MXPA97005165A - Stabilization of liquid compositions of enzi - Google Patents

Abstract

A liquid composition containing enzymes, which is well suited, for example, for subsequent incorporation into a multicomponent composition, such as a liquid laundry detergent composition in laundry or dishwashing, comprises: (1) an enzyme in an amount which exceeds 40%, and (11) a reversible inhibitor of the enzyme in an amount effective to improve the storage stability of the enzyme in a multicomponent composition in which the liquid composition is subsequently incorporated.

Description

STABILIZATION OF LIQUID COMPOSITIONS OF ENZYMES FIELD OF THE INVENTION The present invention relates, inter alia, to liquid enzyme compositions containing (i) one or more enzymes in a concentration from relatively high to very high, and (ii) one or more appropriate enzyme inhibitors, more preferably inhibitors. reversible, which exhibit strong inhibitory properties of enzyme and / or which are present at such a concentration that they cause a strong inhibition of the enzyme (s).

The invention further relates to: a multicomponent composition, which contains enzymes, such as a liquid composition, for example, a cleaning composition such as a detergent composition, prepared using a liquid enzyme composition, stabilized with inhibitors, of the invention as the enzyme source; a process for preparing a multicomponent composition, which contains enzymes, such as a cleaning composition, for example, a detergent composition, wherein the enzyme (s) in question is REF: 24896 introduces (n) in the form of a liquid enzyme composition stabilized with inhibitors of the invention, i.e., the enzyme (s) is (are) in the form of a composition stabilized with inhibitors, when incorporated into the multicomponent composition; and a multicomponent composition, which contains enzymes, such as a cleaning composition, for example a detergent composition, prepared by a process of the above type. The invention further relates to the use of a liquid composition of enzymes according to the invention in the preparation or manufacture of a multicomponent composition, which contains enzymes, such as a cleaning composition, for example, a detergent composition.

BACKGROUND OF THE INVENTION Storage stability problems in conjunction with liquid compositions containing enzymes are well known. Thus, for example, a major problem with liquid detergents containing enzymes, such as liquid detergents containing a protease (peptidase), is that of ensuring the retention of the proper activity of the enzyme during storage. Considerable effort has been devoted to finding ways to improve the storage stability of compositions containing enzymes, for example, liquid compositions such as liquid detergents, for example, by adding a protease inhibitor to compositions containing a protease. The discussion in the remainder of this section refers, predominantly, by way of illustration, to the inhibition of proteases, but the underlying principles are equally applicable to other types of enzymes, for example, lipases, amylases, cellulases and oxidoreductases ( such as peroxidases and oxidases), and the various aspects of the present invention are not limited in any way to the aspects associated with the stabilization of proteases by protease inhibitors (reversible). By way of example, it is known that boronic acids and boric acids reversibly inhibit proteolytic enzymes. A discussion of the inhibition of a serine protease, subtilisin, by boronic acid is given in Molecular & Cellular Biochemistry 51, 1983, pp. 5-32.

Boronic acids have very different abilities as inhibitors of subtilisin. Boronic acids containing only alkyl groups such as methyl, butyl or 2-cyclohexylethyl are poor inhibitors, with methylboronic acid being the poorer inhibitor. However, boronic acids having aromatic groups such as phenyl, 4-methoxyphenyl or 3,5-dichlorophenyl are described as being very good inhibitors, with 3,5-dichlorophenylboronic acid being particularly effective (see Keller et al. Biochem, Biophys, Res. Comm. 176, 1991, pp. 401-405). It is also claimed, in WO 92/19707, that the arylboronic acids which are substituted at the 3-position of the aryl group with respect to boron are unexpectedly good reversible inhibitors of the protease. Thus, for example, "acetamidobenzene boronic acid" is described in the latter document as being a particularly effective inhibitor of proteolytic enzymes. When the inhibitory effect of a reversible enzyme inhibitor is quantified, the so-called "inhibition constant" is commonly used.

(K as a measure of the ability to inhibit the activity of the enzyme, Ki which is normally defined as follows: K, = [E] • [I] / [E] wherein [E], [I] and [El] denote equilibrium concentrations (conventionally molar concentrations) of the enzyme, inhibitor and the enzyme-inhibitor complex, respectively, under the conditions in question. This latter definition of Kx is used in the present specification and claims. In this way, a relatively low Ki value is indicative of a relatively strong inhibitor.

DESCRIPTION OF THE INVENTION In the preparation or manufacture of multicomponent, enzyme containing compositions it would be desirable to employ, as the enzyme source, a pre-prepared enzyme composition containing appropriate enzyme (s) and enzyme inhibitor (s), ( reversible). It would be particularly convenient and advantageous for the pre-prepared enzyme composition to contain a very high concentration of at least one enzyme, and possibly of several enzymes of the type (s) in question, since this would allow, inter alia, the preparation of large volumes or amounts of a multicomponent composition, containing enzymes, using relatively small volumes or amounts of the enzyme / inhibitor composition, pre-prepared. Additionally, it would clearly be an advantage if the satisfactory stability of the enzyme (s) during storage of the multicomponent composition is ultimately assured only by the presence of the enzyme inhibitor (s) in question in the (s). ) amount (s) into which they have been introduced in the form of the enzyme, inhibitor, pre-prepared, original, ie, that the inclusion of the additional stabilizing agent in the multicomponent composition (eg, a detergent composition) In addition, in this pre-prepared enzyme / inhibitor composition, the stability of the enzyme (s) for which it will be present is necessary (although, if desired, additional stabilizing agents may be incorporated). An inhibitor, considering the nature of things, would be expected to be high.Thus, numerous pre-prepared compositions of this type would be expected to be capable of storage during relatively long periods. nons of time after manufacture, without the significant loss, or at least without the unacceptable loss, of enzymatic activity. The present invention thus relates to a liquid composition comprising: (i) an enzyme in an amount exceeding 40 μM; and (ii) a reversible enzyme inhibitor in an amount effective to improve the storage stability of the enzyme in a multicomponent composition (e.g., a detergent composition, such as a liquid detergent composition) in which the composition of Cleaning is subsequently incorporated. A liquid composition of the invention may be, for example, a predominantly aqueous composition, or a predominantly non-aqueous composition (eg, a composition comprising as a solvent a high proportion of one or more non-aqueous liquids, but usually miscible in water, in general, organic liquids (such as alcohols, glycols and the like) The liquid compositions of the invention will normally be by themselves non-detergent compositions Apart from the detergent compositions (for example), for washing in laundry, washing dishes and similar, other types of multicomponent compositions, which contain enzymes, notably in the form of liquid compositions, which are of relevance in the context of the present invention include: composition for cleaning dentures, contact lenses and hard surfaces (e.g. slaughterhouses, or in the food processing industry), compositions for use in the ustria of the skin, (for example, for depilation and / or degreasing of animal hides; compositions for the desizing of textiles; and compositions for washing denim textiles to achieve a "stone wash" appearance. The use of these compositions for these purposes are within the scope of the invention.

Enzymes The enzyme classification numbers (EC numbers) referred to in the. present specification with the claims are in accordance with the Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press Inc., 1992.

A liquid composition of the invention contains at least one enzyme. Suitable enzymes include any commercially available enzyme. Particularly relevant enzymes include enzymes selected from the group consisting of proteases (ie, peptidases, EC 3.4), amylases (classified under EC 3.2.1), lipases (including those classified under EC 3.1.1.3), cellulases (including EC 3.2.1.4) and oxidoreductases (EC 1), for example, peroxidases (EC 1.11) and oxidases [including enzymes classified under EC 1.10.3, such as laccases (EC 1.10.3.2)], and any mixtures thereof. Enzyme mixtures from the same class, (for example, mixtures of different proteases, different lipases (etc.) are also included.The amount (s) of enzyme (s) in the liquid composition will vary according to with the type of enzyme (s) and the proposed use of the liquid composition In general, any enzyme present will preferably be present in an amount in the range of 0.2-50% by weight (w / w) of the liquid composition (calculated based on the enzyme pure protein), frequently 0.5-25% w / w, such as 1-10% w / w, for example 2-8% w / w of the liquid composition, expressed in units of concentration, Preferred concentration range for an enzyme present in a liquid composition of the invention will be from about 50 μM to about 20 mM, frequently from 100 μM to 10 mM, such as from 500 μM to 5 mM, for example from 750 μM to 3 mM (calculated on the basis of the moles of the enzyme pure protein).

Proteases. Any protease (proteolytic enzyme) suitable for use in a liquid composition can be used. Suitable proteases include those of animal, plant, or microbial origin, especially of microbial origin, as well as mutants (variants) produced chemically or engineered by proteins (genetically engineered) in which they have been replaced, inserted and / or deleted or more amino acids relative to the amino acid sequence of an enzyme of one of the above types, and exhibiting proteolytic activity. The protease can be, for example, a serine peptidase, preferably a microbial, alkaline protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). In the commercial Bacillus subtilisins examples are the Alcalase ™, Savinase ™, Espeirase ™ and Durazym ™ products all available from Novo Nordisk A / S. A number of these protease products are available, such as Alcalase ™, Esperase "" and Savinase ™, (eg, Savinase ™ 16.0 L, Type DX and Type EX) which are well suited (vide infra) to the preparation of liquid compositions containing protease according to the present invention. Examples of trypsin-like protease include trypsin (for example of porcine or bovine origin) and Fusarium protease described in WO 89/06270.

Amylases Any amylase (amylolytic enzyme) suitable for use in a liquid composition can be used. Suitable amylases include those of bacterial and fungal origin, as well as mutants (variants) produced chemically or engineered (genetically engineered) in which one or more amino acids have been replaced, inserted and / or deleted relative to the amino acid sequence of an enzyme of one of the above types, and exhibiting amylolitic activity. Amylases include, for example, α-amylases (EC 3.2.1.1), for example, those obtained from a particular strain of B. licheniformis, described in more detail in British Patent Specification No. 1,296,839. A very suitable α-amylase is Termamyl®, which is available (inter alia as a liquid product) from Novo Nordisk A / S.

Lipases Any lipase (lipolytic enzyme) suitable for use in a liquid composition can be used. Suitable lipases include those of bacterial and fungal origin, as well as mutants (variants) produced chemically or engineered (genetically engineered) in which one or more amino acids have been replaced, inserted and / or deleted relative to the amino acid sequence of an enzyme of one of the above types, and exhibiting lipolytic activity. A very suitable lipase is that obtained by cloning the Humicola lanuginosa gene and expressing the gene in Aspergillus oryzae, as described in EP 0 258 068, which is available (inter alia as a liquid product) from Novo Nordisk A / S under the trade name LipolaseMR.

Cellulases Any cellulase (cellulolytic enzyme) suitable for use in a liquid composition can be used. Suitable cellulases include those of bacterial or fungal origin, as well as mutants (variants) produced chemically or engineered (genetically engineered) in which one or more amino acids have been replaced, inserted and / or deleted relative to the amino acid sequence of an enzyme of one of the above types, and exhibiting cellulolytic activity. Suitable cellulases are described, for example, in US 4,435,307. A very suitable cellulase is that produced by a strain of Humicola insolens, available from Novo Nordisk A / S under the tradename Celluzyme ™.

Peroxidases Any suitable peroxidase can be used for the use of a liquid composition, for example, a liquid detergent composition. Peroxidases suitable herein include those of plant, bacterial and fungal origin, as well as mutants (variants) produced chemically or engineered (genetically engineered) in which one or more amino acids have been replaced, inserted and / or deleted in relation to to the amino acid sequence of an enzyme of one of the above types, and exhibiting peroxidase activity.

Examples of suitable peroxidases are those derived from a strain of Coprinus (for example C. cinerius or C. macrorhizus) or from a strain of Bacillus (for example, B. pumilus), particularly a peroxidase according to PCT / DK90 / 00260.

Rusty . Any suitable oxidase can be used for the use of a liquid composition, for example, a liquid detergent composition. Suitable oxidases herein include those of bacterial and fungal origin, as well as mutants (variants) produced chemically or engineered (genetically engineered) in which one or more amino acids have been replaced, inserted and / or deleted relative to the amino acid sequence of an enzyme of one of the above types, and exhibiting oxidase activity. Examples of oxidase are laccases derived from strains of Aspergillus, Neurospora (e.g., N. crassa), Trametes (e.g., Villosa) or Myceliophthora (e.g., M. thermophila).

Enzyme inhibitors (enzyme stabilizers) A liquid composition according to the invention contains at least one reversible enzyme inhibitor, usually which is an inhibitor for at least one enzyme present in the liquid composition. Thus, for example, a liquid composition of the invention can, in addition to containing a first type of enzyme and a reversible inhibitor for that type of enzyme, contain a second, third, etc., type of enzyme (e.g. enzyme of one of the types mentioned above) one or more of which is not accompanied by a reversible inhibitor for the same. It should be mentioned here that it is perfectly feasible to prepare a liquid composition of enzymes containing one or more enzymes, in amounts as described herein, but which instead of containing an inhibitor for one or more of these enzymes contains one inhibitor for another. type of enzyme, with which the liquid composition can subsequently be put in contact. An example thereof would be a liquid composition containing, as the sole enzyme, a lipase together with a protease inhibitor; the protease inhibitor then protects the lipase with respect to degradation by a protease which, deliberately or inadvertently, may come into contact, subsequently with the lipase. The nature and amount (s) of the inhibitor (s) employed will depend inter alia on the nature and concentration of the enzyme (s) present in the composition, and the proposed use of the composition. This is discussed later in the present. With respect to reversible inhibitors (and associated Ki values thereof) of relevance for use in conjunction with various classes / types of enzymes that may be present in a liquid composition of the invention, reference is made to: H, Zollner, Handbook of Enzyme Inhibitors (Parts A and B), 2nd Edition, VCH Verlagsgesellschaft bH, Weinheim, Germany, 1993, for an extensive list. Additional sources of relevance include: S. Patkar and F. Bjdrkling, Lipase inhibitors, in: Lipases - their Structure, Biochemistry and Application, Editors P. Woolley and S.B. Petersen, Cambridge University Press, Cambridge 1994. Selected examples of relevant types of reversible inhibitors are the following: Protease inhibitors. A particularly interesting class of reversible protease inhibitors consists of boronic acids [R'B (OH) 2] and borinic acids [R'R "B (CH)] (wherein R 'and R" are organic substituents, for example, optionally substituted aryl or heterocyclic substituents), some of the examples of which have been mentioned above. Additional examples of relevant compounds of this type can be selected from those mentioned in WO 92/19707, in EP 0 478 050 Al and in EP 0 511 456 Al. Interesting compounds of this type can be found among those described in WO 95/02046 (which was not published on the priority date of the present application), and which comprises compounds of the following general formula: R - (R2) ft - B - OH I I R3 where Ri is an optionally substituted, fused aromatic ring structure containing 14 or 18 carbon atoms in the ring or an optionally substituted heterocyclic, aromatic, fused or oncyclic ring structure containing up to 17 ring carbon atoms, or an optionally substituted, fused or monocyclic quinonoid ring structure containing up to 18 ring carbon atoms; R2 has the formula: where X is the same or different and is selected from hydrogen, alkyl of 1 to 6 carbon atoms, alkyl of 1 to 6 carbon atoms, substituted, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate or phosphonate, I, p and q can be the same or different and can each be 0, 1 or 2; m and n may be the same or different and may each be 0 or 1; R3 is the same or different as Ri and is selected from Ri, or R3 is the hydroxyl group, or Rx and R3 are both optionally substituted cyclic or monocyclic aromatic ring structures. In the above context, the optionally substituted ring structures are such that the substituents in the ring structure are freely chosen, but are preferably selected from hydrogen, alkyl of 1 to 6 carbon atoms, alkyl of 1 to 6 carbon atoms, substituted carbon, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate and phosphonate. The boronic and borinic acid derivatives can be prepared using methods well known to those skilled in the art, for example, by using one of the following methods: a) Hydroboration of unsaturated materials, ie, alkenes and alkynes, using either catecholborane (1, 3, 2-benzodioxaborol) or dichloroborane-dimethyl sulfide complex as the hydroboration agent, for reference see HC Brown, S.K. Gupta In JACS 97, 1975, PP. 5249-5255 and H.C. Brown, N .. Ravindran, S.U. Kuikarni in J. Org. Chem. 45, (1980), p. 384. b) The reaction of a Grignard reagent with either tri-n-butyl borate or trimethyl borate, followed by hydrolysis of the boronic ester thus formed; for reference see < F.R. Bean, J.R. Johnson in Jacs 54, 1932, pp. 4415-4425 and S.H. Dandegaonher, S.P. Ingleshwar in Journal of Shivasi University 6, 1932, pp. 11-13. Bromine-substituted starting materials that are not commercially available can be conveniently prepared in two steps from the corresponding carboxylic acids by reduction with LiAlH4, followed by treatment with CBr4. c) The reaction of an organolithium reagent with butylborate; for reference see S.O. Lauesson, pp. 387-395 in Thiophene Chemistry, Part 7, and D. Florentin, B. Roques in C. R. Acad. Sci. Paris, t.270 (May 11, 1970), pp. 1608-1610. d) The boronic acid derivatives can be prepared according to method b, above. However, the ratio of the Grignard reagent to the borate adopted then is 2: 1. e) Any nuclear substitution or protection of the functional groups is achieved by using normal methods well known to those skilled in the art.

Interesting, additional compounds of this type may be selected from naphthalene boronic acids which are described in WO 95/29223 (which was not published on the priority date of the present application) and which comprises compounds having the following general formulas: where Ri, R2, R3 R4 # -Rsr Re and 7 are the same or different and are selected from hydrogen, alkyl of 1 to 6 carbon atoms, alkyl of 1 to 6 carbon atoms substituted, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate and phosphonate. The boronic acid derivatives of naphthalene can also be prepared using methods well known to those skilled in the art, for example, by using a Grignard preparation as follows: The Grignard reagent is prepared by the slow addition dropwise of the appropriate bromonaphthalene starting material in sodium-dried ether to magnesium chips in sodium-dried ether. The reaction is promoted by the addition of a small crystal of iodine. The trimethyl borate or tri-n-butyl borate in the dry sodium ether is cooled to -70 ° C, and then the Grignard reagent is added dropwise over a period of 2 hours while maintaining the organoborate solution at -70. ° C and stir continuously. The reaction mixture is allowed to warm to room temperature overnight, after which it is hydrolyzed by the dropwise addition of dilute, cold sulfuric acid. The ester layer is separated and the aqueous layer is extracted with ether. The fractions containing ether are combined and the solvent is removed. The residue becomes strongly alkaline, and any methanol or butanol thus formed is removed. The alkaline solution is cooled and the resulting crystals of the desired boronic acid are removed by filtration. All products are recrystallized preferably from distilled water.

The naphthalene boronic acids can also be prepared using either direct lithiation of the naphthalene and / or lithiation of the bromide. Any nuclear substitution or protection of the functional groups can be achieved by using normal methods well known to those skilled in the art. Among the compounds of the types mentioned above, the specific boronic acids of interest in the context of the invention include the following: benzofuran-2-boronic acid; phenyl boronic acid; 4-bromophenyl-boronic acid; 4-formylphenyl boronic acid; 3-acetamidophenyl-boronic acid; 3, 5-dichlorophenyl-boronic acid; 5-chlorothiophen-2-boronic acid; Naphthalene-1-boronic acid; Naphthalene-2-boronic acid; and 6-hydroxynaphthalene-2-boronic acid. Additional specific compounds of this type that are of interest in the context of the invention are mentioned in the table given in Example 2 herein (vide infra). The boronic or borinic acid type inhibitors can be introduced or incorporated into the liquid compositions of the invention in the form of the same acids (for example dissolved in an appropriate water-miscible organic solvent, such as mono-propylene glycol or the like) or it is suitable (for example for reasons of solubility), as salts (for example alkali metal salts, such as sodium or potassium salts). Reversible Amylase Inhibitors. Reversible amylase inhibitors of possible relevance in the context of the invention can be found, for example, between so-called "acarbose" compounds (ie, pseudo-oligosaccharides containing a portion of acarviosin and one or more maltose units) . Other compounds of relevance as inhibitors, for a-amylases, in particular, include maltose and maltotriose. Compounds such as methyl a-glucoside, as well as cycloamylase, for example cyclohexa- and / or cycloheptaamylose, may be of relevance as reversible inhibitors for β-amylases. Reversible Cellulase Inhibitors. Reversible cellulase inhibitors of potential interest in the context of the invention include 4-thiol-cellooligosaccharides [see for example, C. Schou et al., Journal of Carbohydrate Chemistry 12 (1993) pp. 743-752]. Reversible Lipase Inhibitors. Reversible lipase inhibitors of relevance in the context of the invention include boronic and borinic acids selected from those described above and / or subsequently mentioned (such as, for example, phenyl boronic acids, optionally substituted).

Degree of Enzyme Inhibition The desired degree (resistance, degree) of inhibition of an enzyme present in a liquid composition according to the invention will depend, among other things, on the purpose for which the composition is proposed. In general, the molar ratio of the reversible inhibitor to the enzyme will be chosen such that at least one molecule of inhibitor is present per active site of the enzyme in question, which will of course require a molar ratio of inhibitor fl) to enzyme (EJ of However, for certain purposes, a molar ratio I: E, for example of approximately 0.8: 1, of approximately 0.7: 1, approximately 0.6: 1 or even approximately 0.5: 1 may be appropriate. In the context of the invention it will generally be desirable to employ a molar ratio of enzyme inhibitor of at least 5, such as at least 15. In certain cases, the molar ratios I: E of at least 50 or at least 100, or even molar ratios can The molar ratio of the inhibitor to the enzyme will be chosen, inter alia, based on the considerations that relate to the percentage of free (non-inhibited) enzyme that is desired to be present during the actual use of a multicore composition. component, which contains enzymes, based on a liquid composition of the invention. Thus, for example, when a laundry detergent composition is used for washing in laundry, it will clearly be a requirement that a satisfactorily high level of free enzyme (eg, a protease or a lipase) be present in the washing (which is typically water to which the detergent composition in question has been added).

In this context, a parameter of importance is the constant inhibition (defined above) for the reversible inhibition of an enzyme. Thus, for example, when a liquid composition of the invention is to be in a detergent composition, the inhibition constant (Ki), expressed in the conventional manner in mol / 1 (M), for a reversible inhibitor of a enzyme where it will often be suitable such that 3 x 10 ~ 8 M < K < 1. 2 x 10"2, such as 3 x 10" 8 M < Ki < 1 x 1-10"2 M. A more desirable" window of inhibition "in conjunction with detergent enzymes will often, however, be such that one (one) inhibitor (s) associated with a particular enzyme exerts an inhibition corresponding approximately to 3 x 10"7 M < K ± < 1 x 10 ~ 3 M, such as 4. 3 x 10 ~ 7 M < Ki < 4.5 x 10"4 M.

In general, preferred liquid compositions containing enzymes of the invention are compositions in which the ratio between: (a) the total molar concentration (which can be denoted [I] t) of a reversible enzyme inhibitor, I, present in the composition; and (b) the inhibition constant (Ki, * expressed in mol / 1) for the inhibition of an enzyme that is present in the composition and that is reversibly inhibited by the inhibitor in question; is at least 50 (ie, [I] t / Ki> 50) such as at least 100 or frequently 250. A practical upper limit for the ratio [I] t / Ki in the liquid compositions of the invention will normally be approximately 10,000 (that is, 104). For many purposes a value for the ratio [I] t / Ki in the range of 250-5000, often in the range of 250-2500, will be appropriate. The value of the ratio [I]? / Ki can be considered as a measure of the "inhibitory capacity" of a liquid composition of the invention. A high value, and thus a high "inhibitory capacity", can be achieved, for example, by incorporating into a liquid composition, a relatively modest to low concentration of a strongly inhibitory inhibitor, or by incorporating a relatively high concentration of a inhibitor more weakly inhibitory. In the presence of a relatively large molar excess of the inhibitor, I, relative to the inhibited enzyme, E, the main proportion of the inhibitor will normally be in the free form, ie, it will not bind to the enzyme. Denoting the concentration of the free inhibitor (not bound) by [I] (as in conjunction with Kx; vide supra), the relation between [I] t / Kx then will be approximately equal to the relation [I] / Ki, that is, [I] t / Ki «[I] t / x under these conditions. It can be mentioned here that liquid compositions according to the invention, comprising enzyme (s) and reversible enzyme inhibitor (s), can be dried by appropriate methods (eg, by lyophilization or by spray drying) . The dried enzyme / inhibitor product can then be comminuted (for example by milling) and dispersed or slurried in an appropriate concentration in a non-aqueous liquid carrier, for example a non-ionic surfactant (such as BP's Softanol ™). ), to form a thick suspension product.

Detergents For detergent compositions, a typical goal will be to achieve at least 50% of a chosen enzyme (eg, a protease) in the detergent composition per se, and an amount of the free enzyme in the corresponding washing medium. at least 50% of the total amount of that enzyme. A detergent composition incorporating a liquid composition of the invention will comprise, apart from the enzyme (s) and the inhibitor (s) a surfactant, and will normally be a liquid detergent composition. The detergent composition can be, for example, a laundry detergent composition or a dishwashing detergent composition.

A liquid detergent composition can be aqueous, for example, typically containing up to 70% water and 0-30% organic solvent, and substantially non-aqueous. The detergent composition will contain one or more surfactants, each of which may be anionic, nonionic, cationic, or amphoteric (zwitterionic). The detergent will usually contain 0-50% ionic surfactant such as linear alkyl benzene sulphonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES) , secondary alkanesulfonates (SAS), methyl esters of alpha-sulfo acid grades, alkyl- or alkenyl-succinic acid, or soap. It may also contain from 0-40% of a nonionic surfactant such as alcohol ethoxylate (AEO or AE), alcohol propoxylate, carboxylated alcohol ethoxylate, nonylphenyl ethoxylate, alkyl polyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (as described for example in WO 92/06154). Normally, the detergent contains 1-65% of a detergent addition product, but some dishwashing detergents may contain up to 90% of a detergent addition product, complexing product such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenyl-succinic acid, soluble silicates or layered silicates (for example, SKS-6 from Hoechst). The detergent addition products can be subdivided into types that contain phosphorus and types that do not contain phosphorus. Examples of detergent, alkaline, inorganic, phosphorus-containing detergent addition products include water soluble salts, especially pyrophosphates, orthophosphates, polyphosphates and alkali metal phosphonates. Examples of inorganic addition products that do not contain phosphorus include water-soluble alkali metal carbonates, borates and silicates as well as layered disilicates and various types of crystalline or amorphous aluminum silicates, insoluble in water of which the zeolite is the most representative. Examples of suitable organic addition products include alkali metal, ammonium or substituted ammonium salts of succinates, malonates, fatty acid malonates, fatty acid sulfonates, carboxymethoxy succinates, polyacetates, carboxylates, polycarboxylates, aminopolycarboxylates and polyacetyl -carboxylates. The detergent may also have no addition products, ie be essentially free of the detergent addition product. The detergent may comprise one or more polymers. Examples are carboxymethylcellulose (CMC), poly (vinylpyrrolidone) (PVP), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, polymaleates, maleic / acrylic acid copolymers and lauryl methacrylate copolymers. acrylic acid The detergent composition may contain chlorine / bromine type or oxygen type bleaching agents. The bleaching agents can be coated or encapsulated. Examples of chlorine / bromine type inorganic bleaches are hypochlorite or lithium hypobromite, sodium or calcium, as well as chlorinated trisodium phosphate. The bleaching system may also comprise a source of H202 such as perborate or percabonate which may be combined with a bleach activator that forms peracid such as tetraacetylethylenediamine (TAED) or nonanoyloxy-encensulfonate (NOBS). Examples of chlorine / bromine type organic whiteners are heterocyclic N-bromo and N-chloroimides such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water solubilizing cations such as potassium and sodium.

The hydantoin compounds are also suitable. The bleaching system may also comprise peroxyacids of, for example, amide, imide, or sulfone type. In dishwashing detergents, oxygen bleaches are preferred, for example, in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid compound. Typical examples of suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percabonates, persilicates and perfosphates. The preferred activated materials are TAED or NOBS. As already mentioned, the enzymes of the detergent composition of the invention are incorporated into the detergent composition in the form of a liquid enzyme / inhibitor composition according to the invention. If desired, additional, conventional, enzyme stabilizing substances can be incorporated, for example a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as a aromatic borate ester (see, for example, WO 92/19709 and WO 92/19708) The detergent may also contain other, conventional, detergent ingredients, such as, for example, fabric conditioners including clays, deflocculating material, foam enhancers / depressants of foam (in dishwashing detergents, foam depressors), suds suppressors, anti-corrosion agents, dirt-dispersing agents, anti-dirt-replenishing agents, dyes, dehydrating agents, bactericides, optical brighteners, or perfume The pH (measured in the aqueous solution at the use concentration) will generally be neutral or alkaline, for example, in the range of 7-11. Particular forms of laundry detergent compositions within the scope of the invention include: 1) A liquid, aqueous detergent composition, comprising 2) A liquid, structure, aqueous detergent composition comprising 3) A liquid, aqueous detergent composition , comprising 4) A liquid, aqueous detergent composition, comprising ) Detergent formulations as described in l) -4), wherein all part of the linear alkylbenzene sulphonate is replaced by (Ci? -iß) alkyl sulfate. 6) Detergent formulations as described in l) -5) containing a stabilized or encapsulated peracid, either as an additional component or as a substitute for already specified bleaching systems. 7). A detergent composition formulated as a non-aqueous detergent liquid comprising a liquid nonionic surfactant such as, for example, linear alkoxylated primary alcohol, a system of adhesion products (eg, phosphate), enzyme and alkali. The detergent may also comprise a surfactant and / or a bleaching system.

Multicomponent compositions, which contain plate washing enzymes and other Examples of relevant types of formulations of this kind include the following: 1) LIQUID COMPOSITION OF DISHWASHING WITH A SURGICAL CLEANING SYSTEM, COMPRISING: Nonionic Surfactant 0 - 1.5% Octadecyl N-Oxide Dihydrate - 0 - 5% Dimethylamine Mix 80:20 C18 / C16 Weight of Octadecyl-Dimethylamine N-Oxide Dihydrate and Hexadecyl Dimetilamine N-Oxide 0 - 4 % Mixture 70:30 C18 / C16 weight of octadecyl-bis (hydroxyethyl) amine N-oxide, anhydrous and hexadecyl-bis- (hydroxyethyl) amine N-oxide, anhydrous 0 - 5% C13-C15 alkyl ethoxy sulfate with an average degree of ethoxylation of 3 0 - 10% C12-C15 ethoxysulfate alkyl with an average degree of ethoxylation of 3 0 - 5% ethoxylated alcohol of C13-C15 with an average degree of ethoxylation of 12 0 - 5% A mixture of alcohols C12-C15 ethoxylates with an average degree of ethoxylation of 9 0 - 6.5% (Continued) A mixture of C13-C15 ethoxylated alcohols with an average degree of ethoxylation of 30 0 - 4% Sodium disilicate 0 - 33% Sodium tripolyphosphate 0 - 46% Sodium citrate 0 - 28% Citric acid 0 - 29% Carbonate Sodium 0 - 20% Sodium Perborate Monohydrate 0 - 11.5% Tetraacetylethylenediamine (TAED) 0 - 4% Copolymer of maleic acid / acrylic acid 0 - 7.5% Sodium sulfate 0 - 12.5% Enzymes 0. 0001 - 0.2% 2) NON-AQUEOUS LIQUID COMPOSITION OF AUTOMATIC PLATE WASHING, WHICH INCLUDES 3) NON-AQUEOUS COMPOSITION, LIQUID, OF WASHING OF DISHES, COMPRISING 4) LIQUID, THIXOTROPIC COMPOSITION OF AUTOMATIC DISHES OF PLATES, WHICH INCLUDES: 5) LIQUID COMPOSITION OF AUTOMATIC DISHWASHING OF PLATES THAT COMPRISES: 6) LIQUID COMPOSITION OF AUTOMATIC PLATE WASHING CONTAINING WHITENING PARTICLES, PROTECTED, WHICH INCLUDES: 7) Automatic dishwashing compositions as described in 1) and 5), wherein the perborate is replaced by percarbonate. 8) Automatic dishwashing compositions as described in 1), which additionally contains a manganese catalyst. The manganese catalyst may be one, for example, of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639. The content of "enzymes" of the above formulations may include the content of the enzyme inhibitor (s) present. Where appropriate, a particular formulation encompassed within one of the above-mentioned types may contain an organic solvent of a type not specifically mentioned in connection with that type of formulation, but which has been functionalized, for example, as a solvent of solubilization for an inhibitor present in a liquid enzyme / inhibitor composition of the invention that has been incorporated into the particular formulation in question. A liquid enzyme / inhibitor composition of the invention can be incorporated into a detergent composition to give an enzyme concentration that is conventionally employed in detergents. It is contemplated herein that, for a detergent composition of the invention, a liquid enzyme / inhibitor composition of the invention can be incorporated, for example, in an amount that, for a given enzyme present in the enzyme / inhibitor composition , corresponds to an amount of enzyme in the range of 0.00001-1 mg (calculated as enzyme pure protein) of enzyme per liter of wash liquor.

Testing Stabilizers The inhibitory efficacy of an inhibitor can be tested in several ways, illustrated here by the following two tests for the case of a protease / protease inhibitor: a) Storage Stability Test in the Liquid Detergent: The liquid enzyme / inhibitor composition is added to a liquid detergent formulation that is stored under well-defined conditions. The activity of each enzyme is determined as a function of time (for example after 1, 3 and 7 days). To calculate the efficiency of inhibition of storage stability data, a reaction mechanism is proposed. The following reactions give a relatively simple, yet plausive, mechanism for a liquid detergent containing protease (P), lipase (L) and inhibitor (I): I) Protease self-digestion: P + P? DP + P II) Denaturing of the protease: P-DP III) Inhibition of the protease: P + I ¿* Pl IV) Protease digestion of the inhibited enzyme: P + Pl -P + DP + I V) Denaturation of the inhibited enzyme: VI) Protease digestion of lipase: P + L? P + DL VII) Denaturation of lipase: L? DL where DP and DL are denatured (ie, non-active) protease and lipase.

From these reactions, three coupled differential equations are derived that describe the deactivation of P, L and Pl. The constants of the reaction rate were derived from storage stability data by the use of a parameter estimation method (Gauss-Newton with the modification of Levenberg). The storage stability data gives the concentration of (P + Pl) and L as a function of time. Reaction III is much faster than the other reactions and equilibrium is accepted in the calculations. Reaction IV is excluded from the system to reduce the number of parameters thus describing the stability of the inhibited enzyme by only one reaction rate constant (from equation V). When a large surplus of inhibitor molecules (relative to the protease molecules) is present, the concentration of the inhibitor can be assumed, as a reasonable approximation, which is a constant. The specific values of the reaction rate constants are somewhat sensitive to small variations in the data, but the sensitivity is significantly reduced when giving the results in relation to the value for boric acid. The use of boric acid [as well as closely related substances or equivalent such as alkali metal borate (for example borax) and boric acid] for the purpose of inhibiting, in particular, proteases is well known (for example, see EP 0 451 924 A2), but the low inhibitory resistance in combination with the generally poorer solubility of these substances (which is further illustrated for boric acid and in the working examples in this; vide infra) makes them generally unsuitable for use in the context of the present invention [in which the inhibitor (s) and / or amount (s) thereof present in a liquid composition of the invention should be such that they still cause good stabilization of the enzyme when the liquid composition is incorporated as a component of a multicomponent composicron]. Thus, compounds such as boric acid, alkali metal borate (for example, borax) and boric oxide will generally be excluded as inhibitors in the context of the present invention. An "improvement factor" can be defined as follows: Ki (boric acid) K ± (inhibitor) IFi defined in this way thus provides a measure of the inhibition efficiency of a given inhibitor relative to boric acid. b) Determination of KL. The inhibition constant Ki can be determined by using normal methods; for reference see Keller et al., Biochem, Biophys. Res. I ate. 176, 1991, pp. 401-405; J. Bieth in Bayer-Symposium "Proteinase Inhibitors", pp. 463-469, Springer-Verlag, 1974 and Lone Kierstein Hansen in "Determination of Specific activities of Selected Detergent Proteases using Protease Activity, Molecular Weights, Kinetic Parameters and Inhibition Kinetics", PhD thesis, Novo Nordisk A / S and University of Copenhagen, 1991. The determination can be carried out, if desired, in the presence of non-enzyme components (surfactant agent (s), etc.) of a detergent composition. Appropriate conditions for determining Kx values for enzyme inhibition will typically be achieved by employing a buffer system that provides a suitable pH system and does not react or complex with the enzyme (s) or inhibitor (s) in question. A suitable buffer system for the enzyme / inhibitor interactions will be, for example, a glycyl-glycine buffer at a mildly alkaline pH, for example, pH 8.6, and an ambient temperature (typically 25 ° C).

Experimental Section The invention is further illustrated and sustained in the following examples, which are not intended to in any way limit the scope of the invention as claimed.

EXAMPLE 1 The following procedures are illustrative of the approaches suitable for the synthesis of numerous types of boronic and borinic acids: Preparation of thiophene-3-boronic acid: 3-bromothiophene (0.043 m) in sodium-dried ether (100 m) was cooled to -60 ° C. Bultillitio was added quickly (30 ml of 1 M). The mixture was then stirred for 3 minutes, then tri-n-butyl borate (0.043 m) or trimethyl borate (0.043 m) in sodium-dried ether (25 ml) was added. The mixture was stirred for 4 hours and allowed to warm to room temperature. Subsequently, the reaction mixture was treated with hydrochloric acid (1 M) and the ether layer was separated. The aqueous layer was extracted with ether (2 times, 25 ml). The combined ether layers were extracted with sodium hydroxide (1 M). The alkaline solution was then acidified with hydrochloric acid (10%), thereby precipitating the desired boronic acid. The boronic acid was isolated and then recrystallized from water / ethanol and allowed to dry in air. C4H5B02S, p.f. 163-164 ° C.

Preparation of diphenylborinic acid: This was prepared using the above method. The Grignard reagent was prepared from bromobenzene and magnesium shavings. However, two moles of Grignard reagent were used per one mole of tri-n-butyl borate. The borinic acid thus formed was isolated by the reaction with ethanolamine thus producing the complex of diphenylboronic acid, ethanolamine ((C6H5) 2BO.CH2CH2NH2), which is easier to handle, m.p. 192-194 ° C.

Preparation of 4-formylphenyl boronic acid: This compound was prepared as described in: Chem. Ber. 123 (1990), pp. 1841-1843. The 4-bromobenzaldehyde diacetal was reacted with magnesium chips in tetrahydrofuran, after which the tributyl borate dissolved in ether is added and the mixture is worked with sulfuric acid.

EXAMPLE 2 Determination of Ki The inhibition constants, K ±, for the inhibition of Savinase ™ protease were determined using normal methods under the following conditions: Substrate: Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitro-anilide = SAAPFpNA (from Sigma, Catalog No. S-7388).

Shock absorber: glycine glycine 0.1 M pH 8.6; 1.5 ml 15% BrijMR 35 per liter; 25 ° C (glycine glycine from Sigma, catalog No. G-3028).

Enzyme concentration in the assay: Savinase ™: 1 X 10"10 - 3 x 10" 10 M.

The initial rate of substrate hydrolysis was determined at nine substrate concentrations in the range of 0.01 to 2 mM using an automated Cobas' Fara spectrophotometer. The kinetic parameters Vmax and Km were determined using ENZFITTER (a non-linear regression data analysis program). The Kcat of the equation Vmax = Kcat X [E0] was calculated. The concentration of the active enzyme [EO] was determined by the titration of the active site using protein-type protease inhibitors, of strong union. The inhibition constants, Ki, were calculated from Km / Kcat programs as a function of inhibitor concentration. Inhibitors were assumed to be 100% pure, and molar concentrations were determined from heavy amounts and molecular weights. The resulting values of the inhibition constants, Ki, for a series of boronic acid enzyme stabilizers (inhibitors) tested are listed below, together with the values of the ratio [I] t / K? for some of these inhibitors.

Table 1 Inhibition constants and values [I] t / Kj for the inhibition of Savinase ™ by different boronic acid derivatives. Boric acid is included by comparison *% p / p of inhibitor in solution, EXAMPLE 3 Storage stability test in liquid detergent.

In the following, reference is made to KNPU ("Units of Kilo Novo Proteasa" in relation to the preparations of Savinase ™, and to KLU ("Units of Kilo of Lipase") in relation to the preparations of Lipolase ™ A KNPU corresponds to 2.53 mg (approximately 9.4 x 10"5 mole) of pure enzyme protein, reference can be made to a brochure, AF 200 / 1- GB (available through the request of Novo Nordisk A / S under Bagsvaerd, Denmark), which relates to the determination of the activity of Savinase® One unit of lipase (LU) is defined as the amount of enzyme which, under normal conditions (30.0 ° C, pH 7.0, with gum arabic as an emulsifier and tributyrin as a substrate) releases 1 μmol of titratable butyric acid per minute. A brochure (AF 95/5) describing this analytical method in more detail is available at the request of Novo Nordisk A / S, Bagsvaerd, Denmark A KLU corresponds to 0.2 mg (approximately 6.7 x 10 ~ 6 mmol) of pure protein from enzyme, several protease inhibitors were tested in the storage stability tests (retention of enzyme activity) in the liquid detergent, using the method described above, under the conditions as described below. A solution at 4% w / w of each inhibitor (except boronic acid) in mono-propylene glycol (MPG) was prepared. Boronic acid is poorly soluble in the medium in question, and therefore dissolved in the final detergent formulation (to give a boric acid concentration of 1% w / w therein) more than in the liquid enzyme / inhibitor composition.

First test series: For the first test series, equal parts by weight of the inhibitor solution and Savinase ™ 16.0 L, Type EX (liquid protease preparation containing 16 KNPU protease per gram; Novo Nordisk A / S) were mixed , Bagsvaerd, Denmark), giving preparations of Savinase ™ 8 L EX each containing 2% w / w of inhibitor. The storage stability results for the detergent compositions each containing 1% w / w of a Savinase ™ / inhibitor preparation, 98% w / w of "detergent base I" (vide infra) and 1% w / w p of Lipolase ™ 100 L, Type EX (liquid preparation of lipase containing 100 KLU of lipase per gram, Novo Nordisk A / S, Bagsvaerd, Denmark) were as follows: % of residual activity of Lipolase ™ (storage at 30 ° C); Inhibitor Storage period (days 1 3 7 None 34 6 0 1% boronic acid 82 52 21 Phenyl boronic acid 79 4 6 17 Benzofuran-2-boronic acid 83 54 24 4-formylphenyl boronic acid 86 64 41 No Savmase or inhibitor 88 72 55 % Residual activities of Savinase, MR (7 days storage at 30 ° C): Inhibitor% Activity None 10 Boric acid 1% 74 Phenyl boronic acid 67 Benzofuran-2-boronic acid 76 4-formylphenyl boronic acid 85 The above results show, inter alia, that the retention of the activities of Lipolase ™ and Savinase ™ in the detergent composition is significantly improved by the presence of the inhibitors.

Second test series: For the second test series, Savinase ™ 16.0 L EX, 4% w / w of inhibitor in MPG and pure MPG in a weight ratio of 50: 37.5: 12.5, respectively, were mixed giving Savinase ™ 8 preparations L EX each containing 1.5% w / w of inhibitor. The storage stability results for the detergent compositions each containing 1% v / v of this Savinase ™ / inhibitor preparation, 98% w / w of "detergent base II" (vide infra) and 1% w / w p of LipolaseMR 100 L, Type EX, were as follows:% residual activities of Lipolase ™ (storage 30 ° C): Inhibitor Storage period (days) 12 None 53 8 0 4-Formylphenyl boronic acid 58 41 28 3-Acetamidophenyl-boronic acid 24 9 5 No Savinase ™ or inhibitor 76 67 55 Third test series: Preparations of Savinase ™ 8 L EX each containing 1.5% w / w of inhibitor were prepared, in the same way as for the second test series, above. The storage stability results for the compositions of detergent each containing 1% w / w of a Savinase ™ / inhibitor preparation, 98% w / w of OMOMR Micro "inactivated" (vide infra) and 1% w / w of Lipolase ™ 100 L, Type EX were as follows: % of LipolaseMR residual activities (storage at 35 ° C) Inhibitor Storage period (days) 7 14 None 90 88 85 4-formylphenyl boronic acid 98 99 99 No Savm • aseMR or 99 99 99 inhibitor Fourth test series: Essentially as for the series, above, with the exceptions that a storage temperature of 30 ° C was used, and the OMOMR Micro inactivated with "detergent base III" was replaced (vide infra). % residual activities of Lipolase ™ (storage at 30 ° C) Inhibitor Storage period (days) 12 None 45 19 4-Formylphenyl boronic acid 32 20 17 No SavinaseMR or inhibitor 39 28 24 These latter results demonstrate, inter alia, that the retention of Lipolase ™ activity in various detergent compositions is significantly enhanced by the presence of the inhibitors.

Composition of detergent base I (North American type): Component% p / p (as pure components) NansaMR 1169 / p 10.3 (linear alkylbenzene sulfonate, LAS) BerolMR 452 3.5 (alkyl sulphate ether, AES) Oleate acid 0.5 Coconut fatty acid 0.5 DobanolMR 25-7 6.4 (alcohol ethoxylate, AEO) Sodium xylene sulphonate 5.1 Ethanol 0.7 MPG 2.7 Glycerol 0.5 Sodium sulphate 0.4 Sodium carbonate 2.7 Sodium citrate 4.4 Citric acid 1.5 Water for the rest Composition of the detergent base II Component% by weight Nansa ™ 1169 / p 10.30 SulfatolMR NA / 25 1.40 Oleic acid 7.05 Coconut fatty acid 7.05 DobanolMR 25-7 13.10 Triethanolamine 5.50 NaOH, 40% 2.00 Sodium xylenesulfonate 1.00 Ethanol 4.90 MPG 2.70 Sodium sulfate 0.20 Sodium citrate 1.40 DequestTM 2060 S 0.40 Water for the rest Composition of detergent base III Component% p / p NansaMR 1169 / p 7.0 Oleic acid 0.45 Coconut fatty acid 0.45 LutensolMR A04 3.80 MPG 0.50 Glycerol 4.60 Na5P3O10-H2O 22.60 Na2SiO3'5H20 1.70 Sodium carbonate 1.43 Water for the rest The OMOMR Micro was a retail product sold in a Danish supermarket. The enzyme content in it was inactivated by the treatment in a microwave oven (85 ° C, 5 minutes).

EXAMPLE 4 Examples of liquid enzyme / inhibitor compositions Some examples of liquid enzyme / inhibitor compositions according to the present invention are as follows (all compositions were solutions of acceptable appearance): 1) 0.1 g of 4-bromophenyl-boronic acid + 10 g Savinase 16.0 L, Type EX: this composition has an inhibitory: enzyme (I: E) ratio of approximately 33. 2) 0.2 g of 3,5-dichlorophenylboronic acid + 40 g of Savinase 16.0 L, Type EX: this composition had a molar ratio of I: E of approximately 17. 3) 0.1 g 5-chlorothiophen-2-boronic acid + 10 g Savinase 16.0 L, Type EX: this composition had a molar ratio of I: E of approximately 40. 4) 0.1 g phenylboronic acid + 10 g Savinase 16.0 L, Type EX: this composition had a molar ratio of I: E of approximately 54. ) 0.2 g phenylbronic acid + 10 g Savinase 16.0 L, Type EX: this composition had a molar ratio of I: E of approximately 108.

It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (13)

1. A liquid composition, characterized in that it comprises: (i) an enzyme in an amount exceeding 40 μM; and (ii) a reversible inhibitor of the enzyme in an amount effective to improve the storage stability of the enzyme in a multicomponent composition in which the liquid composition is subsequently incorporated.

2. A composition according to claim 1, characterized in that the enzyme is present in an amount of from 500 uM to 5 mM.

3. A composition according to claim 1 or 2, characterized in that the molar ratio of the inhibitor to the enzyme is at least 5.

4. A composition according to any of claims 1-3, characterized in that the ratio [I] t / Ki (as defined herein) is at least 50.

5. A composition according to claim 4, characterized in that the ratio [I.t./Ka. It is in the range of 250-5000.

6. A composition according to any of the preceding claims, characterized in that the enzyme is selected from the group consisting of proteases, amylases, cellulases, lipases and oxidoreductases.

7. A composition according to any of the preceding claims, characterized in that the enzyme is a protease.

8. A composition according to claim 7, characterized in that the inhibitor is selected from the group consisting of boronic acids, borinic acids and salts thereof.

9. A process for preparing a detergent composition comprising an enzyme and an inhibitor to the enzyme, characterized in that a liquid composition according to any of claims 1-8 is combined with the remaining components of the detergent composition.

10. A process according to claim 9, characterized in that the detergent composition is a liquid composition.

11. A detergent composition, characterized in that it is prepared by a process according to claim 9 or 10.

12. The use of a liquid composition according to any of claims 1-8, in the manufacture of a laundry detergent composition in laundry or dish washing.

13. The use according to claim 12 for the manufacture of a liquid detergent composition.