CN1168155A - 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 mu M; 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

The stabilization of liquid enzyme compositions

The present invention be more particularly directed to liquid enzyme compositions, it contains the enzyme of (i) one or more concentration from higher to high, (ii) one or more suitable enzyme inhibitorss, reversible inhibitor particularly, this inhibitor has very strong enzyme rejection characteristic and/or exists with the concentration that can cause strong enzyme inhibition.

The invention still further relates to:

A kind of multicomponent composition that contains enzyme, for example, with of the present invention be the liquid composition of the cleaning combination such as cleaning composition of enzyme source preparation by the stable liquid enzyme compositions of inhibitor;

A kind of preparation is such as cleaning combination, contain enzyme multicomponent method for compositions as cleaning composition and so on, wherein, described enzyme is to sneak into the form by the stable liquid enzyme compositions of inhibitor of the present invention, that is: when described enzyme is impregnated in the described multicomponent composition, for a kind of by inhibitor stable composition form; With

A kind of multicomponent composition such as cleaning combination by a kind of method preparation in back is as cleaning composition.

The invention still further relates to liquid enzyme compositions of the present invention is used to prepare or produce such as cleaning compositions contain enzyme multicomponent method for compositions, as cleaning composition.

Just contain the composition of enzyme, storage stability is well-known problem.Therefore, for instance, contain the enzyme liquid washing agent, as the subject matter that contains the liquid washing agent of proteolytic enzyme (peptase) is to guarantee to keep suitable enzymic activity at lay up period.To contain enzyme composition in order seeking to improve,, to have paid sizable effort, for example, in the composition that contains proteolytic enzyme, add proteinase inhibitor as the method for the package stability that contains enzyme composition such as liquid washing agent.

For example, all the other contents of this part relate generally to the arrestin enzyme, but this principle can be applicable to the enzyme of other type equally, as lipase, amylase, cellulase and oxydo-reductase (as peroxidase and oxydase), and each side of the present invention is not limited to and the relevant problem of usefulness (reversible) proteinase inhibitor stabilize proteins enzyme.

For instance, known boric acid and one replaces reversibly arrestin enzyme of boronic acid containing (boronic acid).At Molecular ﹠amp; Cellular Biochemistry 51,1983 has discussed the problem that suppresses serine protease, subtilisin with boronic acid containing among the PP.5-32.

As the subtilisin inhibitor, various one replaces boronic acid containing has ability far from it.The alkyl that only contains such as methyl, butyl or 2-cyclohexyl ethyl is bad inhibitor, is the poorest inhibitor with the methyl boronic acid containing.Yet, according to reports, have such as phenyl, 4-p-methoxy-phenyl or 3, it is splendid inhibitor that one of the aryl of 5-dichlorophenyl and so on replaces boronic acid containing, wherein, 3,5-dichlorophenyl one replace boronic acid containing for especially effectively a kind of (referring to Keller etc., Biochem, Biophys.Res.Comm.176,1991, PP.401-405).

Point out also that in WO 92/19707 substituted aryl boronic acid containing is beat all good reversible protease inhibitors on the 3-position with respect to boron on the aryl.Therefore, for example, " acetyl amino phenyl one replaces boronic acid containing " mentioned in following document is the special effective inhibitors of proteolytic enzyme.

The inhibition effect of irreversible enzyme inhibitor being carried out when quantitative, usually " inhibition constant " (K _i) as the index of weighing the inhibitory enzyme activity ability, usually, K _iBe defined as follows:

K _i＝[E]·[l]/[El]

Wherein (E), (l) and (El) are illustrated respectively in equilibrium concentration (being generally volumetric molar concentration), inhibitor equilibrium concentration and the enzyme-inhibitor complex equilibrium concentration of enzyme under the described condition.In this specification and claims book, adopt K _iBack a kind of definition.Therefore, lower K _iValue representation is than effective inhibitors.

In preparation or when producing multicomponent and containing enzyme composition, it is desirable to previously prepared contain enzyme and suitably the enzyme composition of (reversible) enzyme inhibitors as the enzyme source.Especially common and advantageously, at least a enzyme that contains high concentration in the previously prepared enzyme composition, and several enzymes that may contain the above-mentioned type, because so, can make large volume or a large amount of enzyme multicomponent composition that contains to use smaller size smaller or previously prepared enzyme/inhibitor combination in a small amount to prepare, but also have other advantage.

In addition, if can be by just guaranteeing to have gratifying stability at the described enzyme of lay up period of finished product multicomponent composition with original previously prepared enzyme/inhibitor combination form and the inhibitor that the consumption that mixes the amount in the said composition adds enzyme, obviously be favourable, that is: at this multicomponent composition (for example, cleaning composition) needn't contain other stablizer (but, also can add other stablizer in case of necessity) in.

And in this previously prepared enzyme/inhibitor combination, the stability prediction of described enzyme is very high under the condition that has its inhibitor.Therefore, estimate that such various previously prepared composition can store considerable time after producing, and its enzymatic activity can be unobvious-or do not have unacceptable loss at least.

Therefore, the present invention relates to a kind of liquid composition, it comprises: (i) a kind of consumption surpasses the enzyme of 40 μ M; (ii) a kind of irreversible enzyme inhibitor, its consumption is for effectively improving the package stability of this enzyme when this liquid composition is impregnated in a kind of multicomponent composition (as cleaning composition, resembling liquid detergent compositions) subsequently.

For instance, liquid composition of the present invention can mainly be an aqueous composition, or mainly is that anhydrous composition (for example, contains a high proportion of one or more compositions anhydrous but the common mixable solvent of water, this solvent is generally organic liquid, as alcohol or glycol etc.).Liquid composition of the present invention itself is non-cleaning composition usually.

Except that cleaning composition (as laundry and wash the dish composition), relevant with content of the present invention, other type of forms of liquid compositions contains enzyme multicomponent composition and comprises: the composition that is used to clean tooth, contact lens or crust (as slaughterhouse or food processing plant); The composition depilation and/or the degreasing of animal hides (as be used for) that is used for leather industry; The composition that is used for fabric desizing; Wash the denim fabric to obtain the composition of " granite-wash " outward appearance with being used to.The purposes that this composition is used for this type of purpose belongs to protection scope of the present invention.

Enzyme

The enzyme classification that is adopted in specification sheets of the present invention and claims number (EC number) meets nomenclature (Recommendation (1992) of the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology of international biological chemistry and the recommendation of the molecular biology council, Academic Press Inc., 1992).

Liquid composition of the present invention contains a kind of enzyme at least.The enzyme that is suitable for comprises all commercially available enzymes.Special involved enzyme comprises that being selected from proteolytic enzyme (is peptase, EC3.4), amylase (branch is gone under the EC3.2.1), lipid acid (be included in EC3.1.1.3 below enzyme), cellulase (comprising EC3.2.1.4) and oxydo-reductase (EC1), (comprise the enzyme below the EC1.10.3 as peroxidase (EC1.11) and oxydase, as laccase (EC1.10.3.2)) enzyme, and the arbitrary composition of above-mentioned enzyme.Also comprise mixture (as different proteolytic enzyme, the mixture of different lipase etc.) from the enzyme of same classification.

The amount of enzyme is different because of type and this liquid composition purposes in the future of enzyme in the liquid composition.Generally, the consumption of all enzymes that had all is advisable to account for liquid composition weight 0.2-50% (W/W) (calculating based on pure enzyme protein), is generally 0.5-25%W/W, as 1-10%W/W, for example, accounts for the 2-8% (W/W) of liquid composition.Represent that with concentration unit the preferred concentration range for of enzyme is the about 20mM of about 50 μ M-in the liquid composition of the present invention, is generally 100 μ M-10mM, as 500 μ M-5mM, for example, 750 μ M-3mM (in the mole number of pure enzyme protein).

Proteolytic enzyme.Anyly be applicable to that the proteolytic enzyme (proteolytic ferment) in the liquid composition all can adopt.Suitable proteolytic enzyme comprises the proteolytic enzyme that comes from animal, plant or microorganism, particularly come from microorganism and mutant (modification) chemical production or protein engineering (genetic engineering), the aminoacid sequence of such relatively a kind of enzyme, there is one or several amino acid to be replaced in this mutant, insert and/or disappearance, and have proteolytic activity.For example, this proteolytic enzyme can be the Serine peptase, is preferably alkaline microbial protease or tryptase.The example of Sumizyme MP is a subtilisin, particularly come from the subtilisin of Bacillus (Bacillus), as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (being disclosed among the WO 89/06279).The example of commercialization Bacillus subtilisin has Alcalase ^TM, Savinase ^TM, Esperase ^TMAnd Durazym ^TMGoods all can be bought from Novo Nordisk A/S.Some above-mentioned proteolytic enzyme goods such as Alcalase ^TM, Esperase ^TMAnd Savinase ^TMCan liquid form provide (as, Savinase ^TM16.0L, Type DX and Type EX), this form extremely is fit to (seeing below) in the preparation proteolytic enzyme liquid composition that contains of the present invention.

The tryptase examples of proteases comprises that trypsin is as deriving from the trypsinase of pig or ox) and be disclosed in neurospora (Fusarium) proteolytic enzyme among the WO 89/06270.

Amylase.Any amylase (amylolytic enzyme) of liquid composition that is applicable to all can adopt.Suitable amylase comprises the amylase that comes from bacterium and fungi, and chemical process mutant (modification) that produce or protein engineering (genetic engineering), compare with the aminoacid sequence of such a kind of enzyme, there is one or several amino acid to be replaced, insert and/or lack in the described modification, and has amylolytic activity.For example, amylase comprises α-Dian Fenmei (EC 3.2.1.1), and the α-Dian Fenmei as obtaining in the strain by bacillus licheniformis (B.Licheniformis) sees british patent specification No.1 for details, 296,839.A kind of very suitable α-Dian Fenmei is Termamyl ^TM, this kind of enzyme (especially liquid product) can be available from Novo Nordisk A/S.

Lipase.Any lipase of liquid composition that is applicable to all can adopt.Suitable lipase comprises the lipase that comes from bacterium and fungi, and by chemical process mutant (modification) that produce or protein engineering (genetic engineering), the aminoacid sequence of such relatively a kind of enzyme has one or several amino acid to be replaced, insert and/or lack in this mutant.A kind of lipase that very is suitable for is to come from the gene of dredging cotton shape humicola lanuginosa (Humicola lanuginosa) by the clone, and this gene of expression obtains in aspergillus oryzae (Aspergillus oryzae), as described in the EP 0,258 068, this kind of enzyme (particularly liquid product) can obtain from Novo Nordisk A/S, and commodity are called Lipolase ^TM

Cellulase.Any cellulase (cellulolytic enzyme) of liquid composition that is applicable to all can adopt.Suitable cellulase Bao Tuo comes from the cellulase of bacterium and fungi, and chemical process mutant (modification) that produce or protein engineering (genetic engineering), the aminoacid sequence of such relatively a kind of enzyme, one or several amino acid in the described mutant is replaced, is inserted and/or lacked, and has cellulolytic activity.For example, suitable cellulase is disclosed in US 4,435, in 307.A kind of very suitable cellulase is by the bacterial strain generation of Humicolainsolens, can buy from Novo Nordisk A/S, and its commodity are called Celluzyme ^TM

Peroxidase.Any liquid composition that is applicable to all can adopt as the peroxidase of liquid detergent compositions.At this, suitable peroxidase comprises the peroxidase that comes from plant, bacterium and fungi, and chemical process mutant (modification) that produce or protein engineering (genetic engineering), the aminoacid sequence of such relatively a kind of enzyme, there is one or several amino acid to be replaced, insert and/or lack in the described mutant, and has peroxidase activity.The example of suitable peroxidase is for a bacterial strain that comes from Coprinus (Coprinus) (as Coprinus cinereus (C.cinerius) or long root ghost umbrella (C.macrorhizus)) or the peroxidase that comes from a bacterial strain of Bacillus (bacillus pumilus (B.pumilus)), particularly as the described a kind of peroxidase of PCT/DK90/00260.

Oxydase.Any liquid composition that is applicable to all can adopt as the oxydase of liquid detergent compositions.Here, suitable oxydase comprises the oxydase that comes from bacterium and fungi, and with chemical process mutant (modification) that produce or protein engineering (genetic engineering), the aminoacid sequence of such relatively a kind of enzyme, one or several amino acid on the described mutant is replaced, is inserted and/or lacked, and has oxidase activity.Suitable oxidasic example is for coming from the laccase of Aspergillus, the mould genus of arteries and veins spore (as coarse arteries and veins spore mould (Neurospore crassa)), trametes (as fine hair shape bolt bacterium (Tramete villosa)) or myceliophthora (Myceliophthora) (as the thermophilic silk mould (M.thermophila) of ruining).

Enzyme inhibitors (enzyme stabilizers)

Liquid composition of the present invention contains a kind of irreversible enzyme inhibitor at least, and usually, this inhibitor is the inhibitor that is present in a kind of enzyme in the liquid composition at least.Therefore, for instance, liquid composition of the present invention is except that the reversible inhibitor that contains a kind of first type enzyme and this kind of enzyme, also contain second, third kind ... etc. the enzyme of type (as in the enzyme of above-mentioned type a kind of), wherein one or more are not attended by its corresponding reversible inhibitor.

Should be noted that at this, it is fully feasible preparing a kind of liquid enzyme compositions that contains one or more enzymes, its consumption is as described herein, but said composition is not the inhibitor that contains above-mentioned one or more enzymes, but containing the inhibitor of the enzyme of another kind of type, this liquid composition will contact such enzyme subsequently.One of its example is to contain the liquid composition that lipase is unique enzyme and a kind of proteinase inhibitor; This proteinase inhibitor can protect described lipase to avoid the degraded of the proteolytic enzyme that will have a mind to or touch unintentionally subsequently.

The character of used inhibitor and consumption especially depend on character and the concentration that is present in the enzyme in the composition, and depend on the purposes of the said composition of imagination.To be described further this below.

About reversible inhibitor (and the relevant K that can use together with the enzyme of various types of/type in the liquid composition of the present invention _iValue), can be with reference to following document: H.Zollner, Handbook ofEnzyme Inhibitors (A part and B part), the 2nd edition, VCHVerlagsgesellschaft mbH, Weinheim, Germany, 1993, wherein enumerated a large amount of inhibitor.Other pertinent literature comprises: S.Patkar and F.Bjokling, Lipase inhibitors, see: Lipases-their Structure, Biochemistry and Application, P.Woolley and S.B.Petersen work, Cambridge University Press, Cambridge 1994.The example of the reversible inhibitor of selected correlation type is as follows:

Proteinase inhibitor.A kind of reversible protease inhibitors of interested especially type replaces boronic acid containing (R ' B (OH) by one ₂) and two replacement boronic acid containings [R ' R " B (OH)] form (wherein, R ' and R " be organic substituent, as aryl or the heterocyclic substituent that was replaced by selectivity), some examples are wherein mentioned hereinbefore.About other example of the compound of this type can be selected from the compound of mentioning in WO 92/19707, EP0478050A1 and EP0511 456A1.

Interested this compounds can be referring to the compound that is disclosed among the WO 95/02046 (this patent is unexposed in the application's priority date fashion), and comprises the compound of following general formula:

Wherein, R ₁Be the fused aromatic ring structure that a selectivity replaces, on this ring, contain 14 or 18 carbon atoms; Or a monocycle or the fused aromatic heterocycle structure that selectivity replaces, on this ring, contain 17 carbon atoms at most; Or the monocycle that replaces of selectivity or condense the quinoid ring structure, on this ring, contain 18 carbon atoms at most;

R ₂Structural formula be:

X wherein is identical or different, and is selected from hydrogen, C ₁-C ₆The C of alkyl, replacement ₁-C ₆The derivative of alkyl, aryl, substituted aryl, hydroxyl, hydroxy derivatives, halogen, amine, alkylated amines, amine, nitro, thiol, sulfur hydroxy derivative, aldehyde, acid, acid salt, ester, sulphonate or phosphoric acid ester, and o, p and q can be identical or different, and can respectively do for oneself 0,1 or 2; M and n can be identical or different, and can respectively do for oneself 0 or 1; R ₃And R ₁Identical or different, and can be selected from R ₁, or R ₃Be hydroxyl, or R ₁And R ₃Be monocycle or bicyclic aromatic ring structure that selectivity replaces.

Hereinbefore, optionally the ring structure of Qu Daiing is such: the substituting group on the ring structure can choose at random, but preferably be selected from hydrogen, C ₁-C ₆The C of alkyl, replacement ₁-C ₆The derivative of alkyl, aryl, substituted aryl, hydroxyl, hydroxy derivatives, halogen, amine, alkylated amines, amine, nitro, thiol, sulfur hydroxy derivative, aldehyde, acid, acid salt, ester, sulphonate and phosphoric acid ester.

Can replace boronic acid containing and two with method preparation one well-known to those skilled in the art and replace the boronic acid containing derivative, for example, adopt one of following method:

A) with catecholborane (1,3,2-benzodioxaborole) or dichloromethane borine-methyl-sulfide title complex be the hydroboration agent to unsaturated material, i.e. alkene and the alkynes hydroboration of carrying out; Referring to 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) Grignard reagent and tri-n-butyl borate or trimethyl borate reaction, what hydrolysis was produced thus then one replaces the boracic acid esters; Referring to F.R.Bean, J.R.Johnson, Jaces 54,1932, pp 4415-4425; And S.H.Dandegaonher, S.P.Ingleshwar, Journal of ShivasiUniversity6,1932, pp.11-13.The raw material that bromine replaces is not purchased, can be synthetic easily with two steps: use LiAlH ₄The reduction corresponding carboxylic acid, and then use CBr ₄Handle.

C) organolithium reagent and butyl borate reaction; Referring to S.O.Lauessen, pp.387-395, Thiophene Chemistry, Part 7; And D.Florentin, B.Roques, C.R.Acad.Sci.Paris, t.270 (on May 11st, 1970), PP.1608-1610.

D) the method preparation two according to above-mentioned b replaces the boronic acid containing derivative, and different is that the Grignard reagent that is adopted and the ratio of boric acid ester are 2: 1.

E) adopt standard method well-known to those skilled in the art can obtain the provide protection of substitution in ring or functional group.

Such other compound of interest can be selected from the naphthalene one that is disclosed among the WO 95/29223 (the priority date fashion in the application is unexposed) and replace boronic acid containing, and comprises the compound with following general formula:

Wherein, R ₁, R ₂, R ₃, R ₄, R ₅, R ₆And R ₇Identical or different, and be selected from hydrogen, C ₁-C ₆The C of alkyl, replacement ₁-C ₆The derivative of alkyl, aryl, substituted aryl, hydroxyl, hydroxy derivatives, halogen, amine, alkylated amines, amine, nitro, sulfenyl, sulfenyl derivative, aldehyde, acid, acid salt, ester, sulphonate and phosphoric acid ester.

Naphthalene one replaces the same available method preparation well-known to those skilled in the art of boronic acid containing derivative, for example, adopts Grignard preparation as described below:

Slowly be added drop-wise to preparation Grignard reagent in the magnesium that in sodium exsiccant ether, rotates by the suitable bromonaphthalene raw material that will be dissolved in the sodium exsiccant ether.Add little iodine crystallization and promote above-mentioned reaction.

The trimethyl borate or the tri-n-butyl borate that will be dissolved in the sodium exsiccant ether are cooled to-70 ℃, Grignard reagent are added drop-wise in the above-mentioned solution with 2 hour time, should remain on-70 ℃ by organic B solution simultaneously, and continue to stir.Allow reaction mixture rise again, spend the night, be hydrolyzed by dripping cold dilute sulphuric acid simultaneously to room temperature.Separate the ether layer, and use the ether aqueous layer extracted.Merge the part that contains ether, remove and desolvate.Making resistates is strong basicity, and all methyl alcohol or the butanols removing thus to be produced.With this basic solution cooling, and by removing by filter resulting purpose one replacement boronic acid containing crystallization.Preferably all products recrystallize from distilled water is come out.

Can prepare naphthalene one replacement boronic acid containing by the direct lithiumation of naphthalene and/or the lithiumation of bromide.

Any ring replaces or the protection method that all available those skilled in the art were familiar with of functional group realizes.

In the compound of the above-mentioned type, the related concrete replacement boronic acid containing of content of the present invention comprises: cumarone-2-one replaces boronic acid containing; Phenyl one replaces boronic acid containing; 4-bromophenyl one replaces boronic acid containing; 4-formylphenyl one replaces boronic acid containing; 3-acetylamino phenyl-one replaces boronic acid containing; 3,5-dichlorophenyl-replaces boronic acid containing; 5-chlorothio benzene-2-one replaces boronic acid containing; Naphthalene-1-one replaces boronic acid containing; Naphthalene-2-one replaces boronic acid containing; Replace boronic acid containing with 6-hydroxyl naphthalene-2-one.In the table (seeing below) that in the example 2 of this paper, provides, listed can be used for of the present invention, such other particular compound.

Can its sour form (as be dissolved in the suitable water miscibility organic solvent, in single propylene glycol), perhaps, necessary words (as reason) owing to solubility with the form of salt (as an alkali metal salt, for example sodium salt or sylvite), replace inhibitor that boronic acid containing or two replaces the boronic acid containing types with one and add or mix in the liquid composition of the present invention.

Reversible amylase inhibitor.Can be used for relevant reversible amylase inhibitor of the present invention, can in the compound that is called as " acarbose (acarbose) " type, seek (that is: containing an acarviosine part and the unitary false oligosaccharides of one or several maltose).As inhibitor, particularly other compound of alpha-amylase inhibitor comprises maltose and trisaccharide maltose.Such as other compound of methyl-α-glucoside and cyclisation starch, as hexanaphthene and/or cycloheptaamylose useful as beta-diastatic reversible inhibitor.

Reversible cellulase inhibitor.Can be used for potential reversible cellulase inhibitor of the present invention and comprise 4-sulfydryl-cell-oligosaccharide (for example, referring to C.Schou etc., Journal ofCarbohydrate Chemistry 12 (1993) PP.743-752).

Reversible lipase inhibitor.Can be used for reversible lipase inhibitor of the present invention and comprise that being selected from an above-mentioned and/or following replacement boronic acid containing and two replaces boronic acid containings (phenyl one that replaces as selectivity replaces boronic acid containing).

Enzyme suppresses degree

The ideal that is present in the enzyme in the liquid composition of the present invention suppresses degree (intensity, degree), depends on also that except that other factors said composition will application aims.Generally, the selection to the mol ratio of reversible inhibitor and enzyme is such: have an inhibitor molecules at least on each avtive spot of relevant enzyme, therefore, require the inhibitor (I) and the mol ratio of enzyme (E) to be at least in the nature of things 1: 1.But,, also can adopt lower I for certain purpose: the E mol ratio, 0.8: 1 according to appointment, about 0.7: 1, about 0.6: 1, even about 0.5: 1.Yet in the present invention, the mol ratio of inhibitor that will adopt and enzyme is at least 5 usually, as is at least 15.In some occasion, I: the E mol ratio may be at least 50 or be at least 100, even adopts higher mol ratio.

The mol ratio of inhibitor and enzyme can be selected, and particularly considers to contain at the multicomponent based on liquid composition of the present invention the per-cent of needed free (not being suppressed) enzyme in the practical application of enzyme composition.Therefore, for instance, when a kind of clothes washing composition is used to do washing, obviously enough high-load resolvase (as proteolytic enzyme or lipase) need be arranged in washing medium (being generally the water that has added relevant cleaning composition).

Thus, but the inhibition constant (defining as indicated above) of the retroactive inhibition that important parameters is a kind of enzyme.Therefore, for instance, when liquid composition of the present invention is used in the cleaning composition, a kind of inhibition constant (K of reversible inhibitor of enzyme _i) when mol/l (M) represents in a usual manner, usually with 3 * 10 ^-8M≤K _i≤ 1.2 * 10 ^-2M is advisable, as 3 * 10 ^-8M≤K _i≤ 1 * 10 ^-2M.But, normally such about detersive enzyme better " suppressing window (window of inhibition) ": the inhibitor relevant with a kind of concrete enzyme can produce and be equivalent to 3 * 10 ^-7M≤K _i≤ 1 * 10 ^-3The inhibition degree of M is as 4.3 * 10 ^-7M≤K _i≤ 4.5 * 10 ^-4M.

Usually, to contain the enzyme liquid composition be such in preferred the present invention:

(a) total mol concentration that is present in the irreversible enzyme inhibitor I in the said composition (can be expressed as (I) _t); With

(b) the inhibition constant (K that is present in the enzyme in the said composition that can reversibly suppress by above-mentioned inhibitor _iRepresent with mol/l) between ratio be at least 50 (i.e. [I] _t/ K _i〉=50), as, be at least 100, or be at least 250 usually.In liquid composition of the present invention, [I] _t/ K _iThe actual upper bound of ratio is about 10000 (promptly 10 usually ⁴).In a lot of occasions, [I] _t/ K _iRatio is advisable in the 250-2500 scope in the 250-5000 scope usually.

Can be [I] _t/ K _iThe value of ratio is considered as an index of liquid composition of the present invention " inhibition ability ".For example, by the medium potent inhibitor of adding in liquid composition to lower concentration, or the more weak inhibitor of adding higher concentration, can obtain high ratio, promptly high " inhibition ability ".

When the mole number of the repressed relatively enzyme E of mole number of inhibitor I exceeds more for a long time, most of inhibitor is generally free form, that is: do not combine with enzyme.Dissociate (non-binding) inhibitor (with K with (I) expression _iRelevant; See above), under this condition [I] _t/ K _iThan approximating [I]/K greatly _iRatio, i.e. [I] _t/ K _i≈ [I]/K _i

Here it is to be noted that the liquid composition that contains enzyme and irreversible enzyme inhibitor of the present invention can be used proper method drying (as lyophilize or spraying drying).Exsiccant enzyme/inhibitor product can be pulverized (as by grinding) then, and suspend or mix and starch in a kind of on-aqueous liquid vehicle with proper concn, as nonionogenic tenside (as Softanol available from BP ^TM) in, to form a kind of pulpous state product.

Washing composition

For cleaning composition, common target is at least 50% the inhibition that obtains selected enzyme (as proteolytic enzyme) in the cleaning composition, and the amount of resolvase is equivalent at least 50% of total enzyme amount in the washing medium.

The cleaning composition that has added liquid composition of the present invention, dezymotize with inhibitor outside, also comprise tensio-active agent, and be generally liquid detergent compositions.For example, this cleaning composition can be the laundry cleaning composition or wash dishwashing and wash composition.

Liquid detergent compositions can be aqueous, for example contains usually up to 70% water and the organic solvent of 0-30%, or anhydrous basically.

Described cleaning composition will contain one or more tensio-active agents, and every kind of tensio-active agent can be anionic, non-ionic type, cationic or amphoteric ion type.This washing composition contains the anion surfactant of 0-50% usually, as linear alkylbenzene sulfonate (LAS), alpha-alefinically sulfonate (AOS), alkyl-sulphate (aliphatic alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary sulfonated alkane (SAS), alpha-sulfo fatty acid methyl ester, alkyl or alkenyl succsinic acid or soap.The nonionic surface active agent that can also contain 0-40%, as fatty alcohol ethoxylate (AEO or AE), Fatty Alcohol(C12-C14 and C12-C18) propoxylated glycerine, carboxylated fatty alcohol ethoxylate, nonyl phenol ethoxylate, alkyl poly glucoside, alkyl dimethyl amine oxide, ethoxylated fatty acid single ethanol amide, fatty monoethanol amide or polyhydroxy alkyl fatty acid amide (for example, described in the WO 92/06154).

Usually, the washing auxiliary detergent that contains 1-65% in this washing composition, but some is washed the dish washing composition even can contain washing auxiliary detergent or coordination agent up to 90%, as zeolite, diphosphate, triphosphate, phosphonate, Citrate trianion, nitrilotriacetic acid(NTA) (NTA), ethylenediamine tetraacetic acid (EDTA) (EDTA), diethylene triaminepentaacetic acid(DTPA) (DTMPA), alkyl or alkenyl succsinic acid, soluble silicate or layered silicate (as SKS-6, available from Hoechst).

Washing auxiliary detergent can be divided into phosphorous type and non-phosphorous type.The example of inorganic phosphor-contained alkaline washing auxiliary agent comprises water-soluble salt particularly alkali metal pyrophosphate, orthophosphoric acid salt, polyphosphate and phosphonate.The example of non-inorganic phosphor-contained auxiliary agent comprises water soluble alkali metal carbonate, borate and silicate, and stratiform two polysilicates and all kinds water-insoluble crystal type or amorphous silicon aluminate, is foremost representative with zeolite wherein.

The example of suitable organic additive comprises an alkali metal salt, ammonium salt or the substituted ammonium salt of following acid: succsinic acid, propanedioic acid, lipid acid propanedioic acid, lipid acid sulfonic acid, carboxyl methoxyl group succsinic acid, poly-acetate, carboxylic acid, poly carboxylic acid, aminopolycarboxylic and poly-ethanoyl carboxylic acid.This washing composition also can be not composite, promptly essentially no washing auxiliary detergent.

This washing composition can contain one or more polymkeric substance.Its example have carboxymethyl cellulose (CMC), poly-(V-Pyrol RC) (PVP), polyoxyethylene glycol (PEG), polyvinyl alcohol (PVA), polycarboxylate, as polyacrylic ester, polymaleic acid ester, toxilic acid/acrylic copolymer and methacrylic acid Lauryl Ester/acrylic copolymer.

Described cleaning composition can contain chlorine/bromine type or oxygenation type SYNTHETIC OPTICAL WHITNER.SYNTHETIC OPTICAL WHITNER can be dressing or seal.The example of inorganic chlorine/bromine type SYNTHETIC OPTICAL WHITNER has lithium salts, sodium salt or the calcium salt of hypochlorous acid or hypobromous acid and Efficacious Disinfeitant.

Described bleaching system can also contain H ₂O ₂The source if can generate bleach-activating agent blended perborate or percarbonate with the peracid such as tetraacetyl ethylene diamine (TAED) or acyloxy benzene sulfonate in the ninth of the ten Heavenly Stems (NOBS).

The example of organochlorine/bromine type SYNTHETIC OPTICAL WHITNER has heterocycle N-bromine and N-chlorine imide, as trichloroisocyanuric acid, tribromo tricarbimide, dibromo isocyanurate and DICHLOROISOCYANURIC ACID, and and water-soluble cationic, as the salt of potassium and sodium.Glycolylurea is suitable equally.Described bleaching system also can contain the peroxy acid just like acid amides, imide or sulfone class.

In washing the dish washing composition, preferred oxygen-bearing bleaching agent as the SYNTHETIC OPTICAL WHITNER of inorganic persalt type, preferably has bleach precursor or is peracetic acid compound.The exemplary of peroxy bleaching compound is the tetrahydrate and the monohydrate of alkali metal perborate, alkali metal percarbonate, persilicate and superphosphate.Preferred active material is TAED or NOBS.

As mentioned above, the enzyme of cleaning composition of the present invention is that form with liquid enzymes/inhibitor combination of the present invention adds in the described cleaning composition.

If necessary, also can add other common enzyme stable material, as polyvalent alcohol, as propylene glycol or glycerine, sugar or sugar alcohol, lactic acid, boric acid or boric acid derivatives are as aromatic borate (for example, referring to WO 92/19709 and 92/19708).

This washing composition also can contain other common cleaning ingredients, as fabric conditioner, comprise clay, deflocculate material, profoamer/suds suppressor (in dishwashing detergent, being suds suppressor), suds suppressor, corrosion inhibitor, soil-suspending agent, anti-soil dirt deposition agent, dyestuff, dewatering agent, sterilant, white dyes or spices again.

It is neutral or alkaline that its pH (recording in the aqueous solution under working concentration) is generally, as in the scope of 7-11.

The specific form of the laundry cleaning composition in the scope of the invention comprises:

1) aqueous liquid detergent composition, contain:

Linear alkylbenzene sulfonate (calculating) with acid	15????-21％
		Fatty alcohol ethoxylate is (as C 12-15Fatty Alcohol(C12-C14 and C12-C18), 7EO or C 12-15Fatty Alcohol(C12-C14 and C12-C18), 5EO)	12????-18％
Soap is as lipid acid (as oleic acid)	3?????-13％
		Alkenyl succinic acid (C 12-14)	0?????-13％
Monoethanolamine	8?????-18％
		Citric acid	2?????-8％
Phosphonate	0?????-3％
		Polymkeric substance (as PVP, PEG)	0?????-3％
Borate is (as B 4O 7)	0?????-2％
		Ethanol	0?????-3％
Propylene glycol	8?????-14％
		Enzyme (in pure enzyme protein)	0.0001-0.2％
Trace ingredients (as dispersion agent, suds suppressor, spices, white dyes)	0?????-5％

2) liquid detergent compositions of water structure, contain:

Linear alkylbenzene sulfonate (in acid)	15????-21％
		Fatty alcohol ethoxylate is (as C 12-15Fatty Alcohol(C12-C14 and C12-C18), 7EO, or C 12-15Fatty Alcohol(C12-C14 and C12-C18), 5EO)	3?????-9％
Soap is as lipid acid (as oleic acid)	3?????-10％
		Zeolite is (as NaAlSiO 4)	14????-22％
Tripotassium Citrate	9?????-18％
		Borate is (as B 4O 7 2-)	0?????-2％
Carboxymethyl cellulose	0?????-2％
		Polymkeric substance is (as PEG, PVD)	0?????-3％
The grappling polymkeric substance is as lauryl methacrylate(LMA)/acrylic copolymer; Mol ratio 25: 1; MW3800	0?????-3％
		Glycerine	0?????-5％
Enzyme (in pure enzyme protein)	0.0001-0.2％
		Trace ingredients (as dispersion agent, suds suppressor, spices, white dyes)	0?????-5％

3. aqueous liquid detergent composition, contain:

Linear alkylbenzene sulfonate (in acid)	15????-23％
		Alcohol ethoxysulfate is (as C 12-15Fatty Alcohol(C12-C14 and C12-C18), 2-3EO)	8?????-15％
Fatty alcohol ethoxylate is (as C 12-15Fatty Alcohol(C12-C14 and C12-C18), 7EO, or C 12-15Fatty Alcohol(C12-C14 and C12-C18), 5EO)	3?????-9％
		Soap is as lipid acid (as dodecylic acid)	0?????-3％
Monoethanolamine	1?????-5％
		Trisodium Citrate	5?????-10％
Hydrotropic agent (as, toluenesulfonic acid sodium salt)	2?????-6％
		Borate is (as B 4O 7 2-)	0?????-2％
Carboxymethyl cellulose	0?????-1％
		Ethanol	1?????-3％
Propylene glycol	2?????-5％
		Enzyme (in pure enzyme protein)	0.0001-0.2％
Trace ingredients (as polymkeric substance, dispersion agent, spices, white dyes)	0?????-5％

4. aqueous liquid detergent composition, contain:

Linear alkylbenzene sulfonate (in acid)	20????-32％
		Fatty alcohol ethoxylate is (as C 12-15Fatty Alcohol(C12-C14 and C12-C18), 7EO, or C 12-15Fatty Alcohol(C12-C14 and C12-C18), 5EO)	6?????-12％
Monoethanolamine	2?????-6％
		Citric acid	8?????-14％
Borate is (as B 4O 7 2-)	1?????-3％
		Polymkeric substance (as toxilic acid/acrylic copolymer, the grappling polymkeric substance is as lauryl methacrylate(LMA)/acrylic copolymer)	0?????-3％
Glycerine	3?????-8％
		Enzyme (in pure enzyme protein)	0.0001-0.2％
Trace ingredients (as hydrotropic agent, dispersion agent, spices, white dyes)	0?????-5％

5) as 1)-4) described cleaning composition, wherein, all or part of linear alkylbenzene sulfonate is by (C ₁₂-C ₁₈) alkyl-sulphate replaces.

6) as 1)-5) described cleaning composition, it contains peracid stabilization or coating, or as another composition or be used to replace above-mentioned bleaching system.

7) a kind of cleaning composition that is formulated into anhydrous washing liq contains a kind of liquid non-ionic surfactant, as straight chain alkoxylate primary alconol, an adjuvant systems (as phosphoric acid salt), enzyme and alkali.This washing composition can also contain anion surfactant and/or bleaching system.

Wash dish with containing enzyme composition with other multicomponent

The relevant example of this type of composition comprises:

1) liquid with detergent surfactant system is washed the dish composition, contains:

Nonionogenic tenside	0???????-1.5％
		Octadecyl N-cacodyl oxide amine dihydrate	0???????-5％
80 of octadecyl N-cacodyl oxide amine dihydrate and hexadecyl N-cacodyl oxide amine dihydrate: 20wt.C18/C16 mixture	0???????-4％
		70 of anhydrous octadecyl N-bis oxide (hydroxyethyl) amine and anhydrous hexadecyl N-bis oxide (hydroxyethyl) amine: 30wt.C18/C16 mixture	0???????-5％
Average degree of ethoxylation is 3 C 13-C 15Alkyl ethoxy sulfate	0???????-10％
		Average degree of ethoxylation is 3 C 12-C 15Alkyl ethoxy sulfate	0???????-5％
Average degree of ethoxylation is 12 C 13-C 15Ethoxylized fatty alcohol	0???????-5％
		Average degree of ethoxylation is 9 C 12-C 15The ethoxylized fatty alcohol mixture	0???????-6.5％
Average degree of ethoxylation is 30 C 13-C 15The ethoxylized fatty alcohol mixture	0???????-4％
		Sodium disilicate	0???????-33％
Tri sodium Phosphate	0???????-46％
		Citrate trianion	0???????-28％
Citric acid	0???????-29％
		Yellow soda ash	0???????-20％
The Sodium peroxoborate monohydrate	0???????-11.5％
		Tetrem acyl ethamine (TAED)	0???????-4％
Toxilic acid/acrylic copolymer	0???????-7.5％
		Sodium sulfate	0???????-12.5％
Enzyme	0.0001??-0.2％

2) anhydrous liq is washed the dish composition automatically, comprising:

Liquid nonionic surfactants (as fatty alcohol ethoxylate)	2.0?????-10.0％
		Alkalimetal silicate	3.0?????-15.0％
Alkali metal phosphate	20.0????-40.0％
		Liquid vehicle is selected from senior dibasic alcohol, polyoxyethylene glycol, polyoxide, glycol ether	25.0????-45.0％
Stablizer is (as phosphoric acid and C 16-C 18The part ester of alkanol)	0.5?????-7.0％
		Suds suppressor (as siloxanes)	0???????-1.5％
Enzyme	0.0001??-0.2％

3) anhydrous liq is washed the dish composition, contains:

Liquid nonionic surfactants (as fatty alcohol ethoxylate)	2.0?????-10.0％
		Water glass	3.0?????-15.0％
Alkaline carbonate	7.0?????-20.0％
		Trisodium Citrate	0.0?????-1.5％
Stable system (as the mixture of siloxanes in small, broken bits and lower molecular weight dialkyl group polyglycol ether)	0.5?????-7.0％
		The low-molecular-weight polypropylene acid polymer	5.0?????-15.0％
Clay gel thickening material (as wilkinite)	0.0?????-10.0％
		Hydroxypropyl cellulose polymer	0.0?????-0.6％
Enzyme	0.0001??-0.2％
		Liquid vehicle is selected from senior dibasic alcohol, polyoxyethylene glycol, polyoxide and glycol ether	Balance

4) thixotropic liquid is washed the dish composition automatically, comprising:

C 12-C 14Lipid acid	0???????-0.5％
		Block copolymer surfactant	1.5?????-15.0％
Trisodium Citrate	0???????-12％
		Tri sodium Phosphate	0???????-15％
Yellow soda ash	0???????-8％
		Aluminium Tristearate Micronized sterile	0???????-0.1％
Cumene sodium sulfonate	0???????-1.7％
		Polyacrylate thickeners	1.32????-2.5％
Sodium polyacrylate	2.4?????-6.0％
		Boric acid	0???????-4.0％
Sodium formiate	0???????-0.45％
		Calcium formiate	0???????-0.2％
Oxidation n-decyl phenylbenzene sodium disulfonate	0???????-4.0％
		Monoethanolamine (MEA)	0???????-1.86％
Sodium hydroxide (50%)	1.9?????-9.3％
		1, the 2-propylene glycol	0???????-9.4％
Enzyme	0.0001??-0.2％
		Suds suppressor, dyestuff, spices, water	Balance

5) liquid is washed the dish composition automatically, comprising:

Fatty alcohol ethoxylate	0???????-20％
		Alpha-sulphonic acid fatty acid ester	0???????-30％
Sodium lauryl sulphate	0???????-20％
		Alkyl poly glucoside	0???????-21％
Oleic acid	0???????-10％
		The sodium disilicate monohydrate	18??????-33％
Trisodium citrate dihydrate	18??????-33％
		Sodium stearate	0???????-2.5％
The Sodium peroxoborate monohydrate	0???????-13％
		Tetraacetyl ethylene diamine (TAED)	0???????-8％
Toxilic acid/acrylic copolymer	4???????-8％
		Enzyme	0.0001??-0.2％

6) contain the liquid of protecting bleaching particle and wash the dish composition automatically, contain:

Water glass	5???????-10％
		Tetrapotassium pyrophosphate	15??????-25％
Tri sodium Phosphate	0???????-2％
		Salt of wormwood	4???????-8％
The protection bleaching particle is as chlorine	5???????-10％
		Polymeric viscosifier	0.7?????-1.5％
Potassium hydroxide	0???????-2％
		Enzyme	0.0001??-0.2％
Water	Balance

7) as 1) and 5) the described dish composition of washing automatically, wherein, peroxyboric acid is replaced by percarbonic acid.

8) as 1) wash the dish composition automatically, it also contains a kind of Mn catalyst.For example, Mn catalyst can be to be disclosed in " Efficient manganese catalysts for low-temperaturebleaching " (Nature 369,1994, pp.637-639) one of the compound in.

" enzyme " content of above-mentioned composition can comprise existing enzyme inhibitors content.If necessary, it is not organic solvent at the above-mentioned type prescription that a kind of concrete composition that belongs to one of the above-mentioned type can contain a kind of, but it can be used as the solvent of inhibitor in the liquid enzymes/inhibitor combination of the present invention that is added in the described particular composition.

Liquid enzymes of the present invention/inhibitor combination can be added in the cleaning composition, to produce a kind of enzyme concn that often is used in the washing composition.For cleaning composition of the present invention, can add liquid enzymes/inhibitor combination of the present invention, for example, its add-on is present in the certain enzyme in this enzyme/inhibitor combination relatively, is equivalent to have in every liter of washings 0.00001-1mg (in pure enzyme protein) enzyme.

The stablizer check

Can check the inhibition effect of inhibitor in every way, be used to check the method for proteolytic enzyme/proteinase inhibitor to be illustrated by following two kinds:

A) in liquid washing agent, carry out storage stability test: liquid enzymes/inhibitor combination is added in the liquid detergent compositions of storing under the clear and definite qualifications.Measure the enzymic activity (for example, after 1,3 and 7 day, measuring) of every kind of enzyme with the functional form of time.

To suppress efficient in order calculating, to have proposed a kind of reaction mechanism by storage stability data.Below reaction has proposed a kind of mechanism that more simply, reasonably is used to contain the liquid washing agent of proteolytic enzyme (P), lipase (L) and inhibitor (I):

I) autodigestion of proteolytic enzyme:

P+P→D _P+P

II) sex change of proteolytic enzyme:

P→D _P

III) inhibition of proteolytic enzyme:

P+I??PI

IV) be subjected to the protease digestion of inhibitory enzyme

P+PI→P+D _P+I

V) be subjected to the sex change of inhibitory enzyme:

PI→D _P+I

VI) protease digestion of lipase:

P+L→P+D _L

VII) sex change of lipase

L→D _L

D wherein _PAnd D _LProteolytic enzyme and lipase for sex change (being non-activity).

Obtain the coupling differential equation of 3 expression D, L and PI inactivation by above reaction.Calculate reaction velocity constant with parameter estimation method (by the improved Gauss Newton of Levenberg method) by storage stability data.Storage stability data provides the concentration of (P+PI) and L with the functional form of time.

III is far faster than other reaction in reaction, and supposes balance in calculating.Reaction IV is excluded outside this reaction, to reduce number of parameters, so that only use a reaction velocity constant (available from equation V) to describe the stability that is subjected to inhibitory enzyme.

When having a large amount of residue inhibitor molecules (with respect to protease molecule), the concentration that can suppose inhibitor is near a rational constant.

The variation sensitivity that the occurrence of reaction velocity constant is small to data to a certain extent, but with respect to the result of boric acid, this susceptibility significantly descends.With boric acid (and closely-related material or equivalent, as alkali metal borate (as borax) and boron oxide) be used to suppress purpose, particularly the arrestin enzyme be well-known (for example, referring to EP 0,451 924 A2), but reaching the solubility of being on duty mutually generally, the low inhibition strength of this material (in following embodiment, will be described further it in conjunction with boric acid; See below), it is not suitable among the present invention (wherein basically, the inhibitor and/or its consumption that are present in the liquid composition of the present invention should be: when a kind of composition form with a kind of multicomponent composition adds described liquid composition, still can produce good enzyme stability).Therefore, go up substantially can not be as inhibitor of the present invention for the compound such as boric acid, alkali metal borate (as borax) and boron oxide.

" improving coefficient " is defined as follows:

With aforesaid method definition IFi, can provide the parameter of the inhibition effect of weighing the relative boric acid of a kind of special inhibitor.

B) measure K _i: can measure with standard method and suppress constant K _iReferring to Keller etc., Biochem.Biophys.Res.Comm.176,1991, PP.401-405; J.Bieth, Bayer-Symposium " Proteinase Inhibitors ", PP.463-469, Springer-Verlag, 1974, with Lone kierstein Hansen, " Determination of Specific Activities ofSelected Detergent Protease Using Protease Activity; Molecular Weight; Kinetic Parameters and Inhibition Kinetics ", Ph D dissertation, Novo NovdiskA/S and University of Copenhagen, 1991.If necessary, this mensuration can be carried out under the condition of the non-enzyme component (tensio-active agent etc.) that has cleaning composition.

Usually obtain to be suitable for measuring the K that enzyme suppresses with a buffering system _iThe condition of value, described buffering system can produce suitable pH and can or not form title complex with involved enzyme or inhibitor reaction.For example, being used for the interactional buffering system of many enzyme/inhibitor is glycyl-glycine buffer, and its pH is a weakly alkaline, and as pH8.6, temperature is envrionment temperature (being generally 25 ℃).

Experimental section

Following examples will the invention will be further described and checking, but these embodiment will limit as the described scope of the present invention of claims from any angle.

Example 1

Following method is that synthetic broad variety one is replaced the model that boronic acid containing and two replaces borated appropriate method:

Preparation thiophene-3-one replaces boronic acid containing: the 3 bromo thiophene (0.043m) that will be dissolved in the sodium exsiccant ether (100ml) is cooled to-60 ℃.Add fourth lithium (30ml, concentration 1M) rapidly.Stir this mixture then 3 minutes, and added tri-n-butyl borate (0.043m) or the trimethyl borate (0.043m) that is dissolved in the sodium exsiccant ether (25ml) again.Stirred this mixture 4 hours, and make it be warming up to room temperature.Use spirit of salt (1M) reaction mixture then, and separate the ether layer.With the extracted with diethyl ether water layer (2 times, 25ml).Ether layer with sodium hydroxide (1M) extraction merging.With spirit of salt (10%) acidifying basic solution, thereby make a replacement boronic acid containing precipitation of wanting.Separate one and replace boronic acid containing, and then from water/ethanol recrystallize, and be allowed to condition at air drying.

C ₄H ₅BO ₂S，mpt.163-164℃。

Preparation phenylbenzene two replaces boronic acid containing: with this material of method for preparing.Prepare Grignard reagent by bromobenzene and magnesium fragment.But, every mole of tri-n-butyl borate uses 2 moles of Grignard reagent.By replacing boronic acid containing, produce phenylbenzene two thus and replace boronic acid containing and thanomin title complex ((C with thanomin Reaction Separation formed thus two ₆-H ₅) ₂BOCH ₂CH ₂NH ₂), this product is easier to handle.Mpt.192-194℃。

Preparation 4-formylphenyl one replaces boronic acid containing: can be with being disclosed in Chem.Ber.123 (1990), and the method among the PP.1841-1843 prepares this compound.Allow 4-bromo benzaldehyde diethyl acetal and magnesium fragment in tetrahydrofuran (THF), react, add the tributyl borate be dissolved in the ether then, and with this mixture of vitriolization.

Example 2

Measure K _i

Use standard method, measure arrestin enzyme Savinase under the following conditions ^TMThe inhibition constant K _i

Substrate: succinyl--Ala-Ala-proline(Pro)-phenylalanine-right-nitro-anilide=SAAPF _PNA (available from Sigma company, classification number S-7388).

Damping fluid: 0.1M glycyl-glycine pH 8.6; Every liter of 1.5ml 15%Brig ^TM35; 25 ℃ (glycylglycine available from Sigma company, classification number G-3028).

The concentration of enzyme in the test:

Savinase ^TM：1×10-10 ^-3×10 ^-10M

Survey the substrate hydrolysis starting velocity of 9 kinds of concentration of substrate in the 0.01-2mM scope with a Cobas Fara automatic spectrophotometer.Measure kinetic parameter V with ENZFITTER (a kind of non-linear regression DAP) _MaxAnd K _mBy equation V _Max=k _Cat* [E _o] calculating k _CatBy carry out avtive spot titration determination organized enzyme (E with extremely strong binding protein proteinase inhibitor _o) concentration.By K _m/ k _CatCurve calculates with the form of inhibitor concentration function and suppresses constant K _iThe purity of supposing inhibitor is 100%, by claim weight and molecular weight determination volumetric molar concentration.

Listed the inhibition constant of a series of proof replacement boronic acid containing enzyme stabilizers (inhibitor) below, and [I] of above-mentioned some inhibitor _t/ K _iValue.

Table 1

Suppress Savinase with different replacement boronic acid containing derivatives ^TMThe inhibition constant and [I] _t/ K _iValue.For relatively, also comprise boric acid.

Inhibitor	10 3×K i(K iRepresent with mol/l)	[I] t/K i2％?????-1.5％ *
			Boric acid	10
Phenyl one replaces boronic acid containing	0.11	1490
			3-acetylamino phenyl one replaces boronic acid containing	0.09	1240????930
Cumarone-2 one replaces boronic acid containing	0.03	4115
			4-formylphenyl one replaces boronic acid containing	0.03	4445????3335
Thiophene-2 one replaces boronic acid containing	0.14
			Thiophene-3 one replaces boronic acid containing	0.17
Naphthalene-1 one replaces boronic acid containing	0.13
			2-formylphenyl one replaces boronic acid containing	0.2
3-formylphenyl one replaces boronic acid containing	0.07

^*The %W/W of inhibitor in solution.

Example 3

Storage stability test in liquid washing agent

Following content relates to KNPU (" K _iLo Novo Protease Units ") and Savinase ^TMPreparation is relevant), and KLU (" K _iLo Lipase Units ") and Lipolase ^TMPreparation is relevant.1 KNPU is equivalent to 2.53mg (about 9.4 * 10 ^-5Mmol) pure enzyme protein.About Savinase ^TMActive mensuration is referring to a brochure, and AF 220/1-GB (can be to NovoNordisk A/S, Bagsvaerd, Denmark asks for).

A lipase unit (LU) be defined under the standard conditions (30.0 ℃, pH7.0; With Gum Arabic is emulsifying agent, is substrate with the tributyrin) per minute discharges the titratable butyro-enzyme amount of 1 μ mol.A brochure (AF 95/5) of telling about this analytical procedure in more detail can be to Novo Nordisk A/S, Bagsvaerd, and Denmark asks for.1 KLU is equivalent to 0.2mg (about 6.7 * 10 ^-6Mmol) pure enzyme protein.

Use aforesaid method, under the condition of Gui Naing, check various proteinase inhibitor in the package stability in liquid washing agent (enzymic activity of the reservation) check below.The 4%W/W solution of the multiple inhibitor of preparation (except that boric acid) in single propylene glycol (MPG).The solubility of boric acid in associated media is relatively poor, and therefore be dissolved in the finished product cleaning composition and (produce the boric acid concentration of 1%W/W), rather than in liquid enzymes/inhibitor combination.

The first check series: in the first check series, will wait the inhibitor solution and the Savinase of weight part ^TM16.0L, Type EX (the liquid protein zymin that contains 16KNPU proteolytic enzyme in every gram; Novo Nordisk A/S, Bagsvaerd Denmark) mixes, and is contained the Savinase of 2%W/W inhibitor separately ^TM8L EX preparation.Contain this Savinase of 1%W/W separately ^TM/ inhibitor formulations, 98%W/W " detergent based 1 " (seeing below) and 1%W/W Lipolase ^TM100L, Type EX (the liquid aliphatic zymin that contains 100KLU lipase in every gram; Novo Nordisk A/S, Bagsvaerd, the package stability result of cleaning composition Denmark) is as follows: the remaining Lipolaste of % ^TMActive (storing down) at 30 ℃: the inhibitor period of storage (my god)

1??????3??????7

34 6 01% boric acid, 82 52 21 phenyl one replace boronic acid containing 79 46 17 cumarones-2 one and replace boronic acid containing 83 54 244-formylphenyls one replacement boronic acid containing 86 64 41 no Savinase ^TMOr inhibitor 88 72 55% remaining Savinase ^TMActive (storing 7 days down at 30 ℃) inhibitor activity % does not have 101% boric acid, 74 phenyl, one replacement boronic acid containing 67 cumarones-2 one replacement boronic acid containing 764-formylphenyl one and replaces boronic acid containing 85

Above result shows that especially the existence of described inhibitor can obviously improve Lipolase in the cleaning composition ^TMAnd Savinase ^TMActive preservation.

The second check series: in the second check series, respectively with 50:37.5: 12.5 weight ratio is mixed Savinase ^TM16.0 L EX, 4%W/W are dissolved in inhibitor and pure MPG among the MPG, are contained the Savinase of 1.5%W/W inhibitor separately ^TM8L EX preparation.Contain this Savinase of 1%W/W separately ^TM/ inhibitor formulations, 98%W/W " detergent based II " (seeing below) and 1%W/W Lipolase ^TM100L, the package stability result of the cleaning composition of Type EX is as follows: the remaining Lipolase of % ^TMActive (storing down) at 30 ℃: the inhibitor period of storage (my god)

159 12 do not have 53 8 04-formylphenyls one replaces boronic acid containing 58 41 283-acetylamino phenyls one replacement boronic acid containing 24 95 no Savinase ^TMOr inhibitor 76 67 55

The 3rd check series: prepare the Savinase that contains the 1.5%W/W inhibitor separately in the mode identical with the above-mentioned second check series ^TM8L EX preparation.Contain this Savinase of 1%W/W separately ^TM/ inhibitor formulations, 98%W/W " inactivation " OMO ^TMMicro (seeing below) and 1%W/W Lipolase ^TM100L, the package stability result of the cleaning composition of Type EX is as follows: the remaining Lipolase of % ^TMActive (storing down) at 35 ℃: the inhibitor period of storage (my god)

57 14 do not have 90 88 854-formylphenyls one replaces boronic acid containing 98 99 99 no Savinase ^TMOr inhibitor 99 99 99

The 4th check series: identical with above-mentioned Tr row basically, different is that the storage temperature that adopts is 30 ℃, and replaces the OMO of inactivation with " detergent based III " (seeing below) ^TMMicro.The remaining Lipolase of % ^TMActive (storing down) at 30 ℃: the inhibitor period of storage (my god)

159 12 do not have 45 194-formylphenyls one replaces boronic acid containing 32 20 17 no Savinase ^TMOr inhibitor 39 28 24

Above result shows that the existence of above-mentioned inhibitor can significantly improve Lipolase in various cleaning composition ^TMActive preservation.Detergent based I prescription (American version) composition %W/W (pure composition) Nansa ^TM1169/p 10.3 (linear alkylbenzene sulfonate, LAS) Berol ^TM452 3.5 (sulfated alkyl ether, AES) oleic acid 0.5 peanut lipid acid 0.5Dobanol ^TM25-7 6.4 (fatty alcohol ethoxylate, AEO) sodium xylene sulfonate 5.1 ethanol 0.7MPG 2.7 glycerine 0.5 sodium sulfate 0.4 yellow soda ash 2.7 Trisodium Citrates 4.4 citric acids 1.5 watering balance detergent based II prescription: composition %W/WNansa ^TM1169/p 10.30Sulfatol ^TMNA/25 1.40 oleic acid 7.05 peanut lipid acid 7.05Dobanol ^TM25-7 13.10 trolamine 5.50NaOH, 40% 2.00 sodium xylene sulfonate, 1.00 ethanol 4.90MPG, 2.70 sodium sulfate, 0.20 Trisodium Citrate 1.40Dequest ^TM2060 S, 0.40 watering balance detergent based III prescription: composition %W/W (pure composition) Nansa ^TM1169/p 7.00 oleic acid 0.45 peanut lipid acid 0.45Lutensol ^TMA04 3.80MPG 0.50 glycerine 4.60Na ₅P ₃O ₁₀H ₂O 22.60Na ₂SiO ₃5H ₂O 1.70 yellow soda ash 1.43 watering balances

OMO ^TMMicro is the Retail commodity that can buy from the supermarket of Denmark.Make enzyme component inactivation wherein by in microwave oven, handling (85 ℃, 5 minutes).

Example 4

The example of liquid enzymes/inhibitor combination

The example of liquid enzymes/inhibitor combination of the present invention following (all compositions are has the solution that can accept outward appearance):

1) 0.1g 4-bromophenyl one replaces boronic acid containing+10g Savinase 16.0L, and Type EX: the inhibitor of said composition: (I: E) mol ratio is about 33 to enzyme.

2) 0.2g 3, and 5-dichlorophenyl one replaces boronic acid containing+40g Savinase 16.0L, and TypeEX: the I of said composition: the E mol ratio is about 17.

3) 0.1g 5-chloro thiophene-2-one replaces boronic acid containing+10g Savinase 16.0L, and TypeEX: the I of said composition: the E mol ratio is about 40.

4) 0.1g phenyl one replaces boronic acid containing+10g Savinase 16.0L, and Type EX: the I of said composition: the E mol ratio is about 54.

5) 0.2g phenyl one replaces boronic acid containing+10g Savinase 16.0L, and Type EX: the I of said composition: the E mol ratio is about 108.

Claims (13)

1. liquid composition, contain:

(i) a kind of content surpasses the enzyme of 40 μ M; With

(ii) a kind of reversible inhibitor of above-mentioned enzyme, its consumption can effectively improve the package stability of enzyme described in the multicomponent composition that adds this liquid composition subsequently.

2. composition as claimed in claim 1, wherein, the consumption of described enzyme is 500 μ M-5mM.

3. as the composition of claim 1 or 2, wherein, the mol ratio of described inhibitor and described enzyme is at least 5.

4. as each composition among the claim 1-3, wherein, described [I] _t/ K _i(as herein defined) than being at least 50.

5. composition as claimed in claim 4, wherein [I] _t/ K _iRatio in the 250-5000 scope.

6. each composition as in the above-mentioned claim, wherein, described enzyme is selected from proteolytic enzyme, amylase, cellulase, lipase and oxydo-reductase.

7. each composition as in the above-mentioned claim, wherein, described enzyme is a proteolytic enzyme.

8. composition as claimed in claim 7, wherein, described inhibitor is selected from one and replaces boronic acid containing, two replacement boronic acid containing and salt thereof.

9. one kind is used to prepare a kind of method that contains the cleaning composition of enzyme and enzyme inhibitors, wherein, each described liquid composition among the claim 1-8 is mixed with all the other compositions of this cleaning composition.

10. method as claimed in claim 9, wherein, described cleaning composition is a kind of liquid composition.

11. cleaning composition with the method preparation of claim 9 or 10.

12. being used to produce laundry, each liquid composition among the claim 1-8 uses or washes the purposes of dish with cleaning composition.

13., be used to produce a kind of liquid detergent compositions as the purposes of claim 12.