CN1168155A - Stabilization of liquid enzyme compositions - Google Patents

Abstract

A liquid enzyme-containing composition, which is highly suitable for subsequent incorporation into a multi-ingredient composition, such as a laundry or dishwashing liquid detergent composition, comprising: (i) an enzyme in an amount greater than 40 μm; and (ii) A reversible inhibitor of the enzyme in an amount effective to increase the storage stability of the enzyme in the multi-ingredient composition into which the liquid composition is subsequently incorporated.

Description

Stabilization of liquid enzyme compositions

The present invention relates in particular to liquid enzyme compositions containing (i) one or several enzymes in a relatively high to very high concentration, and (ii) one or several suitable enzyme inhibitors, especially reversible inhibitors, The inhibitor has strong enzyme-inhibitory properties and/or is present at a concentration which causes a strong enzyme-inhibitory effect.

The invention also relates to:

An enzyme-containing multi-component composition, for example, a liquid composition of a cleaning composition, such as a washing composition, prepared using the inhibitor-stabilized liquid enzyme composition of the present invention as an enzyme source;

A method of preparing an enzyme-containing multi-ingredient composition such as a cleaning composition, such as a washing composition, wherein said enzyme is incorporated in the form of an inhibitor-stabilized liquid enzyme composition of the invention, i.e.: When said enzyme is incorporated into said multi-ingredient composition, it is in the form of an inhibitor-stabilized composition; and

A multi-ingredient composition, such as a cleaning composition, such as a cleaning composition, prepared by the latter method.

The invention also relates to methods of using the liquid enzyme compositions of the invention for the preparation or production of enzyme-containing multi-ingredient compositions, such as cleaning compositions, such as washing compositions.

Storage stability issues are well known problems with enzyme-containing compositions. Thus, for example, a major problem with enzyme-containing liquid detergents, such as protease (peptidase)-containing liquid detergents, is ensuring retention of appropriate enzyme activity during storage. Considerable efforts have been made to find ways to improve the storage stability of enzyme-containing compositions, such as liquid detergents, eg by adding protease inhibitors to protease-containing compositions.

As an example, the remainder of this section deals primarily with the inhibition of proteases, but the principles are equally applicable to other types of enzymes such as lipases, amylases, cellulases and oxidoreductases such as peroxidases and oxidases ), and aspects of the invention are not limited to the problems associated with stabilizing proteases with (reversible) protease inhibitors.

For example, boronic acid and monosubstituted boronic acids are known to reversibly inhibit proteases. In Molecular & Cellular Biochemistry 51, 1983, PP.5-32, the problem of inhibiting serine proteases, subtilisins with boronic acids is discussed.

The various monosubstituted boronic acids have very different abilities as subtilisin inhibitors. Only alkyl groups such as methyl, butyl or 2-cyclohexylethyl are poor inhibitors, with methylboronic acid being the worst inhibitor. However, monosubstituted boronic acids with aryl groups such as phenyl, 4-methoxyphenyl or 3,5-dichlorophenyl have been reported to be excellent inhibitors, where 3,5-dichlorophenyl Chlorophenyl monosubstituted boronic acids are a particularly effective one (see Keller et al., Biochem, Biophys. Res. Comm. 176, 1991, PP. 401-405).

It is also pointed out in WO 92/19707 that arylboronic acids substituted on the aryl group in the 3-position with respect to boron are surprisingly good reversible protease inhibitors. Thus, for example, "acetaminophen-substituted boronic acids" mentioned in the following documents are particularly effective inhibitors of proteases.

When quantifying the inhibitory effect of reversible enzyme inhibitors, the "inhibition constant" (K _i ) is usually used as an index to measure the ability to inhibit enzyme activity. Usually, K _i is defined as follows:

K _i =[E]·[l]/[El]

Wherein [E], [1] and [El] respectively represent the equilibrium concentration of the enzyme (usually molar concentration), the equilibrium concentration of the inhibitor and the equilibrium concentration of the enzyme-inhibitor complex under the conditions described. This latter definition of _Ki applies throughout the specification and claims. Therefore, lower _Ki values indicate more potent inhibitors.

When preparing or producing multi-component enzyme-containing compositions, it is desirable to use as an enzyme source a pre-prepared enzyme composition containing an enzyme and an appropriate (reversible) enzyme inhibitor. It is especially common and advantageous that pre-prepared enzyme compositions contain very high concentrations of at least one enzyme, and possibly several enzymes of the above-mentioned types, since in this way large volumes or large quantities of enzyme-containing multicomponent Compositions can be prepared using smaller volumes or smaller quantities of previously prepared enzyme/inhibitor compositions, among other advantages.

In addition, if the inhibitor of the enzyme can be added from the original pre-prepared enzyme/inhibitor composition form and the amount incorporated into the composition, it can ensure that the enzyme has the desired effect during storage of the finished multi-ingredient composition. Satisfactory stability is clearly advantageous, ie it is not necessary to contain further stabilizers in the multi-ingredient composition (for example, detergent composition) (however, other stabilizers can also be added if desired).

Also, in such pre-prepared enzyme/inhibitor compositions, the stability of the enzyme in the presence of its inhibitor is expected to be high. Accordingly, various pre-prepared compositions of this type are expected to be capable of being stored for substantial periods of time after manufacture without appreciable - or at least without unacceptable - loss of enzymatic activity.

Accordingly, the present invention relates to a liquid composition comprising: (i) an enzyme in an amount greater than 40 μM; and (ii) a reversible enzyme inhibitor in an amount such that when the liquid composition is subsequently incorporated into a The storage stability of the enzyme is effectively improved in multi-ingredient compositions (eg detergent compositions, like liquid detergent compositions).

For example, the liquid compositions of the present invention may be predominantly aqueous compositions, or predominantly anhydrous compositions (e.g., compositions containing a high proportion of one or more anhydrous, but generally water-miscible solvents , the solvent is generally an organic liquid, such as alcohol or glycol, etc.). The liquid compositions of the present invention are typically non-washing compositions themselves.

In addition to cleaning compositions (such as laundry and dishwashing compositions), other types of enzyme-containing multi-ingredient compositions in the form of liquid compositions of relevance to the context of the present invention include: for cleaning teeth, contact lenses or hard surfaces ( Compositions such as slaughterhouses or food processing plants); compositions for the leather industry (such as for dehairing and/or degreasing of animal hides); compositions for desizing fabrics; and for washing denim fabrics Composition to obtain a "stonewashed" look. The use of such compositions for such purposes falls within the scope of the present invention.

enzyme

The enzyme classification number (EC number) adopted in the description of the present invention and the claims conforms to the nomenclature recommended by the International Biochemistry and Molecular Biology Committee (Recommendation (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press Inc., 1992).

The liquid compositions of the present invention contain at least one enzyme. Suitable enzymes include all commercially available enzymes. Particularly relevant enzymes include those selected from proteases (i.e. peptidases, EC3.4), amylases (classified under EC3.2.1), fatty acids (including enzymes under EC3.1.1.3), cellulases (including EC3. 2.1.4) and oxidoreductases (EC1), such as peroxidases (EC1.11) and oxidases [including enzymes below EC1.10.3, such as laccases (EC1.10.3.2)], and the above Any composition of enzymes. Also included are mixtures of enzymes from the same class (eg different proteases, mixtures of different lipases, etc.).

The amount of enzyme in the liquid composition will vary depending on the type of enzyme and the future use of the liquid composition. Generally, it is advisable that all enzymes are used in an amount of 0.2-50% (W/W) by weight of the liquid composition (calculated based on pure enzyme protein), usually 0.5-25% W/W, such as 1- 10% W/W, eg, 2-8% (W/W) of the liquid composition. Expressed in concentration units, the preferred concentration range of the enzyme in the liquid composition of the present invention is about 50 μM to about 20 mM, usually 100 μM to 10 mM, such as 500 μM to 5 mM, for example, 750 μM to 3 mM (based on moles of pure enzyme protein).

Protease. Any protease (proteolytic enzyme) suitable for use in liquid compositions can be used. Suitable proteases include proteases of animal, vegetable or microbial origin, especially of microbial origin and chemically produced or protein engineered (genetic engineered) mutants (variants) relative to the amino acid sequence of an enzyme of this type In other words, the mutant has one or several amino acid substitutions, insertions and/or deletions, and has proteolytic activity. For example, the protease may be a serine peptidase, preferably alkaline microbial protease or tryptase. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (disclosed in WO 89/06279 middle). Examples of commercial Bacillus subtilisins are the Alcalase ^™ , Savinase ^™ , Esperase ^™ and Durazym ^™ preparations, all commercially available from Novo Nordisk A/S. Several of the above-mentioned protease preparations such as Alcalase ^™ , Esperase ^™ and Savinase ^™ are available in liquid form (e.g., Savinase ^™ 16.0L, Type DX and Type EX), which forms are very suitable (see below) for preparing the protease-containing liquid combinations of the present invention things.

Examples of trypsin-like proteases include trypsin (eg, of porcine or bovine origin) and the Fusarium protease disclosed in WO 89/06270.

Amylase. Any amylase (amylolytic enzyme) suitable for use in liquid compositions can be used. Suitable amylases include those of bacterial and fungal origin, as well as chemically produced or protein engineered (genetic engineered) mutants (variants) which, compared to the amino acid sequence of an enzyme of this type, have One or several amino acids are substituted, inserted and/or deleted, and have amylolytic activity. For example, amylases include alpha-amylases (EC 3.2.1.1), such as that obtained from a strain of B. licheniformis, described in British Patent Specification No. 1,296,839. A very suitable alpha-amylase is Termamyl( ^TM) , which is commercially available (especially in liquid form) from Novo Nordisk A/S.

Lipase. Any lipase suitable for use in liquid compositions can be used. Suitable lipases include those of bacterial and fungal origin, as well as chemically produced or protein engineered (genetic engineered) mutants (variants) which, relative to the amino acid sequence of an enzyme of this type, One or several amino acids are substituted, inserted and/or deleted in the body. An extremely useful lipase was obtained by cloning a gene from Humicola lanuginosa and expressing it in Aspergillus oryzae, as described in EP 0258 068, Such an enzyme (in particular a liquid product) is available from Novo Nordisk A/S under the trade name Lipolase ^(TM) .

Cellulase. Any cellulase (cellulolytic enzyme) suitable for use in liquid compositions can be used. Suitable cellulases include cellulases of bacterial and fungal origin, as well as chemically produced or protein engineered (genetic engineered) mutants (variants), relative to the amino acid sequence of an enzyme of this type, One or several amino acids in the mutant are substituted, inserted and/or deleted, and have cellulolytic activity. For example, suitable cellulases are disclosed in US 4,435,307. A very suitable cellulase is produced by a strain of Humicolainsolens, commercially available from Novo Nordisk A/S under the trade name Celluzyme ^(TM) .

peroxidase. Any peroxidase enzyme suitable for use in liquid compositions, such as liquid detergent compositions, may be used. Here, suitable peroxidases include peroxidases of plant, bacterial and fungal origin, as well as chemically produced or protein-engineered (genetic engineered) mutants (variants), relative to an enzyme of this type In terms of the amino acid sequence of the mutant, one or several amino acids are substituted, inserted and/or deleted in the mutant, and have peroxidase activity. Examples of suitable peroxidases are those derived from a strain of Coprinus (such as C. cinerius or C. macrorhizus) or from a strain of Bacillus (brevius). A peroxidase of a strain of Bacillus (B. pumilus), in particular a peroxidase as described in PCT/DK90/00260.

oxidase. Any oxidase enzyme suitable for use in liquid compositions, such as liquid detergent compositions, can be used. Here, suitable oxidases include oxidases derived from bacteria and fungi, as well as chemically produced or protein engineered (genetic engineered) mutants (variants), relative to the amino acid sequence of an enzyme of this type, One or several amino acids on the mutant are substituted, inserted and/or deleted, and have oxidase activity. Examples of suitable oxidases are enzymes from Aspergillus, Neurospora (e.g. Neurospore crassa), Trametes (e.g. Tramete villosa) or Myceliophthora (such as the laccase of Myceliophthora thermophila (M.thermophila)).

Enzyme Inhibitors (Enzyme Stabilizers)

The liquid compositions of the present invention contain at least one reversible enzyme inhibitor. Typically, this inhibitor is an inhibitor of at least one enzyme present in the liquid composition. Thus, for example, the liquid composition of the present invention contains, in addition to a first type of enzyme and a reversible inhibitor of this enzyme, a second, third, ..., etc. type of enzyme (such as the above-mentioned type one of the enzymes), one or several of which are not accompanied by their corresponding reversible inhibitors.

It should be pointed out here that it is entirely feasible to prepare a liquid enzyme composition containing one or several enzymes, the dosage of which is as described herein, but the composition does not contain the above-mentioned one or several enzymes inhibiting Instead, the liquid composition contains an inhibitor of another type of enzyme to which the liquid composition will subsequently be contacted. An example of this is a liquid composition containing lipase as the only enzyme and a protease inhibitor; the protease inhibitor protects the lipase from degradation by proteases to which it is subsequently exposed, intentionally or unintentionally.

The nature and amount of inhibitors used depend inter alia on the nature and concentration of the enzymes present in the composition and on the envisaged use of the composition. This will be further explained below.

For reversible inhibitors (and their associated _Ki values) that can be used with the various classes/types of enzymes in the liquid compositions of the invention, reference can be made to the following literature: H. Zollner, Handbook of Enzyme Inhibitors (Parts A and B Part), 2nd edition, VCH Verlagsgesellschaft mbH, Weinheim, Germany, 1993, which lists a large number of inhibitors. Other relevant literature includes: S. Patkar and F. Bjokling, Lipase inhibitors, see: Lipases-their Structure, Biochemistry and Application, by P. Woolley and SBPetersen, Cambridge University Press, Cambridge 1994. Selected examples of relevant types of reversible inhibitors are as follows:

Protease inhibitor. One class of reversible protease inhibitors of particular interest consists of monosubstituted boronic acids [R′B(OH) ₂ ] and disubstituted boronic acids [R′R″B(OH)] (where R′ and R″ is an organic substituent, such as an optionally substituted aryl or heterocyclic substituent), some examples of which have been mentioned above. Further examples of compounds of this type may be selected from the compounds mentioned in WO 92/19707, EP0478050A1 and EP0511456A1.

Such compounds of interest may be found in the compounds disclosed in WO 95/02046 (which patent was not published at the priority date of this application), and include compounds of the following general formula:

Among them, R ₁ is an optionally substituted fused aromatic ring structure containing 14 or 18 carbon atoms on the ring; or an optionally substituted monocyclic or fused aromatic heterocyclic structure on the ring Containing up to 17 carbon atoms; or an optionally substituted monocyclic or fused quinoid ring structure containing up to 18 carbon atoms in the ring;

The structural formula of _R2 is:

wherein X is the same or different and is selected from hydrogen, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, aryl, substituted aryl, hydroxyl, hydroxy derivatives, halogen, amine, alkylation Amines, derivatives of amines, nitro groups, thiols, thiol derivatives, aldehydes, acids, acid salts, esters, sulfonates or phosphates, while o, p and q may be the same or different and may each be 0, 1 or 2; m and n may be the same or different, and may each be 0 or 1; _R3 and _R1 are the same or different, and may be selected from _R1 , or _R3 is hydroxyl, or _R1 and _R3 All are optionally substituted monocyclic or bicyclic aromatic ring structures.

In the above, the optionally substituted ring structure is such that the substituents on the ring structure can be chosen at will, but are preferably selected from hydrogen, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, Aryl, substituted aryl, hydroxyl, hydroxyl derivatives, halogens, amines, alkylated amines, amine derivatives, nitro, thiol, thiol derivatives, aldehydes, acids, acid salts, esters, sulfonic acids esters and phosphates.

Monosubstituted boronic acid and disubstituted boronic acid derivatives can be prepared by methods well known to those skilled in the art, for example, by one of the following methods:

a) using catechol borane (1,3,2-benzodioxaborole) or dichloroborane-dimethyl sulfide complex as the hydroboration reaction agent for unsaturated materials, i.e. olefins and alkynes; See H.C. Brown, S.K. Gupta, JACS 97, 1975, pp. 5249-5255 and H. C. Brown, N. Ravindran, S. U. Kuikarni, J. Org. Chem. 45, (1980), p. 384.

b) Reaction of Grignard's reagent with tri-n-butyl borate or trimethyl borate followed by hydrolysis of the resulting monosubstituted boronic ester; see FRBean, JR Johnson, Jaces 54, 1932, pp 4415-4425; and SHDandegaonher, SPIngleshwar , Journal of Shivasi University 6, 1932, pp.11-13. Bromine-substituted starting materials are not commercially available and can be conveniently synthesized in two steps: reduction of the corresponding carboxylic acid with LiAlH ₄ followed by treatment with CBr ₄ .

c) Reaction of organolithium reagents with butyl borate; see S.O.Lauessen, pp.387-395, Thiophene Chemistry, Part 7; and D.Florentin, B.Roques, C.R.Acad.Sci.Paris, t.270 (1970 5 11), PP.1608-1610.

d) Prepare disubstituted boronic acid derivatives according to the method in b above, except that the ratio of Grignard reagent to boric acid ester used is 2:1.

e) Protection of ring substitutions or functional groups can be achieved using standard methods well known to those skilled in the art.

Other compounds of interest of this type may be selected from the naphthalene-substituted boronic acids disclosed in WO 95/29223 (unpublished at the priority date of this application), and include compounds of the general formula:

Wherein, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are the same or different, and are selected from hydrogen, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, aromatic group, substituted aryl group, hydroxyl group, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, nitro group, thio group, thio group derivative, aldehyde, acid, acid salt, ester, sulfonate and phosphates.

Naphthalene-substituted boronic acid derivatives can also be prepared by methods known to those skilled in the art, for example, using the Grignard formulation as follows:

Grignard's reagent was prepared by slowly adding the appropriate bromonaphthalene starting material dissolved in sodium-dried ether dropwise to magnesium rotating in sodium-dried ether. Add small iodine crystals to promote the above reaction.

Cool trimethyl borate or tri-n-butyl borate dissolved in sodium-dried ether to -70°C, and add Grignard reagent dropwise to the above solution over 2 hours while keeping the organoboron solution at -70°C , and keep stirring. The reaction mixture was allowed to warm to room temperature overnight while hydrolysis was carried out by dropwise addition of cold dilute sulfuric acid. The ether layer was separated, and the aqueous layer was extracted with ether. The ether fractions were combined and the solvent was removed. The residue was made strongly basic and any methanol or butanol thus produced was removed. The alkaline solution was cooled, and the resulting crystals of the desired monosubstituted boric acid were removed by filtration. All products are preferably recrystallized from distilled water.

Naphthalene-substituted boronic acids can be prepared by direct lithiation of naphthalene and/or lithiation of bromide.

Any ring substitution or protection of functional groups can be accomplished using methods familiar to those skilled in the art.

Among the above-mentioned types of compounds, the specific monosubstituted boronic acid involved in the content of the present invention includes: benzofuran-2-substituted boronic acid; phenyl monosubstituted boronic acid; 4-bromophenyl monosubstituted boronic acid; -Formylphenyl-monosubstituted boronic acid; 3-acetamidophenyl-monosubstituted boronic acid; 3,5-dichlorophenyl-monosubstituted boronic acid; 5-chlorothiobenzene-2-monosubstituted boronic acid; naphthalene-1-substituted boronic acids; naphthalene-2-monosubstituted boronic acids; and 6-hydroxynaphthalene-2-monosubstituted boronic acids. In the table given in Example 2 herein (see below), other specific compounds of this type are listed which are useful in the present invention.

It can be used in its acid form (e.g. in a suitable water-miscible organic solvent, such as monopropylene glycol) or, if necessary (e.g. for reasons of solubility) in the form of a salt (e.g. an alkali metal salt, e.g. sodium or potassium salts), monosubstituted boronic acid or disubstituted boronic acid type inhibitors are added or incorporated into the liquid compositions of the present invention.

Reversible amylase inhibitors. Related reversible amylase inhibitors useful in the present invention can be found in compounds of the so-called "acarbose" type (ie: pseudooligosaccharides containing an acarviosine moiety and one or several maltose units) . Other compounds which are inhibitors, especially alpha-amylase inhibitors, include maltose and maltotriose. Other compounds such as methyl-α-glucoside and cyclized starches such as cyclohexane and/or cycloheptane amylose can be used as reversible inhibitors of β-amylases.

Reversible cellulase inhibitors. Potential reversible cellulase inhibitors useful in the present invention include 4-mercapto-cellooligosaccharides [eg, see C. Schou et al., Journal of Carbohydrate Chemistry 12 (1993) pp. 743-752].

Reversible lipase inhibitors. The reversible lipase inhibitors that can be used in the present invention include monosubstituted boronic acids and disubstituted boronic acids (such as optionally substituted phenyl monosubstituted boronic acids) selected from the above and/or below.

degree of enzyme inhibition

The desired degree of inhibition (strength, degree) of enzymes present in the liquid compositions of the invention depends, inter alia, on the purpose for which the composition is to be used. In general, the molar ratio of reversible inhibitor to enzyme is chosen such that there is at least one molecule of inhibitor per active site of the enzyme in question, thus a logical ratio of inhibitor (I) to enzyme (E) The molar ratio is at least 1:1. However, lower I:E molar ratios such as about 0.8:1, about 0.7:1, about 0.6:1, or even about 0.5:1 may also be used for certain purposes. However, in the present invention generally a molar ratio of inhibitor to enzyme of at least 5, such as at least 15, is employed. In some cases, the I:E molar ratio may be at least 50 or at least 100, with even higher molar ratios employed.

The molar ratio of inhibitor to enzyme can be chosen, in particular taking into account the percentage of free (uninhibited) enzyme required for practical use of the multi-component enzyme-containing composition based on the liquid composition of the invention. Thus, for example, when a laundry detergent composition is used for laundry, it is obviously necessary to have a sufficiently high level of free enzymes (such as proteases or lipases) in the wash medium (usually water to which the relevant detergent composition is added) ).

An important parameter in this regard is the inhibition constant (defined above) for reversible inhibition of an enzyme. Thus, for example, when the liquid composition of the invention is used in a detergent composition, the inhibition constant (K _i ) of a reversible inhibitor of an enzyme, when expressed in the conventional manner in mol/l (M), is usually Preferably, 3×10 ^-8 M≤K _i ≤1.2×10 ^-2 M, such as 3×10 ^-8 M≤K _i ≤1×10 ^-2 M. However, a more ideal "window of inhibition" for washing enzymes is usually such that an inhibitor associated with a specific enzyme produces the equivalent of 3×10 ^-7 M≤K _i ≤1×10 ^{- 3} M suppression degree, such as 4.3×10 ^-7 M≤K _i ≤4.5×10 ^-4 M.

In general, preferred enzyme-containing liquid compositions of the present invention are:

(a) the total molar concentration of reversible enzyme inhibitor I present in the composition (may be expressed as [I] _t ); and

(b) The ratio between the inhibition constants (K _i ; expressed in mol/l) of the enzymes present in the composition which can be reversibly inhibited by the aforementioned inhibitors is at least 50 (i.e. [I] _t /K _i ≥ 50), such as at least 100, or usually at least 250. In the liquid compositions of the present invention, the practical upper limit of the ratio [I] _t /K _i is usually about 10000 (ie 10 ⁴ ). In many cases, the [I] _t /K _i ratio is in the range of 250-5000, usually in the range of 250-2500.

The value of the [I] _t / _Ki ratio can be considered as an index of the "inhibitory ability" of the liquid composition of the present invention. For example, high ratios, ie high "inhibitory power", can be obtained by adding moderate to low concentrations of strong inhibitors, or higher concentrations of weaker inhibitors, to the liquid composition.

When the number of moles of inhibitor I exceeds the number of moles of inhibited enzyme E, most of the inhibitor is usually in free form, ie: not bound to the enzyme. By [I] denoting the free (unbound) inhibitor (relative to K _i ; see above), under such conditions the [I] _t / _Ki ratio is approximately equal to the [I]/ _Ki ratio, i.e. [I] _t /K _i ≈[I]/K _i .

It should be pointed out here that the liquid composition containing the enzyme and the reversible enzyme inhibitor of the present invention can be dried by a suitable method (such as freeze-drying or spray-drying). The dried enzyme/inhibitor product may then be comminuted (e.g. by grinding) and suspended or slurried at an appropriate concentration in a non-aqueous liquid vehicle such as a non-ionic surfactant (e.g. Softanol ^™ from BP) , to form a slurry product.

detergent

For detergent compositions, it is generally aimed to achieve at least 50% inhibition of selected enzymes (eg proteases) in the detergent composition, and the amount of free enzyme in the wash medium corresponds to at least 50% of the total enzyme amount.

Cleaning compositions incorporating the liquid compositions of the present invention comprise, in addition to enzymes and inhibitors, surfactants and are typically liquid cleaning compositions. For example, the detergent composition may be a laundry detergent composition or a dishwashing detergent composition.

Liquid detergent compositions may be aqueous, eg typically containing up to 70% water and 0-30% organic solvent, or substantially anhydrous.

The cleaning composition will contain one or more surfactants, each of which may be anionic, nonionic, cationic or zwitterionic. The detergent usually contains 0-50% of anionic surfactants, such as linear alkylbenzene sulfonate (LAS), alpha-olefin sulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) ( AS), fatty alcohol ethoxysulfates (AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl or alkenyl succinic acids or soaps. Can also contain 0-40% nonionic surfactants such as fatty alcohol ethoxylates (AEO or AE), fatty alcohol propoxylates, carboxylated fatty alcohol ethoxylates, nonylphenol ethoxylates compounds, alkyl polyglycosides, alkyl dimethylamine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides, or polyhydroxyalkyl fatty acid amides (for example, as described in WO 92/06154).

Typically, the detergent contains 1-65% detergency builders, but some dishwashing detergents can even contain up to 90% detergency builders or complexing agents such as zeolites, diphosphates, triphosphates, phosphine salts, citrates, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl or alkenyl succinic acids, soluble silicates or Layered silicates (eg SKS-6 from Hoechst).

Detergent builders can be classified into phosphorus-containing types and non-phosphorus-containing types. Examples of phosphorus-containing inorganic alkaline detergent builders include water-soluble salts, especially alkali metal pyrophosphates, orthophosphates, polyphosphates and phosphonates. Examples of non-phosphorus-containing inorganic builders include water-soluble alkali metal carbonates, borates and silicates, as well as layered dimer silicates and various types of water-insoluble crystalline or amorphous aluminosilicates, Among them, zeolite is the most famous representative.

Examples of suitable organic builders include alkali metal, ammonium or substituted ammonium salts of the following acids: succinic acid, malonic acid, fatty acid malonic acid, fatty acid sulfonic acid, carboxymethoxysuccinic acid, polyacetic acid, carboxylic acid, Polycarboxylates, aminopolycarboxylates and polyacetylcarboxylates. The detergent may also be unformed, ie substantially free of detergent builders.

The detergent may contain one or several polymers. Examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polycarboxylates such as polyacrylates, polymaleic acid esters, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent compositions may contain chlorine/bromine or oxygenated bleaches. The bleaching agent can be coated or encapsulated. Examples of inorganic chlorine/bromine type bleaches are the lithium, sodium or calcium salts of hypochlorous or hypobromous acid, and chlorinated trisodium phosphate.

The bleach system may also contain _{a H2O2} source such as perboron which can be mixed with a peracid generating bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS) salt or percarbonate.

Examples of organochlorine/bromine type bleaches are heterocyclic N-bromo and N-chloroimides such as trichloroisocyanuric acid, tribromoisocyanuric acid, dibromoisocyanuric acid and dichloroisocyanuric acid, and Salts with water-soluble cations such as potassium and sodium. Hydantoin is also suitable. The bleaching system may also contain peroxyacids such as amides, imides or sulfones.

In dishwashing detergents, oxygen-containing bleaches are preferred, such as those of the inorganic persalt type, preferably with bleach precursors or as peroxyacid compounds. Typical examples of peroxygen bleaching compounds are the tetrahydrates and monohydrates of the alkali metal perborates, the alkali metal percarbonates, persilicates and perphosphates. Preferred active materials are TAED or NOBS.

As stated above, the enzymes of the detergent compositions of the present invention are incorporated into said detergent compositions in the form of liquid enzyme/inhibitor compositions of the present invention.

If necessary, other common enzyme stabilizing substances can also be added, such as polyols, like propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, such as aromatic borate esters (see, for example, WO 92/19709 and 92/19708).

The detergent may also contain other common detergent ingredients such as fabric conditioners including clays, deflocculating materials, suds boosters/suds suppressors (in dishwashing detergents), suds suppressors, corrosion inhibitors , soil suspending agents, anti-soil redeposition agents, dyes, dehydrating agents, bactericides, optical brighteners or fragrances.

Its pH (measured in aqueous solution at the concentration used) is generally neutral or basic, such as in the range of 7-11.

Specific forms of laundry detergent compositions within the scope of the present invention include:

1) An aqueous liquid detergent composition comprising: Linear alkylbenzene sulfonate (calculated as acid) 15-21% Fatty alcohol ethoxylates (such as C _12-15 fatty alcohol, 7EO or C _12-15 fatty alcohol, 5EO) 12-18% Soaps, such as fatty acids (such as oleic acid) 3-13% Alkenylsuccinic acid (C _12-14 ) 0-13% aminoethanol 8-18% citric acid 2-8% Phosphonate 0-3% Polymers (such as PVP, PEG) 0-3% Borate (such as B ₄ O ₇ ) 0-2% ethanol 0-3% Propylene Glycol 8-14% Enzymes (calculated as pure enzyme protein) 0.0001-0.2% Minor ingredients (e.g. dispersants, foam suppressors, fragrances, optical brighteners) 0-5%

2) A liquid detergent composition of water structure, comprising: Linear alkylbenzene sulfonate (as acid) 15-21% Fatty alcohol ethoxylates (such as C _12-15 fatty alcohol, 7EO, or C _12-15 fatty alcohol, 5EO) 3-9% Soaps, such as fatty acids (such as oleic acid) 3-10% Zeolite (such as NaAlSiO ₄ ) 14-22% potassium citrate 9-18% Borate (such as B ₄ O ₇ ^2- ) 0-2% carboxymethyl cellulose 0-2% Polymers (e.g. PEG, PVD) 0-3% Anchoring polymer such as lauryl methacrylate/acrylic acid copolymer; molar ratio 25:1; MW3800 0-3% glycerin 0-5% Enzymes (calculated as pure enzyme protein) 0.0001-0.2% Minor ingredients (e.g. dispersants, foam suppressors, fragrances, optical brighteners) 0-5%

3. An aqueous liquid detergent composition comprising: Linear alkylbenzene sulfonate (as acid) 15-23% Fatty alcohol ethoxysulfate (such as C _12-15 fatty alcohol, 2-3EO) 8-15% Fatty alcohol ethoxylates (such as C _12-15 fatty alcohol, 7EO, or C _12-15 fatty alcohol, 5EO) 3-9% Soaps, such as fatty acids (such as lauryl acid) 0-3% aminoethanol 1-5% Sodium citrate 5-10% Hydrotropes (eg, sodium toluenesulfonate) 2-6% Borate (such as B ₄ O ₇ ^2- ) 0-2% carboxymethyl cellulose 0-1% ethanol 1-3% Propylene Glycol 2-5% Enzymes (calculated as pure enzyme protein) 0.0001-0.2% Minor ingredients (e.g. polymers, dispersants, fragrances, optical brighteners) 0-5%

4. An aqueous liquid detergent composition comprising: Linear alkylbenzene sulfonate (as acid) 20-32% Fatty alcohol ethoxylates (such as C _12-15 fatty alcohol, 7EO, or C _12-15 fatty alcohol, 5EO) 6-12% aminoethanol 2-6% citric acid 8-14% Borate (such as B ₄ O ₇ ^2- ) 1-3% Polymers (e.g. maleic/acrylic acid copolymers, anchoring polymers such as lauryl methacrylate/acrylic acid copolymers) 0-3% glycerin 3-8% Enzymes (calculated as pure enzyme protein) 0.0001-0.2% Minor ingredients (e.g. hydrotropes, dispersants, fragrances, optical brighteners) 0-5%

5) The cleaning composition as described in 1)-4), wherein all or part of the linear alkylbenzene sulfonate is replaced by (C ₁₂ -C ₁₈ ) alkyl sulfate.

6) Washing compositions according to 1) to 5), which contain stabilized or coated peracids, either as an additional ingredient or in place of the above-mentioned bleaching systems.

7) A detergent composition formulated as an anhydrous wash liquor comprising a liquid nonionic surfactant such as linear alkoxylated primary alcohol, a builder system such as phosphate, enzymes and alkali. The detergent may also contain anionic surfactants and/or bleaching systems.

Dishwashing and other multicomponent enzyme-containing compositions

Relevant examples of compositions of this type include:

1) A liquid dishwashing composition having a detersive surfactant system comprising: nonionic surfactant 0-1.5% Octadecyl N-Dimethylamine Oxide Dihydrate 0-5% 80:20 wt. C18/C16 mixture of octadecyl dimethylamine N-oxide dihydrate and hexadecyl dimethylamine N-oxide dihydrate 0-4% 70:30 wt. C18/C16 mixture of anhydrous octadecyl N-oxide bis(hydroxyethyl)amine and anhydrous hexadecyl N-oxide bis(hydroxyethyl)amine 0-5% C ₁₃ -C ₁₅ alkyl ethoxy sulfates with an average degree of ethoxylation of 3 0-10% C ₁₂ -C ₁₅ alkyl ethoxy sulfates with an average degree of ethoxylation of 3 0-5% C ₁₃ -C ₁₅ ethoxylated fatty alcohols with an average degree of ethoxylation of 12 0-5% C ₁₂ -C ₁₅ ethoxylated fatty alcohol mixtures with an average degree of ethoxylation of 9 0-6.5% C ₁₃ -C ₁₅ ethoxylated fatty alcohol mixture with an average degree of ethoxylation of 30 0-4% Sodium disilicate 0-33% sodium triphosphate 0-46% Citrate 0-28% citric acid 0-29% Sodium carbonate 0-20% Sodium perborate monohydrate 0-11.5% Tetraacetylethylamine (TAED) 0-4% Maleic acid/acrylic acid copolymer 0-7.5% sodium sulfate 0-12.5% enzyme 0.0001 -0.2%

2) Anhydrous liquid automatic dishwashing compositions comprising: Liquid nonionic surfactants (e.g. fatty alcohol ethoxylates) 2.0 -10.0% Alkali metal silicate 3.0 -15.0% Alkali metal phosphate 20.0-40.0% Liquid carrier selected from higher glycols, polyethylene glycols, polyoxides, glycol ethers 25.0-45.0% Stabilizers (such as partial esters of phosphoric acid and C ₁₆ -C ₁₈ alkanols) 0.5 -7.0% Foam suppressors (such as silicones) 0-1.5% enzyme 0.0001 -0.2%

3) An anhydrous liquid dishwashing composition comprising: Liquid nonionic surfactants (e.g. fatty alcohol ethoxylates) 2.0 -10.0% Sodium silicate 3.0 -15.0% Alkali metal carbonate 7.0 -20.0% Sodium citrate 0.0 -1.5% Stabilizing systems (e.g. mixtures of finely divided siloxanes with low molecular weight dialkyl polyglycol ethers) 0.5 -7.0% Low molecular weight polyacrylic acid polymer 5.0 -15.0% Clay gel thickener (such as bentonite) 0.0 -10.0% Hydroxypropyl Cellulose Polymer 0.0 -0.6% enzyme 0.0001 -0.2% Liquid carrier selected from higher glycols, polyethylene glycols, polyoxides and glycol ethers balance

4) Thixotropic liquid automatic dishwashing compositions, comprising: C ₁₂ -C ₁₄ fatty acids 0-0.5% block copolymer surfactant 1.5 -15.0% Sodium citrate 0-12% sodium triphosphate 0-15% Sodium carbonate 0-8% Aluminum tristearate 0 -0.1% Sodium cumene sulfonate 0-1.7% polyacrylic acid thickener 1.32 -2.5% Sodium polyacrylate 2.4-6.0% boric acid 0-4.0% sodium formate 0-0.45% calcium formate 0-0.2% Oxidized sodium n-decyl diphenyl disulfonate 0-4.0% Monoethanolamine (MEA) 0 -1.86% Sodium Hydroxide (50%) 1.9 -9.3% 1,2-propanediol 0-9.4% enzyme 0.0001 -0.2% Foam suppressor, dye, fragrance, water balance

5) Liquid automatic dishwashing compositions comprising: Fatty Alcohol Ethoxylates 0-20% Sulphonic fatty acid ester 0-30% Sodium dodecyl sulfate 0-20% Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate 18-33% Sodium citrate dihydrate 18-33% sodium stearate 0-2.5% Sodium perborate monohydrate 0-13% Tetraacetylethylenediamine (TAED) 0-8% Maleic acid/acrylic acid copolymer 4-8% enzyme 0.0001 -0.2%

6) A liquid automatic dishwashing composition containing bleach-protecting granules, comprising: Sodium silicate 5-10% Tetrapotassium pyrophosphate 15-25% sodium triphosphate 0-2% potassium carbonate 4-8% Protects against bleaching particles such as chlorine 5-10% polymeric thickener 0.7 -1.5% Potassium hydroxide 0-2% enzyme 0.0001 -0.2% water balance

7) The automatic dishwashing composition according to 1) and 5), wherein perboric acid is replaced by percarbonic acid.

8) The automatic dishwashing composition of 1), which further contains a manganese catalyst. For example, the manganese catalyst may be one of the compounds disclosed in "Efficient manganese catalysts for low-temperature bleaching" (Nature 369, 1994, pp. 637-639).

The "enzyme" content of the above compositions may include the level of enzyme inhibitors present. A particular composition belonging to one of the above-mentioned types may, if necessary, contain an organic solvent not formulated for the above-mentioned type, but which may be added as part of the liquid enzyme/inhibitor composition of the invention to said particular composition. Inhibitor solvent.

The liquid enzyme/inhibitor compositions of the present invention can be added to detergent compositions to produce an enzyme concentration commonly used in detergents. For the cleaning compositions of the present invention, the liquid enzyme/inhibitor compositions of the present invention may be added, for example, in an amount equivalent to 1 liter of wash relative to the particular enzyme present in the enzyme/inhibitor composition. There are 0.00001-1mg (calculated as pure enzyme protein) enzyme in the solution.

Stabilizer inspection

Inhibitor inhibition can be tested in various ways, illustrated by the following two methods for testing proteases/protease inhibitors:

a) Storage Stability Test in Liquid Detergents: The liquid enzyme/inhibitor composition was added to a liquid detergent composition stored under well-defined conditions. The enzymatic activity of each enzyme was measured as a function of time (eg, after 1, 3 and 7 days).

In order to calculate the inhibition efficiency from the storage stability data, a reaction mechanism was proposed. The following reaction proposes a simpler, rational mechanism for liquid detergents containing protease (P), lipase (L) and inhibitor (I):

1) Autodigestion by proteases:

P+P→D _P +P

II) Denaturation of protease:

P→D _P

III) Inhibition of proteases:

P+I PI

IV) Protease Digestion of Inhibited Enzymes

P+PI→P+D _P +I

V) Denaturation of inhibited enzymes:

PI→D _P +I

VI) Protease digestion with lipase:

P+L→P+D _L

VII) Denaturation of lipase

L→D _L

Among them, _DP and _DL are denatured (ie inactive) protease and lipase.

From the above reactions, three coupled differential equations representing the inactivation of D, L and PI were obtained. Reaction rate constants were calculated from storage stability data using a parameter estimation method (Gauss Newton method modified by Levenberg). Storage stability data are presented as a function of time for (P+PI) and L concentration.

Reaction III was much faster than the other reactions and equilibrium was assumed in the calculations. Reaction IV was excluded from this reaction to reduce the number of parameters so that only one reaction rate constant (obtained from Equation V) describes the stability of the inhibited enzyme.

When there is a large amount of remaining inhibitor molecules (relative to protease molecules), the concentration of inhibitor can be assumed to be close to a reasonable constant.

The specific value of the reaction rate constant is somewhat sensitive to small variations in the data, but this sensitivity is significantly reduced relative to the results for boric acid. The use of boric acid (and closely related substances or equivalents such as alkali metal borates (e.g. borax) and boron oxide) for inhibition purposes, especially for the inhibition of proteases is well known (see for example EP 0451 924 A2), but this The low inhibitory strength and overall rather poor solubility of these species (which are further illustrated in the following examples in connection with boric acid; see below) make them essentially unsuitable for use in the present invention (wherein the present invention The inhibitor and/or the amount thereof in the liquid composition of the invention should be such that when added to said liquid composition as an ingredient of a multi-ingredient composition, good enzyme stability is still produced). Therefore, compounds such as boric acid, alkali metal borates (such as borax) and boron oxide are essentially ineffective as inhibitors in the present invention.

The definition of "improvement factor" is as follows:

Defining IFi in the manner described above provides a parameter to measure the inhibitory effect of a particular inhibitor relative to boronic acid.

b) Determination of K _i : The inhibition constant K _i can be determined using standard methods; see Keller et al., Biochem. Biophys. Res. Comm. 176, 1991, pp. 401-405; PP.463-469, Springer-Verlag, 1974, and Lone Kierstein Hansen, "Determination of Specific Activities of Selected Detergent Protease Using Protease Activity, Molecular Weight, Kinetic Parameters and Inhibition Kinetics", PhD thesis, Novo Novdisk A/S and University of Copenhagen , 1991. The assay can be performed in the presence of non-enzymatic components of the detergent composition (surfactants, etc.), if desired.

Conditions suitable for determining _Ki values for enzyme inhibition are generally obtained using a buffer system that produces a suitable pH and that does not react or form complexes with the enzyme or inhibitor of interest. For example, a buffer system for many enzyme/inhibitor interactions is a glycyl-glycine buffer at a slightly alkaline pH, such as pH 8.6, at ambient temperature (typically 25°C).

Experimental part

The following examples will further illustrate and verify the present invention, but these examples are not intended to limit the scope of the present invention as described in the claims from any angle.

example 1

The following method is exemplary of a suitable method for the synthesis of various types of mono- and di-substituted boronic acids:

Preparation of thiophene-3-monosubstituted boronic acids: 3-bromothiophene (0.043m) dissolved in sodium-dried ether (100ml) was cooled to -60°C. Lithium butyl (30ml, concentration 1M) was added rapidly. The mixture was then stirred for 3 minutes before addition of tri-n-butyl borate (0.043m) or trimethylborate (0.043m) dissolved in sodium-dried ether (25ml). The mixture was stirred for 4 hours and allowed to warm to room temperature. The reaction mixture was then treated with hydrochloric acid (1M) and the ether layer was separated. The aqueous layer was extracted with ether (2 times, 25ml). The combined ether layers were extracted with sodium hydroxide (1M). The basic solution was acidified with hydrochloric acid (10%), thereby precipitating the desired monosubstituted boronic acid. A substituted boronic acid was isolated, then recrystallized from water/ethanol and allowed to air dry.

_{C4H5BO2S ,} mpt _. 163-164°C.

Preparation of Diphenyl Disubstituted Boronic Acids: This material was prepared as described above. Preparation of Grignard's reagent from bromobenzene and magnesium fragments. However, 2 moles of Grignard's reagent were used per mole of tri-n-butyl borate. The disubstituted boronic acid thus formed is isolated by reaction with ethanolamine, thereby yielding the diphenyldisubstituted boronic acid and ethanolamine complex ((C ₆ -H ₅ ) ₂ BO·CH ₂ CH ₂ NH ₂ ), which The product is easier to handle. Mpt.192-194°C.

Preparation of 4-formylphenyl monosubstituted boronic acid: This compound can be prepared by the method disclosed in Chem. Ber. 123 (1990), PP. 1841-1843. 4-Bromobenzaldehyde diethyl acetal was reacted with magnesium fragments in tetrahydrofuran, then tributyl borate in ether was added and the mixture was treated with sulfuric acid.

Example 2

Determination of K _i

The inhibition constant K _i of protease Savinase ^™ was determined under the following conditions by standard methods.

Substrate: succinyl-alanine-alanine-proline-phenylalanine-p-nitro-anilide=SAAPF _P NA (purchased from Sigma, catalog number S-7388).

Buffer: 0.1M glycyl-glycine, pH 8.6; 1.5ml per liter of 15% Brig ^TM 35; 25°C (glycylglycine was purchased from Sigma, Cat. No. G-3028).

Concentration of enzyme in assay:

^SavinaseTM : 1× ^10-10-3 × ^10-10 M

The onset rate of substrate hydrolysis was measured at nine substrate concentrations ranging from 0.01 to 2 mM using a Cobas Fara automated spectrophotometer. Kinetic parameters V _max and K _m were determined using ENZFITTER (a non-linear regression data analysis program). k _cat is calculated from the equation V _max =k _cat ×[E _o ]. The concentration of active enzyme [E _o ] was determined by active site titration with very potent binding protein protease inhibitors. Inhibition constants _Ki were calculated from _Km / _kcat curves as a function of inhibitor concentration. Molar concentrations were determined from weighed weights and molecular weights, assuming 100% purity of the inhibitors.

Listed below are the inhibition constants for a series of tested mono-substituted boronase stabilizers (inhibitors), as well as the [I] _t / _Ki values for some of the above inhibitors.

Table 1

Inhibition constants and [I] _t /K _i values of Savinase ^TM inhibited by different monosubstituted boronic acid derivatives. For comparison, boric acid is also included.

Inhibitor 10 ³ ×K _i (K _i expressed in mol/l) [I] _t / _Ki 2% -1.5% ^* boric acid 10 Phenyl-substituted boronic acid 0.11 1490 3-Acetamidophenyl monosubstituted boronic acid 0.09 1240 930 Benzofuran-2 monosubstituted boronic acid 0.03 4115 4-Formylphenyl monosubstituted boronic acid 0.03 4445 3335 Thiophene-2 monosubstituted boronic acid 0.14 Thiophene-3 monosubstituted boronic acid 0.17 Naphthalene-1-substituted boronic acid 0.13 2-Formylphenyl monosubstituted boronic acid 0.2 3-Formylphenyl monosubstituted boronic acid 0.07

^* % W/W of inhibitor in solution.

Example 3

Storage Stability Test in Liquid Detergents

The following refers to KNPU ("K _i lo Novo Protease Units") in relation to the Savinase ^™ formulation), and KLU ("K _i lo Lipase Units") in relation to the Lipolase ^™ formulation. One KNPU is equivalent to 2.53mg (about 9.4×10 ^-5 mmol) of pure enzyme protein. For the determination of Savinase ^(TM) activity see a brochure, AF 220/1-GB (available on request from NovoNordisk A/S, Bagsvaerd, Denmark).

One lipase unit (LU) is defined as the amount of enzyme that releases 1 μmol of titratable butyric acid per minute under standard conditions (30.0°C, pH 7.0; Gum Arabic as emulsifier and tributyrin as substrate) . A brochure (AF 95/5) describing the analytical method in more detail is available on request from Novo Nordisk A/S, Bagsvaerd, Denmark. 1 KLU is equivalent to 0.2 mg (about 6.7×10 ^-6 mmol) of pure enzyme protein.

Using the method described above, various protease inhibitors were tested in a storage stability (retained enzyme activity) assay in liquid detergents under the conditions summarized below. 4% W/W solutions of various inhibitors (except boric acid) were prepared in monopropylene glycol (MPG). Boronic acid is poorly soluble in the relevant medium and is therefore dissolved in the finished wash composition (yielding a boric acid concentration of 1% W/W), rather than in the liquid enzyme/inhibitor composition.

First test series: In the first test series, equal parts by weight of the inhibitor solution and Savinase ^™ 16.0 L, Type EX (liquid protease preparation containing 16K NPU protease per gram; Novo Nordisk A/S, Bagsvaerd, Denmark) Mix to give Savinase ^™ 8L EX formulations each containing 2% W/W inhibitor. Each contained 1% W/W of this Savinase ^™ /inhibitor formulation, 98% W/W "Detergent Base 1" (see below) and 1% W/W Lipolase ^™ 100L, Type EX (100 KLU of fat per gram The storage stability results of the liquid lipase preparation of the enzyme; detergent compositions of Novo Nordisk A/S, Bagsvaerd, Denmark) are as follows: % Residual Lipolaste ^™ activity (stored at 30° C.): inhibitor Storage time (days)

1 3 7

34 6 01% boronic acid 82 52 21 Phenyl monosubstituted boronic acid 79 46 17 Benzofuran-2 monosubstituted boronic acid 83 54 244-Formylphenyl monosubstituted boronic acid 86 64 41 No Savinase ^TM or inhibitor 88 72 55 % Residual Savinase ^TM activity (storage at 30°C for 7 days) Inhibitor Activity % None 101% Boronic acid 74 Phenyl monosubstituted boronic acid 67 Benzofuran-2 monosubstituted boronic acid 764-Formylphenyl monosubstituted boronic acid 85

The above results show in particular that the presence of said inhibitors can significantly improve the preservation of Lipolase ^™ and Savinase ^™ activities in detergent compositions.

Second test series: In the second test series, Savinase ^TM 16.0 L EX, 4% W/W inhibitor dissolved in MPG and pure MPG were mixed at a weight ratio of 50:37.5:12.5 to obtain 1.5% each Savinase ^™ 8L EX formulation of W/W inhibitors. Storage Stability of Detergent Compositions Each Containing 1% W/W of This Savinase ^™ /Inhibitor Formulation, 98% W/W "Detergent Base II" (see below) and 1% W/W Lipolase ^™ 100L, Type EX The results are as follows: % Residual Lipolase ^™ activity (stored at 30°C): Inhibitor Storage time (days)

1 5 9 12 None 53 8 04-Formylphenyl monosubstituted boronic acid 58 41 283-Acetamidophenyl monosubstituted boronic acid 24 9 5No Savinase ^TM or inhibitor 76 67 55

Third test series: Savinase ^™ 8L EX formulations each containing 1.5% W/W inhibitor were prepared in the same manner as the second test series above. Storage of detergent compositions each containing 1% W/W of this Savinase ^™ /inhibitor formulation, 98% W/W "deactivated" OMO ^™ Micro (see below) and 1% W/W Lipolase ^™ 100L, Type EX Stability results are as follows: % residual Lipolase ^™ activity (storage at 35°C): inhibitor Storage time (days)

5 7 14 None 90 88 85 4-Formylphenyl monosubstituted boronic acids 98 99 99 No Savinase ^TM or inhibitors 99 99 99

Fourth Test Series: Essentially the same as the third series above, except that a storage temperature of 30°C was used and "Detergent Base III" (see below) was used instead of the inactivated OMO ^™ Micro. % Residual Lipolase ^™ activity (stored at 30°C): inhibitor Storage time (days)

1 5 9 12 None 45 194-Formylphenyl monosubstituted boronic acids 32 20 17 No Savinase ^TM or inhibitors 39 28 24

The above results show that the presence of the above inhibitors can significantly improve the preservation of Lipolase ^™ activity in various detergent compositions. Detergent Base I Formulation (US Type) Ingredients % W/W (Pure Ingredient) Nansa ^TM 1169/p 10.3 (Linear Alkyl Benzene Sulfonate, LAS) Berol ^TM 452 3.5 (Alkyl Ether Sulfate, AES) Oil Acid 0.5 Peanut Fatty Acid 0.5 Dobanol ^TM 25-7 6.4 (Fatty Alcohol Ethoxylate, AEO) Sodium Xylene Sulfonate 5.1 Ethanol 0.7 MPG 2.7 Glycerin 0.5 Sodium Sulfate 0.4 Sodium Carbonate 2.7 Sodium Citrate 4.4 Citric Acid 1.5 Water Balance Detergent Base II Formula: Ingredient %W/W Nansa ^TM 1169/p 10.30 Sulfatol ^TM NA/25 1.40 Oleic Acid 7.05 Arachis Fatty Acid 7.05 Dobanol ^TM 25-7 13.10 Triethanolamine 5.50 NaOH, 40% 2.00 Sodium Xylene Sulfonate 1.00 Ethanol 4.90 MPG 2.70 Sodium Sulphate 0.20 Sodium Citrate 1.40 Dequest ^TM 2060 S 0.40 Water Balanced Detergent Base III Formulation: Ingredient % W/W (Pure Ingredient) Nansa ^TM 1169/p 7.00 Oleic Acid 0.45 Arachis Fatty Acid 0.45 Lutensol ^TM A04 3.80MPG 0.50 Glycerin 4.60Na ₅ P ₃ O ₁₀ H ₂ O 22.60Na ₂ SiO ₃ 5H ₂ O 1.70 Sodium carbonate 1.43 Water balance

OMO ^™ Micro is a retail item available from supermarkets in Denmark. Enzyme components were inactivated by microwave treatment (85°C, 5 minutes).

Example 4

Examples of liquid enzyme/inhibitor compositions

Examples of liquid enzyme/inhibitor compositions of the invention are as follows (all compositions are solutions with acceptable appearance):

1) 0.1g 4-bromophenyl monosubstituted boronic acid + 10g Savinase 16.0L, Type EX: The inhibitor: enzyme (I:E) molar ratio of this composition is about 33.

2) 0.2g 3,5-dichlorophenyl monosubstituted boronic acid+40g Savinase 16.0L, TypeEX: The I:E molar ratio of the composition is about 17.

3) 0.1g 5-chlorothiophene-2-monosubstituted boronic acid + 10g Savinase 16.0L, TypeEX: The I:E molar ratio of the composition is about 40.

4) 0.1g phenyl-substituted boronic acid + 10g Savinase 16.0L, Type EX: The I:E molar ratio of the composition is about 54.

5) 0.2g phenyl-substituted boronic acid + 10g Savinase 16.0L, Type EX: The I:E molar ratio of the composition is about 108.

Claims (13)

1. A liquid composition comprising: (i) an enzyme present in an amount greater than 40 μM; and (ii) A reversible inhibitor of the above enzyme in an amount effective to increase the storage stability of said enzyme in a multi-ingredient composition subsequently added to such liquid composition. 2. The composition according to claim 1, wherein the enzyme is used in an amount of 500 μM-5 mM. 3. The composition of claim 1 or 2, wherein the molar ratio of said inhibitor to said enzyme is at least 5. 4. The composition of any one of claims 1-3, wherein the [I] _t / _Ki (as defined herein) ratio is at least 50. 5. The composition of claim 4, wherein the _ratio [I] _t /Ki is in the range of 250-5000. 6. A composition according to any one of the preceding claims, wherein the enzyme is selected from the group consisting of proteases, amylases, cellulases, lipases and oxidoreductases. 7. A composition according to any one of the preceding claims, wherein the enzyme is a protease. 8. The composition of claim 7, wherein the inhibitor is selected from the group consisting of monosubstituted boronic acids, disubstituted boronic acids and salts thereof. 9. A process for the preparation of a cleaning composition comprising enzymes and enzyme inhibitors, wherein the liquid composition of any one of claims 1-8 is mixed with the remaining ingredients of the cleaning composition. 10. The method of claim 9, wherein said cleaning composition is a liquid composition. 11. A detergent composition prepared by the process of claim 9 or 10. 12. Use of a liquid composition according to any one of claims 1 to 8 for the production of a laundry or dishwashing cleaning composition. 13. Use according to claim 12 for the production of a liquid detergent composition.