AT401652B - METHOD FOR INCREASING THE ACTIVITY OF LIPASES - Google Patents

Description

AT 401 652 B

Lipases, which form enzymatic bonds with carboxylic acid derivatives or are able to dissolve them, are becoming increasingly important as biocatalysts for the synthesis of valuable chemical compounds, even in the industrial sector. Since the efficiency of an enzymatic process increases, among other things, with the activity of the enzyme used, there has been no lack of attempts to find processes by means of which the activity of a lipase can be increased.

From Enzyme Microb. Technol. Vol. 11 (1989), pp. 44-48, for example, it is known that the activity of a lipase when loosening or forming carboxylic acid ester bonds can be influenced by modification of amino acid side chains of the lipase by reaction with certain aldehydes, carboxylic acids, carboxylic anhydrides, sugars, imides, sulfonyl halides. The reaction of lipases with such reagents is a relatively complex process. In addition, there is always a loss of activity and no increases in carbon ester cleavage.

JP 87/43658 (Chemical Abstracts Vol. 111, 230697), JP 89/185636 (Chemical Abstracts Vol. 115, 227116) and JP 89/322909 (Chemical Abstracts Vol. 115, 202027) propose lipases in the presence of an activator , such as a surfactant, e.g. B. immobilize lecithin, sucrose fatty acid ester, dextrin, polyols, hydrophilic amino acids, peptides. The aim of this is to increase the activity of an immobilized lipase over an esterification or transesterification reaction compared to one which was not immobilized in the presence of one of the activators mentioned above. A disadvantage of this method is that the lipase has to be immobilized to increase the activity, it being known that large amounts of activity are lost through the occupation of the active center by the carrier material, so that the immobilized, activated lipase has less activity than the same amount of non-immobilized, non-activated Lipase.

In J. Chem. Soc. Perkin Trans 2 (1991), pp. 1851-1854 describes that a lipase can be made soluble in organic solvents by adding a detergent and then lyophilizing, while the activity of the lipase should be retained. In fact, however, in the case of lipases which have been treated in this way, there is a loss of activity during the enzymatic hydrolysis of a carboxylic acid derivative. In addition, the detergent present in the lyophilisate can sensitively disrupt or prevent the enzymatic reaction on many substrates.

Several methods are also known in which a lipase is brought into contact with alcohols, detergents or proteins and this mixture carries out the desired reaction, but the activators always remain in the reaction mixture.

DE-OS 35 45 056 relates to the transesterification between an oil or fat and another oil or fat or fatty acid in an organic solvent. In a first step, a mixture of oil or fat, a carrier material, water or a lower dihydroxy or trihydroxy alcohol and lipase is reacted in order to decompose the oil or fat. After filtering off the remaining oil or fat from the decomposition product, an activated lipase preparation is obtained, consisting of lipase and carrier. In a further step, this lipase preparation is mixed with water or a lower dihydroxy or trihydroxy alcohol. After 2 separate activation steps in the non-aqueous medium, the activator remains in the lipase. US Pat. No. 5,100,796 describes a detergent composition consisting of a specific, officially deposited Pseudomonas strain and 1-30% detergent. The enzyme activity is determined by washing various soiled tissues (standard test EMPA). GB-PS 1 574 269 relates to an enzyme preparation containing a lipase of non-animal origin and 5-50% of an animal protein as a stabilizer. Here too the protein remains in the enzyme preparation and is not removed from the lipase.

It has now been found, unexpectedly, that by treating a lipase with an activator selected from an alcohol and / or a detergent and / or a protein, an extraordinary increase in the activity of the lipase in loosening and / or forming bonds of a carboxylic acid derivative is achieved without the Modifying or immobilizing lipase, which is retained even if the activator in the enzymatic reaction mixture is greatly diluted or removed at all.

The invention therefore relates to a method for increasing the activity of a lipase when loosening and / or forming bonds of a carboxylic acid derivative, which is characterized in that a lipase in an aqueous diluent with an activator selected from an alcohol and / or a detergent and / or a protein is brought into contact in a suitable concentration, whereupon the activator is greatly diluted or removed at all and the lipase treated in this way is used in an enzymatic reaction to loosen and / or form bonds of carboxylic acid derivatives.

A lipase that occurs in animals, plants and fungi is an enzyme that is capable of breaking bonds of carboxylic acid derivatives, primarily carboxylic acid ester bonds, by hydrolysis or by 2 ΑΤ 401 652 Β

To loosen and / or tie esterification or transesterification. Preferred lipases which can be activated according to the invention are lipases from fungi, for example the genus Candida, Pseudomonas, Geotrichum, Rhizomucor or from plants, for example from wheat germ. Lipases from fungi are particularly preferably activated.

To carry out the process according to the invention, the lipase is dissolved or at least dissolved in an aqueous diluent and mixed with a water-soluble activator, an activation mixture being formed in which, according to the invention, a high activator concentration is essential.

Possible aqueous diluents are water, an aqueous salt solution, preferably an aqueous buffer solution. The aqueous diluent may optionally contain conventional organic auxiliary solvents which serve to dissolve or dissolve the substrate which is to be reacted with the aid of the lipase. Organic auxiliary solvents are generally used in the reaction solution in concentrations of at most 10% by weight.

An alcohol, a detergent or a protein or mixtures of such compounds are suitable as activators.

Alcohol is to be understood as meaning aliphatic or aromatic alcohols. Aromatic alcohols are preferably those which contain a phenyl group, such as benzyl, phenylethyl alcohol. Aliphatic alcohols are those with 3 to 22 carbon atoms, such as propanol, butanol, hexanol, dodecanol, eicosanol or their isomers such as iso-propanol, iso-pentanol, 2-ethyl-hexanol. Aliphatic alcohols are particularly preferred, very preferably those having 3 to 16 and very preferably those having 3 to 8 carbon atoms. Preferred alcohols are monohydric alcohols and those that are water soluble.

A detergent is a surface-active substance. Detergents and their production are generally known. Detergent is preferably to be understood as an ionic detergent, particularly preferably one with an acidic character or its salts. Alkyl or aryl sulfonic acids or their salts, for example sodium dodecyl sulfate, are very particularly preferred.

A protein is a macropeptide with more than 100 amino acids, which is soluble in water or in aqueous systems, preferably a globular protein, such as a plasma, milk, egg or seed protein, for example albumins, globulins. However, an enzyme with the spatial structure of a protease, for example trypsin, may also be considered as a protein, it being shown that the activity-increasing effect of the enzyme on the lipase is not due to the enzymatic mode of action of the enzyme used.

A protein with the spatial structure of a protease is preferably used as the protein.

The concentration of the lipase in the activation mixture depends essentially on its solubility in the aqueous diluent and is generally not critical. Concentrations of 0.01 to 10% by weight were used in experiments.

The activator is either dissolved in an aqueous diluent as indicated above or added as such to the activation mixture. By high activator concentration in the activation mixture it is meant that the concentration of the activator in the activation mixture must be significantly higher than is the case in the enzymatic reaction solution in which the activated lipase is used. The suitable concentration of the activator in the activation mixture can easily be determined by simple preliminary tests with the aid of a series of dilutions and / or by exposure times of the activator of different lengths and respective activity measurement of the treated lipase. It has been shown that for each activator a certain concentration or a certain action time of the activator on the lipase must not be undercut, since otherwise no activation of the lipase occurs. Too high a concentration or too long exposure time of the activator can lead to complete deactivation of the lipase.

In the case of butanol or iso-propanol as an activator, it has been shown that a concentration of the alcohol of about 15 to 65% by weight in the activation mixture gives excellent results with regard to the increase in activity of the lipase.

Sodium dodecyl sulfate as a detergent is optimally used in concentrations of approximately 0.02 to 0.2% by weight in the activation mixture.

With trypsin as protein, it was found in preliminary tests that a concentration of 0.1 to 1.0% by weight in the activation mixture is advantageous.

The activation mixture, which contains the lipase and the activator in aqueous dilution, is briefly shaken or stirred, preferably at room temperature, that is to say from about 0.5 minutes to about 1 hour, preferably from 1 to 30 minutes. If the activator is used in the appropriate concentration, the activity of the lipase unexpectedly increases.

It has been found that when a very strongly diluted activator solution is used as the enzymatic reaction solution or after the activator has been removed from the activation mixture by 3

AT 401 652 B, for example dialysis, ultrafiltration, lyophilization, the activity of the activated lipase remains just as high as in an activation mixture in which the activator is present in the high concentration required for activation. This apparent stability of the activation, which remains even when the activator is no longer present in the enzymatic reaction mixture, or only in a very dilute form, is completely unexpected, especially because a low activator concentration is not sufficient to activate the lipase . As already described, a certain activator concentration must not be fallen below, since otherwise there is no activation of the lipase. A possible explanation of this phenomenon could be that, upon activation, a change in the conformation of peptide chains in the lipase, which manifests itself in changed fluorescence properties of the lipase, occurs, which gives the substrate better access to the active center during the enzymatic reaction of the lipase .

The lipase activated according to the invention, which is also a subject of the invention, can be used as a biocatalyst with increased activity compared to a non-activated lipase in customary enzymatic reactions which can be catalyzed with the aid of a lipase, thereby saving lipase or increasing the speed of the enzymatic reaction . The process is therefore an enrichment of technology.

example 1

A 3% strength by weight aqueous solution was prepared from a Candida rugosa lipase OF from Meito by shaking for 2 minutes in 0.1 M sodium phosphate buffer, pH 5 and centrifuging undissolved substances. 500 ul of this solution was shaken with 400 ul isopropanol for 1 minute at room temperature. In the same way, the 3% by weight solution of the lipase was treated with water instead of with isopropanol for comparison purposes. A 1:50 dilution of each of these mixtures was prepared using sodium phosphate buffer. The activity of the lipases in these dilutions in the hydrolysis of p-nitrophenyl butyrate was measured and compared. For this purpose, 23 ul of p-nitrophenyl butyrate were shaken with 5 ml of a 1% strength by weight polyvinyl alcohol solution, centrifuged off and the supernatant used. 200 µl of the supernatant was mixed with 1 ml of 0.1 M phosphate buffer (pH 7). For this, 25 μl of the 1:50 activated and the non-activated lipase were added. The reaction mixtures were shaken and the change in absorption was monitored at 405 nm.

The activity of the activated lipase was 300% higher than that of the non-activated lipase.

Example 2 was carried out in the manner described in Example 1, using instead of Candida rugosa lipase a) a Pseudomonas fluorescens lipase b) a Geotrichum candidum lipase. The increase in activity of the activated lipase, which was measured according to Example 1, compared to an inactivated lipase was a) 500% b) 200%.

Example 3 was carried out as in Example 1, using 1-butanol instead of isopropanol. The activity of the activated lipase increased by 260% compared to an unactivated one.

Example 4

A 3% by weight solution of the Candida rugosa OF lipase, prepared as described in Example 1, was a) mixed with a 0.125% by weight aqueous sodium dodecyl sulfate solution and b) for comparison with water in a volume ratio of 1: 1 and shaken for 1 minute. The lipase solutions according to a) and b) were each diluted to 1:50 with phosphate buffer. When determining the activity according to Example 1, the activity of the activated lipase was increased by about 350% compared to the unactivated one. 4th

Claims (8)

AT 401 652 B Example 5 A 0.025% strength by weight solution of a Candida rugosa OF lipase, prepared by the method described in Example 1, was mixed with a 0.5% strength by weight aqueous solution of trypsin from bovine pancreas (Boehringer-Mannheim) in a ratio of 1 : Shaken for 15 minutes. 25 ul of this mixture was diluted 1:20 with phosphate buffer. The same procedure was followed with an aqueous solution of the trypsin, the enzymatic activity of which was inhibited by treatment with N-tosyl-L-lysine-chloromethyl ketone (TLCK). The activity measurement, which was carried out according to Example 1, showed an increase in activity of about 250% for the lipases which had been activated with trpysin and those which had been inhibited with trypsin, compared to an unactivated lipase. Example 6 Candida rugosa lipase was activated with trypsin according to Example 5, the concentration of the aqueous trypsin solution used for the activation being varied. In the activity measurement according to Example 1, a) with a trypsin concentration of 0.025% by weight after activation for 25 minutes, b) with a trypsin concentration of 0.5% by weight after activation for 5 minutes, c) with a trypsin concentration of 2% by weight after 2 minutes an activity increase of 250% compared to a non-activated lipase was found. Example 7 Candida rugasa lipase was activated in the manner described in Example 1. The activator concentration was reduced to less than 0.001% by weight in both cases by dialysis or ultrafiltration, and the activities of these lipase solutions according to Example 1 were measured. An increase in activity of the activated lipase compared to an unactivated lipase was measured by 300%. The activity of the lipase was therefore not reduced by removing the activator. 1. A method for increasing the activity of a lipase when loosening and / or forming bonds of a carboxylic acid derivative, characterized in that a lipase in an aqueous diluent with an activator selected from an alcohol and / or a detergent and / or a protein, in a suitable manner Concentration is brought into contact, whereupon the activator is greatly diluted or removed at all and the lipase treated in this way is used in an enzymatic reaction to loosen and / or form bonds of carboxylic acid derivatives. 2. The method according to claim 1, characterized in that a lipase from plants or from fungi is activated as lipase. 3. The method according to any one of claims 1 or 2, characterized in that a lipase from fungi is activated as lipase. 4. The method according to any one of claims 1 to 3, characterized in that a monohydric alcohol is used as an activator. 5. The method according to claim 4, characterized in that an aliphatic alcohol having 3 to 22 carbon atoms is used as activator. 6. The method according to any one of claims 1 to 3, characterized in that an ionic detergent with an acidic character or its salt is used as an activator. 7. The method according to any one of claims 1 to 3, characterized in that a globular protein is used as an activator. 5 AT 401 652 B 8. The method according to any one of claims 1 to 3, characterized in that an enzyme with the spatial structure of a protease is used as an activator. 6