatic compositions.
• the invention provides methods for the preparation of stabilized liquid enzymatic compositions.
• a formulation for stabilizing a liquid enzymatic composition comprising:
• components (a) and (b) are present in an amount effective to stabilize at least one enzyme contained in a liquid enzymatic composition. More preferably, the polymer (a) and the C 2 -C 8 polyhydric alcohol (b) are present in a combined amount synergistically effective to stabilize at least one enzyme contained in a liquid enzymatic composition.
• the inventive stabilizing formulation can be used with a wide variety of enzymes utilized in liquid enzymatic compositions performing a wide variety of functions.
• the enzymes and classes of enzymes with which- this stabilizing formulation can be used include, but are not limited to, those discussed above.
• the invention also relates to a stabilized liquid enzymatic composition
• a stabilized liquid enzymatic composition comprising:
• components (a) and (b) are present in an amount effective to stabilize at least one enzyme in the liquid enzymatic composition. More preferably, the polymer (a) and the polyhydric alcohol (b) are present in a combined amount synergistically effective to stabilize at least one enzyme contained in the liquid enzymatic composition.
• the invention further relates to a method for the preparation of a stabilized liquid enzymatic composition by combining an enzyme with the stabilizing formulation above. Additionally, the invention relates to a method of using the stabilizing formulation to stabilize a liquid enzymatic formulation comprising the step of combining the stabilizing formulation with a liquid enzymatic composition.
• the invention provides a formulation for stabilizing a liquid enzymatic composition
• a formulation for stabilizing a liquid enzymatic composition comprising: (a) at least one polymer selected from a poly(cellulosic)ether, an acrylic polymer, and a polyamide,
• a water-soluble, or at least partially water-soluble, polymer is used in a formulation for stabilizing a liquid enzymatic composition or a stabilized liquid enzymatic compositions of the invention. That is, the polymer must have sufficient solubility to be miscible with water and form a single phase. Having this solubility, the polymer should not separate out when combined with the 2 -C 8 polyhydric alcohol and water of a stabilizing formulation or with a liquid enzymatic composition.
• the formulation is not required to be a clear solution.
• the formulation is an emulsion having an apparent homogeneous texture.
• 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 required to stabilize an enzyme.
• the polymer may preferably be used in amounts up to about 50% by weight of the stabilizing formulation. More preferably, the polymer is present from 0.05 to 30% by weight, and most preferably from 1% to 10% by weight. In a preferred embodiment, the polymer, of course, is present in an amount that gives the desired synergistic stabilization of a liquid enzymatic composition when combined with the polyhydric alcohol in a water-based or aqueous formulation.
• the polymer employed in the present invention is selected from a poly(cellulosic)ether, an acrylic polymer, and a polyamide.
• the polymer may be substituted or unsubstituted.
• the polymer may have a slight ionic charge, but is preferably non-ionic in nature.
• the polymer employed is a poly(cellulosic)ether
• the polymer is preferably selected from poly(carboxymethylcellulose)ether, poly(hydroxypropyl- methylcellulose)ether, poly(hydroxyethylmethylcellulose)ether, poly(hydroxybutylmethylcellulose)ether , poly(hydroxypropyl- cellulose)ether, and poly(ethylhydroxyethylcellulose)ether.
• the polymer is poly(carboxymethyl ⁇ cellulose)ether.
• Certain poly(cellulosic)ethers used in the present invention, such as poly(carboxymethylcellulose)ether, are sold as salts, i.e. sodium salts, and possess a slight anionic charge.
• Preferable poly(cellulosic)ethers are those with molecular weights ranging from 15,000 to 100,000, but more preferred are those with molecular weights ranging from 20,000 to 75,000.
• the acrylic polymers used in the invention are preferably polymers or copolymers of acrylic acid, methacrylic acid or derivatives thereof such as esters or salts of acrylic acids.
• 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 polymers can range from approximately 5,000 to greater than 4,000,000.
• Preferred acrylic polymers have molecular weights ranging from 100,000 to greater than 4,000,000, but more preferred are those with 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 having these various molecular weights are available from Aldrich Chemical Company.
• Acrylic polymers can possess an anionic charge.
• Preferred acrylic polymers are those polymers or derivatives which have only a slight anionic charge or no charge,
• the polyamide is polyvinylpyrrolidone.
• Preferred polyvinylpyrrolidones are those with molecular weights ranging from 5,000 to 400,000, but more preferred are those with molecular weights ranging from 300,000 to 400,000, with the higher molecular weights being preferred.
• the stabilizing formulation also contains a C 2 ⁇ Cg polyhydric alcohol as a second component.
• the C 2 -Cg polyhydric alcohol acts synergistically with the polymer, described above, to stabilize an enzyme in a liquid enzyme composition.
• the C 2 -C ⁇ polyhydric alcohol is preferably selected from a glycol and a trihydric alcohol. More preferably, the C 2 -C 8 polyhydric alcohol is glycerol, sorbitol, propylene glycol, butylene glycol, hexylene glycol, or ethylene glycol. Most preferably, the'C 2 -C ⁇ polyhydric alcohol is glycerol.
• the stabilizing formulation contains the C 2 -C ⁇ polyhydric alcohol in an amount sufficient, with the polymer, to stabilize at least one enzyme in a liquid enzymatic composition.
• the C 2 -C ⁇ polyhydric alcohol present is about 0.50 to 60% by weight of the stabilizing formulation, more preferably, about 5 to 50% by weight and, even more preferably, between 10 and 30%.
• a stabilizing formulation or enzymatic composition contains the C 2 -C ⁇ polyhydric alcohol in a combined amount with the polymer to achieve synergistic stabilization.
• the formulations of the invention are water-based or aqueous formulations containing sufficient water to allow the polymer to be miscible with the formulation and not separate out.
• the C 2 -C ⁇ polyhydric alcohol is soluble in water, but sufficient water should be present to allow the formulation to form a single phase.
• the formulation is not required to be a clear solution and preferably is an emulsion having an apparent homogeneous texture.
• Water-based formulations may contain additional solvents other than water.
• the stabilizing formulations of the invention can be prepared by mixing the components in any order, the formulations are preferably prepared by adding the desired amount of polymer to a C2-C6 polyhydric alcohol/water mixture.
• the C 2 -C fi polyhydric alcohol/water mixture can be prepared by means known in the art.
• the mixture can be prepared by simply mixing the desired polyhydric alcohol with an appropriate amount of water, or diluting a previously prepared mixture.
• the preferred mixture of the 2 -C ⁇ polyhydric alcohol and water may contain any percentage of C 2 -C ⁇ alcohol sufficient with the polymer discussed above to stabilize, preferably synergistically, at least one enzyme in a liquid enzymatic composition.
• the mixture is 1-95% by weight of the polyhydric alcohol, or water-soluble polymer thereof; more preferably 10-50% by weight; and most preferably, 30-50% by weight.
• the polyhydric alcohol is glycerol
• the mixture is preferably a 50% by weight glycerol/ water mixture.
• the enzyme should possess at least 90% activity after 30 days at 25°C.
• the examples below demonstrate the preferred synergistic stabilization of various enzymes at 50°C after 30 days.
• the stabilizing formulation described here can be employed with a wide variety of enzymes and industrial processes or commercial products.
• the enzymes, industrial processes and commercial products with which this stabilizing formulation can be used include, but are not limited to, those previously discussed.
• the invention also relates to a stabilized liquid enzymatic composition
• a stabilized liquid enzymatic composition comprising at least one polymer selected from a poly(cellulosic)ether, an acrylic polymer, and a polyamide; a C2 ⁇ Cg polyhydric alcohol; water; and at least one enzyme.
• the polymer and C 2 -C 8 polyhydric alcohol are present in a combined amount effective to stabilize, preferably to synergistically stabilize, at least one enzyme contained in the liquid enzymatic composition.
• Preferred compositions according to the invention are capable of developing greater viscosities than quantitatively proportional aqueous mixtures with the same polymers.
• the liquid enzymatic composition of this invention can be practiced with a wide variety of enzymes.
• enzymes include, but are not limited to, the enzyme classes and specific enzymes heretofore discussed.
• Enzymes that may be used are derived from animal, plant, fungal, bacterial, and synthetic sources.
• Preferable water dispersible enzymes for this system are proteases, including acid, alkaline, and neutral proteases, which are widely used in the laundry detergent and cheese making industries; amylases, including acid, alkaline, and neutral amylases, used, for example, in the corn syrup in ⁇ dustry; lipases, used in developing flavors in cheese, and in the pulp and paper and leather making industries; cellulases, and xylases.
• enzymes are often packaged and sold in concentrated liquid enzymatic compositions to be diluted prior to use. Enzymes can also be supplied in powdered or desiccated form.
• the amount of enzyme present after dilution of the concentrated enzyme depends on the form in which the enzyme is supplied. In general, the amount of enzyme preferably may range from 0.05 to 40% by weight of a concentrated liquid enzymatic composi ⁇ tion, more preferably 0.5 to 25%, and most preferably 10 to 20%.
• the stabilizing formulation of this invention is specifically contemplated for use in concentrated liquid enzymatic compositions as well as with compositions already diluted for use.
• the amount of stabilizing formulation needed to stabilize, or to synergistically stabilize, a concentrated solution can, and most likely will, differ from that for a diluted composition. Determining the appropriate quantity of stabilizing formulation or its components can be readily ascertained by one of ordinary skill in the art using the method set out in the examples below. As known in the art, the amount of enzyme present, however, is dependent upon the activity of the particular enzyme and the desired end use.
• the pH of the final stabilized liquid 1 enzymatic composition is preferably from 5.0 to 10.0, but more pref ⁇ erably around 7.0. Most preferably, the system should be allowed to seek its own pH, generally around neutral. But, as understood in the art, adjustment of pH may be necessary with a small amount of acidic or alkaline material.
• the stabilized liquid enzymatic composition may be water-based or aqueous and contain other solvents or additives directed toward the use of the composition in a particular industrial process.
• the stabilized liquid enzymatic composition can contain additives such as surfactants, defoamers, and the like, as are known in the art.
• additives such as surfactants, defoamers, and the like, as are known in the art.
• synergistic stabilization such additives may be added in amounts not interfering with the synergistic stabilization of the liquid enzymatic composition.
• the stabilizing formulation may also act as a dispersant aid for the enzyme in industrial process waters.
• the invention also relates to a method for the preparation of a stabilized liquid enzymatic composition comprising the step of combining at least one enzyme with the inventive stabilizing formulation.
• the invention further relates to a method of using the stabilizing formulation to stabilize a liquid enzymatic composition comprising the step of combining a liquid enzymatic composition with the stabilizing formulation.
• Illustrative and preferred components, as well as the amounts of the components used in the method, are the same as discussed above.
• step (b) adding to the mixture prepared in step (a) at least one polymer selected from a poly(cellulosio)ether, an acrylic polymer, and a polyamide, and
• step (c) adding at least one enzyme to the mixture resulting from step (b) .
• the polymer and C 2 -C ⁇ 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 method for the preparation of a stabilized liquid enzymatic composition comprises the steps of:
• step (b) adding to the mixture prepared in step (a) at least one polymer selected from a poly(cellulosic)ether, an acrylic polymer, and a polyamide, and
• step (c) combining the formulation resulting from steps (a) and (b) with a liquid enzymatic composition containing at least one enzyme.
• the polymer and C 2 -C ⁇ polyhydric alcohol are preferably present in a combined amount synergistically effective to stabilize at least one enzyme in the liquid enzymatic composition.
• the polymer added in step (b) may be added alone, as an aqueous dispersion, or as a solution where the polymer is dissolved in water or a suitable organic solvent.
• additives may be added at any time, but preferably prior to step (c), or in a separate step after the enzyme is added.
• Synergism was demonstrated by testing glycerol, designated as Compound A, and poly(carboxymethylcel- lulose)ether, (CMC), having a 250,000 molecular weight, designated as Compound B, or polyvinylpyrrolidone, (PVP), having a 360,000 molecular weight, designated as Compound B' .
• CMC poly(carboxymethylcel- lulose)ether
• PVP polyvinylpyrrolidone
• Tables 1 and 2 experiments were set up by varying ratios of Compound A to Compound B or B' over a range of concentrations and assaying various types of enzymes for enhanced stabilization of enzymatic activity at 50°C for 30 days. The concentration of each compound required for an assay of 90% of the original enzymatic activity was taken as an end point.
• concentrations are expressed as percent by weight of the compound in the final composition including the added enzyme with the balance being water.
• Water was added to compound A, the glycerol to form a glycerol-water mixture. End points for the compositions containing Compound A and Compound B or B' were then compared with the end points for Compound A alone and Compound B or B' alone.
• Synergism was determined by the method described by Kull, F.C., Eisman, P.C., Sylwestrowicz, H.D., and Mayer, R.L., Applied Microbiology 9: 538-541 (1961) employing the ratio determined by
• Stabilizing formulations of the invention as in Example 1 were tested with another enzyme, Diazyme L-200, a glucoamylase sold by Solvay Enzymes, Inc.
• Diazyme L-200 a glucoamylase sold by Solvay Enzymes, Inc.
• the results, set forth in Tables 3 and 4 also demonstrate enhanced stabilization with this enzyme.
• the sums of QA/Qa + QB/Qb for all compositions containing glycerol (Compound A) and CMC (Compound B) were calculated. Sample calculations are shown for some end points where synergism was evident in this example. As set forth in Table 3 and the sample calculations, these end point values were 0.59, 0.67, 0.67, and 0.84, respectively, indicating the existence of synergism.
• Enzymes with esterase activity were tested with stabilizing formulations according to the invention as in Example 1.
• the results obtained with the enzyme, Lipolase, a lipase sold by Novo Nordisk Bioindustries, Inc. are set forth in Tables 5 and 6 and indicate the enhanced stabilization of this enzyme.
• the sums of QA/Qa + QB/Qb for all compositions containing aqueous glycerol (Compound A) and CMC (Compound B) were calculated. Sample calculations are shown for some end points where synergism was evident in this example. As set forth in Table 5 and the sample calculations, these end point values were 0.43, 0.37, 0.57, and 0.77, respectively, indicating the existence of synergism.
• the hemicellulase, Pulpzyme HB, a xylanase sold by Novo Nordisk Bioindustrials, Inc. was selected for enhanced stability formulation testing as in Example 1.
• the following results obtained for this example are set forth in Tables 7 and 8.
• the sums of QA/Qa + QB/Qb for all compositions containing glycerol (Compound A) and CMC (Compound B) were calculated. Sample calculations are shown for those end points where synergism was evident in this example. As set forth in Table 7 and the sample calculations, these end point values were 0.43, 0.53, 0.57, and 0.77, respectively, indicating the existence of synergism.

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