NZ330424A - Enzyme composition stabilised by a non-ionic poyether-polyol surfactant - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number 330424 0 New Zealand No 330424 International No. PCT/ TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates 10 03 1993, Complete Specification Filed 01 121994 Classification (6) C11D3/386, C12N9/96 Publication date 28 October 1999 Journal No 1445 NO DRAWINGS NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention Enzyme stabilization by block-copolymers Name, address and nationality of applicant(s) as in international application form BUCKMAN LABORATORIES INTERNATIONAL, INC, a Tennessee corporation of 1256 North McLean Boulevard, Memphis, Tennessee 38108, United States of America 1 - lct- DescriPtion ENZYME STABILIZATION BY BLOCK-COPOLYMERS Technical Field The field of the invention is the stabilization of enzymes by means of a non-ionic polyether-polyol block-copolymer surfactant Background Art Enzymes generally are formulated into aqueous-based liquid enzymatic compositions designed for a particular process. These liquid enzymatic compositions, however, t 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 It is not uncommon to have industrial products, such as liquid enzymatic compositions, stored in warehouses m various climates around the world where the product is subjected to a temperature that may range from freezing to above 50°C for extended periods. After storage at temperature extremes ranging from 0°C to 50°C for many months, most liquid enzymatic compositions lose from 20 to 100 percent of their enzymatic activity due to enzyme instability.

Various attempts have been made to stabilize enzymes contained m liquid enzymatic compositions Attempts to increase the stability of liquid enzymatic compositions using formulations containing alcohols, glycerols, dialkylglycolethers, and mixtures of these and other compounds have had only marginal success, even m moderate storage temperature ranges.

In Munk, U.S Patent No 4,801,544, a system o£ ethylene glycol and ethoxylated linear alcohol nomonic ' PCT/US94/13744 surfactant with hydrocarbon solvent was utilized as a stabilizer and the encapsulation of enzymes m micelles within the solvent/surfactant mixture was described The water content of the composition was kept at less than 5 percent, and enzyme stability was checked at 35°, 70° and 100 °F The stabilization of an aqueous enzyme preparation using certain esters has been described by Shaer m 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 m an amount from 0.1 to about 2 5% by weight The enzyme ingredient that is employed according to the patentee is a commercial enzyme preparation sold m a dry powder, solution or slurry form containing from about 2 percent to about 80 percent of active enzymes and a carrier such as sodium or calcium sulfate, sodium chloride, glycerol, non-ionic surfactants or mixtures thereof as the remaining 20 percent to 98 percent Letton et al , 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 Guilbert et al , 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.

Weber, 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 subtilus 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 Dorrit et al , European Patent No 0 352 244 A2 describes stabilized liquid detergent compositions using an amphoteric surfactant - Kammsky et al., U.S Patent No. 4,305,837 describes -stabilized aqueous enzyme compositions containing a stabilizing system of calcium ions and a low molecular weight carboxylic acid or salt and a low molecular weight alcohol. This stabilized enzyme is used m a detergent composition The composition may include non-ionic surfactants having the formula RA(CH2CH20)nH where R is a hydrophobic moiety, A is based on a group carrying a reactive hydrogen atom and n represents the average number of ethylene oxide moieties. R typically contains from about 8 to about 22 carbon atoms but can be formed by the condensation of propylene oxide with a lower molecular weight compound whereas n usually varies from about 2 to about 24 The low molecular weight alcohol employed may be either a monohydric alcohol containing from l to 3 carbon atoms or a polyol containing from 2 to about 6 carbon atoms and from 2 to about 6 hydroxy groups Kammsky et al note that the polyols can provide improved enzyme stability and include propylene glycol, ethylene glycol and glycerine Tai, U.S. Patent No. 4,404,115 describes an aqueous enzymatic liquid cleaning composition which contains as an enzyme stabilizer, an alkali metal pentaborate, optionally with an alkali metal sulfite and/or a polyol The polyol contains 2-6 hydroxy groups and includes materials such as 1,2-propane diol, ethylene glycol, erythntan, glycerol, sorbitol, manmtol, glucose, fructose, lactose, and the like Boskamp, U.S Patent No 4,462,922 also describes an aqueous enzymatic detergent composition with a stabilizer based on a mixture of boric acid or a salt of boric acid with a polyol or polyfunctional amino compound together with a reducing alkali metal salt. Substantially the same polyols are used as m Kammsky et al The present invention is directed to a method for providing stabilized enzymes and a stabilized enzyme composition in which the foregoing and other disadvantages are overcome The advantages sought according to the present invention are to provide a novel method for stabilizing enzymes as well as a stabilized enzyme composition Disclosure of the Invention The present invention is directed to a novel method and composition that substantially obviates one or more of the foregoing and other problems due to limitations and disadvantages of the related art, or at least provides the public with a useful choice Additional features and advantages of the invention will be set forth in the description which follows, and m part will be apparent from the description, or may be learned by practice of the invention The advantages of the invention will be realized and obtained by the method and composition of matter, particularly, pointed out m the written description and claims hereof To achieve these and other advantages and m accordance with the purpose of the invention, as embodied and broadly described, a novel method for stabilizing an enzyme against loss of activity at elevated temperatures or by water is set forth comprising combining the enzyme with j u'klLLUUf '1 ' utl/QFIICE _ 5 _ (* 1 » \ a stabilizing amount of a non-ionic polyether-polyol block-copolymer surfactant Where the enzyme is stabilized against deactivation at elevated temperatures the surfactant is selected to have a cloud point greater than such temperatures.

In one embodiment, the non-ionic polyether-polyol block-copolymer surfactant is a polyoxyalkylene glycol ether all-block, block-heteric, heteric-block or heteric-heteric block copolymer where the alkylene units have from 2 to about 4 carbon atoms and especially those surfactants which contain hydrophobic and hydrophilic blocks where each block is based on at least oxyethylene groups or oxypropylene groups or mixtures of these groups More specifically, in a first aspect the present invention provides a method for stabilizing an enzyme composition containing greater than about 20 weight percent of water against loss of activity evaluated at 50°C, comprising combining said enzyme with stabilizing amounts of a surfactant where the surfactant comprises a) a block polymer surfactant formed from a starting material having the formula I-[Am-Bn]x, wherein I represents an alcohol, A represents a hydrophobe compnsing an alkylene oxide unit in which at least one hydrogen has been replaced by an alkyl group or an aryl group, m is the degree of polymerization which is greater than about 6, B is an aqueous solubilizing group compnsing at least one oxyethylene group, n is the degree of polymerization which is greater than about 6, and x is the functionality of I and is from 1 to 4, or b) a surfactant having the formula R0(CH2CH2O)nH, wherein R is a hydrophobic group, and n is greater than about 5 i Ii'.'LlELTU ' i rillY (jt-FICc | <V J I 4 AUG 1033 \ _J.L.r F|v r"0 - 5a - The invention also comprises a composition of matter based on the foregoing enzyme and surfactant It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed Best Mode for Carrying Out the Invention The present invention is directed to a method for stabilizing an enzyme against loss of activity, either at elevated temperatures or by water, by combining the enzyme v/xth a non-ionic polyether-polyol block-copolymer surfactant.

The use of enzymes and liquid enzymatic compositions m industry and m the commercial marketplace has grown rapidly over the last several years. As is well-known, enzymes can be acid, alkaline or neutral, depending upon the pH range m which they are active Lipase alone or an enzyme comprising lipase, i.e , Lipase is any combination with the following enzymes can be used. All of these tytses of enzymes are contemplated to be useful m connection with the invention disclosed herein.

WO 95/15371 1 PCTAJS94/13744 a stabilizing amount of a non-ionic polyether-polyol block-copolymer surfactant Where the enzyme is stabilized against deactivation at elevated temperatures the surfactant is selected to have a cloud point greater than such temperatures In one embodiment, the non-ionic polyether-polyol block-copolymer surfactant is a polyoxyalkylene glycol ether all-block, block-heteric, heteric-block or hetenc-heteric block copolymer where the alkylene units have from 2 to about 4 carbon atoms and especially those surfactants, f which contain hydrophobic and hydrophilic blocks where each block is based on at least oxyethylene groups or oxypropylene groups or mixtures of these groups The invention also comprises a composition of matter based on the foregoing enzyme and surfactant It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed Best Mode for Carrvmcr Out the Invention The present invention is directed to a method for stabilizing an enzyme against loss of activity, either at elevated temperatures or by water, by combining the enzyme with a non-ionic polyether-polyol block-copolymer surfactant.

The use of enzymes and liquid enzymatic compositions m industry and m the commercial marketplace has grown rapidly over the last several years As is well-known, enzymes can be acid, alkaline or neutral, depending upon the pH range m which they are active. Lipase alone or an enzyme comprising lipase, i.e., Lipase is any combination with the following enzymes can be used All of these types of enzymes are contemplated to be useful m connection with the invention disclosed herein Many enzymes and liquad enzymatic compositions have been associated with liquid detergents and have shown utility as solubilizmg and cleaning formulations In addition to their association with liquid detergents, enzymes and liquid enzymatic compositions have also shown utility m a number of different commercial and industrial areas m which a wide variety of enzyme classes are now used.

Proteases are a well-known class of enzymes frequently utilized m a wide variety of industrial applications where they act to hydrolyze peptide bonds m proteins and proteinaceous substrates. Proteases are used to help to " remove protein based stains such as blood or egg stains Liquid enzymatic compositions containing alkaline proteases have also shown to be useful as dispersants of bacterial films and algal and fungal mats m cooling tower waters and metalworkmg fluid containment bays Proteases can be characterized as acid, neutral, or alkaline proteases depending upon the pH range m which they are active. The acid proteases include the microbial rennets, rennm (chymosm) , pepsin, and fungal acid proteases The neutral proteases include trypsin, papain, bromelam/ficm, and bacterial neutral protease The alkaline proteases include subtilism and related proteases Commercial liquid enzymatic compositions containing proteases are available under the names Rennilase®, "PTN" (Pancreatic Trypsin NOVO), "PEM" (Proteolytic Enzyme Mixture), Neutrase®, Alcalase®, Esperase®, and Savmase™ which are all supplied by Novo Nordisk Biomdustrials, Inc of Danbury, CT. Another commercial protease is available under the name KT-Proteolytic supplied by Solvay Enzyme Products.

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. As a class amylases include a-amylase, /3-amylase, amyloglucosidase (glucoamylase), fungal amylase, and pullulanase Commercial liquid enzymatic compositions containing amylases are available under the names BAN, Termamyl®, AMG, Fungamyl®, and Promozyme™, which are supplied by Novo Nordisk, and Diazyme L-200, a product of Solvay Enzyme Products Other commercially valuable enzyme classes are those which affect the hydrolysis of fiber. These classes include cellulases, hemicelluloses, pectmases, and (3-glucanases Cellulases are enzymes that degrade cellulose, a linear glucose polymer occurring in the cell walls of plants Hemicelluloses are involved in the hydrolysis of hemicellulose which, like cellulose, is a polysaccharide found in plants The pectmases are enzymes involved m the degradation of pectin, a carbohydrate whose main component is a sugar acid. /3-glucanases are enzymes involved m the hydrolysis of /3-glucans which are also similar to cellulose m that they are linear polymers of glucose Collectively, cellulases include endocellulase, exocellulase, exocello-biohydrolase, and cellobiase and for the purpose of the present invention will also include hemicellulase. Commercial liquid enzymatic compositions containing cellulases are available under the names Celluclast® and Novozym®188 which are both supplied by Novo Nordisk Hemicellulases that may be used include the xylanases PULPZYM® product, available from Novo Nordisk, and ECOPULP® product, from Alko Biotechnology, are two examples of commercially available liquid enzymatic compositions containing xylanase-based enzymes 1 PCT/US94/13744 As a class, hemicellulases include hemicellulase mix-* ture and galactomannanase Commercial liquid enzymatic compositions containing hemicoxlulases are available as PULPZYM® from Novo, ECOPULP® from Alko Biotechnology and Novozym®280 and Gamanase*", which are both products of Novo Nordisk The pectmases that tray be used comprise endopolygalacturonase, exopoly-galacturonase, endopectate lyase (transelimmase) , exopectate lyase (transelimmase), and endopectin lyase (transelimmase) . Commercial liquid 4 enzymatic compositions containing pectmases are available' under the names Pectmex"1 Ultra SP and Pectinex"1*, both supplied by Novo Nordisk.

The jS-glucanases that may be used comprise lichenous, laminarmase, and exoglucanase. Commercial liquid enzymatic compositions containing /3-glucanases are available under the names Novozym®234, Cereflo®, BAN, Finizym®, and Ceremix®, all of which are supplied by Novo Nordisk In Edition to lipases, and phospholipases may also be used Lipases and phospholipases are esterase enzymes which hydrolyze fats and oils by attacking the ester bonds in these compounds. Lipases act on triglycerides, while phospholipases act on phospholipids. In the industrial sector, lipases and phospholipases represent the commercially available esterases. Novo Nordisk markets two liquid lipase preparations under the names Resmase™ A and Resmase™ A 2X.

Commercial liquid enzymatic compositions containing lipases are available For example, such compositions are available under the trade names Lipolase 100, Greasex 50L, Palatase^A, Palatase^M, and Lipozyme™ which are all supplied by Novo Nordisk Another commercially valuable class of enzymes are the isomerases which catalyze conversion reactions between -N I isomers of organic compounds 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 m the breakdown and synthesis of many biochemicals Currently, many redox enzymes have not gained a prominent place m industry since most redox enzymes require the presence of a cofactor However, where cofactors are an integral part of an enzyme or do not have to be supplied, redox enzymes are commercially useful.

The redox enzymes, glucose oxidase, and lipoxidase ,T (lipoxygenase) can be used. Other redox enzymes have possible applications ranging from the enzymatic synthesis of steroid derivatives to use m diagnostic tests These redox enzymes include peroxidase, superoxide dismutase, alcohol oxidase, polyphenol oxidase, xanthine oxidase, sulfhydryl oxidase, hydroxylases, cholesterol oxidase, laccase, alcohol dehydrogenase, and steroid dehydrogenases Of the various non-ionic polyether-polyol surfactant block-copolymers available, the preferred materials comprise polyoxyalkylene glycol ethers which contain hydrophobic and hydrophilic blocks, each block preferably being based on at least optionally oxyethylene groups or oxypropylene groups or mixtures of these groups The most common method of obtaining these surfactants is by reacting ethylene oxide with the hydrophobic material which contains at least one reactive hydrogen. Alternative routes include the reaction of the hydrophobe with a preformed polyglycol or the use of ethylene chlorohydrm instead of ethylene oxide The reacting hydrophobe must contain at least one active hydrogen preferably alcohols, and optionally acids, WO 95/15371 " ' PCT/TJS94/13744 amides, mercaptans, alkyl phenols and the like. Primary amines can be used as well Especially preferred non-ionic surfactants are those obtained by block polymerization techniques By the careful control of monomer feed and reaction conditions, a series of surfactants can be prepared m which such characteristics as the hydrophile-lipophile balance (HLB), wetting and foaming power can be closely and reproducibly controlled. The chemical nature of the initial component employed in the formation of the initial polymer block generally determines the classification of the surfactants The initial component does not have to be hydrophobic since hydrophobicity will be derived from one of the two polymer blocks. The chemical nature of the initial component m the formation of the first polymer block generally determines the classification of the surfactants. Typical starting materials or initial components include monohydric alcohols such as methanol, ethanol, propanol, butanol and the like as well as dihydric materials such as glycol, glycerol, higher polyols, ethylene diamine and the like The various classes of preferred surfactants, suitable for practice of the present invention have been described by Schmolka in "Non-Ionic Surfactants," Surfactant Science Series Vol. 2, Schick, M.J., Ed Marcel Dekker, Inc , New York, 1967, Chapter 10 which is incorporated herein by reference The first and simplest is that in which each block is homogeneous which is to say a single alkylene oxide is used m the monomer feed during each step m the preparation Such materials are referred to as all-block surfactants The next classes are termed block-heteric and heteric-block, m which one portion of the molecule (i e , either the hydrophobe or hydrophile) is composed of a single alkylene oxide while the other is a mixture of two or more such materials, one of which may be the same as ) PCT/US9-4/13744 that of the homogeneous block portion of the molecule In the preparation of such materials, the hetero portion of the molecule will be totally random The properties of these non-ionics will be entirely distinct from those of the pure block surfactants The other subclass is that in which both steps m the preparation of the hydrophobe and hydrophile involve the addition of mixtures of alkylene oxides and is defined as a heteric-heteric block copolymer The block polymer surfactant is typified by a mono-functional starting material such as a monohydric alcohol,^ acid, mercaptan, secondary amine or N-substituted amides -Such materials can generally be illustrated by the following formula- I- [A -B ] m n x where I is the starting material molecule as described before The A portion is a hydrophobe comprising an alkylene oxide unit in which at least one hydrogen has been replaced by an alkyl group or an aryl group, and m is the degree of polymerization which is usually greater than about 6 The B moiety is an aqueous solubilizmg group such as oxyethylene with n again being the degree of polymerization. The value of x is the functionality of I Thus, where I is a monofunctional alcohol or amine, x is 1, where I is a polyfunctional starting material such as a diol (e.g., propylene glycol) x is 2 as is the case with the Pluronic® surfactants. Where I is a tetrafunctional starting material such as ethylenediamme, x will be 4 as is the case with Tetronic® surfactants Preferred surfactants of this type are the polyoxypropylene-polyoxyethylene block copolymers.

Multifunctional starting materials may also be employed to prepare the homogeneous block surfactants In the block-heteric and heteric-block materials either A or B will be a mixture of oxides with the 1 PCMJS94/13744 remaining block being a homogeneous block, One block will" be the hydrophobe and the other the hydrophile Either of the two polymeric units will serve as the solubilizing unit but the characteristics will differ depending on which is employed. Multifunctional starting materials can also be employed in materials of this type The heteric-heteric block copolymers are prepared essentially the same way as discussed previously with the major difference being that the monomer feed for the alkylene oxide in each step is composed of a mixture of two or more materials The blocks will therefore be random copolymers of the monomer feed with the solubility characteristics determined by the relative ratios of potentially water soluble and water insoluble materials The average molecular weight of the polyoxyalkylene glycol ether block copolymers utilized according to the present invention is from about 500 to about 30,000 especially from about 800 to about 25,000 and preferably from about 1,000 to about 12,000 The weight ratio of hydrophobe to hydrophile will also vary from about 0.4 1 to 2 5 1, especially from about 0.6 1 to about 18 1 and preferably from about 0.8:1 to about 1.2 1 In an especially preferred embodiment, these surfactants have the general formula RX (CH_CH„0) H 2 2 n where the hydrophobe of the block copolymer has an average molecular weight of from about 500 to about 2,500, especially from about 1,000 to about 2,000 and preferably from about 1,200 to about 1,500 and where R is usually a typical surfactant hydrophobic group but may also be a polyether such as a polyoxypropylene group or a mixture of polyoxypropylene and polyoxyethylene groups In the above formula X is either oxygen or nitrogen or another functionality capable of linking the polyoxyethylene chain to the hydrophobe. In most cases, n, the average number of oxyethylene units in the hydrophilic group, must be greater than about 5 or about 6 to impart sufficient water solubility to make the materials useful The polyoxyalkylene glycol ethers are the preferred non-ionic polyether-polyol block-copolymer surfactants. However, other non-ionic block-coplymer surfactants useful is the invention can be modified block copolymers using the following as starting materials- (a) alcohols, (b) fatty acids, (c) alkylphenol derivatives, (d) glycerol and its derivatives, (e) fatty amines, (f)-1,4-sorbitan derivatives, (g) castor oil and derivatives, and (h) glycol derivatives.

Cloud point is one of the most distinct characteristics for most non-ionic surfactants and depends on the number of oxyethylene, oxypropylene, and/or oxybutylene groups reacted in the formation of the surfactant block copolymers of the present invention Cloud point is also affected by other components m solution, the concentration of surfactants, and the solvents, if any, in the system. Cloud point has been defined as the sudden onset of turbidity of a non-ionic surfactant solution on raising the temperature. When the non-ionic surfactant is dissolved in water, it is theorized that an increase of temperature will increase the activity of the water molecules, which cause the dehydration of ether oxygens m the polyoxyethylene group m the non-ionic surfactant. Molecules with greater percentages of oxyethylene groups have a greater capacity for hydration, and so have a higher cloud point. This is important in the stabilization of enzymes in solution, since the long-term stability of the enzyme is evaluated at a temperature of 50°C. If the cloud point of a non-ionic surfactant is less than 50°C, when the solution reaches that temperature, the ' PCT/US9-4/13744 enzyme will hydrate while the surfactant has coalesced and becomes less water soluble.

Cloud point has also been described as that characteristic of the non-ionic surfactants m which they exhibit an inverse temperature-solubility relationship m water, which is to say that as the temperature of the solution is increased, the solubility of the surfactant decreases. This phenomenon has been attributed to a disruption of specific interactions such as hydrogen bonding between the water and the polyoxyethylene units m( * the molecule The temperature at which components of the polyoxyethylene surfactant begin to precipitate from T solution has also been defined as the "cloud point " In general, the cloud point of the given family of surfactants will increase with the average number of oxyethylene groups.

The cloud point of the non-ionic polyether-polyol surfactant block copolymers and especially the polyoxyalkylene glycol ether surfactant polymers of the present invention is greater than the temperature at which the enzyme or enzyme system degrades and may be anywhere from about 0°C to about 110°C, especially from about 10°C to about 100°C and preferably from about 20°C to about 95eC. These cloud points are for a 1 weight % solution of the surfactant in water.

Although the inventors do not want to be limited by any theory, it is believed that the non-ionic surfactants of the present invention contribute to the stability of the enzyme by increasing the viscosity of the water m the formulation. Generally, high viscosity will lead to poor transport to the Ca++ rich zones in enzymes such as protease, or slower ion transfer This also helps to keep the matrix of the enzyme intact, although in some of the O PCT/TJS94/13744 cases described according to the present invention, the higher viscosity may not be necessary for stability Chelating agents generally deactivate enzymes, decreasing the molecular compactness of the enzyme and causing deformation of the enzyme. Kon-iomc surfactants are not influenced by the electrostatic effect, i.e., by the charged groups on the enzyme, and so do not impact on the special structure of the enzyme.

A suitable polyoxyalkylene glycol ether block-copolymer that may be used according to the present / invention contains a hydrophobe based on a hydrocarbon moiety of an aliphatic monohydric alcohol containing from 1 to about 8 carbon atoms, where the hydrocarbon moiety has attached thereto through an ether oxygen linkage, a heteric mixed chain of oxyethylene and 1,2-oxypropylene groups The weight ratio of oxyethylene groups to 1,2-oxypropylene groups in the hydrophobe is from about 5 95 to about 15:85 and the average molecular weight of the hydrophobe is from about 1,000 to about 2,000. A hydrophile is attached to the mixed chain and is based on oxyethylene groups The weight ratio of hydrophile to hydrophobe is anywhere from about 0.8.1 to about 1.2:1. This polyoxyalkylene glycol ether is further defined by Steele, Junior, et al , U S Patent No 3,078,315 which is incorporated herein by reference.

One of the preferred polyoxyalkylene glycol ethers is Tergitol XD produced according to the method of Steele, Jr , et al. U.S. Patent No. 3,078,315 and available from Union Carbide. This is a non-ionic block copolymer having a cloud point of about 76°C as a 1% solution m water and a molecular weight of about 3120 based on its hydroxyl number Other non-ionic polyoxyalkylene glycol ether block-copolymers can be employed such as those manufactured by ' PCT/US94/13744 the BASF Wyandotte Corporation including Pluronic® and Tetronic® types Pluronic® and Tetronic® polyol surfactants vary from mobile liquids to flakable solids and those with high ethylene oxide contents exhibit no solution cloud point even at 100°C Other similar non-ionic polyoxyalkylene glycol ether block-copolymer surfactants can be employed such as those manufactured by Dow Chemical Company and Witco Chemical Corporation The Pluronic® surfactants that may also be employed according to the present invention are prepared by synthesizing a hydrophobe of desired molecular weight by the controlled addition of propylene oxide to the two f hydroxyl groups of propylene glycol. Ethylene oxide is then added to both ends of the hydrophobe to form oxyethylene chains that constitute from about 10 wt % to about 80 wt.% of the final molecule. The average molecular weight of the Pluronic® surfactant is from about 1,100 to about 12,600 and the HLB (hydrophobe lipophobe balance) is from about 1-7 to about 18-23 or greater than about 24 Pluronic® P-105 employed according to the present invention has an average molecular weight of about 6,500, a melting point of about 35°C, a cloud point of about 91°C and an HLB of about 12-18 Tetronic® surfactants that may also be employed according to the invention are tetra-functional block copolymers derived from the sequential addition of propylene oxide and then ethylene oxide to ethylene- diamine The average molecular weight of these surfactants is from about 1,650 to about 30,000 and have an HLB of from about 1-7 to about 18-23 and greater than about 24 Tetronic® 13 04 employed according to the invention has an average molecular weight of about 10,50 0, a melting point of about 59°C, a cloud point greater than about 100°C and an HLB greater than about 24.

The enzyme and surfactant may also be used in comomation with an organic solvent compatible with the enzyme and which will also act as a solvent for the non-ionic polyether-polyol block-copolymer surfactant The solvent preferably is hydrophilic such as a polyol or a mixture of polyols wnere che polyol has from 2 to about 6 carbon atoms ana from 2 to about 6 hydroxy1 groups and includes materials sucn as 1,2-propane diol, ethylene glycol, erythntan, glycerol, sorbitol, manmtol, glucose, fructose, lactose, and che like / The stabilized enzyme composition according to tne present invention, therefore may contain an enzyme in an amount from about 2 to about 95 parts by weight, especially from about 5 to about 90 parts by weight and preferably from aoout 10 to about 8 0 parts by weight, water m an amount from about 1 to about 90 parts by weight and especially from aoout 2 to about 8 5 parts by weight and preferably from about 5 to aoout 80 parts by weight, a solvent from about 0 to about 70 parts by weight and especially from about 2 to about SO parts ov weight and preferably from about 3 to about 55 parts ov weignt and the non-ionic polyether-polyol block-copolymer surfactant m an amount from about 0 2 to about 40 parts by weignt and especially from about 0 8 to about 30 parts oy weignt and preferably from about 1 to about 25 parts oy weignt The invention also comprises a process for stabilizing an enzyme as well as a stabilized enzyme composition containing from about 1 to 90% by weight of water based on tne aforesaid enzyme and water m combination with tne aforesaid nomonic polyether-polyol block-compolymer surfactant In another embodiment, he invention also comprises a process for stabilizing an enzyme as well as a stabilized enzyme composition containing greater about 20% by weight of water based on the aforesaid enzyme and water in. combination with the aforesaid nonionic polyether-polyol block-compolymer surfactant The following examples are illustrative Example 1 The composition listed below was made from Pulpzyme H3, an aqueous enzyme suspension, commercially available from Novo Nordisk 3iomdustrials, Inc which is a xylanase preparation with a bacterial origin. Tergitol XD, as described aoovs was also employed The glycerol used is a 96% pure material where the impurity is water A higher purity glycerol may also be employed The glycerol acts as a solvent for Tergitol XD, which is a solid at room temperature Viscosity of the formulation is 2,200 cps measured, by using a Brookfield viscosimeter model number LVT, at 30 rpm, spindle number 4 at room temperature (20°C) The formulation dissolves easily m water Enzyme activity, IU per ML, was measured according to the method of Bailey, M J. et al , J Biotech 23. 257-270, 1992. This method entails a five-minute incubation of the xylanase enzyme (suitably diluted m pH 5.3 citrate buffer) wicn a 1% birchwood xvlan substrate. After incubation, tne released sugars are determined by a 5 minute reaction, with tne original DNS reagent of Sumner (1921) Absorbance is measured at 540 nm against a reagent blank comprised of substrate, DNS reagent and buffer Enzyme readings are corrected by subtracting an enzyme blank composed of substrate and DNS reagent to which the diluted enzyme is added with immediate color development/quencnmg ratner than incubation Component Weight Percent Pulpzyme HB 75 Glycerol S Tergitol XD 20 Table 1 below shows the excellent stability of this formulation The enzyme activity increase is within experimental error - 19 -Table 1 Enzyme Stabilization In Example 1 Enzyme Activity (IU per ML)* Original Sample Room Temperature 8°C 50°C 9170 9130 9820 10900 Thirty days at the condition indicated Example 2 1 Example 1 was repeated using Pulpzyme HB, however, Tergitol XD was substituted by Pluronic® P-105® which is a commercial non-ioric block copolymer available from BASF Wyandotte _ -Corprration The cloud point of this copolymer is 91°C (1-4 solution m water) and 94°C (10% solution m water). The average molecular weight of the surfactant is about 6,500 Table 2 shows, within experimental error, the reduction m stability of this formulation when compared to Example 1 which appears to be a function of Pluronic® P-105 compared to Tergitol XD. Stability is nonetheless better than enzymes without Pluronic® P-105. The enzyme will rapidly lose its activity under these conditions without the stabilization provided by Pluronic® P-105 Table 2 Enzyme Stabilization In Example 2 Enzyme Activity (IU per ML)♦ Original Sample Room Temperature 8°C 50°C 8400 8280 8970 7370 * Thirty days at the condition indicated.

Example 3.

Example 1 was repeated using a protease enzyme from Solvay Enzymes, Inc. or a lipase enzyme from Novo Nordisk Biomdustrials, Inc , the results of which are set forth m Table 3. l ' PCT/US94/13744 Table 3 Component HT-Proteolytic L.-175® (protease) Glycerol (96% plus) Tergitol XD Activity (14 davs) at 50°C at Room Temp. (2 0°C) Component Resmase A2XTH (lipase) Glycerol (96% plus) Tergitol XD Water Pluronic® P105 Tetronic 13 04® BASF Wyandotte Activity (30 davs)at 50°C at Room Temp. (20°C) 85 5 10 Weight 70 20 10 45 90 100 24 91 Weight 85 85 5 85 0 0 047 054 0 0 0553 0472 0.049 0.033 0.048 0.067 It will be apparent to those skilled in the art that modifications and variations can be made in the method and composition of the present invention without departing from the spirit or scope thereof It is intended that these modifications and variations and their equivalents are to be included as part of this invention provided they come within the scope of the appended claims. 21

Claims (31)

1. WHAT WE CLAIM IS

   1 A method for stabilizing an enzyme composition containing greater than about 20 weight percent of water against loss of activity evaluated at 50°C, compnsing combining said enzyme with stabilizing amounts of a surfactant where the surfactant comprises a) a block polymer surfactant formed from a starting material having the formula

   I-[Am-Bn]x, wherein I represents an alcohol, A represents a hydrophobe comprising an alkylene oxide unit in which at least one hydrogen has been leplaced by an alkyl group or an aryl group, m is the degree of polymerization which is greater than about 6, B is an aqueous solubilizing group comprising at least one oxyethylene group, n is the degree of polymerization which is greater than about 6, and x is the functionality of I and is from 1 to 4, or b) a surfactant having the formula

   R0(CH2CH2O)nH, wherein R is a hydrophobic group, and n is greater than about 5
2. 2 The method of claim 1, wherem said enzyme is stabilized against decomposition at elevated temperatures by said surfactant which has a cloud point greater than said temperatures
3. 3 The method of claim 2, wherem said temperatures are from about 0°C to about 100°C
4. 4 The method of claim 2, wherem said surfactant is dissolved m an organic solvent compatible with said enzyme

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5. 5 The method of claim 1, wherein said enzyme is a system of an enzyme in combination with water, said enzyme being stabilized against decomposition fiom watei by said non-ionic polyether-polyol block-copolymer-surfactant which raises the viscosity of water in said system
6. 6 The method of claim 5, wherein said surfactant is dissolved in an organic solvent compatible with said enzyme
7. 7 The method of claim 4 or 6, wherein said solvent is hydrophilic
8. 8 The method of claim 7, wherein said solvent is a polyol or a mixture of polyols
9. 9 The method of claim 8, wherem said polyol has from 2 to about 6 carbon atoms and from 2 to about six hydroxyl groups
10. 10 The method of claim 1, wherein said surfactant contains hydrophobic and hydrophilic blocks based on at least oxyethylene groups, oxypropylene groups or mixtures of said groups
11. 11 An en2yme composition containing greater than about 20 weight percent of water stabilized against loss of activity evaluated at 50°C, compnsing an enzyme in combination with a stabilizing amount of a surfactant where the surfactant comprises a) a block polymer surfactant formed from a starting material having the formula

    I-[Am-Bn]x, wherein I represents an alcohol, A represents a hydrophobe comprising an alkylene oxide unit in which at least one hydrogen has been replaced by an alkyl group or an aryl group, m is the degree of polymerization which is greater than about 6, B is an aqueous solubilizmg group comprising at least one oxyethylene group, n is the degree of polymerization which is greater than about 6, and x is the functionality of I and is froni 1

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    b) a surfactant having the formula

    RO(CH2CHoO)nH, wherein R is a hydrophobic group, and n is greater than about 5
12. 12 The composition of claim 11, wherein said enzyme is a system of an en2yme in combination with water, said enzyme bemg stabilized against decomposition from water by said non-ionic surfactant which raises the viscosity of water in said system
13. 13 The composition of claim 12, wherein said surfactant is dissolved in an organic solvent compatible with said enzyme
14. 14 The composition of claim 11, wherein said surfactant is dissolved in an organic solvent compatible with said enzyme
15. 15 The composition of claim 13 or 14, wherein said solvent is hydrophilic
16. 16 The composition of claim 15, wherein said solvent is a polyol or mixtuie of polyols
17. 17 The composition of claim 16, wherein said polyol has from 2 to about 6 carbon atoms and from 2 to about 6 hydroxyl groups
18. 18 The composition of claim 17 compnsing an aqueous enzyme suspension of xylanase, said surfactant and glycerol
19. 19 The composition of claim 14, where said surfactant is a polyoxyalkylene glycol ether block-copolymer having a hydrophobe based on a hydrocarbon moiety of an ahphatic monohydnc alcohol containing from 1 to about 8 carbon atoms, where the hydrocarbon moiety has attached thereto through an ether oxygen linkage, a hetenc mixed chain of oxyethylene and

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    1,2-oxypropylene groups, the weight ratio of oxyethylene groups to 1,2-oxypropylene groups in the hydroprohe is from about 5 95 to about 15 85 and the average molecular weight of the hydrophobe is from about 1,000 to about 2,000, a hydrophile being attached to the mixed chain and is based on oxyethylene groups, and the weight ratio of hydrophile to hydrophobe is from about 0 8 1 to about 12 1
20. 20 The composition of claim 19 optionally including as a solvent, a polyol having from 2 to about 6 carbon atoms and from 2 to about 6 hydroxyl groups
21. 21 -The composition of claim 20, wherein said solvent is glycerol and said enzymes are amylase, protease or lipase
22. 22 The composition of claim 14, where said surfactant is a polyoxyalkylene glycol ether block-copolymer having a hydrophobe based on a propylene oxide adduct of propylene glycol where the propylene glycol has attached thereto through an ether oxygen linkage, oxypropylene groups, a hydrophile being attached to the hydrophobe and is based on oxyethylene groups and the average molecular weight of the surfactant is from 1,100 to about 12,600
23. 23 The composition of claim 22 optionally including as a solvent, a polyol having from 2 to about 6 carbon atoms and from 2 to about 6 hydroxyl groups
24. 24 The composition of claim 23, wherein said solvent is glycerol and said enzymes are amylase, protease or lipase
25. 25 The composition of claim 14 where said surfactant is a polyoxyalkylene glycol ether block-copolymer having a hydrophobe based on a propylene oxide adduct of ethylene diamine where the ethylenediamine has attached thereto through an ether oxygen linkage, 1,2-oxypropylene groups, a hydrophile being attached to the mixed chain and is based on oxyethylene groups, and the average molecular weight of the surfactant;! is^fr9m""about;y 'g j-; 1,650 to about 30,000 j hr 1 1

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26. 26 The composition of claim 25 optionally containing as a solvent, a polyol having from 2 to about 6 carbon atoms and from 2 to about 6 hydroxyl groups
27. 27 The composition of claim 26, wherem said solvent is glycerol and said enzymes are amylase, protease or lipase
28. 28 The composition of claim 11, wherem said surfactant contains hydrophobic and hydrophilic blocks, each block being based on at least oxyethylene groups, or oxypropylene groups or mixtures of said groups
29. 29 The composition of claim 28, wherein the average molecular weight of said surfactant is from about 500 to about 30,000, the weight ratio of hydrophobe to hydrophile is from about 0 4 1 to about 2 5 1 and the cloud pomt of said surfactant is from about 0°C to about 100°C
30. 30 A method as defined in claim 1 for stabilizing an enzyme composition substantially as herem descnbed with reference to any example thereof
31. 31 An enzyme composition as defined m claim 11 substantially as herem described with reference to any example thereof

    END OF CLAIMS

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