WO2017072111A1

WO2017072111A1 - Enzyme stabilizers

Info

Publication number: WO2017072111A1
Application number: PCT/EP2016/075663
Authority: WO
Inventors: André HÄTZELT; Timothy O'connell
Original assignee: Henkel Ag & Co. Kgaa
Priority date: 2015-10-28
Filing date: 2016-10-25
Publication date: 2017-05-04
Also published as: DE102015221052A1

Abstract

The present invention relates to detergents and cleaning agents, containing at least one protease and at least one organic compound of formula (I) (3,4-dihydroisoquinoline derivatives), said compound acting as a protease inhibitor and thus being a suitable enzyme stabilizer, and to the use of these compounds as enzyme stabilizers in protease-containing detergents and cleaning agents. The invention also relates to the corresponding washing and cleaning methods and to the use of the agents described herein. The invention further relates to the use of general structural formula (I) for use in the treatment of diseases from the following groups: respiratory diseases, inflammatory diseases, HIV, hepatitis, parasitic infectious diseases, malaria, Chagas disease, and cancer.

Description

enzyme stabilizers

The present invention relates to detergents and cleaners containing at least one enzyme and at least one organic compound which acts as a protease inhibitor and is thus a suitable enzyme stabilizer, and the use of these compounds as enzyme stabilizer in enzyme-containing detergents and cleaners.

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

The enzymes which are the longest-established and are contained in virtually all modern, powerful detergents and cleaners are proteases and, in particular, serine proteases, to which the subtilases according to the invention are also calculated. They are used to break down proteinaceous soiling on the items to be cleaned. However, they also hydrolyze themselves (auto-proteolysis) and all other proteins contained in the agents concerned, ie in particular enzymes. This happens especially during the cleaning process, ie in the aqueous wash liquor, when comparatively favorable reaction conditions are present. This occurs to a lesser extent but also during storage of the agents concerned, which is why a long storage always accompanied by a certain loss of protease activity and the activities of other enzymes. This is particularly problematic in gel or liquid and especially in water-containing formulations, because in this with the water contained both the reaction medium and the hydrolysis reagent available. One goal in the development of detergent formulations is to stabilize the enzymes contained, especially during storage. This is understood as protection against various unfavorable influences, such as denaturation or decay by physical influences or oxidation. One focus of these developments is the protection of the contained proteins and / or enzymes against proteolytic cleavage. This can be done by the construction of physical barriers, such as by encapsulation of the enzymes in special enzyme granules or by packaging the means in two- or multi-chamber systems. The other many pathway is to add chemical compounds that inhibit the proteases and thus act collectively as stabilizers for proteases and the other proteins and enzymes contained. It must be reversible protease inhibitors, since the protease activity is only temporarily, especially during storage, but not be suppressed during the cleaning process.

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

Other established enzyme stabilizers are amino alcohols such as mono-, di-, triethanol- and -propanolamine and mixtures thereof, aliphatic carboxylic acids up to C12, such as succinic acid, other dicarboxylic acids or salts of said acids. End-capped fatty acid amide alkoxylates are also established for this purpose. Certain organic acids used as builders are capable, as disclosed in WO 97/18287, of stabilizing an enzyme in addition to their builder function.

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

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

Boric acid derivatives occupy an outstanding position among the serine protease inhibitors, which are effective at comparatively low concentrations. However, regardless of their stabilizing effect, the boric acid derivatives have a significant disadvantage: many of them, such as borate, form undesirable by-products with some other detergent ingredients, so that they are no longer available in the agents concerned for the desired cleaning purpose, or even remain as an impurity on the laundry.

The object of the present invention was therefore to identify boron-free compounds which act as protease inhibitors and are suitable for use as enzyme stabilizers in detergents and cleaners. Here, the use in total liquid, gel or pas- tösen washing and cleaning agents was of particular interest, and including in particular those containing water.

This object is achieved by washing or cleaning agents which comprise at least one protease and at least one enzyme stabilizer, the at least one enzyme stabilizer being selected from compounds of the general structural formula (I)

(I) wherein R is an unsubstituted or substituted Ci-20-alkyl radical, wherein the substituents are selected from -OR 'and -OSO3 ^" , wherein R' is independently H or a straight-chain or branched C1-20 alkyl radical.

In preferred embodiments, R is a C 1-6 alkyl radical, especially methyl, or a 2-sulfooxyalkyl radical of 3 to 20 carbon atoms. In various embodiments of the Sulfooxyalkylrest 2- is a radical of formula (II) -CH2-CH (OS03 ^~) -R _a, wherein R _a is a straight or branched C1-12 alkyl radical or a Alkyloxyalkylrest of formula (III) - (CHR " ) m-O- (CHR ") n-CH3, wherein each R" is independently H or a C1-6 alkyl, m is 0, 1 or 2, n is 0 or an integer from 1 to 10, wherein m + n = 1 to 18. In preferred embodiments, the sulpho-oxyalkyl radical is a radical of the formula -CH ₂ -CH (OSO 3 ^" ) -CH ₂ -O- (C 6 -12 -alkyl), in particular -CH ₂ -CH (OSO 3) - CH ₂ -O-CH ₂ -CH (C 2-4 alkyl) - (CH ₂ ) 2-6 CH ₃ .

In various embodiments in which the radical R does not comprise a negatively charged sulfate group, the compound of the formula (I) may further comprise any Y anion. For example, Y may be selected from inorganic anions such as fluoride, chloride, bromide, sulfate, phosphate, or organic anions such as acetate, benzoate, citrate, sulfonate, but is not limited thereto. If polyvalent anions are used, e.g. Sulfate, it is understood that the compound of formula (I) and the anion are used in a charge-balancing stoichiometry. In various embodiments, the anion Y is a sulfonate, for example p-toluenesulfonate.

Exemplary compounds of formulas (I) include, but are not limited to:

(2-sulfooxypropyl- (2-n-butyloctyloxy)) - 3,4-dihydroisoquinoline, (2-sulfooxypropyl- (2-ethylhexyloxy)) - 3,4-dihydroisoquinoline, 3,4-dihydro-2-methylisoquinoline, p Toluenesulfonate and 2-sulfooxydecyl-3,4-dihydroisoquinoline.

Under a detergent or cleaning agent according to the invention are all means that are suitable for washing or cleaning of particular textiles and / or solid surfaces. Other suitable ingredients are described in detail below.

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

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

The first advantage of the compounds relevant to the invention over the prior art is that they have favorable inhibition constants with respect to the proteases which can be used in detergents and cleaners. The inhibitors thus bind reversibly, i. they do not interfere with solid and not too loose transient interactions with the enzyme. Advantageously, the majority of the protease relevant to the invention is thus present during storage in the form of a protease-inhibitor complex. The protease and optionally other proteins contained, in particular other enzymes are protected in this way against proteolysis by this enzyme (stabilized against proteolysis) and are thus fully efficient even after storage. On the other hand, at the moment of dilution of the agent according to the invention with water to prepare an aqueous washing or cleaning liquor, the binding equilibrium is shifted in the direction of dissociation so that the complex dissolves and most of the protease relevant protease can become proteolytically active.

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

Furthermore, they have a good solubility in water, in particular because of the ionic groups present, in particular sulphate groups, so that they can easily be incorporated into corresponding agents and precipitation during storage is avoided.

In principle, the compounds mentioned probably act as reversible inhibitors because, similar to the substrate of the proteases, they are structurally adapted to the conditions of the binding pocket. This applies in particular to serine proteases, as has been shown on the basis of the examples of the present application with the positive effect of the compounds experimentally described there on the basis of serine proteases, specifically subtilases, even more specific subtilisins.

Further objects of the present invention relate to:

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

A washing or cleaning process in which a protease is inhibited which is inhibited and / or stabilized with a compound described above; the use of a washing or cleaning agent according to the invention for washing and / or cleaning textiles and / or hard surfaces; such as

the use of a protease and a compound described above for the preparation of a detergent or cleaner.

In detergents or cleaners according to the invention, which in one embodiment are in predominantly solid form and in another embodiment in predominantly liquid, pasty or gel form, the enzyme, i. the protease in an amount of 0.001 to 2% by weight, preferably 0, 1 to 1, 5% by weight, and the enzyme stabilizer in an amount of 0.001 to 2% by weight, preferably 0.05 to 1, 0 wt .-%, based on the total weight of the detergent or cleaning agent contained in this.

In various embodiments, the enzyme and the enzyme stabilizer may be pre-formulated in an enzyme composition, wherein the enzyme and the enzyme stabilizer are contained in the enzyme composition in amounts such that the above-mentioned final concentrations in the agent are achieved. This enzyme composition, which is also a constituent of the present invention, can then be used in detergents or cleaners according to the invention in amounts which lead to the above-mentioned final concentrations in the washing or cleaning agent.

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

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

the alkaline protease from Bacillus amyloliquefaciens (ΒΡΝ '),

the alkaline protease from Bacillus licheniformis (subtilisin Carlsberg),

the alkaline protease PB92,

Subtilisin 147 and / or 309 (Savinase)

the alkaline protease from Bacillus lentus, preferably from Bacillus lentus (DSM 5483), or an alkaline protease which is at least 90% identical thereto at the amino acid level, more preferably the Bacillus lentus alkaline protease (DSM 5483) with the substitution R99E,

the alkaline protease from Bacillus alcalophilus (DSM 1 1233), the alkaline protease from Bacillus gibsonii (DSM 14391) or an alkaline protease which is at least 70% identical thereto at the amino acid level,

the alkaline protease from Bacillus sp. (DSM 14390) or an at least 98.5% identical at the amino acid level alkaline protease, and

the alkaline protease from Bacillus sp. (DSM 14392) or an at least 98, 1% identical at the amino acid level alkaline protease.

Compositions according to the invention may contain, in addition to the protease, one or more further enzymes, in particular from the following group: one or more further proteases, amylases, hemicellulases, cellulases, lipases and oxidoreductases.

The amylase (s) is preferably an α-amylase. The hemicellulase is preferably a β-glucanase, a pectinase, a pullulanase and / or a mannanase. The cellulase is preferably a cellulase mixture or a one-component cellulase, preferably or predominantly an endoglucanase and / or a cellobiohydrolase. The oxidoreductase is preferably an oxidase, in particular a choline oxidase, or a perhydrolase.

The compositions described herein include all conceivable types of detergents or cleaners, both concentrates and neat agents, for use on a commercial scale, in the washing machine or in hand washing or cleaning. These include detergents for textiles, carpets, or natural fibers, for which the term detergent is used. These include, for example, dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather, for which the term detergent is used, ie in addition to manual and machine Dishwashing agents, for example, scouring agents, glass cleaners, toilet scenters, etc. The washing and cleaning agents in the invention also include washing aids which are added to the actual detergent in the manual or machine textile laundry to achieve a further effect. Furthermore, laundry detergents and cleaners in the context of the invention also include textile pre-treatment and post-treatment agents, ie those agents with which the laundry item is brought into contact before the actual laundry, for example to dissolve stubborn soiling, and also agents which are in one of the actual Textile laundry downstream step to give the laundry further desirable properties such as comfortable grip, crease resistance or low static charge. Among the latter, i.a. calculated the fabric softener.

Embodiments of the present invention include all solid, powdered, liquid, gel or pasty dosage forms of the agents described herein, optionally can also consist of several phases and can be present in compressed or uncompressed form. The agent can be present as a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l. The solid dosage forms of the composition also include extrudates, granules, tablets or pouches. Alternatively, the agent can also be liquid, gelatinous or pasty, for example in the form of a non-aqueous liquid washing or dishwashing detergent or a non-aqueous paste or in the form of an aqueous liquid washing or dishwashing detergent or a water-containing paste. Furthermore, the agent may be present as a one-component system. Such funds consist of one phase. Alternatively, an agent can also consist of several phases. Such an agent is therefore divided into several components.

The detergents or cleaners described herein, which may be in the form of powdered solids, in densified particulate form, as homogeneous solutions or suspensions, may further additionally contain all known ingredients customary in such compositions, preferably at least one further ingredient being present in the composition. In particular, the agents described herein may contain surfactants, builders, bleaches or bleach activators. In addition, they may contain water-miscible organic solvents, sequestering agents, electrolytes, pH regulators and / or further auxiliaries, such as optical brighteners, graying inhibitors, foam regulators, as well as dyes and fragrances, and combinations thereof.

Advantageous ingredients of the compositions described herein are disclosed in International Patent Application WO2009 / 121725, beginning on page 5, penultimate paragraph, and ending on page 13, after the second paragraph. This disclosure is incorporated herein by reference and the disclosure is incorporated herein by reference.

A further subject of the invention is a method for the cleaning of textiles or hard surfaces, which is characterized in that in at least one method step a means described herein is used.

These include both manual and mechanical processes, with mechanical processes being preferred. Methods for cleaning textiles are generally distinguished by the fact that various cleaning-active substances are applied to the items to be cleaned and washed off after the contact time, or that the items to be cleaned are otherwise treated with a detergent or a solution or dilution of this product. The same applies to processes for cleaning all other materials than textiles, especially hard surfaces. All conceivable washing or cleaning methods can be enriched in at least one of the method steps by the use of a washing or cleaning agent described herein and then represent embodiments of the present invention. Another subject of the invention is the use of an agent described herein for cleaning or washing textiles or for cleaning hard surfaces.

Yet another subject of the invention is the use of the compounds described herein for stabilizing an enzyme in a protease-containing detergent or cleaning agent.

Yet another subject of the invention is the use of the compounds described herein in the treatment of diseases selected from the group of respiratory diseases, inflammatory diseases, HIV, hepatitis, parasitic infectious diseases, malaria, Chagas disease and cancer.

All aspects, objects, and embodiments described for means described herein are also applicable to the aforementioned methods and uses. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the above described methods and uses.

Examples

example 1

The storage stability of protease-containing detergents and cleaning agents in the presence of the candidate compounds for use as enzyme stabilizers was investigated. The protease used was the Bacillus lentus alkaline protease (DSM 5483) with the substitution R99E. For this purpose, the candidate compounds (see Table 2) were formulated in parallel reaction mixtures in the presence of 1, 2-propanediol in each case to 1% (w / w) in a detergent formulation (see Table 1). Then the protease was added to the formulation at 1% (w / w) and the formulation stored at 30 ° C for 20h, 140h, 234h and 668h. The control batch contained only the same amount of protease and the detergent formulation (and 1% w / w 1, 2-propanediol).

Following storage, the proteolytic activity of the protease was determined from the described reaction mixtures. For this purpose, the release of the chromophore para-nitroaniline from the substrate succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; Bachem L-1400) was measured. The release of pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of the enzymatic activity. The measurement was carried out at a temperature of 25 ° C, at pH 8.6 and a wavelength of 410 nm. The measuring time was 5 min at a measuring interval of 20 to 60 seconds.

For evaluation, the initial values for the proteolytic activity of the agent in question are compared with the values determined after storage. The higher the activity remaining after storage, the better the protease obtained is inactivated during storage and the better the compound in question is suitable as a stabilizer according to the invention. The stabilizing effect of each tested compound is therefore measured as a relative percentage increase in protease residual activity.

Table 1: Formulation for activity test

Ingredient Amount (wt%)

demineralized water rest

Citric acid 1, 42623

Defoamer (10%) 0.008

Fatty alcohol ether sulphate (70%) 5,6

Fatty alcohol ether 4.4

Alkylbenzenesulfonic acid 4.4

(96%)

C12-18 fatty acid 2,4 NaOH (50%) 0.95

Glycerol (99.5%) 2

Phosphate (DTPMP-Na ₇ H ₃ ) 0.2

(32%)

Preservative 0.10

Ethanol (93%) 1

The pH of the formulation was adjusted to 8.4 by the NaOH. The formulation was clear and colorless.

As candidate compounds were tested:

3,4-dihydro-2-methylisoquinoline p-toluenesulfonate

3,4-dihydro-2-sulfooxydecyl-isoquinoline

3,4-dihydro- (2-sulfooxy-3- (2-ethylhexyloxy) propyl) isoquinoline

3,4-dihydro- (2-sulfooxy-3- (2-n-butyloctyloxy) propyl) isoquinoline

Table 2: Measured protease residual activity of the candidate compounds

Candidate compound (KV) relative residual activity after incubation with KV in%

0h 20h 140h 234h 668h

None 100 100.8 67.9 59.9 38.7

3,4-dihydro- (2-sulfooxy-3- (2-n-butyloctyloxy) propyl) -100.4, 90.4 65.9 64.4 35.3 isoquinoline

3,4-dihydro- (2-sulfooxy-3- (2-ethylhexyloxy) propyl) - 100 92.2 62.6 66.5 37.6 isoquinoline

3,4-Dihydro-2-sulfooxydecyl-isoquinoline 100 84.9 64.0 59.6 32.1

3,4-Dihydro-2-methyl-isoquinoline p-toluenesulfonate 100 91, 1 67, 1 61, 0 32.3

Claims

claims

1. washing or cleaning agent containing at least one protease and at least one enzyme stabilizer, wherein the at least one enzyme stabilizer is selected from compounds of the general structural formula (I)

wherein R is an unsubstituted or substituted Ci-20-alkyl radical, wherein the substituents are selected from -OR 'and -OSO3 ^" , wherein R' is independently H or a straight-chain or branched C1-20 alkyl radical.

2. Washing or cleaning agent according to claim 1, characterized in that R is a C1-6 alkyl radical, in particular methyl, or a 2-sulfooxyalkyl radical having 3 to 20 carbon atoms, is.

3. Washing or cleaning agent according to claim 1 or 2, characterized in that R is a 2-sulfooxyalkyl radical of the formula (II) -CH 2 -CH (OSO 3 ^~ ) -R _a , wherein R _{a is} a straight-chain or branched C1-12 alkyl radical or an alkyloxyalkyl radical of the formula (III) - (CHR ") _m -O- (CHR") n-CH3, where each R "is independently H or a C1-6 alkyl radical, m is 0, 1 or 2, n 0 or an integer from 1 to 10, where m + n = 1 to 18.

4. washing or cleaning agent according to one of claims 1 to 3, characterized in that R is a radical of the formula -CH ₂ -CH (OSO 3 ^" ) -CH ₂ -O- (C6-i2 alkyl), in particular -CH ₂ -CH ( OS03) -CH ₂ -0-CH ₂ -CH (C2-4 alkyl) - _(CH2) 2-6CH. ₃

5. washing or cleaning agent according to one of claims 1 to 4, characterized in that the at least one enzyme stabilizer (2-sulfooxypropyl (2-butyloctyloxy)) - 3,4-dihydroisoquinoline, (2-sulfooxypropyl- (2-ethylhexyloxy) ) -3,4-dihydroisoquinoline, 3,4-dihydro-2-methylisoquinoline, p-toluenesulfonate or 2-sulfooxydecyl-3,4-dihydroisoquinoline.

6. washing or cleaning agent according to one of claims 1 to 5, characterized in that the at least one protease in an amount of 0.001 to 2 wt .-%, preferably 0.1 to 1, 5 wt .-%, and the at least an enzyme stabilizer in an amount of 0.001 to 2 wt .-%, preferably 0.05 to 1, 0 wt .-%, based on the total weight of the washing or cleaning agent, are contained in this.

7. Use of a washing or cleaning agent according to any one of claims 1 to 6 for washing textiles or cleaning of solid surfaces.

8. A method for cleaning textiles or hard surfaces, characterized in that in at least one method step, a washing or cleaning agent according to any one of claims 1-5 is applied.

9. Use of a compound of the general structural formula (I)

(I) where R is an unsubstituted or substituted Ο-20-alkyl radical, the substituents being selected from -OR 'and -OSO3 ^" , where R' is independently H or a straight-chain or branched C1-20 alkyl radical, for stabilizing an enzyme in a protease-containing detergent or cleaner.

10. A compound of the general structural formula (I)

wherein R is an unsubstituted or substituted C1-20 alkyl radical, wherein the substituents are selected from -OR 'and -OSO3-, wherein R' is independently H or a straight or branched C1-20 alkyl radical;

for use in the treatment of diseases selected from the group of respiratory diseases, inflammatory diseases, HIV, hepatitis, parasitic infectious diseases, malaria, Chagas disease and cancer.