EP4098730A1 - Stabilization of enzymes by phenoxyethanol - Google Patents

Abstract

The invention relates to a composition containing at least one peroxidase and phenoxyethanol. The invention also relates to a washing or cleaning agent comprising the composition according to the invention and the use of phenoxyethanol for stabilizing enzymes.

Description

The invention relates to a composition containing at least one peroxidase and phenoxyethanol. The invention also relates to a washing or cleaning agent comprising the composition according to the invention and the use of phenoxyethanol for stabilizing enzymes.

In the production of detergents and cleaning agents, various technical devices are used to dose defined quantities of raw materials. If a specific raw material is to be dosed in particularly small volumes, this can be problematic because the systems used may not be designed for this.

It is therefore fundamentally desirable to combine several raw materials and keep them storable for a few weeks so that fewer dosing stations are required and larger volumes can be used.

In the case of enzymes in particular, dilution often leads to instability of the enzymes over time. Thus, either expensive and sensitive dosing stations have to be purchased to add the very small amounts of the enzymes to a detergent, or solvents such as glycerol or propylene glycol (PG) are added to the diluted enzyme raw materials to increase enzyme stability.

The inventors of the present invention have now surprisingly found that the stability of enzymes, in particular peroxidases, can be significantly improved by adding phenoxyethanol, in particular in aqueous compositions.

Therefore, in a first aspect, the invention relates to a composition containing at least one peroxidase and phenoxyethanol.

In preferred embodiments, the at least one peroxidase comprises or consists of a catalase.

In a further aspect, the invention relates to a washing or cleaning agent comprising the composition according to the invention.

Finally, the invention relates to the use of phenoxyethanol to stabilize at least one enzyme.

These and other aspects, features and advantages of the invention will become apparent to those skilled in the art from a study of the following detailed description and claims. Anybody can Feature from one aspect of the invention can be used in any other aspect of the invention. For example, features or embodiments that are described in connection with the composition according to the invention can also be used for the washing or cleaning agent or for the use. Furthermore, it should be understood that the examples contained herein are intended to describe and illustrate the invention, but not to limit it, and in particular the invention is not limited to these examples.

"At least one" as used herein refers to 1 or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. With regard to an ingredient, the information refers to the type of ingredient and not to the absolute number of molecules. "At least one peroxidase" thus means, for example, at least one type of peroxidase, i.e. one type of peroxidase or a mixture of several different peroxidases can be meant. Together with percentages, the information relates to all compounds of the type specified that are contained in the composition, i.e. the composition does not contain any other compounds of this type beyond the specified amount of the corresponding compounds.

Unless otherwise stated, all percentages are percentages by volume (vol%), based in each case on the total volume of the corresponding composition.

Numerical ranges given in the format "in/from x to y" include the stated values. Where multiple preferred numeric ranges are given in this format, it is understood that any ranges resulting from the combination of the different endpoints are also included.

Numerical values given herein without decimal places refer to the full stated value with one decimal place. For example, "99%" means "99.0%".

The expression "approximately" or "approximately" in connection with a numerical value refers to a variance of ±10% based on the stated numerical value, preferably ±5%, particularly preferably ±1%.

The expression "substantially free from" means that the respective component can in principle be contained in the composition according to the invention, but is then present in an amount which is significantly below the amount usually required or used for the function of this component. In the context of the present invention, the property “substantially free from” of a specific component is therefore preferably a total amount of less than 1% by weight, more preferably less than 0.1% by weight, even more preferably less than 0.01% by weight. -%, more preferably below 0.001% by weight, in particular free, ie below the detection limit, considered thereto, based on the total weight of the composition. It can also mean that the corresponding component was not intentionally added.

In a first aspect, the invention relates to a composition containing at least one peroxidase and phenoxyethanol.

Preferably, the composition according to the invention comprises, based on the total weight of the composition, 0.01 to 5% by weight, more preferably 0.025 to 3% by weight, for example 0.25 to 2.5% by weight, 0.125 to 1 25% by weight or 0.03125 to 0.3125% by weight active enzyme content, i.e. the at least one peroxidase.

In preferred embodiments, the at least one peroxidase comprises or consists of a catalase.

The peroxidase is preferably used as an enzyme liquid formulation, for example as a ready-made liquid preparation such as is commercially available. Here, it can be present in suitable buffer solutions or water. The peroxidase can also be formulated or packaged together with accompanying substances or residues from the fermentation. Examples of this are solvent residues, buffer substances, salts, pH adjusters or enzyme stabilizers, without being limited to them. In preferred embodiments, the peroxidase is essentially free of fermentation residues.

The peroxidase may have been purified using known techniques such as affinity chromatography or size exclusion chromatography, or it may be present, for example, as a crude cell extract.

Typically, the enzyme is in free form, but may also be immobilized (fixed in space) by known techniques, such as, but not limited to, covalent attachment to the surface of a support or cross-linking.

It is also possible for the composition to include one or more other enzymes in addition to the at least one peroxidase. For example, one or more other enzymes may be formulated/packaged along with the peroxidase.

Suitable further enzymes can be selected, for example, from the group consisting of amylases, proteases, cellulases, hemicellulases, lipases, oxidoreductases or mixtures of these enzymes, without being restricted thereto. 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 enzymes mentioned can all be formulated/packaged as described above for the peroxidases.

In preferred embodiments, the composition of the invention is an aqueous composition. Preferably, the composition comprises 0.5 to 99.99% by volume, more preferably 0.5 to 99.9% by volume, even more preferably 0.5 to 99.5% by volume, even more preferably 0 5 to 99% by volume, even more preferably 0.5 to 98% by volume, even more preferably 0.5 to 97% by volume, even more preferably 1 to 96% by volume of water, for example about 1 bis 74% by volume, 40 to 74% by volume, 1 to 75% by volume, 40 to 75% by volume, 1 to 85% by volume, 50 to 85% by volume, 1 to 87% by volume %, 50 to 87% by volume, 1 to 88% by volume, 50 to 88% by volume, 60 to 96% by volume, 60 to 97% by volume, 60 to 98% by volume %, 60 to 99% by volume, 65 to 99.5% by volume, 70 to 99.9% by volume or 75 to 99.99% by volume based on the total volume of the composition. Alternatively, the quantitative data can also relate to % by weight based on the total weight of the composition. Various buffer substances, i.e. in particular salts suitable as buffers, can be contained in the composition. It is also possible for further (organic) solvents to be added to the composition in addition to the water.

The peroxidase used can be a naturally occurring enzyme or an enzyme that has been modified by one or more mutations based on naturally occurring enzymes in order to positively influence desired properties, such as catalytic activity, stability or cleaning performance. For example, a genetically engineered enzyme may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with a naturally occurring parent enzyme over the entire length of the parent protein. In various embodiments, the catalytic activity of a genetically engineered enzyme can be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the catalytic activity of its parent enzyme.

The enzymes included in the composition according to the invention can originate from different types of organisms. Suitable microorganisms are selected, for example, from the group consisting of the genera Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, without being limited thereto.

However, the enzymes included in the composition according to the invention can also originate from eukaryotes. Examples are fungi, such as Actinomycetes, or yeasts, such as Saccharomyces or Kluyveromyces. In various embodiments, the enzymes used can originate from fungi, in particular filamentous fungi, for example those of the genus Trichoderma.

The at least one peroxidase of the composition according to the invention particularly preferably comprises a catalase. A catalase which can be used according to the invention is commercially available, for example, from Novozymes. Catalase available under the trade name Terminox Supreme 1000 BCU from Novozymes is particularly preferred.

In preferred embodiments, the at least one peroxidase, in particular the at least one catalase, which can usually be present pre-formulated in a liquid enzyme formulation, is included mixed with water to obtain an aqueous composition according to the invention. In one embodiment, the phenoxyethanol can already be added to the water used for the dilution. Alternatively, the phenoxyethanol can then be added to the aqueous composition so that the at least one peroxidase and phenoxyethanol are present in the aqueous composition.

In preferred embodiments there is a dilution of a preformulated peroxidase liquid preparation, for example by the addition of water or buffer, the preformulated peroxidase liquid preparation being present in an amount of 1 to 50% by volume, in particular 2 to 30% by volume, for example about 2.5 vol.%, 3.125 vol.%, 5 vol.%, 10 vol.%, 12.5 vol.%, 15 vol.%, 20 vol.%, 25 vol.% or 30 % by volume is used. In various embodiments, the composition according to the invention, optionally diluted, contains the peroxidase in an amount of 0.01 to 5% by weight, preferably 0.025 to 3% by weight, for example 0.25 to 2.5% by weight, 0.125 to 1.25% by weight or 0.03125 to 0.3125% by weight active enzyme content based on the total weight of the composition according to the invention.

In preferred embodiments, the composition according to the invention comprises, based on the total volume of the composition, phenoxyethanol in an amount of 0.01 to 10% by volume, preferably 0.1 to 5% by volume, in particular 0.5 to 2% by volume %, for example 0.9% by volume. Alternatively, these quantities can also relate to % by weight relative to the total weight.

In a particularly preferred embodiment, the composition, in particular the aqueous composition, comprises at least one catalase and phenoxyethanol.

In preferred embodiments, the compositions according to the invention can comprise further additives.

Suitable additives are, for example, without being limited to, selectable from surfactants, builders, bleaches, bleach catalysts, bleach activators, electrolytes, pH adjusters, perfumes, perfume carriers, fluorescent agents, dyes, hydrotropes, complexing agents, foam inhibitors, silicone oils, soil release polymers , graying inhibitors, shrinkage inhibitors, anti-crease agents, antimicrobial active ingredients, germicides, fungicides, antioxidants, corrosion inhibitors, antistatic agents, bittering agents, ironing aids, repellent and impregnating agents, skin-care active ingredients, swelling and non-slip agents, softening components and UV absorbers and mixtures thereof.

In a further aspect, the invention relates to a washing or cleaning agent comprising the composition according to the invention.

The general production and composition of detergents and cleaning agents is known in principle to the person skilled in the art in this field.

Exemplary detergent and cleaning agent compositions and suitable ingredients are, for example, in WO 01/44433 A1 or WO 2016/091650 A1 described.

Finally, in a further aspect, the invention relates to the use of phenoxyethanol for stabilizing at least one enzyme.

In the context of this invention, “stabilization” preferably means a reduced decrease in the initial enzyme activity over time. Typically, enzyme activity continues to decrease over time. Stabilization with phenoxyethanol leads in particular to slowing down this decrease.

In various embodiments, the enzyme is stabilized in an aqueous solution. Preferably, the aqueous solution comprises 0.5 to 99.99% by volume, more preferably 0.5 to 99% by volume, even more preferably 1 to 96% by volume, for example about 1 to 74% by volume, 40 to 74% by volume, 1 to 85% by volume, 50 to 85% by volume, 1 to 87% by volume or 60 to 96% by volume of water or buffer solution, particularly preferably water. Other solvents, for example organic solvents, but also other ingredients, in particular additives for detergents or cleaning agents, as described above, can be added to the solution. Components that have been formulated or packaged together with the enzyme can also be included in the aqueous solution. In particular, at least one enzyme is stabilized in a detergent or cleaning agent.

In preferred embodiments, the at least one enzyme is an amylase, protease, cellulase, hemicellulase, peroxidase, lipase, oxidoreductase, or a mixture of these enzymes. The enzyme preferably comprises or consists of at least one peroxidase, in particular a catalase. Suitable organisms, enzyme formulations or formulations have already been described above for the composition according to the invention and can be used here.

The at least one enzyme, more preferably the peroxidase, in particular the catalase, is preferably present in an amount of 0.01 to 5% by weight, more preferably 0.025 to 3% by weight, for example 0.25 to 2.5% by weight. -%, 0.125 to 1.25% by weight or 0.03125 to 0.3125% by weight active enzyme content in the aqueous solution, based on the total weight of the aqueous solution.

In various embodiments, phenoxyethanol is present in the aqueous solution in an amount of from 0.01 to 10% by volume, preferably from 0.1 to 5% by volume, more preferably from 0.5 to 2% by volume, for example 0 9% by volume, based on the total volume of the aqueous solution.

In preferred embodiments, the aqueous solution is an aqueous composition according to the present invention. In particular, the aqueous solution can be a washing or cleaning agent according to the present invention.

Phenoxyethanol can particularly preferably be used to increase the storage stability of enzymes, preferably peroxidases, in particular catalases, in aqueous solutions. For example, the (storage) stability can be increased for at least 12 weeks. In various embodiments, the stabilization by phenoxyethanol takes place at temperatures between 10 and 41°C, preferably 20 to 40°C, for example at room temperature (approximately 20 to 25°C), 30°C or 40°C.

The invention is illustrated below using examples, but is not limited to these.

examples Example 1: Bioanalytical method used

1. 1. Principle:
   • In the determination method, H ₂ O ₂ reacts in the presence of peroxidase with a system of chromotropic acid and 4-aminoantipyrine to form a blue dye (D = chromophore), the extinction maximum is at 600 nm.
     
   • The concentration of the produced dye (chromophore) is proportional to the H ₂ O ₂ concentration in the sample and to the absorbance. The Lambert-Beer law is therefore applicable to this measurement method (original method according to Meiattini, 1975). The H ₂ O ₂ concentration can be determined using a calibration curve.
   • According to the Lambert-Beer law, the extinction is proportional to the layer thickness and the concentration of the absorbing substances (chromophore).
2. 2. Execution:
   The H ₂ O ₂ concentration is measured photometrically in microtiter plates at a wavelength of 600 nm. First, the enzyme solution is incubated with substrate and buffer for 10 minutes while shaking (25 °C). Then the reaction is stopped by adding the catalase inhibitor hydroxylamine sulfate and the color reaction is initiated by adding the peroxide reagent (chromotropic acid/4-aminoantipyrine in phosphate buffer, pH 6.5) and the horseradish peroxidase solution (HRP, Sigma Aldrich P8125). The reaction is started by adding peroxidase. Color development is complete after about one minute, the dye remains stable for a period of 6 minutes. The absorbance is measured after 3 minutes as an end point determination (600 nm). The dilutions of the sample solutions are chosen so that the concentrations to be determined be between 10 and 50 nmol of hydrogen peroxide. The measurement is carried out against a reaction mixture with buffer as a blank value.

Example 2: Dilution of the enzyme raw material

The catalase (Terminox Supreme 1000 BCU, Novozymes, 10-100 mg/mL active enzyme content) was diluted with water to 25%, 12.5% and 3.125%. For example, a 25% catalase solution is equivalent to a solution containing 25 mL of catalase (commercially available enzyme formulation (enzyme raw material)) and 75 mL of water. 0.9% phenoxyethanol (PHE) was added to the dilutions.

The catalase, the diluted catalase solutions and the diluted catalase solutions + PHE were incubated at different temperatures. The enzyme activity of the starting value at 0 weeks was defined as 100%. The residual activity after storage for 12 weeks was determined. The bioanalytical method described under Example 1 was used to determine the enzyme activities. <b>Table 1: Overview of the enzyme activities after 12 weeks at different temperatures.</b> approaches 0 weeks 12 weeks RT 12 weeks 30°C 12 weeks 40°C Catalase (Enzyme raw material) 100% 74% 65% 73% 25% catalase solution 100% 47% 43% 29% 25% catalase solution + 0.9% PHE 100% 88% 73% 89% 12.5% catalase solution 100% 48% 34% 11% 12.5% catalase solution + 0.9% PHE 100% 75% 68% 69% 3.125% catalase solution 100% 31% 13% 5% 3.125% catalase solution + 0.9% PHE 100% 74% 67% 62%

Process-related deviations of 5 to 10% are expected.

Dilution of the commercially available enzyme formulation results in significantly reduced enzyme activity. Surprisingly, it was found that the addition of 0.9% PHE leads to a significantly improved enzyme activity at all temperatures, which counteracts the reduction in enzyme activity caused by the dilution.

Claims (10)

Composition containing at least one peroxidase and phenoxyethanol. The composition of claim 1, wherein the at least one peroxidase comprises or consists of a catalase. The composition according to claim 1 or 2, wherein the peroxidase is contained in an amount of 0.01 to 5% by weight, preferably 0.025 to 3% by weight of the active enzyme content in the composition, based on the total weight of the composition. The composition according to any one of claims 1 to 3, wherein phenoxyethanol is contained in the composition in an amount of 0.01 to 10% by volume, preferably 0.1 to 5% by volume, based on the total volume of the composition. The composition of any one of claims 1 to 4, wherein the composition is an aqueous composition and preferably comprises from 0.5% to 99%, more preferably from 1% to 96% by volume of water. Detergent or cleaning agent comprising the composition according to any one of claims 1 to 5. Use of phenoxyethanol to stabilize at least one enzyme. Use according to claim 7, wherein the stabilization takes place in an aqueous solution. Use according to Claim 7 or 8 for stabilizing an enzyme in a washing or cleaning agent. Use according to any one of claims 7 to 9, wherein the at least one enzyme comprises or consists of an amylase, protease, cellulase, hemicellulase, peroxidase, lipase, oxidoreductase or mixtures of these enzymes, preferably a peroxidase, more preferably a catalase.