EP2188360A1

EP2188360A1 - Polycyclic compounds as enzyme stabilizers

Info

Publication number: EP2188360A1
Application number: EP08803298A
Authority: EP
Inventors: Robin Ghosh; Andreas Michels; Cornelius Bessler; Daniela Lowis
Original assignee: Henkel AG and Co KGaA
Current assignee: Henkel AG and Co KGaA
Priority date: 2007-09-04
Filing date: 2008-08-28
Publication date: 2010-05-26
Also published as: JP2010538138A; US20100210502A1; DE102007041754A1; WO2009030632A1

Abstract

The present invention relates to laundry and cleaning agents containing polycyclic compounds that act as protease inhibitors and are therefore suitable enzyme stabilizers.

Description

Polycyclic compounds as enzyme stabilizers

The present invention relates to detergents and cleaners containing polycyclic compounds which act as protease inhibitors and are thus suitable enzyme stabilizers.

The use of enzymes in detergents and cleaners has been established in the art for decades. They serve to extend the range of services of the funds concerned according to their specific activities. These include in particular hydrolytic enzymes such as proteases, amylases, lipases and cellulases. The first three hydrolyze proteins, starches and fats and thus contribute directly to soil removal. Cellulases are used in particular because of their tissue effect. Another group of washing and cleaning agent enzymes are oxidative enzymes, in particular oxidases, which, if appropriate, in combination with other components, are preferably used to bleach soiling or to produce the bleaching agents in situ. In addition to these enzymes, which are subjected to constant optimization, further enzymes are constantly being made available for use in detergents and cleaners in order to be able to optimally address particular soiling, such as pectinases, β-glucanases, mannanases or other hemicellulases (glycosidases) Hydrolysis in particular of special vegetable polymers.

The enzymes which are the longest-established and are contained in virtually all modern, powerful detergents and cleaners are proteases and, in particular, serine proteases, to which the subtilases according to the invention are also calculated. They serve the degradation of protein-containing stains on the cleaning material. However, they also hydrolyze themselves (autoproteolysis) and all other proteins contained in the agents concerned, i. especially enzymes. This happens especially during the cleaning process, i. in the aqueous wash liquor, when comparatively favorable reaction conditions are present. This occurs to a lesser extent but also during storage of the agents concerned, which is why during a long storage always a certain loss of protease activity and the activities of other enzymes is accompanied. This is particularly problematic in gel or liquid and in particular in water-containing formulations, because in this with the water contained both the reaction medium and the hydrolysis reagent are available.

One goal in the development of detergent formulations is to stabilize the enzymes contained, especially during storage. This is understood as protection against various unfavorable influences, such as denaturation or decay by physical influences or oxidation. One focus of these developments is the protection of the contained proteins and / or enzymes against proteolytic cleavage. This can be done by building physical barriers, such as by encapsulating the Enzymes in special enzyme granules or by compounding the means in two- or multi-chamber systems. The other many pathway is to add chemical compounds that inhibit the proteases and thus act collectively as stabilizers for proteases and the other proteins and enzymes contained. It must be reversible protease inhibitors, since the protease activity only temporarily, especially during the storage, but not be suppressed during the cleaning process.

Polyols, in particular glycerol and 1,2-propylene glycol, benzamidine hydrochloride, borax, boric acids, boronic acids or their salts or esters are established as reversible protease inhibitors in the prior art. These include, in particular, derivatives having aromatic groups, for example ortho, meta or para-substituted phenylboronic acids, in particular 4-formylphenylboronic acid, or the salts or esters of the abovementioned compounds (see above). A particularly good protection results when boric acid derivatives are used together with polyols, since they can then form a complex stabilizing the enzyme. Also peptide aldehydes, that is, oligopeptides with reduced C-terminus, especially those of 2 to 50 monomers are described for this purpose. Among the peptidic reversible protease inhibitors include ovomucoid and leupeptin. Also, specific, reversible peptide inhibitors and fusion proteins from proteases and specific peptide inhibitors are used for this purpose.

More established enzyme stabilizers are aminoalcohols, such as mono-, di-, triethanol- and - propanolamine and mixtures thereof, aliphatic carboxylic acids up to Ci _2, such as succinic acid, other dicarboxylic acids or salts of said acids. End-capped fatty acid amide alkoxylates are also established for this purpose. Certain organic acids used as builders are capable, as disclosed in WO 97/18287, of stabilizing an enzyme in addition to their builder function.

As detergency and detergent proteases various protease classes are established, for example metalloproteases. However, among the detergent and detergent proteases, subtilisin-type proteases (subtilases, subtilopeptidases, EC 3.4.21.62) occupy an outstanding position due to their favorable enzymatic properties such as stability or pH optimum. They are attributed to the serine proteases due to the catalytically active amino acids. They act as nonspecific endopeptidases, that is, they hydrolyze any acid amide linkages that are internal to peptides or proteins. Their pH optimum is usually in the clearly alkaline range. For an overview of this family, see for example the article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996. Subtilases become natural formed by microorganisms; These include in particular those of To mention Bacillus species formed and secreted subtilisins as the most significant group within the subtilases.

It is therefore particularly worked to provide reversible inhibitors just this class of enzymes available. In this case, polyols such as glycerol and 1, 2-propylene glycol have proved to be unfavorable due to their high concentrations necessary use, because the other active ingredients of the respective agents can thus be contained only in correspondingly lower proportions.

Boric acid derivatives occupy an outstanding position among the serine protease inhibitors, which are effective at comparatively low concentrations. As examples of this, WO 92/19707 A1 discloses meta-substituted phenylboronic acids. Para-substituted phenylboronic acids as protease inhibitors are disclosed in EP 478050 A1. The protease-inhibiting effect of complexes of boric acids and boric acid derivatives with aromatic compounds is disclosed in EP 51 1456 A1. Protease-inhibiting derivatives of boronic acids and borinic acids, including aromatic compounds, disclosed in WO 95/02046 A1. WO 95/29223 A1 discloses the same effect of substituted naphthalene boronic acids.

Furthermore, the applications WO 96/21716 A1 and WO 96/41859 A1 should be mentioned. WO 96/21716 A1 cites the five applications cited above and discloses that all the protease inhibitors listed therein are also suitable for the specific purpose of stabilizing enzymes in detergents and cleaners. A selection of particularly efficient stabilizers thereof discloses WO 96/41859 A1.

Furthermore, there is prior art to further increase the effect of these stabilizers. Thus, the application WO 93/11215 A1 describes the combined use of 1,2-propanediol and boric acid or various boric acid derivatives for stabilizing liquid detergent formulations, and EP 451924 A2 discloses liquid detergent formulations obtained by the combined use of hydroxypolycarboxylic acids, calcium salt and special boron compounds be stabilized.

However, regardless of their stabilizing effect, the boric acid derivatives have a significant disadvantage: many of them, such as borate, form undesirable by-products with some other detergent ingredients, so that they are no longer available in the agents concerned for the desired cleaning purpose, or even remain as an impurity on the laundry.

It was therefore the task of identifying boron-free chemical compounds which act as protease inhibitors and are thus suitable as enzyme stabilizers in detergents and cleaners. Here, the use in total liquid, gel or pasty detergents and cleaning agents of particular interest, and including in particular those containing water.

This task is solved by the following means:

Detergents or cleaning agents containing a protease and a compound of the general

Structural formula:

in the

A is any 5- or 6-membered, mono- or polyunsaturated ring which may optionally also contain at least one heteroatom, in particular selected from O and N, wherein A is preferably selected from the group consisting of benzo, thiopheno , Pyrido, pyrimidino, imidazo, oxazo, pyrrazo and pyrrolo radicals,

X and Y independently of one another represent O, NR ¹ or CR ¹ R ² , preferably O,

R ^1, R ² and Z are independently hydrogen, Ci _-6 alkyl, especially methyl, ethyl,

Propyl, butyl, pentyl or hexyl, Ci _-6 alkenyl, in particular ethenyl, propenyl, butenyl, pentenyl or hexenyl, phenyl, benzyl or halogen, in particular fluorine, chlorine, bromine or iodine, preferably hydrogen, are n = 1 or 2.

In a preferred embodiment stand

A is a radical selected from the group consisting of benzo, thiopheno, pyrido,

Pyrimidino, imidazo, oxazo, pyrrazo and pyrrolo radicals, particularly preferred for a benzo

Thiopheno or pyrido radical, especially for a benzo radical,

X is O or NR ¹ , particularly preferably O,

Y is O or NH, particularly preferably O,

R ¹ for benzyl, Z is hydrogen and n is 1.

In a particularly preferred embodiment, it is a compound of the structural formula

([1- (carboxymethoxy) -6-oxo-6H-benzochromen-3-yl] oxy} acetic acid (CAS: 133540-71-3))

Under a detergent or cleaning agent according to the invention are all means that are suitable for washing or cleaning of particular textiles and / or solid surfaces. Suitable ingredients for this purpose are detailed below.

According to the invention, a protease is to be understood as meaning all enzymes which are capable of hydrolyzing acid amide linkages of proteins. The proteases are also detailed below.

Without wishing to be bound by this theory, it is assumed according to the invention that the compounds relevant to the invention form a complex with the protease to be inhibited / stabilized according to the invention. This probably looks like that the invention-relevant compound is incorporated in the substrate binding pocket of the protease and non-covalently bound there. In this way, the active site of the protease is blocked by a compound which is not hydrolyzable by this enzyme and is not available for hydrolysis of other proteins present. This is a reversible bond, i. for a balance between association and dissociation. The equilibrium coefficient of this reaction is called the inhibition constant or K,.

The first advantage of the compounds relevant to the invention over the prior art, in addition to their lower volume requirement compared to the polyols, is that they provide favorable inhibition constants with respect to the proteases which can be used in detergents and cleaners exhibit. This applies, for example, to serine proteases, but also to metalloproteases. The inhibitors thus bind reversibly, ie they do not undergo solid and not too loose transient interactions with the enzyme. Advantageously, the majority of the protease relevant to the invention is thus present during storage in the form of a protease-inhibitor complex. The protease and possibly other proteins contained, in particular other enzymes are protected in this way against proteolysis by this enzyme (stabilized against proteolysis). On the other hand, at the moment of dilution of the agent according to the invention with water to produce an aqueous washing or cleaning liquor during the cleaning process, the binding equilibrium is shifted towards dissociation, so that the complex dissolves and most of the protease relevant protease is proteolytically active. Thus, the compounds relevant to the invention are functioning protease inhibitors and thus enzyme stabilizers for detergents and cleaners in accordance with the task formulated.

The second advantage of the invention relevant compounds over the prior art is that they have as elements only C, H, N and O and optionally halides and / or sulfur and in particular are free of boron. Thus, they do not form the undesirable boron-derived by-products with other detergent or cleaning ingredients.

Furthermore, they have a good water solubility, in particular due to the carboxyl groups contained in each aromatic ring, so that they can be easily incorporated into corresponding agents and precipitation during storage is avoided.

In principle, the compounds mentioned probably act as reversible inhibitors because, similar to the substrate of the proteases, they are structurally adapted to the conditions of the binding pocket. Conversely, basically all proteases can be inhibited by the compounds relevant to the invention, so that they are suitable according to the invention as protease inhibitors. This applies in particular to serine proteases, as has been shown with reference to the examples of the present application with the positive effect of the compounds experimentally described there on the basis of serine proteases, specifically subtilases, even more specific subtilisins, namely a variant of the subtilisin from Bacillus lentus DSM 5483.

Further objects of the present invention relate to:

the use of a compound described above as a reversible inhibitor and / or stabilizer of a protease, in the context of a detergent or cleaning agent formulation;

A washing or cleaning process in which a protease is inhibited which is inhibited and / or stabilized with a compound described above;

the use of a washing or cleaning agent according to the invention for washing and / or cleaning textiles and / or hard surfaces; such as the use of a protease and a compound described above for the preparation of a detergent or cleaner.

According to the invention, those detergents or cleaners are preferred in which the stabilizing compound has an inhibition constant (Ki) of 0.01 to 10 mM, preferably 0.1 to 5, particularly preferably 0.5 to 2, with respect to the protease contained.

The inhibition constant K, can be determined in the following way: For the characterization of a reversible inhibitor of the enzymatic activity, the inhibition constant K _{1 is} a characteristic and decisive variable. K ₁ describes the equilibrium between enzyme, inhibitor and enzyme-inhibitor complex for reversible binding. The enzyme-inhibitor complex is catalytically inactive and inhibits the reaction by reducing the concentration of free enzyme that is still available for binding of substrate. The ki is accordingly defined as:

K ₁ = [I] x [E] / [EI]

Therein, [E], [I] and [El] denote the respective molar equilibrium concentrations of enzyme (E), inhibitor (I) and the enzyme-inhibitor complex (El). According to this definition, a substance with a small Ki is a good inhibitor under the respective test conditions.

The determination of K ₁ is based on the activity test of the protease in the presence of the corresponding inhibitor. The established Michaelis-Menten kinetics determine the enzymatic parameters K _m and k _cat in the presence of various concentrations of the inhibitor. By determining the initial rate of hydrolysis (v _Anf ) at different substrate concentrations [S] and fitting the experimental data in Equation 1, one obtains K,

Equation 1: v _Anf = k _cat x [S] x E ₀ / (K _m x (1+ [I] / K,) + S)

Here again [I] stands for the inhibitor concentration.

Alternatively, K ₁ can be determined using the Cheng-Prusoff equation (Equation 2) via the IC ₅₀ value. The determination of the IC ₅₀ value is made by determining the proteolytic activity on a substrate in the presence of various concentrations of the inhibitor and fitting the experimental data to a variable slope sigmoid dose-response equation (pseudo-hill slopes). It is the value of half the inhibitor concentration that would be needed to achieve complete inhibition.

K ₁ results from the following equation 2: Equation 2: K, = IC ₅₀ / (1 + [S] / K _d )

Therein, [S] is the substrate concentration in the assay and K _{d is} the equilibrium dissociation constant for the substrate, which in IC ₅₀ can be set to be identical to K _m for the substrate.

The K values which can be determined in this way characterize the compound investigated with respect to the protease used in the experiment. In example 2, this was carried out for the Bacillus lentus alkaline protease F49 (according to WO 95/23221 A1). Since this is a typical subtilisin protease, the values obtained with this enzyme are also typical of other serine proteases, in particular other subtilisin proteases. The exact value for a protease of interest must be determined in doubt on the basis of each specific protease.

In detergents or cleaners according to the invention, which are present in a preferred embodiment in predominantly solid form and in a second embodiment in predominantly liquid, pasty or gel form, the protease is especially in a content of 2 .mu.g to 20 mg per g of the composition, preferably from 5 μg to 17.5 mg per g of the agent, more preferably from 20 μg to 15 mg per g of the agent, most preferably from 50 μg to 10 μg of the agent.

The stabilizer is contained in agents according to the invention in particular in a content of up to 50 mg per g of the agent, preferably up to 10 mg, more preferably up to 7 mg, most preferably up to 5 mg per g of the agent. It is further preferred that the stabilizer is contained in a content of 0.01 to 100 × K, (based on the protease contained), preferably 0.1 to 10 × K _{1, more} preferably 1 to 5 × K ₁ .

The molar ratio of stabilizer to protease is preferably in the range from 1: 1 to 1000: 1, in particular from 1: 1 to 500: 1, particularly preferably from 1: 1 to 100: 1, very particularly preferably from 1: 1 to 20 :1.

In addition to the stabilizer according to the general formula given above, an agent according to the invention may contain at least one further stabilizer, in particular a polyol, such as glycerol or 1,2-ethylene glycol, and / or an antioxidant.

The protease stabilized or reversibly inhibited according to the invention is preferably a serine protease, in particular a subtilase, more preferably a subtilisin. Subtilisin may be a wild-type enzyme or a subtilisin variant, wherein the wild-type enzyme or the starting enzyme of the variant is preferably selected from one of the following:

The alkaline protease from Bacillus amyloliquefaciens (BPN '),

The alkaline protease from Bacillus licheniformis (subtilisin Carlsberg),

- Alkaline Protease PB92,

Subtilisin 147 and / or 309 (Savinase)

The alkaline protease from Bacillus lentus, preferably from Bacillus lentus (DSM 5483),

The alkaline protease from Bacillus alcalophilus (DSM 11233),

- the alkaline protease from Bacillus gibsonii (DSM 14391) or an at least 70% identical alkaline protease, the alkaline protease from Bacillus sp. (DSM 14390) or an at least 98.5% identical alkaline protease, the alkaline protease from Bacillus sp. (DSM 14392) or an at least 98.1% identical alkaline protease,

the alkaline protease from Bacillus gibsonii (DSM 14393) or an at least 70% identical alkaline protease,

- in SEQ ID NO. 4 of the application WO 2005/063974 A1 described alkaline protease or an at least 40% identical alkaline protease,

- in SEQ ID NO. 4 of the application WO 2005/103244 A1 described alkaline protease or an at least 80% identical alkaline protease,

- in SEQ ID NO. 7 of the application WO 2005/103244 A1 described alkaline protease or a thereto at least 80% identical alkaline protease and

- in SEQ ID NO. 2 of the application DE 102005028295.4 described protease or a thereto at least 66% identical protease.

In a preferred embodiment, the protease is a variant with a point mutation in the range of positions 95 to 103 (counting according to subtilisin 309), preferably with an insertion of a single amino acid between position 99 and 100, more preferably starting from subtilisin 147 and / or 309 (subtilisin 309), or a variant thereof. In particular, the protease is a variant with a point mutation in position 217 (count according to the wild-type protease from Bacillus amyloliquefaciens, BPN '), preferably with a substitution of a single amino acid in this position, more preferably with the amino acid substitution X217L, most preferably starting from the wild type protease from Bacillus amyloliquefaciens (BPN '), or a variant thereof. In a preferred embodiment, the protease is a variant having an amino acid change relative to a starting protease homologizable with the Bacillus lentus alkaline protease in one or more of the following positions: 3, 4, 36, 42, 43, 47, 56 , 61, 69, 87, 96, 99, 101, 102, 104, 114, 1, 18, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 250, 253, 255 and 268, in the count the Bacillus lentus alkaline protease, preferably with an amino acid change from the parent molecule in one or more of the following positions: 3, 4, 43, 61, 188, 193, 199, 21 1, 224, 250 and 253, most preferably with one or more several of the amino acid exchanges X3T, X4I, X43V, X61A, X188P, X193M, X199I, X21 1D, X211E, X211G, X211N or X211Q, X224V, X250G and X253N, most preferably starting from the Bacillus lentus DSM alkaline protease 5483, or a variant thereof.

Agents according to the invention may contain, in addition to the protease, one or more further enzymes, in particular from the following group: one or more further proteases, amylases, hemicellulases, cellulases, lipases and oxidoreductases. The amylase is preferably an α-amylase. The hemicellulase is preferably a β-glucanase, a pectinase, a pullulanase and / or a mannanase. The cellulase is preferably a cellulase mixture or a one-component cellulase, preferably or predominantly an endoglucanase and / or a cellobiohydrolase. The oxidoreductase is preferably an oxidase, in particular a choline oxidase, or a perhydrolase.

Agents according to the invention preferably comprise at least one complexing agent and / or builder substances, the builder being in particular a zeolite builder, and / or a nonionic surfactant, the nonionic surfactant preferably being a hydroxy mixed ether, and / or optical Brightener, wherein the optical brightener is diphenyl compounds, in particular distyryl biphenyl derivatives, and / or stilbentriazine derivatives.

Examples

example 1

Examination of protease residual activity in the presence of an inhibitor

In order to demonstrate that the compounds according to the invention exert an activity which inhibits the protease activity, the proteolytic residual activity of the Bacillus lentus alkaline protease F49 (according to WO 95/23221 A1) is determined in the presence of these compounds.

In parallel reactions, in 100 mM Tris buffer, pH 6.8, 0.1% (w / v) BrijTM35, the substrate succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; Bachern L-1400) and 5 x 10 ^{~ 9} or 1 × 10 ^{~ 8} M of the protease. In addition, the compounds to be tested come in a final concentration of 10 mM. They are each dissolved in anhydrous DMSO, with effects of DMSO on the enzymatic activity being corrected by the appropriate reference with the same amount of DMSO but without the compound in question. The incubation was carried out for 5 min at pH 8.6 and 25 ° C. 1 U corresponds to 1 μmol of cleaved substrate per minute.

Using [1- (carboxymethoxy) -6-oxo-6H-benzochromen-3-yl] oxy} acetic acid (CAS: 133540-71-3), inhibition of residual proteolytic activity to a residual activity of less than 50% was thus achieved become.

Example 2

Investigation of the storage stability of protease-containing detergents and cleaners in the presence of

Protease inhibitors

The basic formulation is a liquid detergent having the following composition (all figures in percentages by weight): 0.3-0.5% xanthan gum, 0.2-0.4% anti-foaming agent, 6-7% glycerol, 0.3 -0.5% ethanol, 4-7% FAEOS, 24-28% nonionic surfactants, 1% boric acid, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut fatty acids, 0.5 % HEDP, 0-0.4% PVP, 0-0.05% optical brightener, 0-0.001% dye, balance: demineralized water.

This formulation is supplemented with the inhibiting compounds to be tested and 1,275,000 HPE / I B. lentus alkaline protease F 49. The protease activity (handle-protease units) indicated in HPE is according to van Raay, Saran and Verbeek, according to the publication "For the determination of the proteolytic activity in enzyme concentrates and enzyme-containing detergents, rinses and cleaners" in Tenside (1970), Volume 7, pp. 125-132. The storage is carried out for various periods of time in airtight containers at 30 ⁰ C.

For evaluation, the initial values for the proteolytic activity of the agent in question are compared with the values determined after storage. The higher the activity remaining after storage, the better the protease contained is inactivated during storage and the better the compound in question is suitable as a stabilizer according to the invention.

Claims

claims

1. washing or cleaning agent containing a protease and an enzyme stabilizer of the general structural formula:

in the

A represents any 5- or 6-membered, mono- or polyunsaturated ring which may optionally also contain at least one heteroatom, in particular selected from O and N,

X and Y independently of one another represent O, NR ¹ or CR ¹ R ² ,

R ^1, R ² and Z represents hydrogen, Ci _-6 -alkyl, CI_ are independently ₆ alkenyl, phenyl, benzyl or halogen, n = 1 or is the second

2. Washing or cleaning agent according to claim 1, characterized in that A is a radical selected from the group consisting of benzo, thiopheno, pyrido, pyrimidino, imidazo, oxazo, pyrrazo and pyrrolo radical.

3. washing or cleaning agent according to claim 1 or 2, characterized in that X is O or NR ¹ , wherein R ^{1 is} benzyl.

4. washing or cleaning agent according to one of claims 1 to 3, characterized in that Y is O or NH.

5. washing or cleaning agent according to one of claims 1 to 4, characterized in that it is the enzyme stabilizer to a compound of the structural formula

is.

6. washing or cleaning agent according to one of claims 1 to 5, wherein the stabilizing compound with respect to the protease contained an inhibition constant (K ₁ ) of 0.01 to 10 mM, preferably 0.1 to 5, particularly preferably 0.5 to 2 having.

7. washing or cleaning agent according to one of claims 1 to 6 in predominantly solid form.

8. washing or cleaning agent according to one of claims 1 to 6 in predominantly liquid, pasty or gel form.

9. washing or cleaning agent according to any one of claims 1 to 8, wherein the protease in a content of 2 micrograms to 20 mg per g of the agent, preferably from 5 micrograms to 17.5 mg per g of the agent, particularly preferably 20 micrograms to 15 mg per g of the agent, most preferably from 50 micrograms to 10 micrograms of the agent.

10. Washing or cleaning agent according to one of claims 1 to 9, wherein the stabilizer in a content up to 50 mg per g of the agent, preferably up to 10 mg, more preferably up to 7 mg, most preferably up to 5 mg per g of the Is included by means.

11. Washing or cleaning agent according to one of claims 1 to 10, wherein the molar

Ratio of stabilizer to protease in the range of 1: 1 to 1000: 1, in particular from 1: 1 to 500: 1, more preferably from 1: 1 to 100: 1, most preferably from 1: 1 to 20: 1.

12. Washing or cleaning agent according to one of claims 1 to 11, wherein the stabilizer in a content of 0.01 to 100 x K, (based on the protease contained), preferably 0.1 to 10 x K _{1, more} preferably 1 to 5 x K _{1 is} included.

13. washing or cleaning agent according to any one of claims 1 to 12, wherein it is the protease is a serine protease, preferably a subtilase, more preferably a subtilisin.

14. Washing or cleaning agent according to claim 13, wherein the subtilisin is a wild-type enzyme or a subtilisin variant, wherein the wild-type enzyme or the starting enzyme of the variant is selected from one of the following:

The alkaline protease from Bacillus amyloliquefaciens (BPN '),

The alkaline protease from Bacillus licheniformis (subtilisin Carlsberg),

- Alkaline Protease PB92,

Subtilisin 147 and / or 309 (Savinase)

The alkaline protease from Bacillus lentus, preferably from Bacillus lentus (DSM 5483), the alkaline protease from Bacillus alcalophilus (DSM 11233),

15: washing or cleaning agent according to any one of claims 1 to 12, wherein the protease is a variant to the initial protease at the amino acid level over the entire length of the variant, a sequence identity of at least 90%, preferably at least 92.5%, more preferably at least 95%, and most preferably at least 97.5%, whether derived from itself or from another enzyme.

16. Washing or cleaning agent according to one of claims 1 to 13, wherein the protease is a variant with a point mutation in the range of the positons 95 to 103 (count according to Subtilisin 309), preferably with an insertion of a single amino acid in between Position 99 and 100, particularly preferably starting from subtilisin 147 and / or 309 (subtilisin 309), or a variant thereof.

17. washing or cleaning agent according to one of claims 1 to 14, wherein it is in the

Protease is a variant with a point mutation in position 217 (count according to the wild-type protease from Bacillus amyloliquefaciens; BPN '), preferably with a substitution of a single amino acid in this position, more preferably with the amino acid substitution X217L, most preferably starting from the Wild-type protease from Bacillus amyloliquefaciens (BPN '), or a variant thereof.

18. washing or cleaning agent according to one of claims 1 to 15, wherein it is in the

Protease is a variant having an amino acid change relative to a starting protease homologizable with the Bacillus lentus alkaline protease in one or more of the following positions: 3, 4, 36, 42, 43, 47, 56, 61, 69, 87, 96 , 99, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 21 1, 224, 229, 236, 237, 242, 243, 250, 253, 255 and 268, in the enumeration of the Bacillus lentus alkaline protease, preferably with an amino acid change from the parent molecule in one or more of the following positions: 3,4,43,61,188,193, 199, 211, 224 , 250 and 253, more preferably with one or more of the amino acid substitutions X3T, X4I, X43V, X61A, X188P, X193M, X199I, X211D, X211E, X21 1G, X21 1N or X211Q, X224V, X250G and X253N, more particularly preferably starting from the alkaline protease from Bacillus lentus DSM 5483, or a variant thereof.

19. Washing or cleaning agent according to one of claims 1 to 16, comprising at least one further stabilizer.

20. Washing or cleaning agent according to claim 17, wherein the at least one further stabilizer is a polyol, in particular glycerol or 1, 2-ethylene glycol, and / or an antioxidant.

21. Washing or cleaning agent according to one of claims 1 to 18, containing one or more further enzymes selected from the group consisting of proteases, amylases, hemicellulases, cellulases, lipases and oxidoreductases.

22. A washing or cleaning composition according to claim 19, wherein the amylase is an α-amylase.

23. A washing or cleaning agent according to claim 19 or 20, wherein the hemicellulase is a β-glucanase, a pectinase, a pullulanase and / or a mannanase.

24. Washing or cleaning agent according to one of claims 19 to 21, wherein the cellulase is a cellulase mixture or a one-component cellulase, preferably or predominantly an endoglucanase and / or a cellobiohydrolase.

25. Washing or cleaning agent according to one of claims 19 to 22, wherein the oxidoreductase is an oxidase, in particular a choline oxidase, or a perhydrolase.

26. Washing or cleaning agent according to one of claims 1 to 23, containing at least one complexing agent and / or builders.

The laundry detergent or cleaning composition of claim 24, wherein the builder is a zeolite builder.

28. Washing or cleaning agent according to one of claims 1 to 25, containing a nonionic surfactant.

29. A composition according to claim 26, wherein the nonionic surfactant is a hydroxy mixed ether.

30. Washing or cleaning agent according to one of claims 1 to 27, containing optical brightener.

31. Washing or cleaning agent according to claim 28, wherein the optical brightener is diphenyl compounds, in particular distyryl biphenyl derivatives, and / or stilbentriazine derivatives.

32. Use of a compound of the general structural formula:

in the

X and Y independently of one another represent O, NR ¹ or CR ¹ R ² ,

R ¹ , R ² and Z are independently hydrogen, C _1-6 alkyl, C- |. ₆ alkenyl, phenyl,

Benzyl or halogen, n = 1 or 2, as a reversible inhibitor of a protease in the context of a washing or

Detergent formulation.

33. A washing or cleaning process in which a protease is acted upon, which is inhibited and / or stabilized with a compound of the general structural formula:

in the

X and Y independently of one another represent O, NR ¹ or CR ¹ R ² ,

R ^1, R ² and Z are independently hydrogen, CI_ ₆ alkyl, CI_ ₆ alkenyl, phenyl,

Benzyl or halogen, n = 1 or 2.

34. Washing or cleaning method according to claim 31, wherein a washing or cleaning agent according to one of claims 1 to 29 is used.

35. Use of a washing or cleaning agent according to any one of claims 1 to 29 for washing and / or cleaning of textiles and / or hard surfaces.

36. Use of a protease and a compound of the general structural formula:

in the

X and Y independently of one another represent O, NR ¹ or CR ¹ R ² ,

R ^1, R ² and Z is hydrogen, CI_ ₆ alkyl, are independently CI_ ₆ alkenyl, phenyl, benzyl or halogen, n = 1 or 2 is, for producing a washing or cleaning agent.