CS240818B1 - Process for the production of optically active amino acids from racemic mixtures - Google Patents

Abstract

The invention relates to the production of optically active <f- -amino acids on a laboratory or preparative scale. The production is based on N-acyl derivatives of racemic mixtures of amino acids and their stereospecific hydrolysis is carried out using immobilized aminoacylase■»· The resolution of the racemic mixture is carried out using a catalyst consisting of aminoacylases immobilized on a carrier with excellent chemical and mechanical stability. In the binding immobilization reaction, a simple and effective one-step procedure was found that preserves the high catalytic activity of the immobilized enzyme. The immobilization of the aminoacylase is carried out on a microporous copolymer of 2-hydroxyethyl methacrylate with ethylene dimethacrylate, which has been surface-modified with epoxy groups. The immobilization reaction takes place in the presence of higher concentrations of salts, which are usually used for protein precipitation. In this way, racemic mixtures can be resolved and pure L- and D-amino acids can be obtained from raw materials of natural origin, as well as non-proteinogenic acids, as well as amino acids labeled with radioactive or stable isotopes.

Description

The invention relates to the production of optically active .beta.-amino acids on a laboratory or preparative scale. The production is based on the N-acyl derivatives of racemic mixtures of amino acids and their stereospecific hydrolysis by immobilized aminoacylase is carried out. In the binding immobilization reaction, a simple and effective one-step procedure was found which retains the high catalytic activity of the immobilized enzyme. The aminoacylase immobilization is carried out on a microporous copolymer of 2-hydroxyethyl methacrylate with ethylenedimethacrylate which has been surface modified with epoxy groups. The immobilization reaction takes place in the presence of higher concentrations of salts, which are usually used for protein precipitation. Thus, racemic mixtures can be resolved to obtain pure L- and D-amino acids from raw materials of natural origin as well as non-proteinogenic acids as well as amino acids labeled with radioactive or stable isotopes.

(51) Int ClA

OJ C 12 P 13/04

240 818

The invention relates to a process for the production of optically active amino acids from racemic mixtures by means of an aminoacylase enzyme immobilized on a macroporous copolymer of 2-hydroxyethyl methacrylate with ethylene dimethacrylate.

In the chemical synthesis of <* - amino acids, optically inactive racemates of the L- and D-isomers are obtained. The L-amino acid configuration represents the basic building blocks of natural proteins and thus important intermediates of metabolisms in all organisms. D-amino acids selins are important, for example, as part of some antibiotic molecules. Proteinogenic and non-proteinogenic optically active amino acids are increasingly of interest to the pharmaceutical, food, biochemical research and biotechnology industries.

L-amino acids are prepared in several ways. In addition to the production of biological material and stereospecific synthesis by some microorganisms, the chemical synthesis of racemic mixtures of amino acids and their subsequent distribution is economically very important. A number of physicochemical, chemical and biochemical methods can be used to resolve racemates. Greeneenstein and N.Winitz: Chemistry of the Aminoacids, J.Wiley &

Sons Inc.New York / London, 1961, VolI-III) .The importance of biochemical methods utilizing high stereospecificity of enzymes is steadily increasing, using enzymes in soluble or immobilized form. Enzymes (e.g., typypine, chymotrypsin, carboxypeptidase, pepsin, leucinaminopeptidase , L-amino acid oxydases) catalyze stereospecifically only the reaction of one enantiomer of suitable D, L-amino acid derivatives and leave the other unchanged.

Aminoacylase is characterized by high stereospecificity, broad substrate specificity and high catalysis yield, making it a very suitable enzyme for the resolution of racemates of N-acylated D, L-amino acids. However, since the aminoacylase in solution is relatively unstable and rapidly deactivates, stabilization of this enzyme by immobilization on a solid support is very advantageous. Binding of the aminoacylase to the support substantially increases its stability, allows easy separation of the active enzymatic enzyme

- 2 240 818 supported catalyst from reaction products and its reuse.

Aminoacylase can be immobilized by adsorption on ion exchangers in a cost-effective manner (T.Tosa, T.Mori, N.Fuse, I.Chibata, Enzymologie 31 (1966) 214; T.Tosa, T.Mori, N.Fuse, I.Chibata, Βιοί Bioeng., 9 (1967) 603; T. Tosa, T. Mori, I. Chibata, Agr.Biol. Chem., 33 (1969) 1053.). A disadvantage of the non-covalent binding of aminoacylase to ion exchangers is that the enzyme is released and eluted from the support when eluting buffers and substrate solutions. In addition, it is necessary to work in relatively dilute substrate solutions and the life of the catalysts is greatly reduced.

Covalent immobilization of enzymes on a solid support forms stable chemical bonds so that relatively concentrated substrate solutions can be worked. A number of special techniques are known for the covalent immobilization of aminoacylase or its incorporation into a polymer network (T.SATO, T.Mori, T.Tosa). ,

I.Chibata, Arch.Biochem.Biophys., 147 (1971) 788; I.Chibata, T. Tosa, T.Sato, T. Mori, Y.Matsuo, Proc.IV.IFS / Ferment.Technol.Today,

383 (1972) 383, M. Maskova, T.Barth, B. Jirovsky, I. Rychlik, Coli. Czechosl.Chem.Commun., 38 (1973) 943, H. N. Weetall, C. C. Detar, Biotechnol. 16 (1974) 1537, B.Szajani, K.Ivony, L. Boross, Acta Biochim et Biophys.Acad.Sci.Hung., 15 (196O) 295; I. Willhardt, P. Hermann, DPR Pat. Amm.DD WP 123 188 (1981).

The disadvantage of known and cited covalently bonded aminoacylase preparations is primarily that immobilization to biopolymers is used in part, whose lifetime is limited to e.g. microbial degradation. Another disadvantage is the laboriousness or high costs of the activation reaction (some activation processes are health-related). Harmful), which serve to allow the formation of a covalent bond between the carrier and the enzyme.

This applies, for example, to the activation of polysaccharide carriers with soluble carbodiimides or cyanogen bromide.

The present invention is directed to the preparation of optically active amino acids by resolution of racemates by immobilized aminoacylase using a highly economical process that can be performed on a laboratory scale in micromol or millimol volume. Mixtures of racemates of stable-3 240 818 labeled isotopes or racemic mixtures of radioactive or non-proteinogenic can be processed. In addition, the abovementioned drawbacks of the known methods used to immobilize aminoacylase can be overcome, particularly when using biopolymers as carriers.

According to the invention, a significant increase in mechanical and chemical stability, resistance to microbial hydrolysis is achieved and advantageous hydrodynamic properties of the immobilized enzyme are obtained. Another advantage is the elimination of some of the activation steps they represent, which constitute a technological complication.

It is an object of the present invention to provide an aminoacylase binding method for resolving racemic mixtures into a surface epoxidized, macroporous spherical copolymer of 2-s-hydroxymethacrylate with ethylene dimethacrylate with high yield of enzymatic activity, extending the life and stability of the bound enzyme.

Activation of the carrier to, for example, amino-containing compounds or activation by compounds such as soluble carbodiipid, cyanogen bromide or glutaraldehyde is usually omitted for covalent binding of the aminoacylase.

According to the invention, the aminoacylase immobilization proceeds in a simple process by dissolving the enzyme in a buffer solution (at 7, 0-8.5; 0 ', 05 - 0.2 mol / l), adding a carrier (epoxidation degree 0.7-2, 0 mmol / g; particle size 100-200 µm; molecular weight exclusion limit .200,000-5 »10) and allowed to react for 5 to 40 hours at 0-10 ° C with occasional shaking. immobilized aminoacylase is the presence of a higher salt concentration such as ammonium sulphate or ammonium phosphate (1.0 - 1.7 mol / l) in the reaction mixture is a necessary condition. The salt content in the buffer during the coupling reaction stabilizes the aminoacylase and results in a highly active immobilized enzyme preparation.This immobilized acylase is reacted with a N? -acyl-DyL amino acid in a known manner in a continuous column or batch process. Separation of the active immobilized active aminoacylase The reaction mixture is filtered or centrifuged followed by isolation of the L-amino acid by a known method. N-Acyl + D-amino acids which have not been transformed by aminoacylase can then be chemically hydrolyzed to yield D-amino acids. also

-acyl-D-amino acids are resuspended and resolved again.

Immobilization of aminoacylase on the surface of epoxy-modified copolymer of 2-hydroxyethyl methacrylate with ethylene dimethacrylate allows substantial time savings in the preparation of bound enzyme. One-stage coupling reaction is simple, harmless and very economical.Thanks to the high stability and durability achieved (e.g. starting activity, after 12 weeks 57 (5%, and after 5 months 33.25% of initial activity), the bound enzyme can be used to cleave the racemic mixture multiple times and over a longer period of time.

In the following, the invention is illustrated by way of example, but not by way of limitation.

Example 1.

Pork kidneys were used as aminoacylase sources, from which the enzyme was obtained by a known method (SMBirnbaum, L. Levintow, RPKingsley, JBGrienstein, J.Biol.Chem.194 (1952) 455). After lyophilization, the enzyme was stored as a dry powder. in the refrigerator without observing any change in its activity during storage.

The partially contaminated aminoacylase prepared by the above procedure was dissolved in 0.1 mol / l phosphate buffer pH 7.0-8.5, diammonium hydrogen phosphate was added in an amount less than the concentration at which the protein started to precipitate from solution, and a macroporescent copolymer of 2-hydroxyethyl methacrylate with ethylene dimethacrylate (exclusion limit 200,000-700,000; particle size 100-200 µm; epoxidation degree 1.5-2.0 mmol / g) was added and the mixture was maintained at 0-10 ° C with occasional shaking for 20 hours. After the reaction time, the support was filtered off and washed with 1.0 M NaCl, substrate solution and phosphate240818 buffer solutions to remove adsorbed proteins. The immobilized supported aminoacylase was used in a batch process to digest 0.01-0. 0.05 mol / l chloroacetyl-D, L-phenylalanine. PH 7.0-8.5, at 30-55 ° C.

After complete deacylation of the L-form, the support was aspirated and stored in an aqueous substrate solution with the addition of thymol. The reaction solution containing L-phenylalanine, chloroacetate and chloroacetyl-D-phenylalanine was fed to a strongly acid cation exchanger in H ⁺ form to separate the components. Chloroacetyl-D-phenylalanine and chloroacetic acid pass through the ion exchanger, while the ion-retained L-phenylalanine is desorbed in the next step with aqueous ammonia and is obtained by crystallization from the ammonia solution.

Example 2.

The aminoacylase immobilization was performed under conditions analogous to Example 1 except that the reaction was carried out in the presence of ammonium sulfate instead of phosphate and that the 2-hydroxyethyl methacrylate / ethylene dimethacrylate copolymer had an exclusion limit of 5,000,000, a particle size of 100-300 yum _t epoxy oxidation degree 0 The carrier, washed in the same manner as in Example 1, was packed into a tempered glass column. The column containing L-methionine, acetic acid and acetyl-Dmethionine was determined at the outlet at 100 ° C and processed further in a manner analogous to that described above. Example 1. Acetyl-D-methionine was racemized again after concentration by a known method and returned to the immobilized aminoacylase column. The desorbed L-methionine desorbed with aqueous ammonia from the cation exchanger was obtained after concentration. in crystalline form.

Claims (1)

Object of the invention 240 818 A method for producing optically active LX-amino acids and D-amino acids by resolution of racemic mixtures of N-acyl derivatives of the corresponding D, L-X-amino acids by enzymatic catalysis using immobilized aminoacylase, characterized in that covalently bound aminoacylase is used. a macroporescent copolymer of 2-hydroxyethyl methacrylate with ethylenedimethacrylate and which has been surface-activated by modification with epoxy groups, wherein the coupling of the aminoacylase to the support is carried out in the presence of salts selected from the alkali or ammonium salts of multivalent valions; solution.