EP2756064A2

EP2756064A2 - Method for adapting a hydrolytic enzyme to a component that stabilizes the hydrolytic enzyme

Info

Publication number: EP2756064A2
Application number: EP12755969.8A
Authority: EP
Inventors: Petra Siegert; Stefan Evers; Marion Merkel; Nina Mussmann; Hendrik Hellmuth; Timothy O'connell; Karl-Heinz Maurer; Ulrich Schwaneberg; Felix Jakob; Ronny MARTINEZ; Brian LAUFS; Ayhan AYDEMIR; Thomas Weber
Original assignee: Henkel AG and Co KGaA
Current assignee: Henkel AG and Co KGaA
Priority date: 2011-09-12
Filing date: 2012-08-21
Publication date: 2014-07-23
Also published as: WO2013037609A2; EP3067411A1; DE102011118027A1; US20140186868A1; US9695461B2; WO2013037609A3

Abstract

The stabilization of a hydrolytic enzyme in a liquid preparation is to be improved through a component that stabilizes the hydrolytic enzyme. This is accomplished by a method for adapting a hydrolytic enzyme to a component that stabilizes the hydrolytic enzyme, comprising the following method steps: a) providing a hydrolytic enzyme (starting enzyme) and a component that stabilizes the hydrolytic enzyme and that comprises a reversible inhibitor of the hydrolytic enzyme; b) changing the amino acid sequence of the hydrolytic enzyme in at least one position, by substitution, deletion or insertion; c) determining the relative activity of the hydrolytic enzyme from step b) and the relative activity of the starting enzyme in a liquid preparation; d) selecting the hydrolytic enzyme that has a reduced relative activity by comparison with the relative activity of the starting enzyme.

Description

A method of adapting a hydrolytic enzyme to a hydrolytic enzyme stabilizing component

The invention is in the field of enzyme technology. In particular, the invention relates to a process in which a hydrolytic enzyme, in particular a protease, is adapted to an enzyme stabilizer and consequently optimized for it. Furthermore, the invention relates to hydrolytic enzymes obtainable by such a process.

Problems concerning the storage stability of enzyme-containing preparations, for example of

Enzyme preparations or detergents, cleaners or disinfectants are known from the prior art. This problem is particularly pronounced in the case of liquid enzyme preparations or liquid enzyme-containing surfactant preparations, for example liquid detergents or cleaners. After only a short time, they lose a considerable amount of enzymatic, in particular hydrolytic and, in particular, proteolytic activity. The surfactant preparation, for example the washing, cleaning or disinfecting agent, then shows no optimal cleaning performance. An aim in the development of enzyme-containing preparations is therefore to stabilize the enzymes contained and to protect them especially during storage and / or during application of the preparation from denaturation, cleavage and / or other degradation. In this regard, especially hydrolytic enzymes and in particular proteases, but also, for example, amylases, are of interest. For this purpose, chemical preparations can be added to the preparations which in particular reversibly inhibit proteases and thus act collectively as stabilizers for the proteases and other enzymes contained. It must be reversible inhibitors, since the enzyme activity only temporarily, especially during storage, but not during the

Cleaning process should be prevented.

Boric acid and boric acid derivatives occupy an outstanding position under enzyme stabilizers which are effective in surfactant preparations at a comparatively low concentration. For example, International Patent Application WO 96/21716 A1 discloses that

Protease inhibitors acting boron and boronic acid derivatives are suitable to stabilize enzymes in liquid preparations, including detergents and cleaning agents. A selection of boronic acid derivatives as stabilizers is disclosed, for example, in international patent application WO 96/41859 A1. WO 92/19707 A1 and EP 478050 A1 disclose meta-substituted or para-substituted phenylboronic acids as enzyme stabilizers. Complexes of boric acids and Boric acid derivatives with aromatic compounds as enzyme stabilizers in liquid

Detergent compositions are disclosed in EP 51 1456 A1.

However, boric acids and boric acid derivatives have the disadvantage that they form unwanted by-products with other ingredients of a surfactant preparation, in particular detergents, cleaners or disinfectant ingredients, so that they are no longer available for the desired cleaning purpose in the respective compositions or even as

Contain contamination, for example on the laundry. Furthermore, boric acids and borates are increasingly regarded as environmentally disadvantageous.

Although boric acid-free or boron-free compounds are known from the prior art, which can reversibly inhibit hydrolytic enzymes and thus stabilize. However, these often have the disadvantage that they do not cause satisfactory enzyme stabilization in a preparation of the enzyme, in particular not in liquid surfactant preparations such as in detergents, cleaners or disinfectants. To get a sufficient

To effect stabilization, such compounds would often have to be used in such a large amount, which often makes use impossible, since these amounts can not be satisfactorily incorporated into the enzyme preparation and / or their incorporation is not economically viable.

The object of the present invention is to adapt a hydrolytic enzyme to a hydrolytic enzyme-stabilizing component, in particular a reversible inhibitor, and consequently to improve its stabilization by this component.

Preferably, the enzyme-stabilizing component should comprise as small a quantity as possible of boric acid or boron-containing compounds or preferably be free of boric acid and / or boron.

The invention relates to a method for adapting a hydrolytic enzyme to a hydrolytic enzyme stabilizing component comprising the steps of a) providing a hydrolytic enzyme (starting enzyme) and a hydrolytic enzyme stabilizing component comprising a reversible inhibitor of the hydrolytic enzyme;

b) altering the amino acid sequence of the hydrolytic enzyme at at least one position by substitution, deletion or insertion;

c) determining the relative activity of the hydrolytic enzyme of step b) and the relative activity of the starting enzyme in a liquid preparation;

d) selecting the hydrolytic enzyme having reduced relative activity as compared to the relative activity of the parent enzyme. A process according to the invention may further comprise the following process step e): e) optionally repeating the process steps b) to d) until the hydrolytic enzyme has a reduced relative activity in comparison with the relative activity of the starting enzyme.

In the context of the present invention, therefore, an unusual approach is followed.

Contrary to the procedure established in the prior art for finding a component for a given hydrolytic enzyme which stabilizes this enzyme, according to the invention the reverse route is taken, namely that the hydrolytic enzyme is modified and thereby attached to the hydrolytic enzyme stabilizing component (enzyme stabilizer), in particular to the reversible inhibitor, so that improved stabilization of the altered hydrolytic enzyme by the hydrolytic enzyme stabilizing component is achieved. It is not the right stabilizer for an enzyme sought, but optimized the enzyme for its stabilization by the given stabilizer. It has been found that it is advantageous to attach the hydrolytic enzyme to the hydrolytic enzyme stabilizing

Component, not vice versa. In this way, for example, advantageous combinations of enzyme and stabilizing compound can be identified in a short time. In preferred embodiments, such combinations result in improved enzyme inhibition and / or enzyme stabilization by the stabilizing compound, especially in a liquid detergent, cleaning or disinfecting agent. This opens up the possibility of using less boric acid-containing and / or boron-containing compounds as enzyme stabilizers in liquid preparations, in particular liquid surfactant preparations. In particular, in particularly preferred embodiments, it is possible to dispense partially or preferably completely with boric acid as enzyme stabilizer in such a liquid preparation, so that the preparation can be free from boric acid. In further particularly advantageous embodiments, such a preparation may ideally be free of boron.

Preferably, inventively adapted combinations of enzyme stabilizer and hydrolytic enzyme also have the advantage that the enzyme stabilizer unfolds its stabilizing effect even in low to very low concentrations. Further, it can be selected so that it preferably has good water solubility. Therefore, it can be easily incorporated into an aqueous liquid preparation, in particular a surfactant preparation and, preferably, a liquid washing, cleaning or disinfecting agent, together with the enzyme stabilized by it. Furthermore, precipitation during storage is preferably reduced or completely avoided.

A hydrolytic enzyme is a hydrolase (EC 3.XXX) and therefore an enzyme that hydrolyzes esters, ethers, peptides, glycosides, acid anhydrides or C-C bonds in a reversible reaction splits. The hydrolytic enzyme therefore catalyzes the hydrolytic cleavage of substances according to AB + H ₂ O <-> AH + B-OH. Hydrolases constitute the third major class of EC classification of enzymes. The EC numbers ("Enzyme Commission numbers") form a numerical classification system for enzymes Each EC number consists of four numbers separated by periods, the first digit designating one of the six major enzyme classes and hydrolases corresponding to EC 3.XXX Their representatives are proteases, peptidases, nucleases, phosphatases, glycosidases and esterases, more preferably the hydrolytic enzyme is a protease.

In step a) a hydrolytic enzyme and a hydrolytic enzyme stabilizing component is provided which comprises a reversible inhibitor of the hydrolytic enzyme. A reversible inhibitor of the hydrolytic enzyme in this regard is any compound which is reversibly capable of non-covalent interaction with the hydrolytic enzyme and which reduces the catalytic activity of the hydrolytic enzyme due to its interaction with the enzyme. The stabilization of the hydrolytic enzyme is therefore due to its inhibition by the reversible inhibitor, preferably in an aqueous environment. Water itself is therefore not a hydrolytic enzyme stabilizing component in the context of the present invention. Preferably, the reversible inhibitor of the hydrolytic enzyme has a molar mass of at least 20 g / mol, and more preferably of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 g / mol. More preferably, this is an organic chemical compound.

The reversible inhibitor with regard to the hydrolytic enzyme in process step a) preferably has an inhibition constant (Ki) of 0.01 to 500 mM, preferably 0.01 to 100 mM, particularly preferably 0.01 to 1 mM or 10 to 100 mM on.

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

For the characterization of a reversible inhibitor of enzymatic activity is the

Inhibition constant K a characteristic and decisive size. K, 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, is accordingly defined as:

K = [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 is made on the basis of the activity test of the protease in the presence of the corresponding inhibitor. Michaelis-Menten kinetics (Leonor Michaelis, Maud Menten (1913), The Inverting Effect Kinetics, Biochem., Z. 49: 333-369) established in the prior art describe the enzymatic parameters K _m , and k _cat in the presence of various concentrations of the inhibitor. The following applies for a Michaelis-Menten kinetics:

Herein: E: Enzyme

S: substrate

ES: enzyme-substrate complex

P: product

k-1, k-i, 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) (ie, the substrate concentration present at half-saturation, where the rate of turnover is thus v = Vmax / 2), is increased

K _m = k.-i / k-ι (Michaelis-Menten case, given if k ₂ «k-ι) or more generally

K _m = k.i + k ₂ / k- ₁ (Briggs-Haldane situation, given for the case that k ₂ can not be neglected with respect to k- ₁ ).

The saturation function of a "Michaelis-Menten enzyme" is calculated using the parameters Km and Vmax as follows: Herein: v: rate of formation of P (v = "velocity") [mol 1-1 s-1]

vmax: maximum velocity [mol 1-1 s-1]

Km: Michaelis-Menten constant [mol 1-1]

[S]: substrate concentration [mol 1-1]

By determining the initial catalysis rate (v- _h .) - for proteases of the initial hydrolysis rate - at various substrate concentrations [S] and fitting the experimental data into Equation 1 below, the inhibition constant K i is obtained.

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

Here again [I] stands for the inhibitor concentration.

Alternatively, K, using the Cheng-Prusoff equation (Equation 2, Cheng Y., Prusoff WH (1973) Biochem. Pharmacol., 22, 3099-3108) can be 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 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 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 values which can be determined in this way characterize the reversible inhibitor with regard to the enzyme used, that is to say, for example, the reversible protease inhibitor with respect to the protease used.

The hydrolytic enzyme stabilizing component may consist entirely of the reversible inhibitor such that the hydrolytic enzyme stabilizing component

(Enzyme stabilizer) is the reversible inhibitor. This embodiment is particularly preferred.

Alternatively, the hydrolytic enzyme stabilizing component may contain other compounds so that the particular reversible inhibitor is part of the hydrolytic enzyme stabilizing component. In particular, the hydrolytic enzyme stabilizing component may comprise combinations of different reversible inhibitors.

Preferably, the hydrolytic enzyme stabilizing component (in addition to the inhibitory effect of the reversible inhibitor) also already provides storage stability of the hydrolytic enzyme in an aqueous environment. By an inventive

Processes, the storage stability is then preferably further improved by the hydrolytic enzyme is adapted to the hydrolytic enzyme stabilizing component, in particular to the reversible inhibitor.

A storage stability is when the presence of the hydrolytic enzyme stabilizing component causes a hydrous liquid preparation comprising hydrolytic enzyme and hydrolytic enzyme stabilizing component (stabilized preparation) to have a higher enzymatic residual activity of the hydrolytic enzyme after storage as compared to a control preparation, which differs from the stabilized preparation only by the absence of the hydrolytic enzyme stabilizing component (control). In this regard, the hydrolytic enzyme stabilizing component is contained in an amount of 0.002 to 0.35% by weight in the stabilized preparation. Therefore, after storage, this preparation has a higher residual activity of the hydrolytic enzyme compared to the control, the stabilized preparation and the control being the same enzymatic

Starting activity at the start of storage, both preparations are treated in the same way, in particular with regard to the conditions of storage and the determination of the enzyme activity. The storage is preferably carried out for at least 48 hours at a temperature of 20 ° C, 25 ° C or 30 ° C. More preferably, storage is for at least 72 hours, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks at a temperature of 20 ° C, 25 ° C or 30 ° C over the entire storage period.

Particularly preferably, the presence of a storage stability is determined

i. for a protease-containing liquid preparation using this protease-containing liquid preparation, which is stored for at least 48 hours at a temperature of 30 ° C and whose residual proteolytic activity is determined by the release of the

Chromophore para-nitroaniline (pNA) from the substrate suc-AAPF-pNA as indicated below; and or

ii. for an amylase-containing liquid preparation using this amylase-containing, preferably protease-free, liquid preparation which is stored for at least 48 hours at a temperature of 30 ° C, and whose amylolytic residual activity is determined as indicated below. For clarification, it should be noted at this point that according to the invention in process step a) there is at least one reversible inhibitor of the hydrolytic enzyme whose inhibitory effect preferably-but not necessarily-already effects a storage stability of the hydrolytic enzyme, the storage stability preferably being determined as described above. However, the mere detection of an inhibitory effect of a compound on a hydrolytic enzyme, the presence of a storage stability is not necessary - in this regard, the determination of the activity of the hydrolytic enzyme in the presence and absence of the compound is crucial. It is therefore between inhibitory effect and storage stability too

differentiate. A reversible inhibitor has an inhibitory effect - this requires preferably, but not necessarily, a storage stability.

In order to adapt the hydrolytic enzyme to the hydrolytic enzyme stabilizing component, in particular to the reversible inhibitor, in process step b) the

Amino acid sequence of the hydrolytic enzyme in at least one position by substitution, deletion or insertion changed. It is also possible to make several substitutions, deletions or insertions. Any desired combinations of one or more substitutions, deletions or insertions can also be carried out according to the invention. The introduction of such mutations into an amino acid sequence is familiar to the expert in the field of enzyme technology and is preferably carried out by altering the nucleic acid sequence coding for the hydrolytic enzyme. The altered enzyme itself is then obtained by expression of this nucleic acid sequence in a suitable host and

optionally subsequently purified. In the case of insertion, the amino acid to be inserted is inserted after the respective amino acid (i.e., C-terminal).

In method step c), the relative activity of the hydrolytic enzyme modified in process step b) and of the starting enzyme is determined. For the determination of the relative activity it is necessary to carry out the activity determination for an enzyme both in the presence and in the absence of the hydrolytic enzyme stabilizing component in a liquid preparation, in particular in a water-containing liquid preparation.

Preferably, the liquid preparation is composed as follows (all figures in weight percent): 0.3-0.5% xanthan, 0.2-0.4% anti-foaming agent, 6-7% glycerol, 0.3-0 , 5% ethanol, 4-7% FAEOS (fatty alcohol ether sulfate), 24-28% nonionic surfactants, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut fatty acids, 0.5% HEDP ( 1-hydroxyethane- (1,1-di-phosphonic acid)), 0-0.4% PVP (polyvinylpyrrolidone), 0-0.05% optical brightener, 0-0.001% dye, balance demineralized water. Such a preparation or a 50% aqueous solution thereof is particularly preferably used for the determination of the relative activity. The determination of the enzyme activity takes place - matched to the particular type of enzyme - in the usual way. Methods for determining activity are familiar to the expert in the field of enzyme technology and are routinely used by him. Methods for determining protease activity are disclosed, for example, in Tenside, Vol. 7 (1970), pp. 125-132. Preferably, the proteolytic activity is determined via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA). The protease cleaves the substrate and releases pNA. The release of pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of enzymatic activity (see Del Mar et al., 1979). The measurement is carried out at a temperature of 25 ° C, at pH 8.6 and a wavelength of 410 nm. The measuring time is 5-10 min with a measuring interval of 20 s to 60 s.

The amylolytic activity can be determined as follows: Under defined reaction conditions (tris-maleate buffer pH 6.5, 50 ° C., 15 minutes), the samples to be examined are diluted with 0.67% starch (FLUKA No. 85642; pretreated according to Zulkowsky (treated with glycerol at 190 ° C)) as a substrate. By addition of dinitrosalicylic acid and heating to 100 ° C, this is reduced by glucose and other reducing sugars to an orange-red dye, which is determined photometrically after completion of the reaction at a wavelength of 540 nm. The amount of released sugar corresponding to the color is a measure of the enzyme activity.

The relative activity to be determined in process step c) of the hydrolytic enzyme modified in process step b) is then obtained in accordance with

[Determined activity of the hydrolytic enzyme modified in step b) in the presence of the hydrolytic enzyme stabilizing component] divided by [determined activity of the hydrolytic enzyme altered in step b) in the absence of the hydrolytic enzyme stabilizing component] multiplied by 100.

The relative activity of the starting enzyme to be determined in step c) is then determined according to [determined activity of the starting enzyme in the presence of the hydrolytic enzyme stabilizing component] divided by [detected activity of the starting enzyme in

Absence of the hydrolytic enzyme stabilizing component] multiplied by 100.

The determination of the activities and consequently also the determination of the relative activities of hydrolytic enzyme (derived from process step b, test mixtures, each with and without stabilizing component) and starting enzyme (control mixtures, each with and without stabilizing component) is carried out under identical conditions, so that the comparable relative activities. Test approaches and control approaches therefore differ in each case only by the enzyme used, in each case in the presence or absence of the stabilizing component).

For carrying out the activity determinations, it is also advantageous if the concentration of the component stabilizing the hydrolytic enzyme is chosen such that the

Starting enzyme in the control batch with stabilizing component between 90% -60% of the activity of the control batch without stabilizing component is present.

In method step d), the relative activity of the in

Process step b) modified hydrolytic enzyme with the relative activity of

Starting enzyme and then selecting that hydrolytic enzyme which has reduced relative activity compared to the relative activity of the parent enzyme. Preferably, the one hydrolytic enzyme selected is one containing at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 17 , 5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5% and 40% have decreased relative activity compared with the relative activity of the parent enzyme ,

Optionally, process steps b) through d) may be repeated until the hydrolytic enzyme has reduced relative activity as compared to the relative activity of the

Starting enzyme. Preferably, the hydrolytic enzyme then obtained has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 17, 5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5% and 40% decreased relative activity compared to the relative activity of the parent enzyme.

A hydrolytic enzyme obtained in this way, in particular a protease, is inhibited in an improved manner by the reversible inhibitor of the component stabilizing the hydrolytic enzyme. This improved inhibitory effect also results in improved stabilization of the hydrolytic enzyme. An improved inhibition of the hydrolytic enzyme is thus accompanied by its improved stabilization. The improved stabilization can be determined in a storage test as described above.

In a further embodiment of the invention, a method according to the invention is characterized in that the hydrolytic enzyme is a protease, amylase, cellulase, glycosidase, hemicellulase, mannanase, xylanase, xyloglucanase, xanthanase, pectin-splitting enzyme, β-glucosidase, carrageenase or a lipase or a mixture comprising at least two of these enzymes. Examples of proteases are the subtilisins BPN 'from Bacillus amyloliquefaciens and Carlsberg from Bacillus licheniformis, the protease PB92, the subtilisins 147 and 309, the protease from Bacillus lentus, subtilisin DY and the subtilases, but no longer the subtilisins in the strict sense attributable enzymes Thermitase, proteinase K and the proteases TW3 and TW7. Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from the company Novozymes A / S, Bagsvaerd, 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. Further useful proteases are, for example, those under the trade names Durazym®, Relase®, Everlase®, Nafizym®, Natalase®, Kannase® and Ovozyme® by the company Novozymes, which are among others

Trademarks, Purafect®, Purafect® OxP, Purafect® Prime, Excellase® and Properase® from Danisco / Genencor, sold under the trade name Protosol® by the company

Company Advanced Biochemicals Ltd., Thane, India, sold under the trade name Wuxi® by Wuxi Snyder Bioproducts Ltd., China under the trade names Proleather® and Protease P® by Amano Pharmaceuticals Ltd., Nagoya, Japan, and the enzyme available under the name Proteinase K-16 from Kao Corp., Tokyo, Japan. Also particularly preferred are the proteases from Bacillus gibsonii and Bacillus pumilus disclosed in international patent applications WO 08/086916 and WO 07/131656. Further advantageous proteases are disclosed in patent applications WO 91/02792, WO 08/007319, WO 93/18140, WO 01/44452, GB 1243784, WO 96/34946, WO 02/029024 and WO 03/057246. Other useful proteases are those that are found in the microorganisms Stenotrophomonas maltophilia, in particular

Stenotrophomonas maltophilia K279a, Bacillus intermedius and Bacillus sphaericus

naturally present.

Examples of amylases are the a-amylases from Bacillus licheniformis, from Bacillus

amyloliquefaciens or from Bacillus stearothermophilus and in particular their improved for use in detergents or cleaners further developments. The enzyme from Bacillus licheniformis is available from the company Novozymes under the name Termamyl® and from the company Danisco / Genencor under the name Purastar®ST.

Further development products of this α-amylase are available from the company Novozymes under the trade name Duramyl® and Termamy Dultra, from the company Danisco / Genencor under the name Purastar®OxAm and from the company Daiwa Seiko Inc., Tokyo, Japan, as Keistase®. The α-amylase from Bacillus amyloliquefaciens is sold by the company Novozymes under the name BAN®, and derived variants of the Bacillus stearothermophilus α-amylase under the names BSG® and Novamyl®, also of the

Company Novozymes. Furthermore, for this purpose, the α-amylase from Bacillus sp. A 7-7 (DSM 12368) and cyclodextrin glucanotransferase (CGTase) from Bacillus agaradherens (DSM 9948). Furthermore, the amylolytic enzymes disclosed in International Patent Applications WO 03/00271 1, WO 03/054177 and WO07 / 079938 can be used. Likewise, fusion products of all the molecules mentioned can be used. In addition, those available under the trade name Fungamyl® from the company Novozymes

Further developments of the a-amylase from Aspergillus niger and A. oryzae suitable. Further advantageously usable commercial products are, for example, the amylase-LT® and Stainzyme® or Stainzyme ultra® or Stainzyme plus®, the latter also from the company

Novozymes. Also variants of these enzymes obtainable by point mutations can be used according to the invention.

Examples of cellulases (endoglucanases, EG) is the fungal, endoglucanase (EG) -rich cellulase preparation or its further developments, which is offered by the company Novozymes under the trade name Celluzyme®. Endolase® and Carezyme®, also available from Novozymes, are based on the 50 kD EG or 43 kD EG from Humicola insolens DSM 1800. Further commercial products of this company are Cellusoft®, Renozyme® and Celluclean®. Further usable are, for example, cellulases available from the company AB Enzymes, Finland, under the trade names Ecostone® and Biotouch®, which are based, at least in part, on the 20 kD-EG of melanocarpus. Other cellulases from AB Enzymes are Econase® and Ecopulp®. Other suitable cellulases are from Bacillus sp. CBS 670.93 and CBS 669.93, those derived from Bacillus sp. CBS 670.93 from the company

Danisco / Genencor under the trade name Puradax® is available. Other usable commercial products of the company Danisco / Genencor are "Genencor detergent cellulase L" and lndiAge®Neutra.

Further preferred hydrolytic enzymes are those which are grouped under the term glycosidases (E.C. 3.2.1.X). These include in particular arabinases, fucosidases,

Galactosidases, galactanases, arabico-galactan galactosidases, mannanases (also called mannosidases or mannases), glucuronosidases, agarase, carrageenases, pullulanases, β-glucosidases, xyloglucanases (xylanases), xanthanases and pectin degrading enzymes (pectinases). Preferred glycosidases are also summarized by the term hemicellulases. Hemicellulases include, in particular, mannanases, xyloglucanases (xylanases), β-glucosidases and carrageenases, as well as pectin-splitting enzymes, pullulanases and β-glucanases.

Pectin-splitting enzymes (pectinases) in the context of the invention are enzymes which cleave pectins and / or other galacturonans. Pectins are polysaccharides whose principal ingredient is α-D-galacturonic acid as a monomer, preferably at least 50 wt .-% and particularly preferably at least 65 wt .-%. These galacturonic acid monomers are linked together via o 1, 4, sometimes even to a small extent via β-1, 4-glycosidic bonds, and form the backbone of the pectin molecule, which is periodically blocked by 1, 2 bonds with α-L- Rhamnose is interrupted. A pectin is thus a rhamno-galacturonic acid. A pectin-splitting enzyme is consequently in particular an enzyme which catalyzes the hydrolysis of 1,4-α-galactosiduronic bonds.

Within the EC classification of enzymes, the numerical classification system for enzymes, the pectin-cleaving enzymes belong in particular to the enzyme classes EC 3.1.1.1 1, EC 3.2.1.15, EC 3.2.1.67 and EC 3.2.1.82 and thus belong to the third of the six major enzyme classes, the hydrolases (EC3. -.-.-), among them the

Glycosylases (E.C. 3.2.-.-) and again below to the glycosidases (E.C. 3.2.1.-), i.

Enzymes that hydrolyze O- and / or S-glycosyl compounds. Thus, pectin-splitting enzymes act particularly against residues on dishes containing pectic acid and / or other galacturonans and catalyze their hydrolysis.

For the purposes of the present invention, pectin-destroying enzymes are likewise counted as enzymes with the designations pectinase, pectate lyase, pectin esterase,

Pectin methethoxylase, pectin methoxylase, pectin methyl esterase, pectase, pectin methyl esterase, pectin esterase, pectin-pectyl hydrolase, pectin-polymerase, endopolygalacturonase, pectolase, pectin hydrolase, pectin-polygalacturonase, endo-polygalacturonase, poly-a-1, 4-galacturonide glycanohydrolase, endogalacturonase, endo-D-galacturonase, galacturan 1, 4-a-galacturonidase, exopolygalacturonase, poly (galacturonate) hydrolase, exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase, exo-poly-a-galacturonosidase,

Exopolygalacturonosidase or Exopolygalacturanosidase.

Examples of enzymes suitable for this purpose are, for example, under the name Gamanase®, Pektinex AR®, X-Pect® or Pectaway® from Novozymes, under the name Rohapect UF®, Rohapect TPL®, Rohapect PTE100®, Rohapect MPE®, Rohapect MA plus HC, Rohapect DA12L®, Rohapect 10L®, Rohapect B1 L® from AB Enzymes, and available under the name Pyrolase® from Diversa Corp., San Diego, CA, USA.

Another example is the β-glucanase obtained from Bacillus subtilis, which is available under the name Cereflo® from the company Novozymes. Particularly preferred according to the invention glycosidases and hemicellulases include mannanases, which, for example under the trade name Mannaway® by the company Novozymes or Purabrite® by the company Danisco / Genencor.

Examples of lipases or cutinases are those originally from Humicola lanuginosa

(Thermomyces lanuginosus) available or further developed lipases, especially those with the amino acid exchange D96L. They are sold, for example, by the company Novozymes under the trade names Lipolase®, Lipolase®Ultra, LipoPrime®, Lipozyme® and Lipex®. Another advantageous lipase is available under the trade name Lipoclean® from the company Novozymes. Furthermore, for example, the cutinases can be used, which have been originally isolated from Fusarium solani pisi and Humicola insolens. Also useful lipases are from the company Amano under the names Lipase CE®, Lipase P®, Lipase B®, and Lipase CES®, lipase AKG®, Bacillis sp. Lipase®, Lipase AP®, Lipase M-AP® and Lipase AML®. From the company Danisco / Genencor, for example, the lipases or cutinases can be used, the initial enzymes were originally isolated from Pseudomonas mendocina and Fusarium solanii. Other important commercial products are preparations M1 Lipase® and Lipomax®, originally sold by Gist-Brocades (now Danisco / Genencor), and Lipase MY-30®, Lipase OF®, by Meito Sangyo KK of Japan and Lipase PL® distributed enzymes, further the product Lumafast® from the company Danisco / Genencor.

The enzymes to be used in the context of the present invention can be derived, for example, originally from microorganisms, such as the genera Bacillus, Streptomyces, Humicola or Pseudomonas, and / or produced by suitable biotechnological methods by suitable microorganisms, for example by transgenic expression hosts, for example the genera Escherichia, Bacillus, or by filamentous fungi.

Most preferably, the hydrolytic enzyme is a protease and / or an amylase. Most preferably, the hydrolytic enzyme is a protease. Preferred among the proteases is a serine protease, more preferably a subtilase, and most preferably a subtilisin. It has been found that proteases, in particular those proteases, can be adapted particularly well to a component stabilizing the hydrolytic enzyme by a method according to the invention, in particular in a liquid surfactant preparation. The same applies to amylases, which can be adapted particularly well to a process according to the invention to a component stabilizing the hydrolytic enzyme, in particular in a liquid surfactant preparation. Because especially for washing, cleaning or

Disinfectants is the storage stability of the enzymes and in particular also of proteases and / or amylases a general problem. Thus, with respect to proteases as preferred hydrolytic enzymes, the stabilizing component comprises a reversible protease inhibitor. Proteases are often responsible for the lack of enzyme stability in liquid preparations by hydrolyzing the enzymes contained therein. Consequently, with a reversible protease inhibitor, it is possible to improve the enzyme stability in liquid preparations by inhibiting the proteolytic activity in the preparation and only removing the inhibition again when necessary for the proteolytic activity, for example by diluting the preparation, in particular with water. It must be reversible protease inhibitors, since the protease activity only temporarily, especially during storage, but not during the intended use of the liquid preparation, for example during the cleaning process, should be prevented. Increased inhibition of the proteases in the liquid preparation leads to a stronger

Stabilization of proteases and other enzymes. Protease inhibitors according to the invention thus reversibly inhibit the protease activity and in this way bring about a stabilization of the proteases as well as of other enzymes, in particular due to the proteolytic activity of the proteases, which is reduced by the inhibitor. Increased inhibition of the proteases leads to greater stabilization of the proteases and of other enzymes, for example because of the increased protection against proteolytic degradation. In a further preferred embodiment of the invention, a method according to the invention is characterized in that the hydrolytic enzyme is a protease and the hydrolytic enzyme-stabilizing component comprises a reversible protease inhibitor.

In a further embodiment of the invention, a method according to the invention is characterized in that the protease is changed at a position which corresponds to the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 , 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 , 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 18 0, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 , 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 , 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274 or 275 in SEQ ID NO. 1, assigned in an alignment with SEQ ID NO. 1, or that the amylase on a Position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,

45, 46, 47, 48 49, 50, 51, 52 53, 54, 55, 56 57, 58, 59, 60 61, 62, 63, 64 65, 66, 67, 68 69, 70, 71, 72, 73, 74 75, 76, 77, 78 79, 80, 81, 82 83, 84, 85, 86 87, 88, 89, 90 91, 92, 93, 94 95, 96,

97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 11 1, 12, 13, 114, 15, 16

SEQ ID NO. 2, corresponds.

The assignment of amino acid positions as well as the determination of the identity of

Nucleic acid or amino acid sequences are made by a sequence comparison. Such a comparison is made by having similar sequences in the nucleotide sequences or

Amino acid sequences are assigned to each other. This sequence comparison takes place

preferably based on the BLAST algorithm established and commonly used in the prior art (see, for example, Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ. (1990) "Basic local alignment search tool Biol. 215: 403-410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): " Gapped BLAST and PSI-BLAST: a new generation of protein database search programs "; Nucleic Acids Res., 25, pp.3389-3402) and is done in principle by similar sequences of nucleotides or amino acids in the nucleic acid or Amino acid sequences are assigned to each other. A tabular assignment of the respective positions is referred to as alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, are usually created using computer programs. For example, the Clustal series (see, for example, Chenna et al., 2003: Multiple sequence alignment with the Clustal series of programs, Nucleic Acid Research 31, 3497-3500), T-Coffee (see, for example, Notredame et al (2000): T-Coffee: A novel method for multiple sequence alignments, J. Mol. Biol. 302, 205-217) or programs based on these programs or algorithms. Within the scope of the present invention

Sequence comparisons and alignments are preferably created using the Vector NTI® Advance 10.3.0 software package (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with default default parameters.

Such a comparison allows on the one hand a statement about the similarity of the compared

Sequences to each other. It is usually given in percent identity, that is to say the proportion of identical nucleotides or amino acid residues at the same positions or in an alignment with one another. The broader concept of homology involves conserved amino acid substitutions in the consideration of amino acid sequences, that is, amino acids with similar properties, since these usually perform similar activities or functions within the protein. Therefore, the similarity of the sequences compared may also be stated as percent homology or percent similarity. Unless stated otherwise, identity or homology information in the present application refers to the total length of the respectively indicated nucleic acid or amino acid sequence.

On the other hand, a sequence comparison (alignment) allows the accurate determination of the position in an amino acid sequence that is associated with a particular position in another amino acid sequence. Consequently, it can be determined with the help of an alignment exactly which

Amino acid in which position in the invention to be modified protease one of the above positions in said reference sequences. SEQ ID NO. 1 is the amino acid sequence of the protease BPN ^' , SEQ ID NO. 2 is the amino acid sequence of the amylase disclosed in SEQ ID NO. 4 of WO 2000/60060.

In a further preferred embodiment of the invention, the method according to the invention is characterized in that the hydrolytic enzyme stabilizing component in an amount of 0.000001 to 10 wt .-%, from 0.0001 to 5 wt .-%, from 0.001 to 1 Wt .-% and particularly preferably from 0.002 to 0.35 wt .-% is contained in the liquid preparation, and / or the hydrolytic enzyme in an amount of 1 x 10 ^"8 -10 wt .-%, of 0.00001 -2 wt%, from 0.001-1 wt%, from 0.007-0.8 wt%, from 0.025-0.5 wt%, and especially preferably from 0.04 to 0.38 wt .-%, based on the total protein content of the hydrolytic enzyme. 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 (AG Gornall, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766).

In a further preferred embodiment of the invention is an inventive

A process characterized in that the liquid preparation in process step c) is a surfactant preparation.

In the context of the present invention, a type of surfactant preparation is any kind of

To understand composition containing at least one surfactant. Preferably, such a composition contains a surfactant as described below.

For the purposes of the present application, "flowable" formulations are those which are pourable and can have viscosities of up to several tens of thousands of mPas The viscosity can be determined by conventional standard methods (for example Brookfield viscometer LVT-II at 20 rpm and 20 ° C., spindle 3) and is preferably in the range from 5 to 10000 mPas Preferred agents have viscosities from 10 to 8000 mPas, values between 120 and 3000 mPas being particularly preferred. A liquid surfactant preparation in the context of the present invention can therefore also be gelatinous or paste-like, it can be present as a homogeneous solution or suspension, and can be sprayed, for example, or formulated in other customary administration forms.

A liquid surfactant preparation according to the invention can be used as such or after dilution with water, in particular for the cleaning of textiles and / or hard

Surfaces. Such dilution can be readily made by diluting a measured amount of the surfactant preparation in a further amount of water in certain weight ratios of surfactant preparation: water and optionally shaking this dilution to ensure uniform distribution of the surfactant formulation in the water. Possible weight or volume ratios of the dilutions are from 1: 0 surfactant preparation: water to 1: 10,000 or 1: 20000 surfactant preparation: water, preferably from 1:10 to 1: 2000

Surfactant preparation: water.

A surfactant preparation in the sense of the present invention can therefore also be the washing or cleaning liquor itself. Under washing or cleaning liquor that is the washing or Detergent-containing use solution understood that acts on textiles or fabrics (wash liquor) or hard surfaces (cleaning liquor) and thus comes into contact with the existing on textiles or fabrics or hard surfaces stains.

Usually, the washing or cleaning liquor arises when the washing or cleaning process begins and the washing or cleaning agent is diluted, for example, in a washing machine or other suitable container with water.

In a preferred embodiment, the surfactant preparation is a washing, cleaning or disinfecting agent. The detergents include all conceivable types of detergents, in particular detergents for textiles, carpets or natural fibers. They can be provided for manual and / or machine application. The detergents also include washing aids, which are added to the actual detergent in the manual or machine textile washing, in order to achieve a further effect. The cleaning agents are all, also in all of these forms of administration occurring means for cleaning and / or

Hard surface disinfection, manual and machine dishwashing detergents, carpet cleaners, abrasives, glass cleaners, toilet scents, etc. counted. Textile pre- and post-treatment are finally on the one hand such means with which the garment is brought into contact before the actual laundry, for example, for solving stubborn dirt, on the other hand, those in one of the actual textile laundry downstream step the laundry further desirable Give properties such as a comfortable grip, crease resistance or low static charge. Among the latter, i.a. calculated the fabric softener. Disinfectants are, for example, hand disinfectants, surface disinfectants and instrument disinfectants, also mentioned in the

Dosage forms may occur. A disinfectant preferably causes a germ reduction by a factor of at least 10 ⁴ , that is to say that of originally 10,000 proliferating germs (so-called colony-forming units - CFU) survived no more than a single, with viruses in this regard are not considered as germs, since they have no cytoplasm and have no own metabolism. Preferred disinfectants cause a

Germ reduction by a factor of at least 10 ⁵ .

As surfactant (s) it is possible to use anionic, nonionic, zwitterionic and / or amphoteric surfactants. From an application point of view, preference is given to mixtures of anionic and nonionic surfactants. The total surfactant content of the liquid surfactant preparation is preferably below 60% by weight, and more preferably below 45% by weight, based on the total liquid surfactant formulation. Suitable nonionic surfactants include alkoxylated fatty alcohols, alkoxylated fatty acid alkyl esters, fatty acid amides, alkoxylated fatty acid amides, polyhydroxy fatty acid amides, alkylphenol polyglycol ethers, amine oxides, alkyl polyglucosides, and mixtures thereof.

The nonionic surfactants used are preferably alkoxylated, advantageously ethoxylated, in particular primary, alcohols having preferably 8 to 18 carbon atoms and on average 1 to 12 moles of ethylene oxide (EO) per mole of alcohol, in which the alcohol radical can be linear or preferably methyl-branched in the 2-position or linear and methyl-branched radicals in the mixture can contain, as they are usually present in Oxoalkoholresten. In particular, however, alcohol ethoxylates with linear radicals of alcohols of native origin having 12 to 18 carbon atoms, for example of coconut, palm, tallow or oleyl alcohol, and on average 2 to 8 EO per mole of alcohol are preferred. The preferred ethoxylated alcohols include, for example, C12-14-alcohols with 3 EO, 4 EO or 7 EO, _C9- n-alcohol with 7 EO, C-ms-alcohols with 3 EO, 5 EO, 7 EO or 8 EO, Ci ₂ -i8 alcohols with 3 EO, 5 EO or 7 EO and mixtures of these, such as

Mixtures of Ci ₂ -i4-alcohol with 3 EO and Ci ₂ -i8-alcohol with 7 EO. The specified

Levels of ethoxylation represent statistical averages, which may be an integer or a fractional number for a particular product. Preferred alcohol ethoxylates have a narrow homolog distribution (narrow rank ethoxylates, NRE). In addition to these nonionic surfactants, fatty alcohols with more than 12 EO can also be used. Examples include tallow fatty alcohol with 14 EO, 25 EO, 30 EO or 40 EO. Nonionic surfactants containing EO and PO groups together in the molecule can also be used according to the invention. Also suitable are also a mixture of a (more) branched ethoxylated fatty alcohol and an unbranched ethoxylated fatty alcohol, such as a mixture of a C16-18 fatty alcohol with 7 EO and 2-propylheptanol with 7 EO. Particularly preferably, the

Surfactant preparation a C ₁₂ _ ₁₈ fatty alcohol with 7 EO or a C ₁₃ . ₁₅ -oxoalcohol with 7 EO as nonionic surfactant.

The content of nonionic surfactants is preferably 3 to 40 wt .-%, preferably 5 to 30 wt .-% and in particular 7 to 20 wt .-%, each based on the total surfactant.

In addition to the nonionic surfactants, the surfactant preparation may also contain anionic surfactants. As anionic surfactant are preferably sulfonates, sulfates, soaps,

Alkyl phosphates, anionic silicone surfactants and mixtures thereof used.

As surfactants of the sulfonate type preferably come C ₉ .i ₃ -Alkylbenzolsulfonate,

Olefinsulfonate, ie mixtures of alkene and hydroxyalkanesulfonates and disulfonates, as they are for example from Ci ₂ _i ₈ monoolefins with terminal or internal double bond by sulfonating with gaseous sulfur trioxide and subsequent alkaline or acid hydrolysis the sulfonation obtained. Also suitable are C ₁₂ -i ₈ -alkanesulfonates and the esters of α-sulfo fatty acids (ester sulfonates), for example the α-sulfonated methyl esters of hydrogenated coconut, palm kernel or tallow fatty acids.

The alkali and especially the sodium salts of the Schwefelsäurehalbester _C12 alk (en) yl sulfates ₁₈ fatty alcohols, for example coconut fatty alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol, or C ₁₀ -C ₂ o Oxo alcohols and those half-esters of secondary alcohols of these chain lengths are preferred. From the washing the Ci2-Ci ₅ alkyl sulfates are preferred ₆ alkyl sulfates and C ₂ -C ₅ alkyl sulfates and C ₄ -C. 2,3-alkyl sulfates are also suitable anionic surfactants.

Also, the sulfuric acid monoesters of straight-chain or branched C ₇ ethoxylated with 1 to 6 moles of ethylene oxide. ₂ i-alcohols, such as 2-methyl-branched Cg-n-alcohols having an average of 3.5 moles of ethylene oxide (EO) or C ₁₂ . ₁₈ fatty alcohols with 1 to 4 EO are suitable.

Also preferred anionic surfactants are soaps. Suitable are saturated and unsaturated fatty acid soaps, such as the salts of lauric acid, myristic acid, palmitic acid, stearic acid, (hydrogenated) erucic acid and behenic acid and, in particular, soap mixtures derived from natural fatty acids, for example coconut, palm kernel, olive oil or tallow fatty acids.

The anionic surfactants including the soaps may be in the form of their sodium, potassium or magnesium or ammonium salts. Preferably, the anionic surfactants are in the form of their sodium salts. Further preferred counterions for the anionic surfactants are also the protonated forms of choline, triethylamine or methylethylamine.

The content of a surfactant preparation of anionic surfactants can be from 1 to 40% by weight, preferably from 5 to 30% by weight and very particularly preferably from 10 to 25% by weight, based in each case on the total surfactant preparation.

In another embodiment, the surfactant preparation is characterized by further comprising at least one other ingredient selected from the group consisting of builder, nonaqueous solvent, acid, water soluble salt, thickener, disinfecting ingredient, and combinations thereof.

The addition of one or more of the further ingredients (e) proves to be advantageous, as this further improved cleaning performance and / or disinfection is achieved.

Preferably, the improved cleaning performance and / or disinfection is based on a synergistic interaction of at least two ingredients. In particular by the Combination of the hydrolytic enzyme, preferably a protease, with one of the builders described below and / or with one of the non-aqueous solvents described below and / or with one of the acids described below and / or with one of the water-soluble salts described below and / or Such a synergy can be achieved with one of the thickening agents described below and / or with one of the disinfecting ingredients described below.

Builders which may be present in the surfactant preparation include, in particular, silicates, aluminum silicates (in particular zeolites), carbonates, salts of organic di- and polycarboxylic acids and mixtures of these substances.

Organic builders which may be present in the surfactant preparation are, for example, the polycarboxylic acids which can be used in the form of their sodium salts, polycarboxylic acids meaning those carboxylic acids which carry more than one acid function. For example, these are citric acid, adipic acid, succinic acid, glutaric acid, malic acid, tartaric acid, maleic acid, fumaric acid, sugar acids, aminocarboxylic acids,

Nitrilotriacetic acid (NTA), methylglycine diacetic acid (MGDA) and their derivatives and mixtures thereof. Preferred salts are the salts of polycarboxylic acids such as

Citric acid, adipic acid, succinic acid, glutaric acid, tartaric acid, sugar acids and

Mixtures of these.

As builders further polymeric polycarboxylates are suitable. These are, for example, the alkali metal salts of polyacrylic acid or polymethacrylic acid, for example, those having a molecular weight of 600 to 750,000 g / mol.

Suitable polymers are in particular polyacrylates, which preferably have a molecular weight of from 1,000 to 15,000 g / mol. Because of their superior solubility, the short-chain polyacrylates, which have molecular weights of from 1,000 to 10,000 g / mol, and particularly preferably from 1,000 to 5,000 g / mol, may again be preferred from this group.

Also suitable are copolymeric polycarboxylates, in particular those of acrylic acid with methacrylic acid and of acrylic acid or methacrylic acid with maleic acid. To improve the water solubility, the polymers may also contain allylsulfonic acids, such as allyloxybenzenesulfonic acid and methallylsulfonic acid, as a monomer.

However, preference is given to using soluble builders, such as, for example, citric acid, or acrylic polymers having a molar mass of from 1,000 to 5,000 g / mol, preferably in the liquid surfactant preparation. For the purposes of this document, the molecular weights stated for polymeric polycarboxylates are weight-average molecular weights M w of the particular acid form, which were determined in principle by means of gel permeation chromatography (GPC), a UV detector being used. The measurement was carried out against an external polyacrylic acid standard, which provides realistic molecular weight values due to its structural relationship with the polymers investigated. These data differ significantly from the molecular weight data in which

Polystyrene sulfonic acids are used as standard. The molar masses measured against polystyrenesulfonic acids are generally significantly higher than the molecular weights specified in this document.

If desired, such organic builder substances may be present in amounts of up to 40% by weight, in particular up to 25% by weight and preferably from 1% by weight to 8% by weight. Amounts close to the stated upper limit are preferably used in paste-form or liquid, in particular water-containing, surfactant preparations.

The surfactant preparations according to the invention are liquid and preferably contain water as the main solvent. In addition or alternatively, non-aqueous solvents may be added to the surfactant preparation. Suitable non-aqueous solvents include mono- or polyhydric alcohols, alkanolamines or glycol ethers, provided that they are miscible with water in the specified concentration range. The solvents are preferably selected from ethanol, n-propanol, i-propanol, butanols, glycol, propanediol, butanediol, glycerol, diglycol, propyldiglycol, butyldiglycol, hexylene glycol, ethylene glycol methyl ether, ethylene glycol ethyl ether, ethylene glycol propyl ether, ethylene glycol mono-n-butyl ether, diethylene glycol methyl ether,

Diethylene glycol ethyl ether, propylene glycol methyl ether, propylene glycol ethyl ether,

Propylene glycol propyl ether, dipropylene glycol monomethyl ether, dipropylene glycol monoethyl ether, diisopropylene glycol monomethyl ether, di-isopropylene glycol monoethyl ether, methoxy triglycol, ethoxy triglycol, butoxy triglycol, 1-butoxyethoxy-2-propanol, 3-methyl-3-methoxybutanol, propylene glycol t-butyl ether, di-n-octyl ether and Mixtures of these solvents. However, it is preferred that the surfactant formulation contain a polyol as a nonaqueous solvent. The polyol may in particular comprise glycerol, 1, 2-propanediol, 1, 3-propanediol, ethylene glycol, diethylene glycol and / or dipropylene glycol. Most preferably, the surfactant formulation contains a mixture of a polyol and a monohydric alcohol. Non-aqueous solvents may be used in the surfactant preparation in amounts of between 0.5 and 15% by weight, but preferably below 12% by weight.

To set a desired, by the mixture of the other components not automatically resulting pH value, the surfactant formulations system and environmentally friendly Acids, in particular citric acid, acetic acid, tartaric acid, malic acid, lactic acid,

Glycolic acid, succinic acid, glutaric acid and / or adipic acid, but also mineral acids, in particular sulfuric acid, or bases, in particular ammonium or alkali metal hydroxides. Such pH regulators are present in the surfactant preparations in amounts of preferably not more than 20% by weight, in particular from 1.2% by weight to 17% by weight.

A surfactant preparation according to the invention may further contain one or more water-soluble salts which serve, for example, for adjusting the viscosity. These may be inorganic and / or organic salts. Useful inorganic salts are preferably selected from the group consisting of colorless water-soluble halides, sulfates, sulfites, carbonates, bicarbonates, nitrates, nitrites, phosphates and / or oxides of the alkali metals, alkaline earth metals, aluminum and / or transition metals; Furthermore, ammonium salts can be used. Particularly preferred are halides and sulfates of the alkali metals; Preferably, therefore, the inorganic salt is selected from the group comprising sodium chloride, potassium chloride, sodium sulfate, potassium sulfate and mixtures thereof. Useful organic salts are, for example, colorless water-soluble alkali metal,

Alkaline earth metal, ammonium, aluminum and / or transition metal salts of carboxylic acids. Preferably, the salts are selected from the group comprising formate, acetate, propionate, citrate, malate, tartrate, succinate, malonate, oxalate, lactate and mixtures thereof.

For thickening, a surfactant preparation according to the invention may contain one or more

Contain thickener. Preferably, the thickener is selected from the group comprising xanthan, guar, carrageenan, agar-agar, gellan, pectin, locust bean gum and mixtures thereof. These compounds are effective thickeners even in the presence of inorganic salts. In a particularly preferred embodiment, the

Xanthan gum as thickening agent, since xanthan gum effectively thickens even in the presence of high salt concentrations and prevents macroscopic separation of the continuous phase. In addition, the thickener stabilizes the continuous, low surfactant phase and prevents macroscopic phase separation.

Alternatively or additionally, it is also possible to use (meth) acrylic acid (co) polymers as thickeners. Suitable acrylic and methacrylic (co) polymers include, for example, the high molecular weight homopolymers of acrylic acid crosslinked with a polyalkenyl polyether, in particular an allyl ether of sucrose, pentaerythritol or propylene (INCI name according to "International Dictionary of Cosmetic Ingredients" of "The Cosmetic, Toiletry and Fragrance Association (CTFA) ": carbomer), also referred to as carboxyvinyl polymers. Such polyacrylic acids are available, inter alia, under the trade names Polygel® and Carbopol®. Furthermore, for example, the following acrylic acid copolymers are suitable: (i) Copolymers of two or more monomers from the group of acrylic acid, methacrylic acid and their simple, preferably with C |. _4- alkanols formed esters (INCI Acrylates Copolymer), which are available, for example, under the trade names Aculyn®, Acusol® or Tego® polymer; (ii) crosslinked high molecular weight acrylic acid copolymers, such as those crosslinked with an allyl ether of sucrose or pentaerythritol copolymers of Cio-30-alkyl acrylates with one or more monomers from the group of acrylic acid, methacrylic acid and their simple, preferably with Ci-4 Alkanols formed, esters (INCI acrylates / C 10-30 alkyl acrylate crosspolymer) include and which are available, for example, under the trade name Carbopol®. Other suitable polymers are (meth) acrylic acid (co) polymers of the Sokalan® type.

It may be preferred that the surfactant preparation according to the invention comprises

(Meth) acrylic acid (co) polymer in combination with another thickener, preferably xanthan. The surfactant preparation may contain from 0.05 to 1, 5 wt .-% and preferably 0.1 to 1 wt .-%, each based on the total surfactant, thickener. The amount of thickener used depends on the type of thickener and the desired degree of thickening.

In particular, ingredients which have an antimicrobial or antiviral activity, ie kill germs, are understood to be a disinfectant ingredient. The germicidal effect is dependent on the content of the disinfecting ingredient in the

Surfactant preparation, wherein the germicidal effect with decreasing content of

disinfecting ingredient or increasing dilution of the surfactant preparation decreases.

A preferred disinfecting ingredient is ethanol or propanol. These monohydric alcohols are often used because of their solvent properties and their germicidal activity

Disinfectants and also commonly used in detergents. The term "propanol" includes both the 1-propanol (n-propanol) and the 2-propanol ("isopropanol"). Ethanol and / or propanol, for example, in an amount of from 10 to 65 wt .-%, preferably 25 to 55 wt .-% in the surfactant preparation. Another preferred disinfecting ingredient is tea tree oil. These are the essential oil of the Australian Tea Tree (Melaleuca alternifolia), a native of New South Wales and Queensland evergreen shrub of the genus Myrtenheiden (Melaleuca), and other tea tree species from different genera (eg Baeckea, Kunzea and Leptospermum) in the family of myrtle family (Myrtaceae). The tea tree oil is obtained by steam distillation from the leaves and branch tips of these trees and is a mixture of about 100 substances; its main constituents include (+) - terpinene-4-ol, α-terpinene, terpinolene, terpineol, pinene, myrcene, phellandrene, p-cymene, limonene and 1,8-cineole. Tea tree oil is, for example, in an amount of 0.05 to 10 wt .-%, preferably 0, 1 to 5.0 wt .-%, in the contain virucidal treatment solution. Another preferred disinfecting ingredient is lactic acid. The lactic acid or 2-hydroxypropionic acid is a fermentation product produced by various microorganisms. She is weakly active in antibiotics. Lactic acid is for example in amounts of up to 10 wt .-%, preferably 0.2 to 5.0 wt .-% in the

Containing surfactant preparation.

Further disinfectant ingredients are, for example, active compounds from the groups of alcohols, aldehydes, antimicrobial acids or their salts, carboxylic esters, acid amides, phenols, phenol derivatives, diphenyls, diphenylalkanes, urea derivatives, oxygen, nitrogen acetals and formals, benzamidines, isothiazoles and derivatives thereof such as isothiazolines and isothiazolinones, phthalimide derivatives, pyridine derivatives, antimicrobial surface active compounds, guanidines, antimicrobial amphoteric compounds, quinolines, 1, 2-dibromo-2,4-dicyanobutane, iodo-2-propynyl-butyl-carbamate, iodine, iodophores and peroxides. Among these, preferred active ingredients are selected from the group comprising 1, 3-butanediol, phenoxyethanol, 1, 2-propylene glycol, glycerol, undecylenic acid, citric acid, lactic acid, benzoic acid, salicylic acid, thymol, 2-benzyl-4-chlorophenol, 2,2 '. -Methylenebis (6-bromo-4-chlorophenol), 2,4,4'-trichloro-2'-hydroxydiphenyl ether, N- (4-chlorophenyl) -N- (3,4-dichlorophenyl) -urea, N , N '- (1, 10-decanediyldi-1-pyridinyl-4-ylidene) bis (1-octanamine) dihydrochloride, N, N'-bis (4-chlorophenyl) -3,12-diimino-2 , 4, 1 1, 13-tetraazatetradecandiimidamide, quaternary surface-active compounds, guanidines. Preferred quaternary surface active compounds contain an ammonium, sulfonium, phosphonium, iodonium or arsonium group. Furthermore, disinfectant essential oils can be used, which at the same time for a scenting of the virucidal

Provide treatment solution. However, particularly preferred active compounds are selected from the group comprising salicylic acid, quaternary surfactants, in particular benzalkonium chloride, peroxo compounds, in particular hydrogen peroxide, alkali metal hypochlorite and mixtures thereof. Such another disinfecting ingredient is, for example, in an amount of 0.01 to 1 wt .-%, preferably 0.02 to 0.8 wt .-%, in particular 0.05 to 0.5 wt .-%, particularly preferably 0 , 1 to 0.3 wt .-%, most preferably 0.2 wt .-% in the surfactant preparation.

Liquid surfactant preparations according to the invention in the form of customary solvent-containing solutions are generally prepared by simply mixing the ingredients, which can be added in bulk or as a solution in an automatic mixer.

In a further preferred embodiment of the invention is an inventive

A method characterized in that the hydrolytic enzyme stabilizing component is substantially free of boric acid or has a boron content of less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%, based on the total amount of the hydrolytic enzyme stabilizing component. Most preferably, the hydrolytic enzyme stabilizing component is boron-free.

In a further preferred embodiment of the invention is an inventive

A method characterized in that the hydrolytic enzyme stabilizing component comprises a reversible inhibitor selected from

a) monosaccharide glycerate, in particular an aldose glycerate, a hemiacetal of an aldose glycerate, a ketose glycerate or a hemi ketal of a ketose glycerate, in particular wherein the

Monosaccharide in monosaccharide glycerate

a. a trioester, in particular glyceraldehyde or dihydroxyacetone, or b. a tetroserol, in particular erythrose, threose or erythrulose, or c. a pentose residue, in particular ribose, arabinose, xylose, lyxose, deoxyribose, ribulose or xylulose, or

d. a hexose residue, in particular allose, altrose, glucose, mannose, gulose, idose, galactose, talose, fucose, rhamnose, chinovose or fructose;

b) oligoamino-biphenyl-oligocarboxylic acid, in particular a diamino-biphenyl-dicarboxylic acid, in particular 3,3-diamino [1, 1-biphenyl] -2,4-dicarboxylic acid;

c) aminophthalic acid, in particular 4-aminophthalic acid;

d) phthaloylglutamic acid;

e) polysubstituted benzenecarboxylic acid which has a carboxyl group on at least two carbon atoms of the benzene radical, in particular a poly-substituted one

Benzenecarboxylic acid which is a benzene carboxylic acid having four carboxyl groups on the benzene moiety, especially pyromellitic acid;

f) phenylalkyldicarboxylic acid, in particular phenylmalonic acid or benzylmalonic acid;

g) polyol, in particular glycerol, 1, 2-ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol; h) antioxidant or glyceric acid;

i) calcium compound, lactate or a lactate derivative;

j) boric acid;

k) phenylboronic acid derivative having the structural formula

in which R is hydrogen, a hydroxyl, a C 1 -C 6 -alkyl, a substituted C 1 -C 6 -alkyl, a C 1 -C 6 -alkenyl or a substituted C 1 -C 6 -alkenyl group, preferably 4-formyl-phenylboronic acid ( 4-FPBA); I) mono- or polysubstituted phenylboronic acid which has a hydroxyl group on at least one carbon atom of the phenyl radical, in particular a phenylboronic acid which has a hydroxyl group on a carbon atom of the phenyl radical, especially where the radicals on the other carbon atoms of the phenyl radical are -H, in particular one

Hydroxyphenylboronic acid, preferably 3-hydroxy-phenylboronic acid (3-HPBA).

The hydrolytic enzyme stabilizing component may comprise a monosaccharide glycerate as a reversible inhibitor. This is understood to mean a substance in which a

Monosaccharide residue is covalently bonded via an acetal bond to a glycerol residue. A monosaccharide glycerate in the context of the present invention is accordingly described by the following formula (I):

(I) HOOC-CH-O-R

I

CH ₂ OH, where R is a monosaccharide residue.

A monosaccharide in the present invention is a product of the partial oxidation of a polyhydric alcohol. A monosaccharide has a chain of at least three

Carbon atoms as a backbone and has a carbonyl group and at least one hydroxy group. The number of carbon atoms distinguishes between trioses (3), tetroses (4), pentoses (5), hexoses (6), heptoses (7), etc. In principle, the length of the carbon chain is unlimited, preferably the carbon chain has between three and nine carbon atoms.

The monosaccharide may also be in open-chain (non-cyclic) or cyclic form. On one of the carbon atoms of the open-chain form is a doubly bonded oxygen atom, so that a carbonyl group is present. Is this one?

Carbonyl group at one end of the carbon chain, is an aldehyde group and the monosaccharide is an aldose. If the carbonyl group is within the carbon chain, a keto group is present and the monosaccharide is a ketose. The cyclic monosaccharides are hemiacetals or hemiketals derived from the corresponding aldoses or ketoses. Such monosaccharides are preferably furanoses (five-membered rings) or pyranoses (six-membered rings), each having one oxygen atom in the ring, each of which may have o or β configuration.

The monosaccharide may also be in any stereoisomeric form. Due to the spatial arrangement of the hydroxyl groups of the monosaccharide, the monosaccharide in D or L-configuration, wherein the configuration is determined in the usual manner by the Fischer projection of the monosaccharide, in which the CC bonds in the imaginary (thermodynamically unfavorable) eclipsed position are shown vertically stacked and rolled on the paper plane, and Substituents (here hydrogen atoms and hydroxyl groups) are listed depending on the configuration right or left, so that there is a clear configuration. The chiral carbon atom farthest from the anomeric C atom forms the D configuration in the right-hand position, and the L configuration in the left-hand position.

Preferred monosaccharides correspond to the general empirical formula: C _n H ₂ n O n, where n is a number between three and nine, preferably between four and six, and particularly preferably six.

The monosaccharides further include derivatives of these compounds which do not conform to the general formula above. Such derivatives have further chemical

In particular, they may contain one or more methyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butyloxycarbonyl, benzoyl, 4- Methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4-toluenesulphonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4, 5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl, 2,2,5,7,8-pentamethylchroman-6-sulphonyl radicals or combinations thereof. For example, D-glucuronic acid (6-carboxy-D-glucose), D-galacturonic acid (6-carboxy-D-galactose), N-acetyl-D-glucosamine (also N-acetylchitosamine), D-glucosamine (also chitosamine), N-acetyl-D-galactosamine (also N-acetylchondrosamine) or D- and L-fucose (6-deoxy-D and -L-galactose) monosaccharides according to the present invention.

Preferably, the monosaccharide glycerate is an aldose glycerate, a hemiacetal of one

Aldose glycerate, a ketose glycerate or a half ketal of a ketose glycerate.

Particularly preferred is the monosaccharide in the monosaccharide glycerate

a. a trioester, in particular glyceraldehyde or dihydroxyacetone, or

b. a tetroester, in particular erythrose, threose or erythrulose, or

c. a pentose residue, in particular ribose, arabinose, xylose, lyxose, deoxyribose, ribulose or xylulose, or

d. a hexose residue, especially allose, altrose, glucose, mannose, gulose, idose, galactose, talose, fucose, rhamnose, chinovose or fructose. Such monosaccharide glycerates are particularly effective as compounds provided in the present invention as the hydrolytic enzyme stabilizing component. In a particularly preferred embodiment of the invention, the monosaccharide glycerate is glucosylglycerate. A particularly preferred glucosylglycerate is given in the following formula (II):

The component stabilizing the hydrolytic enzyme may comprise an oligoamino-biphenyl-oligocarboxylic acid as a reversible inhibitor, in particular a diamino-biphenyl-dicarboxylic acid, in particular 3,3-diamino [1,1-biphenyl] -2,4-dicarboxylic acid.

An oligoamino-biphenyl-oligocarboxylic acid in the present invention is described by the following formula (III):

wherein R ₁ , R ₂ , R ₃ , R ₄ , R 5, R 6, R 7, R 8, R 9 and R 10 are each -NH ₂ , -COOH or -H, wherein at least two of R ₁ to R 10 are - NH ₂ and at least two of R ₁ to

R10 are -COOH and the other radicals are -H. Preferably, there are those radicals R ₁ to R 10 which are --NH ₂ and those radicals R ₁ to R 10 which are -COOH on adjacent carbon atoms (C atoms), ie in ortho position to one another.

Further preferred are exactly two of the radicals R ₁ to R 10 -NH ₂ and two of the radicals R ₁ to

R10 -COOH and the other radicals -H. Such an embodiment of the invention is therefore characterized in that the oligoamino-biphenyl-oligocarboxylic acid is a diamino-biphenyl- dicarboxylic acid is. Particularly preferred is R1 is -COOH, R2 _-NH2, -NH ₂ R7, R8 COOH, and R3, R4, R5, R6, R9, R10 are each -H. In such a particularly preferred embodiment of the invention, the oligoamino-biphenyl-oligocarboxylic acid is 3,3-diamino [1, 1-biphenyl] -2,4-dicarboxylic acid, which is given in the following formula (IV):

In the context of the invention, an oligoamino-biphenyl-oligocarboxylic acid also includes derivatives of these compounds. Such derivatives have further chemical modifications, in particular they may be glycosylated, oxidized, N-methylated, N-formylated, N-acetylated on one or more of the amino groups or one or more methyl, amino, nitro, chloro, fluoro -, bromo-, hydroxyl, carboxyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butyloxycarbonyl, benzoyl, 4-methylbenzyl -, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4-toluenesulphonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5 trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl, 2,2,5,7,8-pentamethylchroman-6-sulphonyl radicals or groups or combinations thereof.

The hydrolytic enzyme stabilizing component may comprise aminophthalic acid as a reversible inhibitor, especially 4-aminophthalic acid.

This is understood as meaning a benzene dicarboxylic acid which has an -NH ₂ radical at at least one further position of the phenyl radical. The two carboxyl groups of the benzenedicarboxylic acid can be in ortho, meta or para position to each other. Preferably, they are in ortho position to each other. The -NH ₂ radical is located on one of those carbon atoms (C atoms) of the phenyl radical on which no carboxyl group is present. The radicals at the remaining positions of the phenyl radical are preferably -H.

A preferred such compound is described by the following formula (V):

(V):

Therein the -NH ₂ -group is at the carbon atom (C-atom) C3 or C4 of the

Phenylrestes, particularly preferably at C4 of the phenyl radical.

For aminophthalic acid in the invention also include derivatives of this compound. Such derivatives have further chemical modifications, in particular they may be glycosylated, oxidized, N-methylated, N-formylated or N-acetylated at the amino group. Alternatively or additionally, they may contain one or more methyl, amino, nitro, chloro, fluoro, bromo, hydroxyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl -, N-hydroxysuccinimide, t-butyloxycarbonyl, benzoyl, 4-methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4-toluenesulphonyl , Pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl, 2,2,5,7,8-pentamethylchroman-6 -sulphonyl radicals or groups or combinations thereof.

The hydrolytic enzyme stabilizing component may comprise phthaloylglutamic acid as a reversible inhibitor.

This is understood to mean a substance which is described by the following formula (VI) (N-phthaloyl-L-glutamic acid):

(VI) Alternatively or additionally, the hydrolytic enzyme stabilizing component may comprise a phthaloyl aspartic acid as a reversible inhibitor. This is understood to mean a substance which is described by the following formula (VII) (N-phthaloyl-L-aspartic acid):

Phthaloylglutamic acid or phthaloylaspartic acid in the context of the invention also include derivatives of these compounds. Such derivatives have further chemical modifications, in particular they may be glycosylated or on the phthaloyl radical one or more methyl, amino, nitro, chloro, fluoro, bromo, hydroxyl, carboxyl, formyl, ethyl , Acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butyloxycarbonyl, benzoyl, 4-methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, Benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4-toluenesulphonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl , 2,2,5,7,8-pentamethylchroman-6-sulphonyl radicals or groups or combinations thereof.

The hydrolytic enzyme stabilizing component may comprise a multi-substituted benzenecarboxylic acid as a reversible inhibitor.

This is understood to mean a benzenecarboxylic acid which has a carboxyl group (-COOH radical) on at least two carbon atoms (C atoms) of the benzene radical. The benzenecarboxylic acid preferably has three, four, five or six carboxyl groups on the benzene radical. More preferably, the benzene carboxylic acid has a hydrogen radical on those carbon atoms which do not carry a carboxyl group.

The carboxyl groups of the di-substituted benzenecarboxylic acid may be ortho, meta or para to each other. Other carboxyl groups can be located on any intermediate carbon atoms (carbon atoms). The poly-substituted benzenecarboxylic acid is particularly preferably a benzene carboxylic acid having four carboxyl groups on the benzene radical. Most preferably, the carboxyl groups are located at the C atoms C1, C2, C4 and C5 of the benzene radical. Such a compound is pyromellitic acid. It has carboxyl groups on the C atoms C1, C2, C4 and C5 of the benzene radical and hydrogen on the C atoms C3 and C6 and represents a very particularly preferred embodiment of the hydrolytic enzyme stabilizing component. It is in the following formula (VIII ) indicated:

For a multiply substituted benzene carboxylic acid in the invention also include derivatives of these compounds. Such derivatives have further chemical modifications, in particular they may contain one or more methyl, amino, nitro, chloro, fluoro, bromo, hydroxyl, formyl, ethyl, acetyl, t-butyl, anisyl , Benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butyloxycarbonyl, benzoyl, 4-methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2- Nitrophenylsulphenyl, 4-toluenesulphonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl, 2,2,5,7,8 - Contain pentamethyl-chroman-6-sulphonyl radicals or groups or combinations thereof.

The hydrolytic enzyme stabilizing component may comprise a phenylalkyl dicarboxylic acid as a reversible inhibitor, especially phenylmalonic acid.

A phenylalkyldicarboxylic acid in the present invention is described by the following formula (IX):

(IX) C ₆ H ₅ - (CH ₂ ) _n -CH (COOH) ₂ , where n is an integer between 0 and 14, and more preferably between 0 and 10, between 0 and 5, between 0 and 4, between 0 and 3, between 0 and 2, and most preferably 0 or 1. Particularly preferred embodiments of the invention are therefore characterized in that the phenylalkyldicarboxylic acid is phenylmalonic acid or benzylmalonic acid. Phenylmalonic acid is shown in the following formula (X), benzylmalonic acid in the following formula (XI):

(X): C ₆ H ₅ CH (COOH) ₂ (XI):

A phenylalkyldicarboxylic acid in the invention also includes derivatives of this compound. Such derivatives have further chemical modifications, in particular they may be glycosylated or oxidized or on the phenyl radical one or more methyl, amino, nitro, chloro, fluoro, bromo, hydroxyl, carboxyl, formyl, ethyl , Acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butyloxycarbonyl, benzoyl, 4-methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, Benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4-toluenesulphonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl , 2,2,5,7,8-pentamethylchroman-6-sulphonyl radicals or groups or combinations thereof.

The hydrolytic enzyme stabilizing component may further comprise as a reversible inhibitor a polyol, in particular glycerol, 1,2-ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol. The hydrolytic enzyme stabilizing component may further comprise, as the reversible inhibitor, an antioxidant or glyceric acid, a calcium compound, lactate or a lactate derivative. The hydrolytic enzyme stabilizing component may further comprise boric acid as a reversible inhibitor.

The hydrolytic enzyme stabilizing component may further comprise, as a reversible inhibitor, a phenylboronic acid derivative having the structural formula

in which R is hydrogen, a hydroxyl, a C 1 -C 6 -alkyl, a substituted C 1 -C 6 -alkyl, a C 1 -C 6 -alkenyl or a substituted C 1 -C 6 -alkenyl group, preferably 4-formyl-phenyl- boronic acid (4-FPBA).

The hydrolytic enzyme-stabilizing component may further comprise as a reversible inhibitor a mono- or polysubstituted phenylboronic acid having at least one carbon atom of the phenyl group a hydroxyl group, in particular a phenylboronic acid having a hydroxyl group on a carbon atom of the phenyl group, especially wherein the groups at the other carbon atoms of the phenyl radical are -H. Particular preference is given to a hydroxyphenylboronic acid, very particularly preferably to 3-hydroxy-phenylboronic acid (3-HPBA). It is described by the following formula (XII):

All compounds provided in the context of the present invention as the hydrolytic enzyme stabilizing component may be present in all protonated or deprotonated forms. Furthermore, all such compounds, in particular their deprotonated forms, may be associated with cations. Preferred cations in this regard are divalent cations, in particular Ca ions (Ca ²⁺ ), Mg ions (Mg ²⁺ ) and Zn ions (Zn ²⁺ ). Particularly preferred are Ca ions (Ca ²⁺ ). Furthermore, the compounds can be present in all possible stereoisomeric forms.

Another object of the invention are hydrolytic enzymes by a

inventive method as described above are available. Preferred hydrolytic enzymes are proteases and amylases, in particular proteases. More preferred hydrolytic enzymes are selected from the group consisting of

a) protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Amino acid substitution S166A; b) Protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Amino acid substitution S166V;

c) protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Amino acid substitution S166Y;

d) protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Amino acid substitution S166G;

e) protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Amino acid substitution A187D;

f) protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Amino acid substitution F189R;

g) protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Amino acid substitution Q191 R;

h) Protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Amino acid substitutions S166G and Q191 R;

i) protease having an amino acid sequence corresponding to SEQ ID NO. 3 is at least 90% and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical over its entire length and is counted in accordance with SEQ ID NO. 1 the

Has amino acid substitutions A187D and F189R.

The identity value, in particular the identity value of 100%, refers here to the respective protease without the required substitution (s), ie to the basic structure (basic sequence) of the protease. In this particular framework then the substitutions are as indicated in each case contribute. For example, a protease having an amino acid sequence corresponding to SEQ ID NO. 3 is identical over its entire length to 100% and in the count according to SEQ ID NO. 1 the Amino acid substitution S166A, a protease according to SEQ ID NO. 3 with the amino acid substitution S166A (in the counting manner according to SEQ ID NO. 1). The same applies to the other proteases.

In a further preferred embodiment, the abovementioned proteases have only the respectively indicated amino acid substitution or the respectively indicated amino acid substitutions.

In another preferred embodiment, the hydrolytic enzyme is thereby

characterized in that it has a reduced relative activity in comparison with the relative activity of the starting enzyme towards the reversible inhibitor, especially towards one of the reversible inhibitors as indicated above. Preferably, the hydrolytic enzyme is a protease or an amylase, in particular a protease and most preferably a protease as described above. Further preferred embodiments of these proteases are characterized in that the

a) protease according to a) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid, 4-formyl-phenyl-boronic acid (4-FPBA) or 3-hydroxy-phenyl-boronic acid (3-HPBA), especially 4-formyl-phenyl-boronic acid (4-FPBA);

b) protease according to b) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid, 4-formyl-phenyl-boronic acid (4-FPBA) or 3-hydroxy-phenyl-boronic acid (3-HPBA), especially 3-hydroxy-phenyl-boronic acid (3-HPBA);

c) protease according to c) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid or 3-hydroxy-phenylboronic acid (3-HPBA), especially 3-hydroxy-phenylboronic acid (3-HPBA);

d) protease according to d) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 has boric acid;

e) protease according to e) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 has boric acid;

f) protease according to f) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid or benzylmalonic acid, especially boric acid;

g) protease according to g) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid or benzylmalonic acid, especially benzylmalonic acid; h) protease according to h) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid or benzylmalonic acid, especially benzylmalonic acid;

i) protease according to i) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid or benzylmalonic acid, especially benzylmalonic acid.

In this regard, the determination of relative activities is preferably as described in Example 1.

Examples:

Example 1: Adaptation of a protease to given compounds which inhibit the protease and thus stabilize

All molecular biology procedures are followed by standard methods such as those described in the Handbook of Fritsch, Sambrook and Maniatis "Molecular Cloning: a Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1989, or similar works ) were used according to the instructions of the respective manufacturers.

The hydrolytic enzyme (starting enzyme) was a protease according to SEQ ID NO. 3 used. Further, the reversible protease inhibitors indicated below were each provided as the hydrolytic enzyme stabilizing component:

1. boric acid

2. 4-formyl-phenylboronic acid (4-FPBA)

3. 3-Hydroxy-phenylboronic acid (3-HPBA)

4. Benzylmalonic acid (BMA)

The nucleic acid sequence of the hydrolytic enzyme present on a plasmid was quantitated using site-directed mutagenesis using specific, matched primers using a Phusion HotStart polymerase at positions S166, A187, F189 or Q191 as enumerated in SEQ ID NO individually or in combination as indicated in Tables 1 and 2 below and transforming the resulting variants into a suitable Bacillus host Active clones were selected by means of a protease-sensitive medium and the DNA sequence sequenced after plasmid isolation for identification the Bacillus host and provided for activity determination. The activity was determined in a liquid surfactant preparation composed as follows (all figures in weight percent): 0.3-0.5% xanthan gum, 0.2-0.4% anti-foaming agent, 6-7% glycerol, 0.3-0.5% ethanol, 4-7% FAEOS (fatty alcohol ether sulfate), 24-28% nonionic surfactants, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut fatty acids, 0 , 5% HEDP (1-hydroxyethane- (1, 1-di-phosphonic acid)), 0-0.4% PVP (polyvinylpyrrolidone), 0-0.05% optical brightener, 0-0.001% dye, balance demineralized water.

To a 50% liquid preparation of the liquid surfactant preparation was added 0.92 mM AAPF. Also included was the amount of reversible inhibitor to be tested, which is needed to inhibit the parent enzyme by 20-40%. 20 μΙ of the respective protease (starting enzyme and modified protease variants) were added and the proteolytic activity was determined by the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe- p-nitroanilide (suc-AAPF-pNA). The protease cleaves the substrate and releases pNA. The release of pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of enzymatic activity (see Del Mar et al., 1979). The measurement was carried out at a temperature of 25 ° C, at pH 8.6 and a wavelength of 410 nm.

The measured activity was compared with the activity of a reference batch of the same protease sample without inhibitor and the respective relative activities of the modified proteases and the starting enzyme were determined. A greater decrease in the relative activity of a variant in the particular inhibitor than in the starting enzyme shows better inhibition. The

Results are shown in Tables 1 and 2 below (each indicates the relative activities).

Table 1 :

Table 2:

Enzyme Without inhibitor Boric acid BMA

S166 100% 79% 81%

A187D 100% 47% 79%

F189R 100% 53% 80% Q191 R 100% 76% 63%

S166G 100% 65% 80%

S166G / Q191 R 100% 57% 35%

A187D / F189R 100% 37% 43%

It becomes clear that the proteases obtained after carrying out a process according to the invention are better adapted to the component stabilizing the hydrolytic enzyme, in particular the reversible inhibitor, and consequently are improved and / or stabilized by it. An improved inhibition after performing a

Method obtained proteases was also achieved for the variants shown in Table 1 with benzylmalonic acid (BMA) as a reversible inhibitor.

Example 2: Inhibition Measurement in the Biochemical Test

The variants S166V and S166G / Q191 R and the starting enzyme according to SEQ ID NO. 3 were expressed in a suitable expression system by means of

purified by ion exchange chromatography and provided for activity determinations in 0.1 M Tris / HCl buffer pH 8.6 containing 0.1% Brij35. The batches were given increasing amounts of the reversible inhibitor 3-HPBA or BMA (see Tables 3 and 4) and the residual proteolytic activity of the respective protease via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA) as described above. The activities obtained are shown in Tables 3 and 4.

Table 3:

Table 4:

Enzyme Without BMA (in% by weight)

inhibitor

0.05 0.5 1, 0 1, 7 2.5 3.3 4.0 5.0

Starting enzyme 100% 96% 95% 93% 94% 74% 73% 78% 64%

S166G / Q191 R 100% 88% 55% 42% 35% 38% 32% 36% 32% It becomes clear that the respective variant shows a stronger inhibitability by the reversible inhibitor than the starting enzyme. The implementation of a method according to the invention thus resulted in obtaining a variant which is better adapted to a predetermined reversible inhibitor, here 3-HPBA or BMA.

Example 3 Determination of Storage Stability in a Liquid Surfactant Preparation

The variants S166V and S166G / Q191 R and the starting enzyme according to SEQ ID NO. 3 were added in concentrations to reaction mixtures which contained a surfactant preparation as described in Example 1 and a reversible inhibitor in concentrations as indicated in Table 5. The batches were stored at 30 ° C and the residual proteolytic activity of the protease via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA) as described above at regular time intervals. The addition of the inhibitors led to the increase factors of the half-lives given in Table 5 (half-life with inhibitor divided by half-life without inhibitor).

Table 5:

(nb: not determined)

It becomes clear that the respective variant also shows a stronger inhibitability by the reversible inhibitor than the starting enzyme in a surfactant preparation. The implementation of a method according to the invention consequently resulted in obtaining a variant which, in the context of a surfactant preparation, is better adapted to a predetermined reversible inhibitor, here in addition to boric acid, in particular 3-HPBA and BMA.

Claims

claims

A method of adapting a hydrolytic enzyme to a hydrolytic enzyme

stabilizing component comprising the process steps

a) providing a hydrolytic enzyme (starting enzyme) and a hydrolytic enzyme stabilizing component comprising a reversible inhibitor of the hydrolytic enzyme;

d) selecting the hydrolytic enzyme having reduced relative activity as compared to the relative activity of the parent enzyme.

2. The method according to claim 1, characterized in that the reversible inhibitor with respect to the hydrolytic enzyme in process step a) an inhibition constant (K,) of 0.01 to 500 mM, preferably from 0.01 to 100 mM, particularly preferably from 0, 01 to 1 mM or from 10 to 100 mM.

3. Process according to claim 1 or 2, characterized in that the hydrolytic enzyme is a protease, amylase, cellulase, glycosidase, hemicellulase, mannanase, xylanase, xyloglucanase, xanthanase, pectinase, β-glucosidase, carrageenase or a lipase or a mixture, which comprises at least two of these enzymes.

4. The method according to any one of claims 1 to 3, characterized in that the hydrolytic enzyme is a protease or an amylase, in particular a protease.

5. The method according to claim 4, characterized in that the protease is changed at a position corresponding to the position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 11, 11, 12 , 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227 , 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 , 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274 or 275 in SEQ ID NO , 1, assigned in an alignment with SEQ ID NO. 1, corresponds, or

that the amylase is changed at a position corresponding to the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

SEQ ID NO. 2, assigned in an alignment with SEQ ID NO. 2, corresponds.

6. The method according to any one of claims 1 to 5, characterized in that the

hydrolytic enzyme stabilizing component in an amount of 0.000001 to 10% by weight is contained in the liquid preparation, and / or the hydrolytic enzyme is contained in an amount of 1 × 10 ^-8 to 5 weight-percent in the liquid preparation based on the total protein content of the hydrolytic enzyme.

7. The method according to any one of claims 1 to 6, characterized in that the liquid

Preparation is a surfactant preparation.

8. The method according to any one of claims 1 to 7, characterized in that the

hydrolytic enzyme stabilizing component is free of boric acid or has a boron content of less than 50 wt .-% or is boron-free.

9. The method according to any one of claims 1 to 8, characterized in that the

hydrolytic enzyme stabilizing component comprises a compound selected from

a) monosaccharide glycerate, in particular an aldose glycerate, a hemiacetal of a

Aldoseglycerats, a Ketoseglycerat or a Halbketal a Ketoseglycerats, in particular where. the monosaccharide in the monosaccharide glycerate

c) aminophthalic acid, in particular 4-aminophthalic acid;

d) phthaloylglutamic acid;

g) polyol, in particular glycerol, 1, 2-ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol h) antioxidant or glyceric acid;

i) calcium compound, lactate or a lactate derivative;

j) boric acid;

k) phenylboronic acid derivative having the structural formula

in which R represents hydrogen, a hydroxyl, a C 1 -C 6 -alkyl, a substituted C 1 -C 6 -alkyl, a C 1 -C 6 -alkenyl or a substituted C 1 -C 6 -alkenyl group, preferably 4-formyl-phenylboronic acid ( 4-FPBA);

I) mono- or polysubstituted phenylboronic acid, the at least one

Carbon atom of the phenyl radical has a hydroxyl group, in particular a

Phenylboronic acid which has a hydroxyl group on a carbon atom of the phenyl radical, especially where the radicals on the other carbon atoms of the phenyl radical are -H, in particular a hydroxyphenylboronic acid, preferably 3-hydroxy-phenylboronic acid (3-HPBA).

10. Hydrolytic enzyme obtainable by a process according to any one of claims 1 to 9, characterized in that the hydrolytic enzyme is selected from the group consisting of

a) protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Amino acid substitution S166A;

b) Protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Amino acid substitution S166V;

c) protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Amino acid substitution S166Y;

d) protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Amino acid substitution S166G;

e) protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Amino acid substitution A187D;

f) protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Amino acid substitution F189R;

g) protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Amino acid substitution Q191 R; h) Protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Amino acid substitutions S166G and Q191 R;

i) protease having an amino acid sequence corresponding to SEQ ID NO. 3 over the total length of which is at least 90% identical and in the count according to SEQ ID NO. 1 the

Has amino acid substitutions A187D and F189R. 1. Hydrolytic enzyme according to claim 10, characterized in that the

a) protease according to a) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 with respect to boric acid, 4-formyl-phenylboronic acid (4-FPBA) or 3-hydroxy-phenylboronic acid (3-HPBA), in particular 4-formyl-phenylboronic acid (4-FPBA);

g) protease according to g) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid or benzylmalonic acid, especially benzylmalonic acid;

h) protease according to h) a reduced relative activity in comparison with the relative activity of a protease according to SEQ ID NO. 3 versus boric acid or benzylmalonic acid, especially benzylmalonic acid;