CA2208705A1 - Stabilization of liquid enzyme compositions - Google Patents

Abstract

A liquid, enzyme-containing composition which is well suited, e.g., for subsequent incorporation into a multi-component composition, such as a liquid detergent composition for laundry washing or dishwashing, comprises: (i) an enzyme in an amount exceeding 40 µM; and (ii) a reversible inhibitor of the enzyme in an amount effective to enhance the storage stability of the enzyme in a multi-component composition into which the liquid composition is subsequently incorporated.

Description

CA 0220870~ 1997-06-2~

WO 96/21716 PCTIDK~''., _CS

STArll I7~TION OF LIQUID ENZYME COMPOSITIONS

FIELD OF THE INVENTION

The present invention relates, interalia, to liquid enzyme compositions containing (i) one or more enzymes in from relatively high to very high concentration, and 5 (ii) one or more appropriate enzyme inhibitors, more particularly reversible inhibitors, which exhibit strong enzyme-inhibitory properties and/or which are present at a concentration such as to cause strong enzyme inhibition.

The invention further relates to:

an enzyme-containing, multi-component composition, such a liquid composition, 10 e.g. a cleaning composition such as a detergent composition, prepared using an inhibitor-stabilized liquid enzyme composition of the invention as the source ofenzyme;

a process for preparing an enzyme-containing, multi-component composition, such as a cleaning composition, e.g. a detergent composition, wherein the 5 enzyme(s) in question is/are introduced in the form of an inhibitor-stabilized liquid enzyme composition of the invention, i.e. the enzymes(s) is/are in the form of an inhibitor-stabilized cornposition when incorporated into the multi-component composition; and an enzyme-containing, multi-component composition, such as a cleaning 20 composition, e.g. a detergent composition, prepared by a process of the latter type.

The invention moreo~er relates to the use of a liquid enzyme composition according to the inv0ntion in the preparation or manufacture of an enzyme-CA 0220870~ 1997-06-2 WO 96/21716 PCT/I)K9"~~~~

containing, multi-component composition, such as a cleaning composition, e.g.
a detergent composition. r BACKGROUND OF THE INVENTION

Storage stability problems are well known in connection with liquid compositions5 containing enzymes. Thus, for example, a major problem with enzyme-containing liquid detergents, such as liquid detergents containing a protease (peptidase), is that of ensuring retention of adequate enzyme activity during storage.
Considerable effort has been devoted to finding ways of improving the storage stability of enzyme-containing compositions, e.g. Iiquid compositions such as 0 liquid detergents, for example by adding a protease inhibitor to compositions containing a protease.

The discussion in the remainder of this section relates, by way of illustration,predominantly to inhibition of proteases, but the underlying principles are equally applicable to other types of enzymes, e.g. Iipases, amylases, cellulases and 15 oxidoreductases (such as peroxidases and oxidases), and the various aspects of the present invention are in no way limited to aspects associated with stabilization of proteases by (reversible) protease inhibitors.

By way of example, boric acid and boronic acids are known to reversibly inhibit proteolytic enzymes. A discussion of the inhibition of a serine protease, subtilisin, 20 by boronic acid is given in Molecular & Cellular Biochemistry 51, 1983, pp. 5-32.

Boronic acids have very different capacities as subtilisin inhibitors. Boronic acids containing only alkyl groups such as methyl, butyl or 2-cyclohexylethyl are poorinhibitors, with methylboronic acid being the poorest inhibitor. However, boronic acids bearing aromatic groups such as phenyl, 4-methoxyphenyl or 3,5-25 dichlorophenyl are described as being very good inhibitors, with 3,5-dichlorophenylboronic acid as a particularly effective one (see Keller et al, Biochem. Bioohvs. Res. Comm. 176, 1991, pp. 401-405).

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WO 96121716 PCT/I)K~ ,C5 It is also claimed, in WO 92/19707, that aryl boronic acids which are substituted at the 3-position of the aryl group relative to boron are unexpectedly good reversible protease inhibitors. Thus, for example, "acetamidobenzene boronic acid" is described in the latter document as being a particularly effective inhibitor 5 of proteolytic enzymes.

When quantifying the inhibitory effect of a reversible enzyme inhibitor, the so-called "inhibition constant" (K;) is ordinarily used as a measure of capacity toinhibit enzyme activity, K; normally being defined as follows:

K; = [E] ~ [I]/[EI]

10 where [E], [I] and [El] denote equilibrium concentrations (conventionally molar concentrations) of enzyme, inhibitor and enzyme-inhibitor complex, respectively,under the conditions in question. The latter definition of K; is employed in thepresent specification and claims. Thus, a relatively low K; value is indicative of a relatively potent inhibitor.

In the preparation or manufacture of multi-component enzyme-containing compositions it will be desirable to employ, as source of enzyme, a pre-preparedenzyme composition containing enzyme(s) and appropriate (reversible) enzyme inhibitor(s). It will be particularly convenient and advantageous that the pre-20 prepared enzyme composition contains a very high concentration of at least oneenzyme, and possibly of several enzymes of the type(s) in question, since this will, among other things, enable the preparation of large volumes or quantities of an enzyme-containing, multi-component composition using relatively small volumes or quantities of pre-prepared enzyme/inhibitor composition.

25 Furthermore, it will clearly be an advantage that satisfactory stability of the enzyme(s) during storage of the final multi-component composition is ensured CA 0220870~ 1997-06-2 WO 96/21716 PCT/DK96'~

solely by the presence of the enzyme inhibitor(s) in question in the amount(s) in which they have been introduced in the form of the original pre-prepared enzyme/inhibitor composition, i.e. that inclusion of further stabilizing agent in the multi-component composition (e.g. a detergent composition) is unnecessary 5 (although further stabilization agents may, of course, be incorporated if so desired) .

Moreover, in such a pre-prepared enzyme/inhibitor composition, the stability of the enzyme(s) for which an inhibitor is present will, in the nature of things, be expected to be high. Thus, numerous pre-prepared compositions of this type will 0 be expected to be capable of storage for relatively prolonged periods of time a~ter manufacture without significant - or at least without unacceptable - loss of enzymatic activity.

The present invention thus relates to a liquid composition comprising: (i) an enzyme in an amount exceeding 40 IJM; and (ii) a reversible enzyme inhibitor in 15 an amount effective to enhance the storage stability of the enzyme in a multi-component composition (e.g. a detergent composition, such as a liquid detergent composition) into which the liquid composition is subsequently incorporated.

A liquid composition of the invention may, for example, be a predominantly aqueous composition, or a predominantly non-aqueous composition (e.g. a 20 composition comprising as solvent a high proportion of one or more non-aqueous - but normally water-miscible - liquids, generally organic liquids (such as alcohols, glycols or the like). Liquid compositions of the invention will themselves normally be non-detergent compositions.

Apart from detergent compositions (e.g. for laundry washing, dishwashing and 25 the like), other types of enzyme-containing, multi-component compositions, notably in the form of liquid compositions, which are of relevance in the context of the present invention include: compositions for cleaning dentures, contact lenses or hard surfaces (e.g. in slaughterhouses, or in the food processing CA 0220870~ 1997-06-25 W O96/21716 PCTADh~-'00005 industry); compositions for use in l:he leather industry (e g. for de-hairing and/or de-fatting of animal hides); compositions for desizing textiles; and compositions for washing denim l:extiles to achieve a "stone-washed" appearance. The use of such compositions for such purposes is within the scope of the invention.

5 Enzvmes Enzyme classification numbers (EC numbers) referred to in the present specification with claims are in accordance with the Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistrv and Molecular Biology, Academic Press Inc., 1992.

10 A liquid composition of the invention contains at least one enzyme. Suitable enzymes include any commercially available enzyme. Particularly relevant enzymes include en;~ymes selected from the group consisting of proteases (i.e.
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 15 oxidoreductases (EC, 1), e.g. peroxidases (EC 1 .1 1 ) and oxidases [including enzymes classified under EC 1.10.3, such as laccases (EC 1.10.3.2)], and any mixture thereof. Mixtures of enzymes from the same class (e.g. mixtures of different proteases, different lipases, etc.) are also included.

The amount(s) of enzyme(s) in the liquid composition will vary according to the 20 type of enzyme(s) and the intended 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 on the basis of pure enzyme protein), often 0.5-25 % w/w, such as 1-10% w/w, e.g. 2-8% w/w of the liquid composition. Expressed in concentration units, a preferred 25 concentration range for an enzyme present in a liquid composition of the invention will be from about 50 ,uM to about 20 mM, often from 100 ~M to 10 mM, such as from 5()0 ~M to 5 mM, e.g. from 750 ,uM to 3 mM (calculated on the basis of moles of pure enzyme protein).

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W O96121716 PCT~DK~f~S

Proteases. Any protease (proteolytic enzyme) suitable for use in a liquid composition can be used. Suitable proteases include those of animal, vegetable or microbial origin, especially microbial origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or 5 more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit proteolytic activity. The protease may, e.g., be a serine peptidase, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g. subtilisin 0 Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in W0 89/06279). Examples of commercial Bacillus subtilisins are Alcalase~, SavinaselM, EsperaselM and Durazym~ products, all available from NovoNordisk A/S. A number of these protease products, such as Alcalase~, Esperase~
and SavinasenA, are available as liquids (e.g. Savinase~ 16.0 L, Type DX and Type EX) which are well suited (vide infra) to the preparation of protease-containingliquid compositions according to the present invention.

Examples of trypsin-like proteases include trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in W0 89/06270.

Amvlases. Any amylase (amylolytic enzyme) suitable for use in a liquid 20 composition can be used. Suitable amylases include those of bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit amylolytic activity.
25 Amylases include, for example, a-amylases (EC 3.2.1.1), e.g. obtained from a particular strain of B. Iicheniformis, described in more detail in British Patent Specification No. 1,296,839. A very suitable a-amylase is TermamyllU, which is available (inter alia as a liquid product) from Novo Nordisk A/S.

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WO 96/21716 PCT/DK~G~?~~--5 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 chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted 5 and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit lipolytic activity. A very suitable lipase is that obtained by cloning the gene from Humicola lanuqinosa and expressing the gene in Aspergillus orvzae, as described in EP 0 258 068, which is available (interalia as a liquid product) from Novo Nordisk A/S under the tradename Lipolase~.

0 Cellulases. Any cellulase (cellulolytic enzyme) suitable for use in a liquid composition can be used. Suitable cellulases include those of bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of a 15 enzyme of one of the latter types, and which exhibit cellulolytic activity. Suitable cellulases are disclosed, 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 peroxidase suitable for use in a liquid composition, e.g. Iiquid 20 detergent composition, can be used. Suitable peroxidases herein include those of plant, bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the aminoacid sequence of an 0nzyme of one of the latter types, and which exhibit 25 peroxidase activity. Examples of suitable peroxidases are those derived from a strain of Coprinus (e.g. C. cinerius or C. macrorhizus) or from a strain of Bacillus (e.g. B. oumilus), particularly a peroxidase according to PCT/DK90/00260.

Oxidases. Any oxidase suitable for use in a liquid composition, e.g. Iiquid detergent composition, can be used. Suitable oxidases herein include those of CA 0220870~ 1997-06-25 W O 96/21716 PCT~Dh~6/0000S

bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit oxidase 5 activity. Examples of suitable oxidases are laccases derived from strains of AsPerqillus, Neurospora (e.g. N. crassa), Trametes (e.g. T. villosa) or MvcelioPhthora (e.g. M. thermoPhila).

Enzvme inhibitors (enzvme stabilizers) A liquid composition according to the invention contains at least one reversible10 enzyme inhibitor, normally one which is an inhibitor for at least one enzyme present in the liquid composition. Thus, for example, a liquid composition of the invention may, in addition to containing a first type of enzyme and a reversibleinhibitor for that type of enzyme, contain a second, third...etc. type of enzyme(e.g. an enzyme of one of the types mentioned above) one or more of which is 15 not accompanied by a reversible inhibitor therefor.

It should be mentioned here that it is perfectly feasible to prepare a liquid enzyme composition which contains one or more enzymes, in amounts as disclosed herein, but which instead of containing an inhibitor for one or more of these enzymes contains an inhibitor for another type of enzyme with which the liquid 20 composition may subsequently be contacted. One example hereof would be a liquid composition containing, as the only enzyme, a lipase together with a protease inhibitor; the protease inhibitor would then protect the lipase with respect to degradation by a protease which - deliberately or inadvertently - maysubsequently be brought into contact with the lipase.

25 The nature and amount(s) of the inhibitor(s) employed will depend, in~eralia, on the nature and concentration of the enzyme(s) present in the composition, and on the intended use of the composition. This is discussed further below.

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WO 96/21716 PCT/DK~ 5 With respect to rever~iible inhibitors (and associated Kj values therefor) of relevance for use in connection with various classes/types of enzymes which may be present in a liquid composition of the invention, reference is made to: H.
Zollner, Handbook of E nzyme Inhibitors (Parts A and B), 2nd edition, VCH
5 Verlagsgesellschaft mbH, Weinheim, Germany, 1993, for an extensive listing.
Further sources of relevance include: S. Patkar and F. Bjorkling, LiPase inhibitors, in: Li~ases - their Structure. Biochemistry and Ac~lication. Editors P. Woolley and S.B. Petersen, Cambridge University Press, Cambridge 1994. Selected examples of relevant types of reversible inhibitors are the following:

0 Protease inhibitors. One particularly interesting class of reversible proteaseinhibitors is constitutecl by the boronic acids [R'B(OH)2] and borinic acids [R'R''B(OH)] (where R' and R'' are organic substituents, e.g. optionally substituted aryl or heterocyclic substituents), some examples of which have beenmentioned above. Further examples of relevant compounds of this type may be 15 selected among those mentioned in WO 92/19707, in EP 0 478 050 A1 and in EP0511 456A1.

Interesting compounds of this type may be found among those described in WO 95/02046 (which was unpublished at the priority date of the present application), and which c:omprise compounds of the following general formula:

R, - (R2)n- B - OH
I

25 where R1 is an optionally substituted fused aromatic ring structure containing 14 or 18 carbon atoms in the ring, or an optionally substituted monocyclic or fusedaromatic heterocyclic ring structure containing up to 17 carbon atoms in the ring, or an optionally substituted monocyclic or fused quinonoid ring structure containing up to 18 carbon atoms in the ring;

-CA 0220870~ 1997-06-2~

WO 96/21716 PCT/I)K~ ~r R2 has the formula:

CH ~o I C=C ~p ~ CH ~ q 5 where X is the same or different and selected from hydrogen, C,-C6 alkyl, substituted C1-C6 alkyl, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate or phosphonate, and o, p and q may be the same or different and may each be 0, 1 or 2;
10 m and n may be the same or different and may each be 0 or 1;
R3 is the same or different as R1 and selected from R1, or R3 is a hydroxyl group, or R~ and R3 are both optionally substituted monocyclic or dicyclic aromatic ring structures.

In the above context, optionally substituted ring structures are such that the 15 substituents on the ring structure are freely chosen, but they are preferablyselected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative,nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate and phosphonate .

20 Boronic and borinic acid derivatives may be prepared using methods well knownto those skilled in the art, for example by using one of the following methods:

a) Hydroboration of unsaturated materials, i.e. alkenes and alkynes, using either catecholborane (1,3,2-benzodioxaborole) or dichloroborane-dimethyl-sulphide complex as the hydroborating agent; for reference see H.C.Brown, S.K.Gupta in 25 IACS 97, 1975, pp. 5249-5255 and H.C.Brown, N.Ravindran, S.U.Kuikarni in 1. Org.Chem . 45, (1980), p. 384.

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WO 96/21716 PCT/DK9G/0~~~;

b) The reaction of a Grignard reagent with either tri-n-butylborate or trimethylborate, 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.lngleshwar in Journal of Shivasi Universitv 6, 1932, pp.
5 11-13. Bromo-substituted starting materials that are not commercially available may be prepared conveniently 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 Chemistrv, Part 7, and D.Florentin, 10 B.Roques in C.R.Acad.Sci.Paris, t.270 (11 May 1970), pp. 1608-1610.

d) Borinic acid derivatives may be prepared according to method b, above.
However, the ratio of Grignard reagent to borate adopted is then 2:1.

e) Any nuclear substitution or protection of functional groups is achieved by using standard methods ~vell known to those skilled in the art.

15 Further interesting compounds of this type may be selected among naphthalene boronic acids which are clescribed in WO 95/29223 (which was unpublished at the priority date of the present application) and which comprise compounds having the following general formulas:

6 ~ or R7 B (OH) z R~4 R~3 Rs~ Rz ( O~ ) z R7 R~

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where R" R2, R3, R4, R5, R6 and R7 are the same or different and are selected from hydrogen, C1 C6 alkyl, substituted C1-CG alkyl, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative,nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate and phos-5 phonate.

Naphthalene boronic acid derivatives may be likewise 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 dropwise addition of the appropriate 10 bromonaphthalene starting material in sodium dried ether to magnesium turningin sodium dried ether. The reaction is promoted by the addition of a small iodine crystal.

Trimethylborate or tri-n-butylborate in sodium dried ether is cooled to -70~C, and the Grignard reagent is added dropwise over a period of 2 hours while keeping 15 the organoborate solution at -70~C and continuously agitating. The reaction mixture is allowed to warm to room temperature overnight, whereupon it is hydrolysed by the dropwise addition of cold dilute sulphuric acid. The ether layer is separated and the aqueous layer extracted with ether. The ether-containing fractions are combined and the solvent removed. The residue is made strongly 20 alkaline, and any methanol or butanol so formed is removed. The alkaline solution is cooled and the resulting crystals of the desired boronic acid are removed by filtration. All products are preferably recrystallized from distilled water.

The naphthalene boronic acids may also be prepared using either direct lithiation of the naphthalene and/or lithiation of the bromide.

25 Any nuclear substitution or protection of functional groups may be achieved by using standard methods well known to those skilled in the art.

CA 0220870~ 1997-06-2 WO 96/21716 PCTlI)K9' Among compounds of the above-mentioned types, 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 5-chlorothiophene-2-boronic acid; naphthalene-1-boronic acid; naphthalene-2-boronic acid; and 6-hydroxynaphthalene-2-boronic acid. Further specific compounds of this type which are of interest in the context of the invention arementioned in the table given in Example 2 herein (vide infra).

Inhibitors of the boronic or borinic acid type may be introduced or incorporated0 into liquid compositions of the invention in the form of the acids themselves (e.g.
dissolved in an appropriate water-miscible organic solvent, such as mono-propylene glycol or the like), or, if appropriate (e.g. for solubility reasons), as salts (e.g. alkali metal salts, such as sodium or potassium salts).

Reversible amvlase inhibitors. Reversible amylase inhibitors of possible relevance 15 in the context of the invention may be found, e.g., among compounds of the so-called "acarbose" type (i.e. pseudo-oligosaccharides containing an acarviosine moiety and one or more maltose units). Other compounds of relevance as inhibitors for, in particular, a-amylases include maltose and maltotriose.
Compounds such as methyl-a-glucoside, as well as cycloamyloses, e.g.
20 cyclohexa- and/or cycloheptaamylose, may be of relevance as reversible inhibitors for ,B-amylases.

Reversible cellulase inhibitors. Reversible cellulase inhibitors of potential interest - in the context of the invention include 4-thiol-cellooligosaccharides [see, e.g., C. Schou et al., Journal of Carbohydrate Chemistrv 12 (1993) pp. 743-752].
A

25 Reversible liDase inhibitors. Reversible lipase inhibitors of relevance in the context of the invention include boronic and borinic acids selected among those described above and/or mentioned below (such as, e.g., optionally substituted phenyl boronic acids).

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WO 96/21716 PCT/I)K9G/000 5 Degree of enzYme inhibition The desired degree ~strength, extent) 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 intended. In general, the molar ratio 5 of reversible inhibitor to enzyme will be chosen to be such that at least about one inhibitor molecule is present per active site of the enzyme in question, which will of course require a molar ratio of inhibitor (I) to enzyme (E) of at least 1:1. For certain purposes a lower l:E molar ratio, e.g. about 0.8:1, about 0.7:1, about 0.6:1 or even about 0.5:1, may, however, be appropriate. However, in the 10 context of the invention it will generally be desirable to employ a molar ratio of inhibitor to enzyme of at least 5, such as at least 15. In certain cases, I:E molar ratios of at least 50 or at least 100, or even higher ratios, may be appropriate.

The molar ratio of inhibitor to enzyme will be chosen, inter a/ia, on the basis of considerations relating to the percentage of free (uninhibited) enzyme which it is desired to have present during the actual use of a multi-component, enzyme-containing composition based on a liquid composition of the invention. Thus, forexample, when using a laundry detergent composition for laundry washing, it willclearly be a requisite that a satisfactorily high level of free enzyme (e.g. a protease or a lipase) is present in the washing medium (which is typically water20 to which the the detergent composition in question has been added).

In this connection a parameter of importance is the inhibition constant (definedabove) for reversible inhibition of an enzyme. Thus, for example, when a liquid composition of the invention is to be in a detergent composition, the inhibitionconstant (K;), expressed in the conventional manner in mol/l (M), for a reversible 25 inhibitor of an enzyme therein will often suitably be such that 3 ~ 1 o-8 M ' K; ' 1.2 ~ 1 o-2 M, such as 3 ~ 1 o-8 M ~ K; c 1 ~ 1 o-2 M. A more desirable "window of inhibition" in connection with detergent enzymes will, however, often be such that an inhibitor(s) associated with a particular enzyme exerts an inhibition CA 0220870~ 1997-06-2~

WO 96/21716 PcT/DK9~ 5 correspondingto about '3-1O-7 M ~ K~ 1O-3 M, such as 4.3 ~ 1O-7 M ~ K; c 4.5-10-4 M.

In general, preferred enzyme-containing liquid compositions of the invention arecompositions in which the ratio between:
5 (a) the total molar concentration (which may be denoted [I]t) of a reversible enzyme inhibitor, I, present in the composition; and (b) the inhibition constant (Kj; expressed in mol/l) for inhibition of an enzymewhich is present in the composition and which is reversibly inhibited by the inhibitor in question;
10 is at least 50 (i.e. [I]t/Kj ~- 50), such as at least 100 or, frequently, at least 250.
A practical upper limit for the ratio [I]t/Kj in liquid compositions of the invention will normally be about 10000 (i.e. 1 O4). For many purposes, a value for the ratio [I]t/K; in the range of 250-5000, often in the range of 250-2500, will be appropriate .

The value of the ratio [l~t/Ki may be regarded as a measure of the "inhibitory capacity" of a liquid composition of the invention. A high value - and thereby ahigh "inhibitory capacity" - may be achieved, for example, by incorporating, in a liquid composition, a re3atively modes~ to low concentration of a strongly inhibiting inhibitor, or by incorporating a relatively high concentration of a more 20 weakly inhibiting inhibitor.

In the presence of a relatively large molar excess of the inhibitor, I, relative to the inhibited enzyme, E, the major proportion of the inhibitor will normally be in free form, i.e. will not be bound to enzyme. Denoting the concentration of free (unbound) inhibitor by [I] (as in connection with Kj; vide supr~), the ratio rl]t/K;
25 will then be approximately equal to ~he ratio [I]/Kj, i.e. [I]t/Kj ~ [I]/K; under such conditions.

It may be mentioned here that liquid compositions according to the invention, comprising enzyme(s) and reversible enzyme inhibitor(s), may be dried by CA 0220870~ 1997-06-2 WO 96t21716 PCT/DK~)G/C~

appropriate methods (e.g. by Iyophilization or by spray-drying. The dried enzyme/inhibitor product may be then be comminuted (e.g. by milling) and suspended or slurried at an appropriate concentration in a non-aqueous liquid vehicle, e.g. a non-ionic surfactant (such as SoftanollM from BP), to form a slurry 5 product.

Detergents For detergent compositions, a typical goal will be attainment of at least 50%
inhibition of a chosen enzyme (e.g. a protease) in the detergent composition perse, and an amount of free enzyme in the washing medium corresponding to at 10 least 50% of the total amount of that enzyme.

A detergent composition incorporating a liquid composition of the invention will, apart from enzyme(s) and inhibitor(s), comprise a surfactant, and will normally be a liquid detergent composition. The detergent composition may, e.g., be a laundry detergent composition or a dishwashing detergent composition.

15 A liquid detergent composition may be aqueous, e.g. typically containing up to 70% of water and 0-30% of organic solvent, or substantially non-aqueous.

The detergent composition will comprise one or more surfactants, each of which may be anionic, nonionic, cationic, or amphoteric (zwitterionic). The detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzene-20 sulfonate (I~AS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE), alcohol propoxylate, carboxylated alcohol ethoxylates, nonylphenol 25 ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).

_ CA 0220870~ 1997-06-2~

WO 96/21716 PCT/I)K96/00005 Normally the detergent contains 1-65 % of a detergent builder, but some dishwashing detergents may contain even up to 90% of a detergent builder, or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), 5 diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent builders may be subdivided into phosphorus-containing and non-phosphorous-containing types. Examples of phosphorus-containing inorganic alkaline detergent builders include the water-soluble salts, especially alkali metal 0 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 disilicates and the varioustypes of water-insoluble crystalline or amorphous alumino silicates of which zeolites is the best known representative.

15 Examples of suitable organic builders include alkali metal, ammonium or substituted ammonium salts of succinates, malonates, fatty acid malonates, fattyacid sulphonates, carbloxymethoxy succinates, polyacetates, carboxylates, polycarboxylates, aminopolycarboxylates and polyacetyl carboxylates. The detergent may also be unbuilt, i.e. essentially free of detergent builder.

20 The detergent may comprise one or more polymers. Examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, - polymaleates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers .

25 The detergent composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type. The bleaching agents may be coated or encapsulated.
Examples of inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite or hypobromite as well as chlorinated trisodium phosphate.

CA 0220870~ 1997-06-2~
WO 96/21716 PCTlDEV-'O~:lOS

The bleaching system may also comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS) .

5 Examples of organic chlorine/bromine-type bleaches are heterocyclic N-bromo and N-chloro imides such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water solubilizing cations such as potassium and sodium. Hydantoin compounds are also suitable. The bleaching system may also comprise peroxyacids of, e.g., the 10 amide, imide, or sulfone type;

In dishwashing detergents the 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 15 percarbonates, persilicates and perphosphates. Preferred activator materials are TAED or NOBS.

As already mentioned, the enzymes of the detergent composition of the invention are incorporated in the detergent composition in the form of a liquid enzyme/inhibitor composition according to the invention.

20 If so desired, further conventional enzyme-stabilizing substances may be incorporated, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as an aromaticborate ester (see, e.g., WO 92/19709 and WO 92/19708).

The detergent may also contain other conventional detergent ingredients such as,25 e.g., fabric conditioners including clays, deflocculant material, foam boosters/foam depressors (in dishwashing detergents foam depressors), suds CA 0220870~ 1997-06-2~

WO 96/21716 PCT/I)K96/00005 suppressors, anti-corrosion agents, soil-suspending agents, anti-soil-redeposition agents, dyes, dehydrating agents, bactericides, optical brighteners, or perfume.
The pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. in the range of 7-11.

5 Particular forms of laundry detergent compositions within the scope of the invention include:

1) An aqueous liquid detergent composition comprising Linear alkylbenzenesulfonate (calculated as 15 - 21%
acid) 0 Alcohol ethoxylate (e.g. C1215 alcohol, 7 E0 or C12 15 alcohol, 5 E0) 1 2 - 1 8%
Soap as fatty acid (e.g. oleic acid) 3 - 13%
Alkenylsuccinic acid (C12 14) ~ - 13%
Aminoethanol 8 - 18%
Citric acid 2 - 8%
Phosphonate 0 - 3%
Polymers (e.g. PVP, PE--G) 0 - 3%
Borate (as B407) ~ - 2%
Ethanol 0 - 3 %
Propylene glycol 8 - 14%
Enzymes (calculated as pure enzyme 0.0001 - 0.2%
protein) - Minor ingredients (e.g. dispersants, suds suppressors, perfume, optical brightener) 0 - 5%

CA 0220870~ l997-06-2 WO 96/2171G PCT/I)K~)G~

2) An aqueous structured liquid detergent composition comprising Linear alkylbenzenesulfonate (calculated as 1 5 - 21%

Alcohol ethoxylate (e . g . C1 2 1 5 alcohol, 7 or C12 ,5 alcohol, 5 EO) Soap as fatty acid (e.g. oleic acid) 3 - 10%
Zeolite (as NaAlSiO4) 14 - 22%
Potassium citrate 9 - 1 8%
0 Borate (as B4072-) 0 - 2%
Carboxymethylcellulose O - 2%
Polymers (e.g. PEG, PVP) O - 3%
Anchoring polymers such as, e.g., lauryl methacrylate/acrylic acid copolymer; molar O - 3%
5 ratio 25:1; MW 3800 Glycerol o - 5%
Enzymes (calculated as pure enzyme pro- 0.0001 - 0.2%
tein) Minor ingredients ~e.g. dispersants, suds 20 suppressors, perfume, optical brighteners) O - 5%

WO 96/21716 PCTIDK~ DC-~:

3) An aqueous liquid detergent composition comprising Linear alkylbenzenesulfonate (calculated as acid) 1 5 - 23 %
Alcohol ethoxysulfate (e.g. C~2 15 alcohol, 2-3 E0) 8 - 15%
Alcohol ethoxylate (e.g. C,2.15 alcohol, 7 EO, 3 - 9 %
or C,2,5 alcohol, 5 E0) Soap as fatty acid (e.g. Iauric acid) 0 - 3%
Aminoethanol 1 - 5 %
Sodium citrate 5 - 10%
Hydrotrope (e.g. sodium toluensulfonate) 2 - 6%
Borate (as B4072-) 0 - 2%
Carboxymethylcellulose 0 - 1%
Ethanol 1 - 3 %
Propylene glycol 2 - 5%
Enzymes (calculated as pure enzyme pro- 0.0001 - 0.2%
tein) Minor ingredients (e.g. polymers, dispersants, perfume, optical brighteners) 0 - 5%

CA 0220870~ 1997-06-2~

O 9 6 /2 1 7 1 6 P C T/ I ) K 9 4) An aqueous liquid detergent composition comprising Linear alkylbenzenesulfonate (calculated as acid) 20 - 32%
Alcohol ethoxylate (e.g. C12 15 alcohol, 7 E0, or C12 15 alcohol, 5 E0) 6 - 1 2%
Aminoethanol 2 - 6%
Citric acid 8 -1 4%
Borate (as B4072-) 1 - 3%
Polymer (e.g. maleic/acrylic acid copolymer, 0 anchoring polymer such as, e.g., lauryl methacrylate/acrylic acid copolymer) 0 - 3%
Glycerol 3 - 8%
Enzymes (calculated as pure enzyme 0.0001 - 0.2%
protein) Minor ingredients (e.g. hydrotropes, dispersants, perfume, optical brighteners) 0 - 5%

5) Detergent formulations as described in 1 ) - 4) wherein all or part of the linear alkylbenzenesulfonate is replaced by (C12-C18) alkyl sulfate.

6) Detergent formulations as described in 1 ) - 5) which contain a stabilized or20 encapsulated peracid, either as an additional component or as a substitute for already specified bleach systems.

7) A detergent composition formulated as a non-aqueous detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali. The detergent may25 also comprise anionic surfactant and/or a bleach system.

Dishwashing and other multi-comPonent. enzvme-containing comPositions Examples of relevant types of formulations of this kind include the following:

CA 0220870~ 1997-06-2~

WO 96/21716 PCT/I)K96/00005 1 ) LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT
SYSTEM, COMPRISING:
- Nonionic surfactant O - 1.5%
Octadecyl dimethylamine N-oxide dihydrate 0 - 5%
80: 20 wt. C 1 8/C 1 6 blend of octadecyl dimethyiamine N-oxide dihydrate and hexadecyldimethyl amine N-oxide dihydrate 0 - 4%
70: 30 wt. C 1 8/C 1 6 blend of octadecyl bis (hydroxyethyl)amine N-oxide anhydrous and 0 hexadecyl bis 0 - 5%
(hydroxyethyl)amine N-oxide anhydrous C,3-C15 alkyl ethoxysulfate with an average degree of ethoxylation of 3 O - 10%
C12-C15 alkyl ethoxysulFate with an average degree of ethoxylation of 3 O - 5%
C~3. C~5 ethoxylated alcohol with an average degree of ethoxylation of 12 0 - 5%
A blend of C,2-C15 ethoxylated alcohols with an average degree of ethoxylation of 9 O - 6.5%
A blend of C13-C15 ethoxylated alcohols with an average degree of ethoxylation of 30 0 - 4%
Sodium disilicate 0 - 33%
Sodium tripolyphospha~e O - 46%
Sodium citrate O - 28%
Citric acid 0 - 29%
Sodium carbonate 0 - 20%
Sodium perborate monohydrate 0 - 11.5%
Tetraacetylethylenediamine (TAED) 0 - 4%
Maleic acid/acrylic acid copolymer 0 - 7.5%
Sodium sulphate 0 - 12.5%
Enzymes 0.0001 - 0.2%

CA 0220870~ 1997-06-2~
WO 9612 17 16 PCTIDK9~;/C _ _ 2) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION
COMPRISING:

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0 - 10.0 %
Alkali metal silicate 3.0 - 1 5.0 %
Alkali metal phosphate 20.0 - 40.0%
Liquid carrier selected from higher glycols, polyglycols, polyoxides, glycolethers 25.0 - 45.0 %
Stabilizer (e.g. a partial ester of phosphoric 0 acid and a C16-C18 alkanol) 0.5 - 7.0%
Foam suppressor (e.g. silicone) 0 - 1.5 %
Enzymes 0.0001 - 0.2%

3) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION COMPRISING:

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0 -1 0.0%
Sodium silicate 3.0 - 1 5 .0%
Alkali metal carbonate 7.0 - 20.0 %
Sodium citrate 0.0 - 1.5%
Stabilizing system (e.g. mixtures of finely divided silicone and low molecular weight dialkyl polyglycol ethers) 0.5 - 7.0%
Low molecule weight polyacrylate polymer 5.0 - 15.0%
Clay gel thickener (e.g. bentonite) 0.0 - 10.0%
Hydroxypropyl cellulose polymer 0.0 - 0.6%
Enzymes 0.0001 - 0.2%
Liquid carrier selected from higher Iycols, polyglycols, polyoxides and glycol ethers Balance CA 0220870~ 1997-06-2~

wo 96/21716 PCT/I)K96/00005 4) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION
~ COMPRISING:

- C12-C14 fatty acid O - 0.5%
Block co-polymer surfactant 1.5 - 15.0%
Sodium citrate O - 12%
Sodium tripolyphosphate O - 1 5 %
Sodium carbonate O - 8%
Aluminium tristearate O - 0.1%
Sodium cumene sulphonate O - 1 .7%
0 Polyacrylate thickener 1 .32 - 2.5%
Sodium polyacrylate 2.4 - 6.0%
Boric acid O - 4.0%
Sodium formate O - 0.45%
Calcium formate O - 0.2%
15 Sodium n-decydiphenyl oxide disulphonate O - 4.0%
Monoethanol amine (MEA) O - 1.86%
Sodium hydroxide (50%) 1.9 - 9.3%
1,2-Propanediol O - 9.4%
Enzymes 0. 0001 - O . 2 %
20 Suds suppressor, dye, perfumes, water Balance CA 0220870~ 1997-06-2S
W O96121716 PCTADh~6/00005 5) LIQUID AUTOMATIC DISHWASHING COMPOSITION COMPRISING:

Alcohol ethoxylate O - 20%
Fatty acid ester sulphonate 0 - 30%
Sodium dodecyl sulphate 0 - 20%
Alkyl polyglycoside 0 - 21%
Oleic acid 0 - 1 0%
Sodium disilicate monohydrate 1 8 - 33%
Sodium citrate dihydrate 18 - 33%
Sodium stearate O - 2.5%
Sodium perborate monohydrate 0 - 13%
Tetraacetylethylenediamine (TAED) 0 - 8%
Maleic acid/acrylic acid copolymer 4 - 8%
Enzymes 0.0001 - 0.2%

6) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING
15 PROTECTED BLEACH PARTICLES, COMPRISING:

Sodium silicate 5 - 1 0%
Tetrapotassium pyrophosphate 1 5 - 25 %
Sodium triphosphate 0 - 2%
Potassium carbonate 4 - 8%
20 Protected bleach particles, e.g. chlorine 5 - 10%
Polymeric thickener 0.7 - 1.5%
Potassium hydroxide O - 2%
Enzymes 0.0001 - 0.2%
Water Balance CA 0220870~ 1997-06-2~

WO 96/21716 PCT/I)K~G/'~,0-S5 7) Automatic dishwashing compositions as described in 1 ) and 5), wherein perborate is replaced by percarbonate.

8) Automatic dishwashing compositions as described in 1), which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the 5 compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639.

The "enzymes" content of the above formulations may include the content of enzyme inhibitor(s) pre!sent. Where appropriate, a particular formulation encompassed within one of the types recited above may contain an organic 0 solvent of a type which i~s not specifically mentioned above in connection with that type of formulation, but which has functioned, e.g., as a solubilizing solvent for an inhibitor present in a liquid enzyme/inhibitor composition of the invention which has been incorporated in the particular formulation in question.

A liquid enzyme/inhibitor composition of the invention of the invention may be 15 incorporated in a detergent composition so as to give an enzyme concentrationwhich is conventionally employed in detergents. It is at present contemplated that, for a detergent cornposition of the invention, a liquid enzyme/inhibitor composition of the invention may, e.g., be incorporated in an amount which - fora given enzyme present in the enzyme/inhibitor composition - corresponds to an 20 amount of enzyme in the range 0.00001-1 mg (calculated as pure enzyme protein) of enzyme per liter of wash liquor.

CA 0220870~ 1997-06-2~
WO 96/21716 PCT/DK9~'..~~~,5 Tests of Stabilizers The inhibitory effectiveness of an inhibitor may be tested in various ways, illustrated here by the following two tests for the case of a protease/protease inhibitor:

5 a) Storaqe Stability Test in Liquid Detergent: Liquid enzyme/inhibitor composition is added to a liquid detergent formulation which is stored under well-defined conditions. The enzyme activity of each enzyme is determined as a function of time (e.g. after 1, 3, and 7 days).

To calculate the inhibition efficiency from the storage stability data, a reaction 10 mechanism is proposed. The following reactions give a relatively simple, yet plausible, mechanism for a liquid detergent containing protease (P), lipase (L), and inhibitor (I):

I) Autodigestion of protease:
P + P~ Dp + P

15 Il) Denaturation of protease:
P ~ Dp lll) Inhibition of protease:
P + I ~ Pl IV) Protease digestion of inhibited enzyme:
P + Pl ~ P + Dp + I

V) Denaturation of inhibited enzyme:
Pl ~ Dp + I

Vl) Protease digestion of lipase:

CA 0220870~ 1997-06-2~
Wo 96/21716 PCT/DK96/00005 P + L~ P + D, Vll) Denaturation of lipase:
L- D, where Dp and D, are denatured (i.e. non-active) protease and lipase.

5 From these reactions three coupled differential equations are derived describing the deactivation of P, L and Pl. The reaction rate constants are derived from storage stability data by the use of a parameter estimation method (Gauss-Newton with the Levenberg modification). The storage stability data give the concentration of (P+ Pl) and L as a function of time.

10 Reaction lll is much faster than the other reactions and equilibrium is assumed in the calculations. Reaction IV is excluded from the system to reduce the number of parameters thereby 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 protease molecules) is 5 present, the concentration of inhibitor may be assumed, as a reasonable approximation, to be constant.

The specific values of the reaction rate constants are somewhat sensitive to small variations in the ciata, but the sensitivity is reduced significantly by giving the results relative to the value for boric acid. The use of boric acid [as well as 20 closely related or equivalent substances such as alkali metal borates (e.g. borax) and boric oxide] for the purpose of inhibiting, in particular, proteases is wellknown (see, e.g., EP 0 451 924 A2), but the low inhibitory strength in combination with the generally rather poor solubility of such substances (which is further illustrated for boric acid in the working examples herein; vide infra) 25 renders 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 =- ~
CA 0220870~ 1997-06-25 W O96/21716 PCT~DX~G~ CD~

of the invention should be such as to still give rise to good enzyme stabilization when the liquid composition is incorporated as a component of a multi-component composition]. Thus, compounds such as boric acid, alkali metal borates (e.g. borax) and boric oxide will generally be excluded as inhibitors in the 5 context of the present invention.

An "improvement factor" may be defined as follows:

Ki (boric acid) IFj =
Ki ( inhibitor) 10 IFj defined in this manner thus provides a measure of the inhibition efficiency of a given inhibitor relative to boric acid.

b) Determination of Kj: The inhibition constant Kj may be determined by using standard methods; for reference see Keller et al, Biochem. Biophys. Res. Comm.
176, 1991, pp.401-405; J. Bieth in Baver-Sym~osium "Proteinase Inhibitors", 15 pp. 463-469, Springer-Verlag, 1974, and Lone Kierstein Hansen in "Deter-mination of Soecific Activities of Selected Detergent Proteases usin~ Protease Activitv. Molecular Weights, Kinetic Parameters and Inhibition Kinetics", PhD
thesis, Novo Nordisk A/S and University of Copenhagen, 1991. The determination may, if desired, be performed in the presence of non-enzyme 20 components (surfactant(s), etc.) of a detergent composition.

Conditions appropriate for determining Kj values for inhibition of enzymes will typically be achieved by employing a buffer system which provides a suitable pH
and which does not react or form complexes with the enzyme(s) or inhibitor(s) in question. A suitable buffer system for many enzyme/inhibitor interactions will, 25 for example, be a glycyl-glycine buffer at a mildly alkaline pH, e.g. pH 8.6, and at ambient temperature (typically 25~C).

CA 0220870~ 1997-06-2~

Exr~erimental section The invention is further illustrated and substantiated in the following examples, which are not intended to be in any way limiting to the scope of the invention as claimed .

The following procedures are illustrative of suitable approaches to the synthesis of numerous types of boronic and borinic acids:

PreParation of thio~hene-3-boronic acid: 3-Bromothiophene (0.043 m) in sodium dried ether (100 ml) was cooled to -60~C. Butyllithium (30 ml of 1 M) was added 10 rapidly. The mixture was ~hen stirred for 3 minutes, thereafter tri-n-butyl borate (0.043 m) or trimethylborate (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.
Thereafter the reaction mixture was treated with hydrochloric acid (1 M) and theether layer was separated. The aqueous layer was extracted with ether (2 times 15 25 ml). The combined ether layers were extracted with sodium hydroxide (1 M).The alkaline solution was then acidified with hydrochloric acid (10%), thus pre-cipitating the desired boronic acid. The boronic acid was isolated and then recrystallized from water//3thanol and allowed to dry in air.
C4H5B02S, mpt. 163-164"C.

20 Preparation of diphenvlborinic acid: This was prepared using the above method.
The Grignard reagent was prepared from bromobenzene and Magnesium turnings.
However, two moles of Grignard reagent were used per one mole of tri-n-butylborate. The borinic acid so formed was isolated by reaction with ethanolamine thus yielding the diphenylborinic acid, ethanolamine complex 25 ((C6H5)2BO.CH2CH2NH2), which is easierto handle. Mpt.192-194~C.

CA 02208705 1997-06-2~
W O96/21716 PCT~DK96/00005 Preparation of 4-formvlphenvl-boronic acid: This compound may be prepared as described in Chem. Ber. 123 (1990), pp. 1841-1843. 4-Bromobenzaldehyde diethylacetal is reacted with magnesium turnings in tetrahydrofuran, after whichtributyl borate dissolved in ether is added and the mixture is worked up with 5 sulfuric acid.

Determination of K;

Inhibition constants, Kj, for the inhibition of the protease SavinaselM were determined using standard methods under the following conditions:

10 Substrate: Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitro-anilide =
SAAPFpNA (from Sigma, catalogue No. S-7388).
Buffer: 0.1 M Glycyl-glycine pH 8.6; 1.5 ml 15% Brij~ 35 per litre; 25~C (glycyl-glycine from Sigma, catalogue No. G-3028).
Enzvme concentration in assay:
15 Savinase~: 1 x1 o-lo - 3X1 o-lo M

The initial rate of substrate hydrolysis was determined at nine substrate concentrations in the range of 0.01 to 2 mM using a Cobas Fara automated spectrophotometer. The kinetic parameters Vmax and Km were determined using ENZFITTER (a non-linear regression data analysis program). kCat was calculated 20 from the equation Vm~X = kCat x [Eo]~ The concentration of active enzyme [Eo] was determined by active site titration using very strongly binding protein-type protease inhibitors. Inhibition constants Kj were calculated from plots of Km/kCat as a function of the concentration of inhibitor. The inhibitors were assumed to be 100% pure, and the molar concentrations were determined from weighed 25 amounts and molecular weights.

CA 0220870~ 1997-06-2~
Wo 96121716 PcT~ 1Gloooos The resulting values of the inhibition constants K; for a series of boronic acidenzyme stabilizers (inhibitors) tested are listed below, together with values of the ratio [I]t/K; for some of these inhibitors.

Table 1.
5 Inhibition constants and ~llt/Kj values for the inhibition of SavinaseTM bv different boronic acid derivatives. Boric acid is included for comparison Inhibitor 103 x Kj [I]t/K;
(K; in mol/l) 2% 1.5%
Boric Acid 1 0 Phenyl-boronic acid 0.1 1 1490 0 3-Acetamidophenyl-boronic acid 0.09 1240 930 Benzofuran-2-boronic acid 0.03 4115 4-Formylphenyl-boronic acid 0.03 4445 3335 Thiophene-2-boronic acid 0.14 Thiophene-3-boronic acid 0.17 Naphthalene-1-boronic acid 0.13 2-Formylphenyl-boronic acid 0 . 2 3-Formylphenyl-boronic acid 0.07 % w/w of inhibitor in solution.

CA 0220870~ 1997-06-2~
WO 96/21716 PCTIDK~ ~5 Storaae stability tests in liauid detergent In the following, reference is made to KNPU ("Kilo Novo Protease Units" in relation to the SavinaseW preparations, and to KLU ("Kilo Lipase Units") in relation 5 to LipolaseTM preparations. One KNPU corresponds to 2.53 mg (ca. 9.4-10-5 mmol) of pure enzyme protein. Reference may be made to a pamphlet, AF 220/1-GB (available on request from Novo Nordisk A/S, Bagsvaerd, Denmark), concerning determination of SavinaseW activity.

One Lipase Unit (LU) is defined as the amount of enzyme which, under standard 10 conditions (30.0~C, pH 7.0; with Gum Arabic as emulsifier and tributyrin as substrate) liberates 1 ~mol of titrable butyric acid per minute. A pamphlet (AF
95/5) describing this analytical method in more detail is available upon requestfrom Novo Nordisk A/S, Bagsvaerd, Denmark. One KLU corresponds to 0.2 mg (ca. 6.7 ~ 1 o-6 mmol) of pure enzyme protein.

5 Various protease inhibitors were tested in storage stability (enzyme activity retention) tests in liquid detergent, using the method described above, under conditions as summarized below. A 4 ~/O w/w solution of each inhibitor (except boric acid) in mono-propylene glycol (MPG) was prepared~ Boric acid is poorly soluble in the medium in ques~ion, and was therefore dissolved in the final 20 detergent formulation (to give a boric acid concentration of 1% w/w therein) rather than in the liquid enzyme/inhibitor composition.

First test series: For the first test series, equal parts by weight of inhibitorsolution and of SavinaseW 16.0 L, Type EX (liquid protease preparation containing 16 KNPU of protease per gram; Novo Nordisk A/S, Bagsvaerd, Denmark) were 25 mixed, giving Savinase~ 8 L EX preparations each containing 2% w/w of inhibitor. The storage stability results for detergent compositions each containing 1% w/w of such a Savinase~/inhibitor preparation, 98% w/w of "detergent base _ _ CA 0220870~ 1997-06-2~

wo 96r21716 PCT/I)~96/OOOOS

I" (videinfra) and 1 % w/w of LipolaseTM 100 L, Type EX (liquid lipase preparation containing 100 KLU of lipase per gram; Novo Nordisk A/S, Bagsvaerd, Denmark) were as follows:

% Residual LipolaserU activities ~storage at 30~C):

Inhibitor Period of storage (days) 10 None 34 6 0 1% Boric acid 82 52 21 Phenyl-boronic acid 79 46 17 Benzofuran-2-boronic acid 83 54 24 4-Formylphenyl-boronic acid 86 64 41 No Savinase~ or inhibitor 88 72 55 % Residual SavinaseTM acltivities (7 days storage at 30~C):

Inhibitor % Activity None 1 0 1% Boric acid 74 Phenyl-boronic acid 67 Benzofuran-2-boronic acid 76 25 4-Formylphenyl-boronic acid 85 The above results demonstrate, infer alia, that the retention of LipolaselM and Savinase~ activities in the detergent composition is significantly improved by the presence of the inhibitors.

CA 0220870~ 1997-06-2~

Second test series: For the second test series, Savinase~ 16.0 L EX, 4% w/w inhibitor in MP~i, and pure MPG were mixed in a weight ratio of 50:37.5:12.5, respectively, giving SavinaselM 8 L EX preparations each containing 1.5% w/w of inhibitor. The storage stability results for detergent compositions each 5 containing 1% w/w of such a SavinaseW/inhibitor preparation, 98% w/w of "detergent base ll" (vide infra) and 1 % w/w of Lipolase~ 100 L, Type EX were as follows:

% Residual LipolaserM activities (storage at 30~C):

10 Inhibitor Period of storage (days) None 53 8 0 15 4-Formylphenyl-boronicacid 58 41 28 3-Acetamidophenyl-boronic acid 24 9 5 No Savinase~ or inhibitor 76 67 55 Third test series: SavinaselM 8 L EX preparations each containing 1.5% w/w of 20 inhibitor were prepared in the same manner as for the second test series, above.
The storage stability results for detergent compositions each containing 1% w/w of such a SavinaseTM/inhibitor preparation, 98% w/w of "inactivated" OMO~
Micro (vide infra) and 1% w/w of LipolaseTM 100 L, Type EX were as follows:

CA 0220870~ 1997-06-2~

WO 96/21716 PCTlDK9~ 5 % Residual Lipolase~ activities (storage at 35~C):

Inhibitor Period of storage (days) None 90 88 85 4-Formylphenyl-boronic acid 98 99 99 No SavinaselM or inhibitor 99 99 99 Fourth test series: Essentially as for the third series, above, with the exceptions that a storage temperature of 30~C was employed, and inactivated OMOTM micro was replaced with "detergent base lll" (vide jnfra).

~/0 Residual Lipolase~ acl:ivities (storage at 30~C):

Inhibitor Period of storage (days) 20 None 45 19 4-Formylphenyl-boronic acid 32 20 17 No Savinase~ or inhibitor 39 28 24 The latter results demonstrate, inter alia, that the retention of Lipolase~ activity 25 in the various detergent compositions is significantly improved by the presence of the inhibitors.

WO 96/21716 PCT/I)K96/00005 Composition of deterqent base I (US tvpe):

Component % w/w (as pure components) Nansa~ 1 1 69/p 1 0. 3 (Linear Alkylbenzene Sulfonate, LAS) BerollM 452 3.5 (Alkyl Ether Sulfate, AES) Oleic acid 0.5 Coconut fatty acid 0.5 Dobanol'M 25-7 6.4 (Alcohol Ethoxylate, AEO) 0 Sodium xylenesulfonate 5.1 Ethanol 0.7 MPG 2.7 Glycerol 0.5 Sodium sulfate 0.4 15 Sodium carbonate 2.7 Sodium citrate 4.4 Citric acid 1.5 Water to balance WO 96/21716 PCTIDK~)G~ 5 Composition of deters~ent base il:

Component % w/w Nansa~ 1169/p 10.30 SulfatollM NA/25 1.40 Oleic acid 7.05 Coconut fatty acid 7.05 DobanolTM 25-7 13.10 0 Triethanolamine 5.50 NaOH, 40% 2.00 Sodium xylenesulfonate 1.00 Ethanol 4.90 MPG 2.70 5 Sodium sulfate 0.20 Sodium citrate 1.40 Dequest~ 2060 S 0.40 Water to balance CA 0220870~ 1997-06-25 Composition of detergent base lll:

Component % w/w Nansa'M 11 69/p 7.00 Oleic Acid 0.45 Coconut fa~ty acid 0.45 LutensollM AO4 3.80 MPG 0.50 0 Glycerol 4.60 Na5P3010 H20 22.60 Na2SiO3 5H2O 1.70 Sodium carbonate 1.43 Water to balance 15 OMOlM Micro was a retail product purchased in a Danish supermarket. The enzyme content therein was inactivated by treatment in a microwave oven (85~C, 5 minutes).

CA 0220870~ 1997-06-2~

WO 96/21716 PCT/I)K96~0000S

Examples of liquid enzyme/inhibitor compositions Some examples of iiquid enzymelinhibitor compositions according to the invention are as follows (all compositions were solutions of acceptable 5 appearance):

1) 0.1 9 4-bromophenyl boronic acid + 10 g Savinase 16.0 L, Type EX: this composition has a molar inhibitor:enzyme (I:E) ratio of about 33.

2) 0.29 3,5-dichlorophenyl boronic acid + 40 9 Savinase 16.0 L, Type EX: this composition has a molar l:E ratio of about 17.

0 3) 0.1 9 5-chlorothiophene-2-boronicacid + 10 9 Savinase 16.0 L, Type EX: this composition has a molar l:E ratio of about 40.

4) 0.1 9 phenyl boronic acid + 10 9 Savinase 16.0 L, Type EX: this composition has a molar l:E ratio of about 54.

5) 0.2 9 phenyl boronic acid + 10 9 Savinase 16.0 L, Type EX: this composition 15 has a molar l:E ratio of about 108.

Claims (13)

1. A liquid composition comprising:

(i) an enzyme in an amount exceeding 40 µM; and (ii) a reversible inhibitor of said enzyme in an amount effective to enhance thestorage stability of said enzyme in a multi-component composition into which said liquid composition is subsequently incorporated.

2. A composition according to claim 1, wherein said enzyme is present in an amount of from 500 µM to 5 mM.

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

4. A composition according to any one of claims 1-3, wherein the ratio [I]t/K i (as defined herein) is at least 50.

5. A composition according to claim 4, wherein the ratio [I]t/K i is the range of 250-5000.

6. A composition according to any one of the preceding claims, wherein said 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 said enzyme is a protease.

8. A composition according to claim 7, wherein said 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 enzyme inhibitor, wherein a liquid composition according to any one of claims1-8 is combined with the remaining components of said detergent composition.

10. A process according to claim 9, wherein the detergent composition is a liquid composition.

11. A detergent composition prepared by a process according to claim 9 or 10.

12. Use of a liquid composition according to any one of claims 1-8 in the manufacture of a detergent composition for laundry washing or dishwashing.

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