EP2220204B1 - Washing agent having stabilized enzymes - Google Patents

Abstract

The present application relates to washing and cleaning agents, containing phosphate compounds having aliphatic and/or aromatic residues, which act as enzyme stabilizers. Further subjects are the use of such compounds as reversible inhibitors of enzymes, in particular of proteolytic enzymes, and thus as stabilizers in washing or cleaning agents, and further methods and uses correlated therewith.

Description

The present application relates to detergents and cleaners containing phosphate compounds having aliphatic and / or aromatic radicals which act as enzyme stabilizers.

The use of enzymes in detergents and cleaners is well established in the art. 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 for hydrolysis, in particular more specific vegetable polymers.

The enzymes that have long been established and are contained in virtually all modern, high-performance detergents and cleaners are proteases, and in particular serine proteases, which include the subtilases. They cause the degradation of protein-containing stains on the items to be cleaned. However, they also hydrolyze themselves (autoproteolysis) and all other proteins contained in the agents concerned, i. especially other enzymes. This happens especially during the cleaning process, i. in the aqueous wash liquor, if comparatively favorable reaction conditions are present. However, this also happens during the storage of the respective agent, which is why with increasing storage time always a certain loss of enzyme activities, such as the protease activity, accompanied. In general, the enzyme activity in the washing or cleaning agent is inversely proportional to the storage time, with increasing storage time, the enzyme activity decreases more and more. 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 detergents and cleaners is therefore to stabilize the enzymes contained, especially during storage. Below that is the protection understood against various unfavorable influences, such as against 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 the construction of physical barriers, such as by encapsulation of the enzymes in special enzyme granules or by packaging the means in two- or multi-chamber systems. Another frequently approached approach is to add chemical compounds to the agents which inhibit the proteases and thus act collectively as stabilizers for the proteases and the other proteins and enzymes contained. It must be reversible protease inhibitors, since the protease activity is only temporarily, especially during 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 with aromatic groups, for example ortho, meta or para-substituted phenylboronic acids, in particular 4-formylphenylboronic acid (4-FPBA) or the salts or esters of the abovementioned compounds. 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.

However, polyols such as glycerol and 1,2-propylene glycol have proved to be unfavorable due to their high necessary use concentrations, because the other active ingredients of the respective agents can thus be contained only in proportionally smaller proportions.

Boric acid derivatives occupy an outstanding position among the serine protease inhibitors, which are effective at comparatively low concentrations. For example, the international patent application discloses WO 96/21716 A1 that boric acid derivatives acting as protease inhibitors are also suitable for stabilizing enzymes in detergents and cleaners. A selection of particularly powerful stabilizers are disclosed in the international patent application WO 96/41859 A1 ,

Regardless of their stabilizing effect, however, the boric acid derivatives have a decisive disadvantage. Many boric acid derivatives, such as borate, form with some other detergent or detergent ingredients unwanted by-products, so that they are no longer available in the relevant agents for the desired cleaning purpose or even remain as an impurity on the laundry.

It was therefore the task of providing boron-free chemical compounds which act as enzyme inhibitors and are thus suitable as enzyme stabilizers in detergents and cleaners. Another object of the present invention is to provide boron-free chemical compounds which act as protease inhibitors and are thus suitable as stabilizers for proteases and / or for other enzymes in detergents and cleaners. In particular, a washing or cleaning agent should be provided with such a boron-free chemical compound.

Here, the use in total liquid, gel or pasty detergents and cleaning agents of particular interest, and including in particular those containing water.

in der X ein aliphatischer oder ein aromatischer Rest ist und Y ein aliphatischer oder ein aromatischer Rest ist, wobei der Rest X ausgewählt ist aus der Gruppe bestehend aus -CH₂-CH₂-CH₃, Phenyl.An object of the invention is a washing or cleaning agent containing an enzyme and a compound of the general structural formula

wherein X is an aliphatic or an aromatic radical and Y is an aliphatic or an aromatic radical, wherein the radical X is selected from the group consisting of -CH ₂ -CH ₂ -CH ₃ , phenyl.

It has surprisingly been found that such compounds surprisingly effectively stabilize the enzymes contained in the detergents and cleaning agents according to the invention, for example, in particular proteases, and this compared to borate as enzyme stabilizer already at a lower or the same high concentration. These compounds thus provide scope for formulation of laundry detergents and cleaners by the ability of enzyme stabilizers known in the art, such as 1,2-propylene glycol or boron compounds (for example, borates and other boric acid derivatives or 4-FPBA), in lesser To be able to use concentration or to completely dispense with it.

In a preferred embodiment of the invention, the washing or cleaning agent is characterized in that the radicals X and Y of the compound are identical, wherein the radical X is selected from the group consisting of: -CH ₂ CH ₂ CH ₃ , phenyl, and the Rest Y as well is selected from the group consisting of: -CH ₂ -CH ₂ -CH ₃ , phenyl. A particularly preferred compound therefore has as radical X and as radical Y a: -CH ₂ -CH ₂ -CH ₃ - group. A further particularly preferred compound accordingly has as radical X and as radical Y a phenyl group. However, all detergents and cleaners containing compounds of the general structural formula given above constitute further embodiments of the present invention, provided that the radicals X and Y are each aliphatic or aromatic and this compound effects a stabilization of an enzyme in the washing or cleaning agent. It is also possible that the radical X is aliphatic and the radical Y is aromatic or the radical X is aromatic and the radical Y is aliphatic.

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.

For the purposes of the present application, an enzyme is to be understood as meaning a protein which has a specific biocatalytic function. For the purposes of the present application, protease is understood as meaning an enzyme which catalyzes the hydrolysis of peptide bonds and is thereby able to cleave peptides or proteins.

The present invention encompasses the compounds mentioned in all protonated and / or deprotonated forms. Optionally, oppositely charged cations (H ⁺ , Na ⁺ , K ⁺ or the like) or anions (Cl ^- , Br ^- , formate, acetate, etc.) are present. In all these forms, the present invention can be realized. Decisive in each case is the interaction between the invention-relevant compound which stabilizes the enzyme and the enzyme to be stabilized according to the invention.

Without having to be bound by this theory, it is assumed according to the invention that the compounds relevant for the invention, ie the stabilizing compounds, form a complex with the enzyme to be stabilized. It is anticipated that non-covalent binding of the stabilizing compound will occur in or at the substrate binding pocket of the enzyme. In this way, the active site of the enzyme is blocked by a compound which is not hydrolyzable by this enzyme and is therefore no longer available for the catalysis of other substrates present. The stabilizing effect of the compound is therefore due to an inhibition of the enzyme. This is a reversible bond, ie a balance between association and dissociation. The equilibrium coefficient of this reaction is called the inhibition constant or K _i . Due to the reversible binding of the stabilizing compound to the enzyme, the enzyme is inhibited and thus stabilized in the washing or cleaning agent, whereas the complexes are increasingly dissociated by the changed conditions when using the detergent and cleaning agent, for example by dilution of the detergent Washing or cleaning agent in the washing or cleaning solution, by the presence of further and / or higher affinity enzyme substrates or by changing the pH in the washing or cleaning solution. The terms "stabilizing" and "inhibiting" or "inhibiting" as well as "stabilizer" and "inhibitor" or "inhibitor" are therefore to be regarded as synonymous in the present patent application.

Since the equilibrium coefficient or the inhibition constant K _{i is} of essential importance for the functionality of the compound as an enzyme stabilizer, a further subject of the invention is a washing or cleaning agent which is characterized in that the compound has an inhibition constant with respect to the enzyme. K _i ) of 0.01 to 10 mM. In a further preferred embodiment of the invention, the washing or cleaning agent is characterized in that the stabilizing compound has an inhibition constant (K _i ) of 0.03 to 5 mM with respect to the enzyme.

• Für die Charakterisierung eines reversiblen Inhibitors der enzymatischen Aktivität ist die Inhibitionskonstante K_i eine charakteristische und entscheidende Größe. K_i beschreibt das Gleichgewicht zwischen Enzym, Inhibitor und Enzym-Inhibitor Komplex für eine reversible Bindung. Der Enzym-Inhibitor Komplex ist dabei katalytisch nicht aktiv und inhibiert die Reaktion durch Herabsetzung der Konzentration von freiem Enzym, das noch zur Bindung von Substrat zur Verfügung steht. Der K_i ist dementsprechend definiert als: $${\mathrm{K}}_{\mathrm{i}}=\left[\mathrm{I}\right]\mathrm{x}\left[\mathrm{E}\right]/\left[\mathrm{EI}\right]$$
  

The inhibition constant K _i can be determined in the following way:

• For the characterization of a reversible inhibitor of enzymatic activity, the inhibition constant K _{i is} a characteristic and decisive factor. K _i 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 K _i is accordingly defined as: $${\mathrm{K}}_{\mathrm{i}}=\left[\mathrm{I}\right]\mathrm{x}\left[\mathrm{e}\right]/\left[\mathrm{EGG}\right]$$
  

Therein, [E], [I] and [EI] represent the respective molar equilibrium concentrations of enzyme (E), inhibitor (I) and the enzyme-inhibitor complex (EI). According to this definition, a substance with a small K _{i is} a good inhibitor under the respective test conditions.

The determination of K _i is carried out on the basis of the activity test of the protease in the presence of the corresponding inhibitor. About the Michaelis-Menten kinetics established in the prior art and known to those skilled in the art ( Leonor Michaelis, Maud Menten (1913). The kinetics of invertin action, Biochem. Z. 49: 333-369 ), the enzymatic parameters K _m , and k _{cat are determined} in the presence of various concentrations of the inhibitor. For a Michaelis-Menten kinetics is simplified: $$\mathrm{e}+\mathrm{S}\underset{{\mathrm{k}}_{\mathrm{1}}}{\overset{{\mathrm{k}}_{\mathrm{1}}}{\begin{array}{c}\to \\ \leftarrow \end{array}}}\mathrm{IT}\stackrel{{\mathrm{k}}_{\mathrm{2}}}{\to }\mathrm{e}+\mathrm{P}$$

• E: Enzym
• S: Substrat
• ES: Enzym-Substrat-Komplex
• P: Produkt
• k₁,k_-1,k₂: Geschwindigkeitskonstanten

Herein mean:

• E: enzyme
• S: substrate
• ES: enzyme-substrate complex
• P: product
• k ₁ , k _-1 , k ₂ : rate constants

Here, k _{2 is} a measure of the maximum reaction rate at substrate saturation (Vmax), also called turnover number, molecular activity, turnover number or kcat (kcat = Vmax / [Eo], where [Eo] is the initial concentration of the enzyme). The Michaelis constant (ie the substrate concentration present at half-saturation, where the rate of turnover is thus v = Vmax / 2), is too high

K _m = k _-1 / k ₁ (Michaelis-Menten case, given k2 << k1) or more generally K _m = k _-1 + k ₂ / k ₁ (Briggs-Haldane situation, given for the Case that k 2 can not be neglected compared to k 1).

The saturation function of a "Michaelis-Menten enzyme" is calculated using the parameters Km and Vmax as follows: $$\mathrm{v}=\frac{{\mathrm{v}}_{\mathrm{Max}}\cdot \left[\mathrm{S}\right]}{{\mathrm{K}}_{\mathrm{m}}+\left[\mathrm{S}\right]}$$

• v: Bildungsgeschwindigkeit von P (v = "Geschwindigkeit") [mol I-1 s-1]
• vmax: maximale Geschwindigkeit [mol I-1 s-1]
• Km: Michaelis-Menten-Konstante [mol I-1]
• [S]: Substratkonzentration [mol I-1]

Hereby means:

• v: formation rate of P (v = "velocity") [mol I-1 s-1]
• vmax: maximum velocity [mol I-1 s-1]
• Km: Michaelis-Menten constant [mol I-1]
• [S]: substrate concentration [mol I-1]

Determination of the initial catalysis rate (v _Anf. ) - for proteases of the initial hydrolysis rate - at various substrate concentrations [S] and fitting of the experimental data into Equation 1 below gives the inhibition constant K _i . $${\mathrm{v}}_{\mathrm{anf}\mathrm{,}}={\mathrm{k}}_{\mathrm{cat}}\times \left[\mathrm{S}\right]\mathrm{x}{\mathrm{e}}_{\mathrm{0}}/\left({\mathrm{K}}_{\mathrm{m}}\mathrm{x}\left(\mathrm{1}+\left[\mathrm{I}\right]/{\mathrm{K}}_{\mathrm{i}}\right)+\mathrm{S}\right)]$$

Here again [I] stands for the inhibitor concentration.

Alternatively, K _i can be calculated using the Cheng-Prusoff equation ( Equation 2, Cheng Y., Prusoff WH (1973) Biochem. Pharmacol. 22, 3099-3108 ) are determined via the IC ₅₀ value. The determination of the IC ₅₀ via the determination of the catalytic activity to a substrate in the presence of various concentrations of the inhibitor and the fitting of the experimental data to a sigmoidal dose-response with variable slope equation (pseudo-Hill slopes). It is the inhibitor concentration needed to achieve 50% inhibition.

K _i results from the following equation 2: $${\mathrm{K}}_{\mathrm{i}}={\mathrm{IC}}_{\mathrm{50}}/\left(\mathrm{1}+\left[\mathrm{S}\right]/{\mathrm{K}}_{\mathrm{d}}\right)$$

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

The K _i values that can be determined in this way characterize the compound with respect to the enzyme used. In Example 1, the residual activity of a protease, namely the Bacillus lentus alkaline protease F49 (according to WO 95/23221 A1 ) in the presence of an inhibitor. 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 an enzyme of interest must be determined in case of doubt based on the specific enzyme.

As already explained above, an advantage of the compounds according to the invention which effect the stabilization of at least one enzyme in detergents and cleaners is that they have favorable inhibition constants with respect to the enzymes to be used in detergents and cleaning agents compared with the prior art. This is especially true for proteases, but also for other enzymes. A largely reversible binding of the inhibitors is thus ensured, ie they do not enter into solid and not too loose transient interactions with the enzyme. Advantageously, the majority of the enzymes are thus present in the form of an enzyme-inhibitor complex during storage of the detergents and cleaners. The enzymes, in particular proteases, and optionally other proteins present are protected in this way, for example against catalytic, in particular hydrolytic activities of these enzymes themselves. In the case of proteases, these are therefore protected from proteolysis by these enzymes, ie they are stabilized against proteolysis , At the moment of dilution of the washing or cleaning agent according to the invention with water to prepare an aqueous washing or cleaning solution during the cleaning process, the binding equilibrium shifted in the direction of dissociation, so that the complex dissolves and the majority of the invention relevant enzymes is catalytically active. Thus, the compounds relevant to the invention are functional enzyme inhibitors according to the task formulated and thus enzyme stabilizers for detergents and cleaners.

Furthermore, compounds according to the invention have a low volume requirement in comparison with established prior art enzyme stabilizers, for example with respect to polyols.

Another advantage of the invention relevant compounds over the prior art is that they have as elements only carbon (C), hydrogen (H), phosphorus (P) and oxygen (O) 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 solubility in water, so that they can be easily incorporated into appropriate detergents and cleaners. Furthermore dadruch a precipitation during storage is reduced or completely avoided.

In principle, the compounds mentioned are presumed to act as reversible inhibitors because they structurally resemble the substrate of the enzymes, concerning proteases, in particular with regard to the acid amide bond to be hydrolyzed. Conversely, the enzymes, preferably proteolytic enzymes, can thus be inhibited by the compounds relevant to the invention. This applies in particular to serine proteases, as shown by the examples of the present application with the positive effect of the described compounds on the basis of subtilisins.

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 enzyme is in particular 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 compound, ie 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, very particularly preferably up to 5 mg per g of the agent. Furthermore, it is preferred that the amount of the compound contained in the agent, ie the stabilizing the enzyme Compound, based on the stabilized enzyme of 0.01 to 100 x the inhibition constant K _i , preferably 0.1 to 10 x K _i , more preferably 1 to 5 x K _i .

The molar ratio of the stabilizing compound to the enzyme is preferably 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 1 to 20: 1.

Another object of the invention are detergents or cleaners characterized in that the enzyme is selected from the group consisting of: protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, Oxidoreductase, lipase, esterase or mixtures thereof. In a particularly preferred embodiment of the invention, the washing or cleaning agent is characterized in that the enzyme is a protease, preferably a serine protease, more preferably a subtilase and most preferably a subtilisin. Because in particular for detergents and cleaners or generally enzyme-containing compositions such as enzyme concentrates containing at least one proteolytic enzyme (protease), the storage stability of the enzymes is a general problem. Due to their enzymatic activity, proteolytic enzymes lead to the hydrolysis of proteins, such as enzymes and peptides, which are contained in the composition, be it other proteins or enzymes or also the proteases themselves. Hydrolysis of the protease by its own proteolytic activity becomes referred to as autoproteolysis. The degree of storage stability of all proteins / enzymes contained in a protein / enzyme composition is thus dependent, in particular, on the proteolytic activity of a protease within this composition. In order to increase the storage stability of all enzymes of such a composition, it is therefore necessary to precisely inhibit the proteolytic activity of the protease during storage. This is best done by the addition of a compound of the invention which is a specific and reversible inhibitor with a high affinity for the protease for the protease contained in the composition.

Examples of such proteases are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, subtilisin DY and the enzymes thermitase, proteinase K which can no longer be assigned to the subtilisins in the narrower sense and the proteases TW3 and TW7. Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from Novozymes A / S, Bagsværd, Denmark. The subtilisins 147 and 309 are sold under the trade names Esperase®, and Savinase® by the company Novozymes. From the protease from Bacillus lentus DSM 5483 derived under the name BLAP® protease variants derived.

Other proteases are, for example, under the trade names Durazym ^®, relase ^®, Everlase® ^®, Nafizym, Natalase ^®, Kannase® ^® and Ovozymes ^® from Novozymes, under the trade names Purafect ^®, Purafect ^® OxP and Properase.RTM ^® from Genencor , under the trade name Protosol® ^® from Advanced Biochemicals Ltd., Thane, India, under the trade name Wuxi ^® from Wuxi Snyder Bioproducts Ltd., China, under the trade names Proleather® ^® and protease P ^® from Amano Pharmaceuticals Ltd., Nagoya, Japan, and the enzyme available under the name Proteinase K-16 from Kao Corp., Tokyo, Japan.

Surprisingly, it has been found that such proteases are particularly well stabilized or reversibly inhibited by the compounds described. In addition, certain variants of proteases, i. also variants of said proteases, stabilized by these compounds particularly advantageous. Such protease variants are part of the invention described below.

A protease stabilized or reversibly inhibited according to the invention can be a wild-type enzyme or a protease variant. Under wild-type enzyme is to be understood that the enzyme is present in a naturally occurring organism or in a natural habitat can be isolated from this. However, enzymes are modifiable and sometimes selectively modified, in particular in order to adapt their properties to the intended uses or to influence their catalytic activity. These changes often occur by altering the amino acid sequence of the enzyme. Such changes can be targeted and thus local or random, for example, by random mutagenesis done. An enzyme variant is understood as meaning enzymes which have been generated from an initial enzyme, for example a wild-type enzyme, by altering the amino acid sequence. The alteration of the amino acid sequence is preferably carried out by mutations, wherein amino acid substitutions, deletions, insertions or combinations thereof may be made. The incorporation of such mutations into proteins is well known in the art and to those skilled in the art of enzyme technology. In principle, all enzymes can be changed in this way. Protease variants are preferred according to the invention. These were generated from an initial protease, for example a wild-type protease, by altering the amino acid sequence, preferably amino acid substitutions, deletions, insertions or combinations thereof. However, the starting protease does not necessarily have to be a naturally occurring wild-type protease; a protease known from the prior art in which changes have already been made can also be further developed and therefore again serve as an initial protease for generating further protease variants.

Thus, for example, all of the proteases described above may be used unchanged in agents according to the invention and be stabilized by the compounds described. However, they can also be the starting enzyme for a variant which is then contained in an agent according to the invention and stabilized by the compounds described.

• Die Alkalische Protease aus Bacillus amyloliquefaciens (BPN'),
• Die Alkalische Protease aus Bacillus licheniformis (Subtilisin Carlsberg),
• Die Alkalische Protease PB92,
• Subtilisin 147 und/oder Subtilisin 309 (Savinase)
• Die Alkalische Protease aus Bacillus lentus, vorzugsweise aus Bacillus lentus DSM 5483,
• Die Alkalische Protease aus Bacillus alcalophilus (DSM 11233),
• die Alkalische Protease aus Bacillus gibsonii (DSM 14391) oder eine hierzu mindestens zu 70% identische Alkalische Protease,
• die Alkalische Protease aus Bacillus sp. (DSM 14390) oder eine hierzu mindestens zu 98,5% identische Alkalische Protease,
• die Alkalische Protease aus Bacillus sp. (DSM 14392) oder eine hierzu mindestens zu 98,1 % identische Alkalische Protease,
• die Alkalische Protease aus Bacillus gibsonii (DSM 14393) oder eine hierzu mindestens zu 70% identische Alkalische Protease.

Among all proteases or variants described further preferred is the wild-type enzyme or the starting enzyme of the variant:

• The alkaline protease from Bacillus amyloliquefaciens (BPN '),
• The alkaline protease from Bacillus licheniformis (subtilisin Carlsberg),
• Alkaline Protease PB92,
• Subtilisin 147 and / or subtilisin 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 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 a further embodiment of the invention, the washing or cleaning agent is therefore characterized in that the protease was obtained from an initial protease by at least one change of an amino acid, the change being a substitution, insertion or deletion of an amino acid, and adding it to the parent protease Amino acid level is at least 90%, preferably at least 92.5%, more preferably at least 95% and most preferably at least 97.5% identical.

Methods for carrying out and producing sequence comparisons, so-called alignments, are known to the person skilled in the art of enzyme technology. By means of such sequence comparisons, for example, the identity or homology values are determined for sequences to be compared. Such a comparison is accomplished by associating similar sequences in the nucleotide or amino acid sequences of the proteins of interest. This is called homologization. A tabular assignment of the respective positions is referred to as alignment. In the analysis of nucleotide sequences, in turn, both are complementary Strands and in each case all three possible reading frames to be considered; as well as the degeneracy of the genetic code and the organism-specific use of codons (codon usage). Meanwhile, alignments are created using computer programs, such as the algorithms FASTA or BLAST; This procedure is for example by DJ Lipman and WR Pearson (1985) in Science, Vol. 227, pp. 1435-1441 described.

A summary of all matching positions in the compared sequences is called a consensus sequence.

Such a comparison also allows a statement about the similarity or homology of the compared sequences to each other. This is represented in percent identity, that is the proportion of identical nucleotides or amino acid residues at the same or in an alignment corresponding positions. A broader concept of homology includes the conserved amino acid substitutions in this value. It then speaks of percent similarity. Such statements can be made about whole proteins or genes or only over individual areas.

Homologous regions of different proteins are defined by matches in amino acid sequence. These can also be identified by identical function. It goes as far as complete identities in the smallest areas, so-called boxes, which contain only a few amino acids and usually perform essential functions for the overall activity. The functions of the homologous regions are to be understood as the smallest partial functions of the function carried out by the entire protein, such as, for example, the formation of individual hydrogen bonds for the complexation of a substrate or transition complex.

In particular, such sequence comparisons or alignments also serve to determine mutually corresponding positions in different molecules. Thus, for example, in an alignment of different enzymes, it can be determined which positions in the respective amino acid or nucleic acid sequence correspond to one another, even if the respective sequences have, for example, different total lengths or different domains or partial sequences or if additional amino acids or nucleotides are present within a sequence are. A specific position in a first sequence can therefore be concretely assigned to a corresponding position in a second sequence, whereby it is quite possible for the positions corresponding to one another to be located at different locations in the molecule. Further, different amino acid residues may be present at the corresponding positions. Therefore, for such sequence comparisons or for the determination Specifically specified a position, which position it is and which enzyme is assumed, that is, which counting method of determining position is to be based.

For the following subjects of the invention, the amino acid sequence of the mature protein of the alkaline protease from Bacillus lentus DSM 5483 is used for determining the position, which is described in International Published Patent Application WO 91/02792 A1 and has a length of 269 amino acid residues (referred to in the present application as Bacillus lentus alkaline protease).

In a further embodiment of the invention, the washing or cleaning agent is characterized in that the protease was obtained from an initial protease by at least one change of an amino acid, the change being a substitution or insertion of an amino acid in that region of the amino acid sequence corresponding to the positions 95 to 103 of the alkaline protease from Bacillus lentus is assigned in an alignment.

Such a protease variant is particularly preferably a variant with an insertion of a single amino acid according to one or more of the positions 95, 96, 97, 98, 99, 100, 101, 102 and / or 103 and very particularly preferably between positions 97 and 98 and / or positions 99 and 100.

In a further embodiment of the invention, the washing or cleaning agent is characterized in that the protease has been obtained from an initial protease by at least one modification of an amino acid which corresponds to the 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, 211, 224, 229, 236, 237, 242 , 243, 250, 253, 255 and 268 of the Bacillus lentus alkaline protease in an alignment, wherein the alteration is a substitution, insertion or deletion of an amino acid.

More preferably, an amino acid change relative to the parent molecule occurs in one or more of the following positions: 3, 4, 43, 61, 188, 193, 199, 211, 224, 250 and 253 (count according to Bacillus lentus alkaline protease), more preferably with one or more of the amino acid substitutions X3T, X4I, X43V, X61A, X188P, X193M, X199I, X211L, X211D, X211E, X211G, X211N or X211Q, X224V, X250G and / or X253N. In particular, the protease is a variant with a point mutation in position 211, preferably with a substitution of a single amino acid in this position, particularly preferably with the amino acid substitution X211 L. The above position information relate turn to those amino acid residues that are assigned to the said positions of the alkaline protease from Bacillus lentus in an alignment.

Detergents or cleaning agents according to the invention may contain only one enzyme. Alternatively, they may also contain other enzymes in a concentration effective for the effectiveness of the agent. A further subject of the invention thus represents agents which further comprise one or more further enzymes, wherein in principle all enzymes established in the prior art for these purposes can be used. Other enzymes which can be used as further enzymes are all enzymes which can develop catalytic activity in the agent according to the invention, in particular proteases, amylases, cellulases, hemicellulases, mannanases, tannases, xylanases, xanthanases, β-glucosidases, carrageenases, oxidases, oxidoreductases, lipases or esterases and preferably mixtures thereof. 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.

These enzymes are basically of natural origin; but starting from the natural molecules, improved variants are also available for use in detergents and cleaners, which are preferably used accordingly. Agents ^according to the invention preferably contain enzymes in total amounts of 1 × 10 ^-8 to 5 percent by weight, based on active protein. Preferably, the enzymes are from 0.001 to 5% by weight, more preferably from 0.01 to 5% by weight, even more preferably from 0.05 to 4% by weight and most preferably from 0.075 to 3.5% by weight. % contained in agents according to the invention, wherein each enzyme contained can be present in the stated proportions.

The protein concentration can be determined by known methods, for example the BCA method (bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method ( Gornall AG, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766 ). The further enzymes particularly preferably support the effect of the agent, for example the cleaning performance of a washing or cleaning agent, with regard to certain stains or stains. Particularly preferably, the enzymes show synergistic effects with respect to their action against certain stains or stains, ie the enzymes contained in the middle composition mutually support each other in their cleaning performance. Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the composition according to the invention.

The enzymes used in agents of the invention are either originally from microorganisms, such as the genera Bacillus, Streptomyces, Humicola, or Pseudomonas, and / or are produced by biotechnological methods known per se by suitable microorganisms, for example by transgenic expression hosts of the genera Bacillus or by filamentous fungi ,

In agents according to the invention, the enzyme (s) and the stabilizing compound are preferably combined with one or more of the following ingredients: nonionic, anionic and / or cationic surfactants, bleaches, bleach activators, bleach catalysts, builders and / or cobuilders, acids, alkaline substances, Hydrotropes, solvents, thickeners, sequestering agents, electrolytes, optical brighteners, grayness inhibitors, corrosion inhibitors, in particular silver protectants (silver corrosion inhibitors), soil release agents, color transfer (or transfer) inhibitors, foam inhibitors, abrasives, dyes, fragrances, perfumes, antimicrobial agents, UV protectants or absorbents, antistatic agents, pearlescing agents and skin protection agents, further stabilizers, in particular enzyme stabilizers, and other components known from the prior art. A further subject of the invention therefore is washing or cleaning agents which are characterized in that they contain at least one further component which is selected from the group consisting of surfactants, builders, acids, alkaline substances, hydrotropes, solvents, thickeners, bleaching agents, Dyes, perfumes, corrosion inhibitors, sequestering agents, electrolytes, optical brighteners, grayness inhibitors, silver corrosion inhibitors, dye transfer inhibitors, foam inhibitors, abrasives, UV absorbers, solvents, antistatic agents, pearlescers and skin protection agents. 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.

The ingredients to be selected as well as the conditions under which the agent is used, such as temperature, pH, ionic strength, redox ratios or mechanical influences, should be optimized for the respective cleaning problem. For example, the usual temperatures for detergents and cleaners in the range of 10 ° C for manual agents over 20 ° C, 30 ° C, 40 ° C and 60 ° C up to 95 ° for mechanical means or in technical applications. Since the temperature is usually infinitely adjustable in modern washing machines and dishwashers, all intermediate stages of the temperature are included. Preferably, the ingredients of the respective agents are coordinated. Synergies are preferred in terms of cleaning performance. Particularly preferred in this regard are synergies which are present in a temperature range between 20.degree. C. and 60.degree. C., since the enzyme or the enzymes contained in the agents according to the invention are also catalytically active in this temperature range.

In a further embodiment of the invention, the washing or cleaning agent is characterized in that it contains at least one further stabilizer. In such an agent, therefore, there are at least two compounds which cause stabilization of a contained enzyme, preferably a protease. Preferably, these compounds act synergistically, ie the stabilization effect achieved by both compounds exceeds the sum of the two individual stabilization effects. In a preferred embodiment, the stabilizer (s) is one or more polyols, in particular glycerol or 1,2-ethylene glycol, an antioxidant, lactate or one or more lactate derivatives or combinations thereof. It is likewise preferably one or more of those enzyme-stabilizing or inhibiting compounds which are described in the international patent applications WO 07/113241 A1 or WO 02/008398 are disclosed.

in der X ein aliphatischer oder ein aromatischer Rest ist und Y ein aliphatischer oder ein aromatischer Rest ist, als reversibler Inhibitor eines Enzyms in einem Wasch- oder Reinigungsmittel. Denn wie vorstehend bereits ausgeführt, bewirken diese Verbindungen auf Grund ihres Wirkmechanismus als reversibler Inhibitor eine vorteilhafte Stabilisierung des Enzyms in dem Wasch- oder Reinigungsmittel. In einer bevorzugten Ausführungsform der Erfindung ist die Verwendung dadurch gekennzeichnet, dass die Reste X und Y identisch sind. In einer weiteren bevorzugten Ausführungsform der Erfindung ist die Verwendung dadurch gekennzeichnet, dass der Rest X ausgewählt ist aus der Gruppe bestehend aus: -CH₂-CH₂-CH₃, Phenyl. In einer weiteren Ausführungsform der Erfindung ist die Verwendung dadurch gekennzeichnet, dass der Rest Y ausgewählt ist aus der Gruppe bestehend aus: -CH₂-CH₂-CH₃, Phenyl.Another object of the invention is the use of a compound of the general structural formula

wherein X is an aliphatic or an aromatic radical and Y is an aliphatic or an aromatic radical, as a reversible inhibitor of an enzyme in a washing or cleaning agent. Because, as already stated above, cause these compounds due to their mechanism of action as a reversible inhibitor advantageous stabilization of the enzyme in the washing or cleaning agent. In a preferred embodiment of the invention, the use is characterized in that the radicals X and Y are identical. In a further preferred embodiment of the invention, the use is characterized in that the radical X is selected from the group consisting of: -CH ₂ -CH ₂ -CH ₃ , phenyl. In a further embodiment of the invention, the use is characterized in that the radical Y is selected from the group consisting of: -CH ₂ -CH ₂ -CH ₃ , phenyl.

In a further preferred embodiment of this subject of the invention, the enzyme is selected from the group consisting of: protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, Oxidoreductase, lipase, esterase or mixtures thereof. The enzyme is particularly preferably a protease, preferably a serine protease, more preferably a subtilase and particularly preferably a subtilisin.

in der X ein aliphatischer oder ein aromatischer Rest ist und Y ein aliphatischer oder ein aromatischer Rest ist, zur Herstellung eines Wasch- oder Reinigungsmittels. Insbesondere handelt es sich bei der Verbindung um eine solche, in der die Reste X und Y identisch sind und/oder der Rest X ausgewählt ist aus der Gruppe bestehend aus: -CH₂-CH₂-CH₃, Phenyl, und/oder der Rest Y ausgewählt ist aus der Gruppe bestehend aus: -CH₂-CH₂-CH₃, Phenyl.Due to the described advantageous uses according to the invention of such a compound for stabilizing an enzyme in a washing or cleaning agent, such a combination of enzyme and stabilizer is likewise advantageously used in the preparation of a corresponding washing or cleaning agent. Another object of the invention is therefore the use of an enzyme and a compound of the general structural formula

wherein X is an aliphatic or an aromatic radical and Y is an aliphatic or an aromatic radical, for the preparation of a washing or cleaning agent. In particular, the compound is one in which the radicals X and Y are identical and / or the radical X is selected from the group consisting of: -CH ₂ -CH ₂ -CH ₃ , phenyl, and / or the Rest Y is selected from the group consisting of: -CH ₂ -CH ₂ -CH ₃ , phenyl.

In a preferred embodiment, this invention is characterized in that the enzyme is selected from the group consisting of: protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, oxidoreductase, lipase, esterase or mixtures thereof. The enzyme is particularly preferably a protease, preferably a serine protease, more preferably a subtilase and particularly preferably a subtilisin.

in der X ein aliphatischer oder ein aromatischer Rest ist und Y ein aliphatischer oder ein aromatischer Rest ist. Insbesondere handelt es sich bei der Verbindung um eine solche, in der die Reste X und Y identisch sind und/oder der Rest X ausgewählt ist aus der Gruppe bestehend aus: - CH₂-CH₂-CH₃, Phenyl, und/oder der Rest Y ausgewählt ist aus der Gruppe bestehend aus: -CH₂-CH₂-CH₃, Phenyl.Another object of the invention is a washing or cleaning process in which an enzyme acts, which is inhibited and / or stabilized with a compound of the general structural formula

wherein X is an aliphatic or an aromatic radical and Y is an aliphatic or an aromatic radical. In particular, the compound is one in which the radicals X and Y are identical and / or the radical X is selected from the group consisting of: CH ₂ -CH ₂ -CH ₃ , phenyl, and / or the radical Y is selected from the group consisting of: -CH ₂ -CH ₂ -CH ₃ , phenyl.

Because, as already stated above, these compounds cause due to their mechanism of action as a reversible inhibitor advantageous stabilization of the enzyme in the washing or cleaning agent, so that for the washing or cleaning process, a higher enzyme activity is available in comparison with a detergent or cleaning agent in which the enzyme was not stabilized. In a further preferred embodiment, this subject matter of the invention is characterized in that the enzyme is selected from the group consisting of: protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, oxidoreductase, lipase, Esterase or mixtures thereof. The enzyme is particularly preferably a protease, preferably a serine protease, more preferably a subtilase and particularly preferably a subtilisin.

In a preferred embodiment of the invention, the washing or cleaning method is further characterized in that a washing or cleaning agent according to the invention is used, as described above.

From the advantageous use of detergents or cleaning agents according to the invention in washing or cleaning processes, it is consequently clear that the washing or cleaning agents according to the invention can advantageously be used for cleaning purposes. A further subject of the invention is therefore the use of a washing or cleaning agent, as described above, for washing and / or cleaning textiles and / or hard surfaces.

It is expressly emphasized at this point that all facts, objects and embodiments which are described for washing or cleaning agents according to the invention are also applicable to all abovementioned uses according to the invention and to all methods according to the invention. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to all above uses and methods according to the invention.

The following examples further illustrate the invention without, however, limiting it thereto.

Examples example 1 Examination of protease residual activity in the presence of an inhibitor

To demonstrate that the compounds listed below exert a protease activity-inhibiting action and thus have a stabilizing effect, the residual proteolytic activity of Bacillus lentus alkaline protease F49 (according to US Pat WO 95/23221 A1 ) 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; Bachem L-1400) and 5 x 10 ^-9 and 1 x 10 ^-8 M of the protease presented. Added to this were the following compounds to be tested in a final concentration of 10 mM. They were each dissolved in anhydrous DMSO, with effects of DMSO on the enzymatic activity being corrected by the corresponding 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. As a comparison, the corresponding reaction mixture was used with protease, but without the compound in question. The protease activity in these comparisons was set as 100%.

• V1: Dibutylphosphat
• V2: Dibenzylphosphat

In this way, the following compounds were investigated:

• V1: dibutyl phosphate
• V2: dibenzyl phosphate

The compounds resulted in a residual activity of the protease of less than 60%. Among them, V1 is the strongest and thus most suitable protease inhibitor or stabilizer, followed by V2. The residual activities of the protease were 45% for V1 and 56% for V2.

Due to these results, these compounds are very well suited to stabilize the enzymatic activities in protease-containing detergents and cleaning agents during storage.

Example 2

Investigation of the storage stability of protease-containing detergents and cleaners in the presence of enzyme stabilizers according to the invention

As a base formulation, a liquid detergent was prepared with the following composition (all figures in percent 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 was admixed with the inhibiting compounds of Example 1 to be tested and 1,275,000 HPE / I B. lentus alkaline protease F 49. The protease activity (Henkel protease units) reported in HPE was calculated after van Raay, Saran and Verbeek, according to the publication " For the determination of the proteolytic activity in enzyme concentrates and enzyme-containing detergents, dishwashing detergents and cleaners "in Tenside (1970), Volume 7, pp. 125-132 , certainly.

The storage took place for different lengths of time in airtight containers at 30 ° C.

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

All investigated compounds showed a clearly stabilizing effect.

Claims (15)

1. A washing or cleaning agent containing an enzyme and a compound of the general structural formula

   

   in which X is an aliphatic or an aromatic residue, and Y is an aliphatic or an aromatic residue, wherein residue X is selected from the group consisting of: -CH₂-CH₂-CH₃, phenyl, in particular one in which the residues X and Y are identical, and/or the residue Y is selected from the group consisting of: -CH₂-CH₂-CH₃, phenyl.
2. The washing or cleaning agent according to Claim 1, characterized in that the compound has, with respect to the enzyme, an inhibition constant (K_i) from 0.01 to 10 mM, in particular an inhibition constant (K_i) from 0.03 to 5 mM.
3. The washing or cleaning agent according to one of Claims 1 or 2, characterized in that the enzyme is contained at a concentration from 2 µg to 20 mg per gram of the agent, 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, particularly preferably from 50 µg to 10 mg of the agent,
   and/or in that the compound is contained at a concentration of up to 50 mg per g of the agent, preferably up to 10 mg, more preferably up to 7 mg, particularly preferably up to 5 mg per gram of the agent,
   and/or in that the molar ratio of the compound to the enzyme is from 1:1 to 1,000:1, preferably from 1:1 to 500:1, more preferably from 1:1 to 100:1, particularly preferably from 1:1 to 20:1.
4. The washing or cleaning agent according to one of Claims 1 to 3, characterized in that the quantity of the compound contained in the agent, based on the stabilized enzyme, is from 0.01 to 100 times the inhibition constant K_i, preferably 0.1 to 10 times K_i, more preferably 1 to 5 times K_i.
5. The washing or cleaning agent according to one of Claims 1 to 4 in predominantly solid form, or in predominantly liquid, pasty, or gel form.
6. The washing or cleaning agent according to one of Claims 1 to 5, characterized in that the enzyme is selected from the group consisting of: protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, oxidoreductase, lipase, esterase, or mixtures thereof.
7. The washing or cleaning agent according to one of Claims 1 to 6, characterized in that the enzyme is a protease, preferably a serine protease, more preferably a subtilase, and particularly preferably a subtilisin.
8. The washing or cleaning agent according to Claim 7, characterized in that the protease was obtained from an initial protease by means of at least one modification of an amino acid, the modification being a substitution, insertion, or deletion of an amino acid, and it is at least 90%, preferably at least 92.5%, more preferably at least 95%, and particularly preferably at least 97.5% identical to the initial protease at the amino acid level,
   and/or in that the protease was obtained from an initial protease by means of at least one modification of an amino acid, the modification being a substitution or insertion of an amino acid in that region of the amino acid sequence that is associated, in an alignment, with positions 95 to 103 of the alkaline protease from Bacillus lentus,
   and/or in that the protease was obtained from an initial protease by means of at least one modification of an amino acid that are associated,
   in an alignment, with 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, 211, 224, 229, 236, 237, 242, 243, 250, 253, 255, and 268 of the alkaline protease from Bacillus lentus, the modification being a substitution, insertion, or deletion of an amino acid.
9. The washing or cleaning agent according to one of Claims 1 to 8, characterized in that it contains at least one further stabilizer, in particular one that involves one or more polyols, in particular glycerol or 1,2-ethylene glycol, an antioxidant, lactate or one or more lactate derivatives, or combinations thereof.
10. Use of a compound of the general structural formula

    

    in which X is an aliphatic or an aromatic residue, and Y is an aliphatic or an aromatic residue, wherein residue X is selected from the group consisting of: -CH₂-CH₂-CH₃, phenyl, as a reversible inhibitor of an enzyme in a washing or cleaning agent, in particular in one in which the residues X and Y are identical, and/or the residue Y is selected from the group made up of: -CH₂-CH₂-CH₃, phenyl.
11. Use of an enzyme, in particular of one that is selected from the group consisting of protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, oxidoreductase, lipase, esterase, or mixtures thereof, and of a compound of the general structural formula

    

    in which X is an aliphatic or an aromatic residue, and Y is an aliphatic or an aromatic residue, wherein residue X is selected from the group made up of: -CH₂-CH₂-CH₃, phenyl, to manufacture a washing or cleaning agent, in particular one in which the residues X and Y are identical, and/or the residue Y is selected from the group made up of: -CH₂-CH₂-CH₃, phenyl.
12. The use according to Claim 11, characterized in that the enzyme is a protease, preferably a serine protease, more preferably a subtilase, and particularly preferably a subtilisin.
13. A washing or cleaning method in which an enzyme, in particular one that is selected from the group consisting of protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, oxidoreductase, lipase, esterase, or mixtures thereof, is effective, which is inhibited and/or stabilized with a compound of the general structural formula

    

    in which X is an aliphatic or an aromatic residue, and Y is an aliphatic or an aromatic residue, wherein residue X is selected from the group consisting of: -CH₂-CH₂-CH₃, phenyl, in particular one in which the residues X and Y are identical, and/or the residue Y is selected from the group consisting of: -CH₂-CH₂-CH₃, phenyl.
14. A washing or cleaning method, characterized in that a washing or cleaning agent according to one of Claims 1 to 9 is used.
15. Use of a washing or cleaning agent according to one of Claims 1 to 9 to wash and/or clean textiles and/or hard surfaces.