WO1991016424A1

WO1991016424A1 - Process for specific alteration of the properties of enzymes by chemical modification and chemically modified enzymes

Info

Publication number: WO1991016424A1
Application number: PCT/EP1991/000705
Authority: WO
Inventors: Hans Ulrich Geyer; Gerhard Konieczny-Janda
Original assignee: Hans Ulrich Geyer; Konieczny Janda Gerhard
Priority date: 1990-04-25
Filing date: 1991-04-12
Publication date: 1991-10-31
Also published as: DE4013142C2; DE4013142A1

Abstract

The description relates to a process for specific alteration of the properties of enzymes by chemical modification. In addition, novel chemically modified enzymes, their production and use are described.

Description

Process for the targeted modification of the properties of enzymes by chemical modification and

chemically modified enzymes

The invention relates to a method for deliberately changing the properties of enzymes by chemically modifying them, to the production of chemically modified enzymes and to the chemically modified enzymes and their use.

In the prior art, it is already known to modify and improve the pharmacological properties of enzymes - such as, for example, adenosine deaminase, asparaginase, uricase, superoxide dismutase, catalase - by reaction with polyethylene glycol. European patent application 154 316 also describes the reaction of lymphokinin with monoalkylene glycol aldehydes. From European patent application 128 975 it is known to produce water-soluble, biologically active conjugates by coupling proteins, eg enzymes, with alkylene glycol esters of a polyuronic acid under alkaline conditions. The polyuronic acid is bound to the protein by amide bonds and to the alkylene glycol residue by ester bonds. German Offenlegungsschrift 29 19 622 discloses water-soluble stabilized enzyme derivatives, in particular protease derivatives, which, by reacting the enzyme with polysaccharide derivatives containing aldehyde groups, for example dialdehyde starch or dialdehyde cellulose, form an immobilized, ie insoluble Enzyme product and subsequent reduction of the immobilized enzyme product with complex hydrides were obtained. Here, at least two of the available amino groups of the enzyme portion and at least two of the available aldehyde groups of the polysaccharide portion have to be crosslinked and the product obtained in this way has to be additionally hydrogenated.

Furthermore, subtilisin has already been nitrated, carbamylated, glutarylated and succinylated in the prior art in order to investigate the effects of these chemical modifications on the enzymatic activity. Α-Amylases have also already been treated with organic acid anhydrides - e.g. Acetic acid, propionic acid, butyric anhydride etc. - modified to the corresponding acyl-derivatized α-amylase. Chymotrypsinogen was also treated with acid anhydrides such as e.g. Acetic or succinic anhydride, also converted into acylated derivatives and, for example, with ethylenediamine, into amidated derivatives. The reaction of glucose isomerase or D-xylose isomerase with diethyl pyrocarbonate and of glucose isomerase with 2,4,6-trinitrobenzenesulfonic acid is also known.

The methods known in the prior art for the chemical modification of enzymes are only limited in their applicability to special enzymes and types of chemical modification with respect to the residues which can be introduced into the enzyme. In addition, the known methods allow only little scope in terms of the manner and in terms of a graduated feasibility of the chemical modification of enzymes. The reagents used in the prior art to modify the enzymes (e.g. for nitration) are often too aggressive or the functional groups (such as aldehyde groups or acid anhydride groups) contained in these reagents which are used to bind the modification reagents to the enzyme are too chemically reactive and make it difficult to control the Degree of modification. Or they are badly compatible with the enzymes themselves. The chemical modification of enzymes in the prior art is therefore difficult to control according to the type and degree of modification. The types of bonds formed between the enzyme and the modification reagent can also prove to be insufficiently stable to hydrolysis (such as, for example, imine, ester or amide bonds) and thereby restrict the use of the modified enzymes.

The object was therefore to provide a simple, mild and at the same time versatile process for the targeted change in the properties of enzymes by derivatization and for the chemical modification of enzymes, which can be used for a large number of enzymes. This procedure should take into account. The type and degree of modification of the enzymes can be light, controllable and thus enable a customized adaptation of enzymes for specific requirements. Another task was to provide new, valuable enzyme derivatives optimized by chemical modification for various applications.

The objects are achieved by the methods according to the invention, the enzymes chemically modified according to the invention (enzyme derivatives) and their use.

The invention relates to a method for specifically changing the properties of an enzyme by chemical modification, which is characterized in that the enzyme is reacted with a terminal epoxy or halohydrin compound which specifically changes the cationic, biochemical and application properties of the enzyme, Contains amphiphilic, anionic and / or lipophilic or hydrophobic organic radical, converted into a water-soluble or non-water-insoluble, chemically modified enzyme (enzyme derivative). The modification reagents used according to the invention for the chemical modification of enzymes contain a terminal epoxy group as a reactive group or a halohydrin group as their precursor. This epoxy or halohydrin group is a mild, chemically reactive group that preferably reacts with free basic groups of the enzyme to be modified. Such basic groups in the enzyme to be modified are preferably free, primary amino groups of polyfunctional amino acids which are contained in the polypeptide chain making up the enzyme and are accessible to the reagent due to their position on the enzyme surface. These are in particular, for example, the ε-amino group of the amino acid lysine or, if appropriate, also the amino function in the guanidinium group of the amino acid arginine. The basicity or nucleophilicity of these amino groups is favorably matched to the reactivity of the epoxy or halohydrin compounds used and the degree of modification can therefore be easily controlled by adjusting the reaction parameters pH, temperature and reaction time. In addition, the epoxides or halohydrins used according to the invention are cheap and can be industrially produced in large quantities and are therefore readily available. A variety of these epoxies are also commercially available. In addition to the terminal epoxy or halohydrin group, which is important according to the invention for the connection to the enzyme, the epoxides can, by their nature, carry a large number of residues which, through their respective specific properties, modify the properties of the enzyme derivatives obtained and thereby the targeted variation of the biochemical and application properties enable these enzyme derivatives.

The invention also provides a process for the production of water-soluble or non-water-insoluble, chemically modified enzymes (enzyme derivatives), which is characterized in that little is done by reaction taking place under basic conditions at least one of the free primary amino groups of an enzyme not located in the activity center with a) epoxides of the formula I /

wherein

R represents a cationic, amphiphilic, anionic or lipophilic or hydrophobic organic radical, or with b) halohydrins of the formula II

wherein

X represents halogen, preferably chlorine or bromine, and

R has the meaning given above, converts the enzyme into a water-soluble or non-water-insoluble, chemically modified enzyme (enzyme derivative), in which one or more of the free amino groups mentioned of the enzyme with a radical of the formula A

where R has the above meaning, are substituted. The molecular size of the enzymes used is hardly changed by the process according to the invention for the chemical modification of enzymes. The water solubility of the enzymes used, which is regarded here in particular as the solubility in an application medium containing water, is therefore hardly impaired by the chemical modification according to the invention. There are thus water-soluble or non-water-insoluble, ie

in particular in water-containing application media, soluble or non-insoluble, chemically modified enzyme derivatives are obtained, the water solubility of which essentially corresponds to that of the underlying enzymes or, in contrast, is possibly reduced to the extent that they are not completely water-insoluble. The terms “water-soluble” or “water-solubility” here also include fine dispersions, such as, in particular, emulsions.

When carrying out the above-mentioned process for producing chemically modified enzyme derivatives, it is possible to start from enzymes in any form. The procedure is independent of the previous history, e.g. the fermentation and isolation conditions, of the enzyme. It is possible to start from solid spray-dried, crystallized, granulated enzymes or from enzyme solutions or concentrates of any concentration. It proves advantageous here that intermediate stages, i.e. solutions or concentrates of enzymes, normal enzyme production can be assumed.

The reaction is usually carried out in solution, preferably in an aqueous solution. The reaction solutions can optionally contain enzyme-compatible organic solvents, for example alcohols, diols etc., or enzyme stabilizers. Solid enzyme starting materials are therefore dissolved in the desired concentration in water or in the water / solvent mixture. If one starts directly from enzyme solutions from a normal enzyme production, then can be used in the resulting concentration or after concentration to the desired concentration. In a preferred embodiment of the process according to the invention for the production of chemically modified enzyme derivatives, however, aqueous enzyme concentrates are used which have a dry substance content of 10 to 35% by weight. In a further embodiment of the process according to the invention, it may prove expedient to carry out the reaction in the presence of enzyme stabilizers and / or solubilizers. The reaction is then preferably carried out in the presence of propanediol, which may be present in amounts of up to about

84% by weight (based on the entire reaction mixture) can be present.

The process according to the invention for producing chemically modified enzymes (enzyme derivatives) is carried out under basic conditions. This means that the reaction is carried out at pH values above 7. In a preferred embodiment of the production process according to the invention, the reaction is carried out at pH values from 9 to 11. The reaction can either be started at a high initial pH value, which gradually shifts to smaller alkaline pH values (down to a maximum of pH 7) in the course of the reaction, or by constant readjustment of the pH value to a constant alkaline pH -Value to be held. The pH value can be adjusted by all inorganic bases that are compatible with enzymes, e.g. in particular alkali oxides or hydroxides. Sodium hydroxide, in particular in the form of an aqueous solution (sodium hydroxide solution), is preferably used for pH adjustment.

The temperature of the process is only limited by the temperature stability of the enzymes used. The implementation is therefore carried out in appropriate embodiments of the manufacturing method according to the invention Temperatures up to a maximum of 35 ° C. However, the reaction is preferably carried out at temperatures of up to a maximum of 30 ° C. At the bottom, the reaction temperature is generally limited by ambient temperatures of approximately 25 ° C., since no or only inadequate conversions can be observed below these temperatures.

Depending on the type of the modifying enzyme and the type of modification reagent chosen and the desired degree of modification, the reaction time can range from a few hours to a whole day. As a rule, in expedient embodiments of the manufacturing process according to the invention, reaction times become. from 1 to 8 hours. The degree of modification of the enzyme derivatives according to the invention obtained can be set in a wide range from low to medium and medium to strong. Instead of longer reaction times, it may also be appropriate, depending on the enzyme used, to shorten the reaction time by using an excess of modification reagent, which is not only advantageous from a process engineering point of view, but also from an economic point of view (higher value of the enzymes; cheap and in large amount of readily available modification reagents). In expedient embodiments of the production process according to the invention, therefore, a 5 to 10-fold excess of modification reagent (based on the free, substitutable amino groups contained in the enzyme to be modified) is generally used. If desired, in order to achieve higher degrees of modification, it may also be desirable to work with an even greater excess of modification reagent; the addition of the modification reagent to the solution of the enzyme to be modified can be distributed over the reaction time either continuously or in several doses or even only once at the beginning the implementation of the enzyme to be modified. The one-time addition at the beginning of the reaction leads to higher reaction rates and also to higher ones Degree of modification and is therefore particularly useful when short reaction times or overall higher degrees of modification are to be achieved.

To terminate the reaction, the reaction mixture is generally cooled to 20 ° C. and adjusted to a pH of 7 with an aqueous solution of an enzyme-compatible inorganic and / or organic acid. An aqueous solution of citric acid may be mentioned as an example. The reaction mixtures obtained can either be used as such for further use or can be subjected to conventional measures in a manner which is customary per se, such as in the case of normal enzyme production / packaging. Thus the mixtures containing the enzyme derivative according to the invention can be admixed with enzyme stabilizers which are conventional per se, worked up to concentrates or in a conventional manner, e.g. by spray drying, converted into a solid product and also granulated.

The invention provides a chemically very mild and enzyme-friendly process for the targeted chemical change in the properties of an enzyme by means of chemical modification, which is outstandingly suitable for the production of chemically modified enzymes. The invention obtained by the process

Enzyme derivatives can be chemically modified enzymes, the underlying enzymes of which can be chemically modified in their properties by substitution with various types of organic residues. Thus, the organic radicals R introduced into the enzyme with the epoxide of the formula I or the halohydrin of the formula II can be cationic, amphiphilic, anionic or hydrophobic.

This enables the properties of an enzyme to be changed in a variety of ways. For example, the isoelectric points, the pH optima, the surface properties (charge, hydrophobicity), the interfacial activity etc. can be influenced in a targeted manner. In a variant of the process according to the invention for the production of water-soluble, chemically modified enzymes, epoxides of the formula I or halohydrins of the formula II are used for the chemical modification of the underlying enzyme, in which R is a cationic organic radical of the formula - (CH ₂ ) _n -N ⁺ R ¹ R ² R ³ , in which R ¹ , R ² and / or R ^{π are} a lower alkyl radical having 1 to 2 carbon atoms, preferably methyl, and n is an integer from 1 to 3. This gives chemically modified enzyme derivatives which, owing to the cationic radicals R, have an increased positive surface charge. In this variant of the production process according to the invention, the radicals R are then, for example, trimethylammoniummethylene, trimethylammoniumethylene, trimethylammoniumtrimethylene groups, the trimethylammonium ethylene group being very particularly preferred. An example of a modification reagent used in this variant is, for example, 2,3-epoxypropyltrimethylammonium chloride.

In another variant of the process according to the invention for the production of water-soluble, chemically modified enzymes, epoxides of the formula I or halohydrins of the formula II are used for the chemical modification of the underlying enzyme, in which R is an amphiphilic organic radical of the formula

- (CH ₂ ) _n -N ⁺ R ¹ R ² R ⁴ , in which R ¹ and / or R ^{2 is} a lower alkyl radical having 1 to 2 carbon atoms, preferably methyl, R ^{4 is} a straight-chain C10 bis

C18 alkyl radical and n is an integer from 1 to 3. This gives chemically modified enzymes whose amphiphilic radicals R contain not only the positive ammonium group but also a hydrophobic substituted R ⁴ . Examples of modification reagents used in this variant are, for example, 3-chloro-2-hydroxypropyldimethyllaurylammonium chloride and 3-chloro-2-hydroxypropyldimethylcoco (C12-C16) ammonium chloride. In a further variant of the process according to the invention for the production of water-soluble, chemically modified enzyme derivatives, epoxides of the formula I or halohydrins of the formula II are used for the chemical modification of the underlying enzyme, in which R represents an anionic organic radical of the formula

- (CH ₂ ) _n -SO ₃ -, where n is an integer from 1 to 3. In this way chemically modified enzymes are obtained which, due to the anionic radicals R, have an increased negative one. Possess surface charge. The chemical modification reagents used in this variant of the production process according to the invention are, for example, 2-chloro-1-hydroxyethylalkylene sulfonates, preferably in the form of the sodium salt. Chemical modification reagents which are particularly preferred for this variant are, for example, the 3-chloro-2-hydroxypropylsulfonates, in particular that

Sodium salt.

In a further different variant of the process according to the invention for the production of water-soluble, chemically modified enzymes, the chemical

Modification of the underlying enzyme, epoxides of the formula I or halohydrins of the formula II, in which R represents a hydrophobic organic radical of the formula - (CH ₂ ) _n -CH ₃ , where n is an integer from 1 to 17. In this way chemically modified enzymes are obtained which, due to the hydrophobic radicals R, have an increased hydrophobicity. In an expedient embodiment of this variant of the production process according to the invention, the reaction is carried out using enzyme concentrates, in particular with very high enzyme concentrations, and using solubilizers, preferably propanediol. In this variant of the process according to the invention, the epoxides of 1-alkenes in particular can be used as chemical modification reagents. Examples include α-epoxides such as hexen-1-oxide, octene-1-oxide, decen-1-oxide, dodecen-1-oxide, tetradecen-1-oxide, hexadecen-1-oxide and octadecen-1-oxide. In the process according to the invention for the production of water-soluble, chemically modified enzyme derivatives, any enzymes per se, in particular those with industrial importance for various applications such as detergent enzymes, enzymes for saccharification of starch and other applications can be used. In a preferred embodiment of the production method according to the invention, proteases, amylases,

Glucose isomerases or lipases used. However, other enzymes such as e.g. Pectinases, cellulases or hemicellulases etc. can be used.

The process according to the invention for the production of water-soluble, chemically modified enzyme derivatives allows targeted, chemically very mild tailoring of enzyme derivatives with properties which are adapted to the particular application in the desired manner. The method according to the invention thus enables in a simple manner, e.g. to vary the surface properties with regard to charge and hydrophobicity in the desired manner. Enzyme properties such as pH optimum, pH stability, temperature optimum, temperature stability, oxidation stability or compatibility with other enzymes (e.g. stability against digestion by protease, if present), interfacial activity etc. can be easily changed and adjusted to the respective technical application of the enzyme in the desired manner.

The invention therefore also relates to those water-soluble or non-water-insoluble, chemically modified enzymes (enzyme derivatives) which, in relation to their respective technical fields of application, are improved in their biochemical and application properties, these chemically modified enzymes being characterized in that they are present in at least one the free primary amino groups of the underlying enzyme which are not in the activity center and have a radical of the formula A

where R is a cationic, amphiphilic, anionic or hydrophobic organic radical, are substituted. Appropriate enzyme derivatives according to the invention are characterized in that the underlying enzyme is a protease, amylase, glucose isomerase or lipase. However, the underlying enzyme can also be another, e.g. a pectinase, cellulase or hemicellulase etc.

In a variant of the enzymes chemically modified according to the invention, they are distinguished by the fact that they are substituted by a radical of the formula A in which R represents a cationic radical of the formula - (CH ₂ ) _n -N ⁺ R ¹ R ² R ³ , wherein R ¹ , R ² and / or R ^{3 represent} a lower alkyl radical having 1 to 2 carbon atoms, preferably methyl, and n is an integer from 1 to 3. In preferred chemically modified enzymes of this variant of the invention, the underlying enzymes are proteases, as are used in particular for washing and cleaning compositions. However, those water-soluble, chemically modified proteases (protease derivatives) whose protease is based on an alkaline or highly alkaline protease are particularly preferred. These alkaline or highly alkaline proteases can be obtained, for example, by cultivating Bacillus strains such as Bacillus subtilis, Bacillus alcalophilus, Bacillus amyloliquefaciens or Bacillus licheniformis etc. In the preferred embodiment of this variant of the invention, there are therefore enzyme derivatives which are distinguished by the fact that they are water-soluble or non-water-insoluble, chemically modified proteases which have a cationic on at least one of the free primary amino groups of the underlying protease which are not in the activity center organic radical of formula B.

wherein R ¹ , R ² and / or R ^{3 are} a lower alkyl radical having 1 to 2 carbon atoms, preferably a methyl radical, and n is an integer from 1 to 3, are substituted. This cationic modification of proteases gives protease derivatives in which the positively charged ε-amino groups of the lysine are quaternary

Ammonium groups are replaced. This increases the pK value (and also the isoelectric point). In particular, strongly cationically modified proteases (proportion of multiple modifications) show somewhat increased temperature stability. Furthermore, for example, the washing action of the modified, highly alkaline proteases is not as strongly dependent on the detergent dosage as the underlying unmodified proteases. Such chemically modified proteases can be used with particular advantage for commercial laundries, where very high wash liquor pH values are often present. Under such conditions, cationically modified highly alkaline proteases are clearly superior to the underlying unmodified proteases. Cationically modified proteases also show better degradability of egg proteins in spots.

In another variant of the enzyme derivatives according to the invention, they are distinguished by the fact that they are substituted by a radical of the formula A in which R represents an amphiphilic radical of the formula - (CH ₂ ) _n -N ⁺ R ¹ R ² R ⁴ , wherein R ¹ and / or R ^{2 is} a lower alkyl radical having 1 to 2 carbon atoms, preferably a methyl radical, R ^{4 is} a straight-chain C10 to C18 alkyl radical and n is an integer from 1 to 3. There are chemically modified enzymes with an amphiphilic character. In a further variant of the chemically modified enzymes according to the invention, they are distinguished by the fact that they are substituted by a radical of the formula A in which R represents an anionic radical of the formula - (CH ₂ ) _n -SO ₃ -, in which n is a means integer from 1 to 3. In a group of expedient, chemically modified enzymes of this variant of the invention, the underlying enzymes are proteases, in particular alkaline or highly alkaline proteases for washing and cleaning compositions, as have already been described in more detail above. In a preferred embodiment of this variant of the invention, therefore, there are water-soluble or non-water-insoluble, chemically modified enzymes which are distinguished by the fact that they are chemically modified proteases, in particular chemically modified alkaline or highly alkaline proteases, which are present in at least one of those which are not in the activity center located free primary amino groups of the underlying protease with an anionic organic radical of the formula CC

where n is an integer from 1 to 3, are substituted. In another group of expedient, chemically modified enzymes of the variant of the invention with anionic modification of the underlying enzyme, the underlying enzyme is an α-amylase. The underlying α-amylases are in particular those as are used for washing and cleaning compositions or, on the other hand, for example also for starch saccharification. Thermostable α-amylase, as can be obtained, for example, from BacillUs licheniformis, is particularly preferred. In a further preferred embodiment of the variant of the invention with anionic modification of enzymes, there are therefore water-soluble or non-water-insoluble, chemically modified enzyme derivatives, which are characterized in that they are chemically modified α-amylases, preferably chemically modified thermostable α-amylases, which have at least one of the free primary amino groups of the underlying amylase which are not in the center of activity and have an anionic radical of the formula CC

where n is an integer from 1 to 3, are substituted.

The anionically modified enzymes described above are particularly suitable for low-pH detergent systems such as heavy-duty liquid detergents and certain mild detergents. The enzyme derivatives from mild alkaline proteases (for example from Bacillus licheniformis or Bacillus subtilis) are particularly advantageous here, especially for bleach-free and largely builder-free liquid detergents. Furthermore, it proves to be extremely advantageous that the anionically modified enzymes have an increased stability compared to conventional liquid detergent components such as linear alkyl sulfonates, soaps, paraffin sulfonates, fatty acid ethoxysulfates etc. and are also advantageous in concentrated liquid detergents, especially those with a high water content and higher pH values Have storage stability. The pH optimum of the anionically modified enzymes is generally shifted towards the acidic range by 0.5 to 1 pH unit compared to the underlying unmodified enzyme. Another effect of the anionic modification is the greatly improved turbidity stability in liquid detergent formulations (ie the tendency of the enzyme dissolved in the liquid detergent or other constituents of the enzyme preparation dissolved therein to be stored flocculation is reduced). The anionically modified enzymes described above, in particular also anionic α-amylases, are also suitable for use in environmentally friendly enzyme-containing dishwashing detergents. In addition, for example, anionically modified α-amylase derivatives in the low pH range from 5.0 to 6.0 at higher temperatures (70 ° C.) have increased stability compared to the underlying unmodified α-amylases.

Another variant of the chemically modified enzymes according to the invention is characterized in that the underlying enzyme is substituted with a radical of the formula A in which R is a whole number for a hydrophobic organic radical of the formula - (CH ₂ ) _n -CH ₃ where n from 1 to 17 means are substituted. There are modified enzymes with a hydrophobic character.

The following examples are intended to explain the invention further, but without restricting its scope.

Example 1 :

A total of 40 kg of an aqueous enzyme concentrate of the following composition was first manually placed in a storage container

Enzyme: Highly alkaline protease

Bacillus alcalophilus

Protein (biuret): 15.1% by weight

Dry matter: 32.2% by weight

Water: 67.8% by weight

Activity (DU / g) *: 600,000

(* 1,000 Delft Units (DU) is the proteolytic activity which, with a volume of 1 ml of a 2% by weight enzyme solution, results in an extinction after casein has been broken down difference of 0.400 results; 1 cm light path, 275 nm; ßestimmung against blind sample test) with sodium hydroxide solution to a pH 10 and at ambient temperature (about 20 ° C) with about 2 kg (= 3.5 wt .-% active substance based on the enzyme contained in the enzyme liquid concentrate used) a 70 to 71 wt. -% aqueous solution of 2,3-epoxypropyl-trimethylammonium chloride (commercially available liquid preparation) mixed. The mixture obtained was transferred to a 50 1 stirred reactor by means of a pump and heated to an inlet temperature of 30 ° C. by means of a plate heat exchanger.

The course of the reaction was followed for the quantification and detection of the chemically modified proteases by cation exchange chromatography (Ultropak TSK CM-2-SW 250 x 4.6 mm = cation exchanger based on carboxymethyl from Pharmacia LKB GmbH, Freiburg). 0.05 molar pH 6.0 sodium acetate buffer was used as the starting luent. Elution was carried out by adding a 0.4 molar sodium acetate buffer with likewise pH 6.0. The results of the course of the reaction are summarized below as a function of the reaction time. The degree of modification here relates to the underlying protease (native protease), multiple substitutions not being taken into account, and is defined as the relative ratio of the concentration of the enzyme derivatives to the sum of the concentrations of the native enzyme and the enzyme derivatives. The degree of modification is given in percent and a degree of modification of 100% is therefore synonymous with the absence of native enzyme. The activity stated in% is volume-corrected and relates to the activity (delft units) of the native enzyme used.

* Filling the reactor

The reaction was exothermic, causing the temperature set at the beginning to increase from 30 ° C. to approx. 33 to 35 ° C. After 4 hours, the reaction was stopped by cooling the reaction mixture to 20 ° C. (plate heat exchanger) and by adding aqueous 20% by weight citric acid with a pH of 7 being set.

The water-soluble, chemically modified protease was isolated by methods known per se for the isolation of enzymes, such as ion exchange chromatography.

The product obtained could be converted into enzyme granules without difficulty by measures known per se, such as extrusion or spray drying.

80 kg of a highly alkaline Bacillus alcalophilus protease chemically modified by substitution with 3-trimethylammonium-2-hydroxypropyl residues were obtained in granular form. The properties of the protease derivative granules obtained are summarized below.

Unmodified protease content <5% by weight multiple substitution low

(* Based on the activity of the native protease (Delft Units) = 100%; high molecular weight substrate: casein; 40 ° C, 40 min .; average values from various measurements.)

The temperature profile of the chemically modified protease (ie the activity in% as a function of the temperature) showed essentially the same profile as the temperature profile for the native protease (20 minutes in each case, pH 8, substrate = acetyl casein, temperature range 40 to 70 ° C.) . The results show that the active center of the chemically modified highly alkaline protease from Bacillus alcalophilus was not affected by the mild chemical modification process.

Analogous modifications with further concentrates of the highly alkaline protease from Bacillus alcalophilus showed that any fluctuations in the composition which may occur during the preparation of the concentrate had practically no influence on the modification rate, the modification rate and the product identity. The protease properties were not adversely affected by the mild chemical modification. The effects of the stronger cationization of the chemically modified protease derivatives compared to the native protease can be seen via secondary effects in the phenomena to be observed from an application point of view described below.

Example 2:

Analogous to Example 1 was an aqueous enzyme concentrate of the following composition Enzyme: Alkaline protease from Bacillus licheniformis

Dry matter: 22.4% by weight

Water: 77.6% by weight

Activity (DU / g): 921,000 adjusted to a pH of 10 with sodium hydroxide solution and mixed at ambient temperature (approx. 20 ° C.) with such an amount of a 70 to 71% by weight aqueous solution of 2,3-epoxypropyltrimethylammonium chloride, that amount of 5% by weight of active substance (= modification reagent) based on the amount of enzyme used. The reagent was added within 5 minutes. The mixture was heated to 25 ° C. and the course of the reaction was followed by cation exchange chromatography as in Example 1. The results of the course of the reaction are summarized below as a function of the reaction time. The degree of modification is defined as in Example 1, the activity in percent (analogously to Example 1) is also volume-corrected and relates to the activity (delft units) of the native protease used.

(* see example 1)

After 4 hours, the reaction mixture was cooled and stopped by adding aqueous 20% by weight citric acid with a pH of 7.

The slight decrease in activity of the chemically modified protease compared to the native protease is due to a higher alkalinity of the alkaline Bacillus licheniformis protease used. Analogous modifications with other Bacillus licheniformis protease concentrates showed that this drop in activity can be avoided when using protanediol-containing protease concentrates. Propanediol-stabilized protease concentrates can be modified almost without loss. The liquid end product of this example was used directly for further experiments.

Example 3:

Analogous to the preceding examples, an aqueous enzyme concentrate of the composition given in example 1 (highly alkaline protease from Bacillus alcalophilus) was reacted with 3-chloro-2-hydroxydimethylcoco (C12-C16) ammonium chloride and the course of the reaction was monitored analogously to example 1 by cation exchange chromatography. The concentration of the protease was adjusted so that there was an activity of 200 kDU / ml in the reaction mixture. The chlorine used as a chemical modification reagent hydrine was used in the form of the diluted, commercially available solution (Degussa) and used in an amount such that 2% by weight of active substance, based on the enzyme used, was present. The reaction was carried out at 30 ° C. and the pH for activating and converting the chlorohydrin into the epoxide was adjusted constantly to pH 11 (trapping the hydrogen chloride released). The degree of modification achieved as a function of the reaction time is summarized below.

Example 4:

In each case, 0.8 kg of aqueous enzyme concentrate of the highly alkaline protease from Bacillus alcalophilus with a propanediol content of 40% by weight and the following further composition

Protein (biuret): 6% by weight

Inorganic salts: 3% by weight (e.g. CaCl ₂ )

Non-symbol components: 1% by weight

Water: 50% by weight with an initial pH of 12 at 30 ° C. under various reaction conditions specified below with sodium 3-chloro-2-hydroxypropyl sulfonate (solid reagent from Shell Chemie).

After the start of the reaction, the initial pH fell to pH 11 within a short time due to the activation of the chlorohydrin used to form the epoxide, with the simultaneous formation of hydrogen chloride, and was adjusted to the desired pH of 11 to 12 by the continuous addition of 4 N sodium hydroxide solution . The formation of the active reagent (ie the epoxide) - and therefore also the course of the reaction - could easily be followed on the basis of the consumption of sodium hydroxide solution. The course of the reaction with the formation of weakly to moderately strongly anionically modified enzyme derivatives (ie in reactions with up to

5% by weight of reagent based on the enzyme used) could also be monitored analogously to Example 1 by means of cation exchange chromatography (Ultropak TSK-2-CM 250 x 4.6 mm, pH = 6). Higher reagent concentrations of 5 to 10% by weight (based on the enzyme used) made it possible to remove the highly alkaline protease used

Modify Bacillus alcalophilus several times (i.e. the number of substituents introduced into a protease molecule was> 1).

To complete the reaction, the respective

Cooled reaction mixture to 20 ° C and neutralized to pH 7 with aqueous 20 wt .-% citric acid.

The isolation / concentration of the chemically modified proteases was carried out according to methods known per se for the isolation of enzymes, such as ion exchange chromatography.

Depending on the set conditions, water-soluble, chemically modified enzyme derivatives with a moderate to high degree of modification were obtained.

For the application tests, the were ionically modified enzymes in the form of a solution stabilized with 40% by weight of propanediol.

Example 5:

Analogous to Example 4, alkaline proteases from Bacillus licheniformis were used in different ways. Form - evaporator concentrate containing propanediol for the production of the alkaline protease (P1), regular product solution of the alkaline protease (P2) or concentrate of the alkaline protease with an activity of 750,000 DU / g - with

Sodium 3-chloro-2-hydroxypropyl sulfonate reacted under the following conditions. Here, depending on the reaction conditions set, chemically modified enzyme derivatives with a medium to high degree of modification were obtained.

The modified proteases obtained in this example have activities of over 90% of the starting activity (activity of the protease used).

Example 6:

Analogously to Example 4, a Bacillus subtilis protease was reacted with sodium 3-chloro-2-hydroxyproylsulfonate under the following conditions. Enzyme: 38 g protease powder

Propanediol: 220 g

Water: 180 g

Activity: 71,000 DU / g (after dilution of the protease with propanediol and water as described above)

Reagent: 8 g

pH: 11

Temperature: 30 ° C

Response time: 4 h

The course of the reaction was monitored by means of cation exchange chromatography (Ultropak TSK-2-CM, pH = 5.2). The reaction was terminated as indicated in Example 4 by cooling to 20 ° C. and adding 20% by weight citric acid to adjust the pH to 7.

This gave water-soluble, anionically modified proteases with a moderate degree of modification, the activity of which was above 93% of the starting activity (activity of the protease used).

Example 7:

An aqueous enzyme concentrate made from highly alkaline protease from Bacillus alcalophilus was reacted with decen-1-oxide (Peroxid-Chemie) with intensive stirring and with the addition of a stabilizing and solvent-imparting amount of propanediol under the following conditions.

Protease concentrate: 15% by weight

Propanediol: 81% by weight

Reagent: 2% by weight at the beginning of the

Response (0 h)

2% by weight after 3 h

pH: 11

Temperature: 30 ° C The course of the reaction could be followed by cation exchange chromatography (Ultropak TSK-CM-2SW 250 x 4.6 mm). After 22 hours, the reaction was carried out as usual by cooling and adjusting the pH

20% by weight citric acid stopped at pH 7 and the product formed with a clear proportion of hydrophobic protease derivatives with a moderate degree of modification by gel filtration (G 25 medium; PD 10 columns from the company

Pharmacia LKB GmbH, Freiburg) and analyzed by reversed phase as follows.

Pillar: Waters Nova Pak C18.

(150 x 4.6 mm; Millipore GmbH, Eschborn).

Solvent A: 0.1% by weight aqueous tri-fluoroacetic acid solution

Solvent B: 0.1% by weight aqueous trifluoroacetic acid solution / acetonitrile in a volume ratio of 40/60

Gradient: from 5 to 55 min., From 0% to 100% solvent B.

Detection: UV light at 280 nm

Example 8:

Analogous to Example 2 was an aqueous standard amylase concentrate of the following composition

Enzyme: Thermostable α-ylmylase from

Bacillus licheniformis

Protein (biuret): 5% by weight

Inorganic salts: 3% by weight (e.g. CaCl ₂ )

Dextrins: 5% by weight

Water: 87% by weight

Activity (MWU / g) *: 820,000 (* 1 Modified Wohlgemuth Unit (MWU) is the

Amount of enzyme which breaks down 1 mg of soluble starch into a dextrin of defined size in 30 minutes; 40 ° C; the size of the dextrin formed is indicated by the color of a dextrin-iodine complex.) adjusted to pH 10 with sodium hydroxide solution and at ambient temperature (20 ° C.) with such an amount of a 70 to 71% by weight aqueous solution of 2 , 3-Epoxypropyltrimethylammoniumchlorid mixed, which corresponded to an amount of 5 wt .-% active substance based on the amount of enzyme used. The mixture was heated to 26 ° C. and the course of the reaction was followed in analogy to Example 1 by catalytic exchange chromatography (Ultropak TSK-CM-2SW 250 x 4.6 mm; pH 5.2).

The reaction could be stopped after 1 hour by cooling to 20 ° C. and adding aqueous 20% by weight citric acid with a pH of 7. The liquid end product, corrected to pH 7 and stabilized with propanediol (amount 50% by weight, based on the finished formulation), was used directly for further experiments.

When using insoluble substrates (Phadebas = blue-colored starch polymer from Pharmacia LKB GmbH, Frsiburg) to determine activity, the temperature and pH activity profile of the product differed only insignificantly from the temperature and pH activity profile of the underlying, unmodified α-amylase; these results show that these two essential characteristics, which are already excellent in the underlying unmodified enzyme, have hardly been affected by the mild chemical modification process.

Example 9:

Analogously to Example 5, an aqueous enzyme concentrate of the thermostable α-amylase from Bacillus licheniformis with the addition of a stabilizing amount of 50% by weight of propanediol to the enzyme concentrate (based on the total batch) and with the continuous addition of 10% by weight sodium hydroxide solution at a constant pH of 11 with sodium 3-chloro-2-hydroxypropylsulfonate (2.5 wt .-% active substance based on the amount of enzyme used) at 30 ° C. After 2 hours 84% and after 4 hours 97% of the amylase used had reacted. To terminate the reaction, the mixture was cooled to 20 ° C. and adjusted to a pH of 7 using aqueous 20% by weight citric acid. The liquid product of the anionically modified amylase was used directly for further experiments.

The pH activity profile of the water-soluble; anionically modified amylass was not impaired by the mild chemical modification process compared to the pH activity profile of the native amylase or it even shows a slightly increased activity profile in the pH range from 4 to 5 (40 ° C, substrate = soluble starch, activity in MWU ). At higher temperatures

(70 ° C) the anionically modified amylase derivative has a significantly increased stability compared to the native amylase at pH values in the range from 5.2 to 5.6.

Example 10:

Pure glucose isomerase was chemically modified with 2,3-epoxypropyl-trimethylammonium chloride. For this purpose, glucose isomerase crystals were dissolved in propanediol / water and reacted with the reagent (70 to 71% by weight aqueous solution of 2,3-epoxypropyltrimethylammonium chloride) under the following conditions.

Enzyme: 160 g (38.4% by weight protein and

95 GlU / mg *)

Propanediol: 400 g

Water: 240 g

Reagent: 5% by weight based on the gas charge Temperature: 25 ° C

Response time: 6 h

pH value: 10 at 0 to 5 h (1 N sodium hydroxide solution)

11 at 5 to 6 h

(* 1 glucose isomerase unit (GIU) is defined as the amount of enzyme that forms 1 mg fructose under the following incubation conditions; incubation conditions: 65 ° C, 1 h, substrate from 0.1 m glucose in 0.05 m phosphate buffer, pH 8, 0 with 0.0004 m MgSO _4. )

The course of the reaction could be determined by anion exchange chromatography (Ultropak DEAE-3-SW 150 x 4.6 mm,

pH 5.8 = column material based on diethylaminoethyl from Pharmacia LKB GmbH, Freiburg; Starting lumen 0.05 m NaH ₂ PO ₄ ; Eluens 0.05 m NaH ₂ PO ₄ / 0.8 m sodium chloride) are followed.

To terminate the reaction, the pH was adjusted to 7.0. In the course of the reaction, no loss of activity compared to the activity of the unmodified enzyme was observed. This in turn shows that the mild chemical modification process for the glucose isomerases used is very well tolerated.

Example 11:

Analogously to Example 10, pure glucose isomerase was reacted with sodium 3-chloro-2-hydroxypropylsulfonate, but the pH was kept constant at pH 11. No loss of activity for the chemically modified glucose isomerase compared to the native glucose isomerase was observed here either. This shows that the mild anionic modification for the native glucose isomerase is also very well tolerated. The liquid end product neutralized after termination of the reaction to pH 7 could be used directly for further experiments. Example 12:

To demonstrate the behavior of the chemically modified enzymes in the washing process, the washing performance was determined by washing tests on test fabrics in laboratory washing machines (type: Linitest). For this purpose, test fabric was washed with standardized detergent formulations which contained the chemically modified enzymes to be tested (enzyme derivatives) or, in comparative experiments, the native enzymes. The tests were carried out at the specified pH values and with water at 15 ° dH in the temperature range from 15 to 60 ° C. specified in each case. The washing performance was determined by measuring the lightening of soiled test fabrics as the remission difference (remission of the dry test fabric after the washing test minus the reflectance of the test fabric before the washing test). The bigger one

Remission difference indicates the better washing performance. The washing tests were carried out as follows: The detergent solution containing enzymes worked in a rotating manner

Sample container onto the test tissue. After completion of the washing process, the test fabric was rinsed twice with tap water and, after ironing, the lightening of the fabric was recorded via the reflectance measurement. a) Conditions: 6 g / l detergent, 15 - 60 ° C, 45 minutes,

15 ° dH.

Test fabric: EMPA 116 = cotton, soiled with

Blood, milk and ink.

Detergent: Commercially available European powder detergent I, containing 25% by weight

Zeolite, 10% by weight sodium carbonate,

6.5% by weight of linear alkyl sulfonates,

4.5% by weight of soap, 2% by weight of carboxymethyl cellulose, 0.5% by weight of phosphonates,

2.0% by weight magnesium silicate, 25% by weight sodium perborate, 1.5% by weight tetraacetylethylene diamine and sodium sulfate ad

100% by weight. The results of this washing test are shown in the table below.

* V = comparison test

* * pH measured before washing b) Conditions: European commercial heavy-duty detergent II, own pH value, 15 - 60 ° C, 1 kDU / 100 ml, 45 minutes.

Test fabric: EMPA 117 = cotton / polyester blend, soiled with blood, milk and ink.

The following results were obtained.

This example shows the low dependence of the cationically modified protease from Example 1 on the detergent dosage. In contrast, the detergent dosage has a much stronger effect on the native protease. c) Conditions: 6 g / 1 detergent, pH = 11.5, 15 ° dH,

15 - 60 ° C, 45 minutes.

Detergent: composition see under a) this

Example.

The following results were obtained.

|

The chemically modified used in this example ∑ated protease derivative shows excellent washing effectiveness at very high wash liquor pH values. This

chemically modified enzyme derivative is therefore particularly suitable for commercial laundry. This cationically modified protease derivative, in particular, degrades proteins such as egg proteins very well, d) conditions: 2 g / l heavy-duty liquid detergent, 30 ° C.,

30 minutes, 15 ° dH, 0.5 kDU / 100ml.

Test fabric: ox. EMPA 117 = cotton / polyester blend, soiled with blood, milk and ink; the mixed fabric was pre-treated with perborate (2 g / l) at pH 10 without the addition of detergent and then subjected to an intensive rinse; This gave test fabrics with aged soiling, which are much more sensitive to differences in the activity of the enzymes and enzyme derivatives in the wash liquor.

Detergent: European heavy duty liquid detergent, containing 10.5% by weight of linear alkyl sulfonates, 13% by weight of sodium fatty acid salt, 5% by weight of alkyl sulfate, 10.3% by weight of fatty alcohol ethoxylate, 10% by weight of triethanolamine; 47% by weight of volatile components (water, propanediol).

The results of this washing test are summarized in the table below.

* V = comparison test

** pH measured before washing

This example clearly shows the shift of the pH optimum by approx. 0.5 - 1 pH unit for the chemically, anionically modified protease derivative compared to the native protease. The anionic modification leads in addition to a greatly improved turbidity stability in heavy duty liquid detergent formulations.

Example 13:

Analogously to the general procedure of Example 12, the anionically modified enzyme derivatives were tested for their stability against individual liquid detergent constituents and against a complete liquid detergent formulation and for their storage stability. a) The stability of the anionically modified enzyme derivatives against various common constituents of liquid detergents, such as linear alkyl sulphonates (LAS) soaps, paraffin sulphonates and fatty acid ethoxysulphates (FAES), was determined under the following conditions: 2.5 g / l of the respective liquid detergent constituent, 40 ° C., Enzyme derivative concentration 4 kDU / ml, addition of 100 ppm calcium. The concentrations chosen here correspond approximately to those in the wash liquor. The anionically modified enzyme derivatives show significantly higher stabilities in the presence of the liquid detergent components compared to the native enzymes. While, for example, there is a rapid decrease in activity in the presence of anionic surfactants in the case of the native proteases, the moderately strongly anionically modified protease from Example 4b and the strongly anionically modified protease from Example 4c show considerably better stabilities than conventional anionic surfactants. Anionically modified enzyme derivatives are very well suited for use in such liquid detergents due to their higher stability towards liquid detergent components. The results of the stability tests are summarized in the table below.

* based on the original activity of the enzyme or enzyme derivative b) The stability of anionically modified enzyme derivatives with respect to the ingredients of a conventional, complete liquid detergent formulation with the following analytical composition

Sodium alkylbenzenesulfonate: 4.5% by weight

Fatty acid / salt: 35.3% by weight

Fatty alcohol ethoxylate: 9% by weight

Triethanolamine: 11.5% by weight

Water: 31% by weight

(volatile components total 38 wt., alcohols,

Water) was tested under the following conditions: 6 g / l liquid detergent formulation, pH = 9.5, 40 ° C., enzyme concentration 4 kDU / ml, 100 ppm calcium content. The results obtained are summarized in the table below.

This example clearly shows the higher stability of the chemically modified proteases in liquid detergent formulations. c) The storage stability of some anionically modified enzyme derivatives in concentrated liquid detergent formulations was tested under the following conditions.

Liquid detergent formulation containing:

- 10 wt .-% linear alkyl sulfonates

- 10% by weight of ionic surfactants

- 5% by weight ethanol

- 5% by weight propanediol

- 70% by weight of water

- 100 ppm calcium content

- pH = 9.5

Storage temperature: 37 ° C

The results summarized in the table below were obtained.

* based on the original activity of the enzyme or

Enzyme derivatives This example shows the good stability of anionically modified proteases in concentrated liquid detergents of defined composition and with high water contents. Similarly good storage stabilities were also observed with the anionically modified protease from Example 6. Washing results with the stored samples confirmed the differences in storage stability found above through activity measurements. In contrast to the anionically modified proteases, after 50 days of storage the native enzymes had virtually no washing action.

Claims

1. A method for specifically changing the properties of an enzyme by chemical modification, characterized in that the enzyme is reacted with a terminal epoxy or halohydrin compound, which specifically changes the biochemical and application properties, cationic, amphiphilic, anionic and / or liponhilic or contains a hydrophobic organic residue, converted into a water-soluble or non-water-insoluble, chemically modified enzyme (enzyme derivative).

2. A process for the preparation of water-soluble or non-water-insoluble, chemically modified enzymes (enzyme derivatives), characterized in that at least one of the free primary amino groups of an enzyme with a) epoxides of the formula I, which is not in the activity center, is reacted under basic conditions

wherein

wherein

X represents halogen, preferably chlorine or bromine, and

where R has the above meaning, are substituted.

3. The method according to claim 2, characterized in that aqueous enzyme concentrates with 10 to 35 wt .-% dry matter content are used.

4. The method according to any one of claims 2 to 3, characterized in that the reaction is carried out in the presence of enzyme stabilizers and / or solubilizers, preferably in the presence of propanediol.

5. The method according to any one of claims 2 to 4, characterized in that the reaction is carried out at pH values from 9 to 11.

6. The method according to any one of claims 2 to 5, characterized in that the reaction is carried out at temperatures up to a maximum of 35 ° C, preferably up to a maximum of 30 ° C.

7. The method according to any one of claims 2 to 6, characterized in that epoxides of the formula I or

Halohydrins of the formula II are used in which R is a cationic organic radical of the formula - (CH ₂ ) _n -N ⁺ R ¹ R ² R ³ , in which R ¹ , R ² and / or R ^{3 is} a lower alkyl radical having 1 are up to 2 carbon atoms, preferably methyl, and n is an integer from 1 to 3.

8. The method according to any one of claims 2 to 6, characterized in that epoxides of the formula I or

Halohydrins of the formula II are used in which R is an amphiphilic organic radical of the formula

C18 alkyl radical and n is an integer from 1 to 3.

9. The method according to any one of claims 2 to 6, characterized in that epoxides of the formula I or

Halohydrins of the formula II are used in which R is an anionic organic radical of the formula

- (CH ₂ ) _n -SO ₃ -, where n is an integer from 1 to 3.

10. The method according to any one of claims 2 to 6, characterized in that epoxides of the formula I or

Halohydrins of formula II are used in which

R represents an organic radical of the formula - (CH ₂ ) _n -CH ₃ , where n is an integer from 1 to 17.

11. The method according to any one of claims 2 to 10, characterized in that proteases, amylases, glucose isomerases or lipases are used as enzymes.

12. Water-soluble or non-water-insoluble, chemically modified enzymes (enzyme derivatives), characterized in that they are in at least one of the not Center of activity, free primary amino groups of the underlying enzyme with a residue of formula A.

where R is a cationic, amphiphilic, anionic or hydrophobic organic radical, are substituted.

13. Enzyme derivatives according to claim 12, characterized in that the underlying enzyme is a protease, amylase, glucose isomerase or lipase.

14. Enzyme derivatives according to claim 12, characterized in that the underlying enzyme is substituted with a radical of formula A, wherein R is a cationic organic radical of the formula - (CH ₂ ) _n -N ⁺ R ¹ R ² R ³ , wherein R ¹ , R ² and / or R ^{3 represent} a lower alkyl radical having 1 to 2 carbon atoms, preferably methyl, and n is an integer from 1 to 3.

15. Enzyme derivatives according to claim 12, characterized in that the underlying enzyme is substituted with a radical of the formula A, in which R is an amphiphilic organic radical of the formula - (CH ₂ ) _n -N ⁺ R ¹ R ² R ⁴ , in which R ⁴ and / or R ^{2 represent} a lower alkyl radical having 1 to 2 carbon atoms, preferably methyl, R ^{4 represents} a straight-chain C10 to C18 alkyl radical and n is an integer from 1 to 3.

16. Enzyme derivatives according to claim 12, characterized in that the underlying enzyme is substituted with a radical of the formula A, in which R represents an anionic organic radical of the formula - (CH ₂ ) _n -SO ₃ -, in which n is an integer from 1 to 3 means.

17. Enzyme derivatives according to claim 12, characterized in that the underlying enzyme is substituted with a radical of the formula A, where i R stands for an organic radical of the formula - (CH ₂ ) _n -CH ₃ , where n is an integer from 1 means to 17.

18. Water-soluble or non-water-insoluble, chemically modified proteases, characterized in that they contain at least one of the free primary amino groups of the underlying protease which is not in the activity center with a cationic organic radical of the formula B.

- B

wherein R ¹ , R ² and / or R ^{3 is} a lower alkyl radical having 1 to 2 carbon atoms, preferably a methyl radical, and n is an integer from 1 to 3, are substituted.

19. Water-soluble or non-water-insoluble, chemically modified proteases, characterized in that they contain an anionic organic radical of the formula CC on at least one of the free primary amino groups of the underlying protease which is not in the activity center

where n is an integer from 1 to 3, are substituted.

20. Water-soluble or non-water-insoluble, chemically modified α-amylase, characterized in that it contains an anionic organic radical of the formula CC on at least one of the free primary amino groups of the underlying α-amylase which is not in the activity center

where n is an integer from 1 to 3, are substituted.