CS263016B1 - The granular biocatalyst based on the immobilized enzyme hydrolase 7 - (4-carboxybutanamido), cephalosporanic acid for the enzymatic production of 7-aminooephalosporanic acid and its preparation - Google Patents

Abstract

The solution relates to a granular biocatalyst based on the immobilized enzyme hydrolase 7/3-(4-carboxybutanamido) cephalosporanic acid for the enzymatic production of 7-amino-cephalosporanic acid according to Czechoslovak patent certificate No. 231 656 and a method of its production. The biocatalyst can be prepared on the basis of cross-linked hydrolase of 7-[beta]-(4-carboxy butanamido) cephalosporanic acid by subsequent treatment with an aqueous solution of glutaraldehyde and/or polyethyleneimine or proteins of microbial, plant or animal origin and an aqueous solution of glutaraldehyde, separation and mechanical homogenization of the resulting product and its possible partial drying, or its subsequent suspension in cooled organic solvents that are completely miscible with water and/or their mixtures with water, repeated separation and possibly further partial drying and finally its washing with water or buffer.

Description

Granular biocatalyst based on immobilized enzyme hydrolase 7- (4-carboxybutanamido), cephalosporic acid for enzymatic production of 7-aminooephalosporanic acid and process for its preparation

The present invention relates to a granular biocatalyst based on immobilized 7/3 - (4-carboxybutanamido) oephalosporanic acid hydrolase enzyme for the enzymatic production of 7-aminocephalosporanoic acid according to U.S. Pat. No. 231 656 and method of its production. The biocatalyst can be prepared on the basis of cross-linked 7β- (4-carboxybutanamido) cephalosporic acid hydrolase by subsequent treatment with an aqueous solution of glutaraldehyde and / or polyethyleneimine or proteins of microbial, vegetable or animal origin and an aqueous solution of glutaraldehyde, separation and mechanical homogenization of the product, optionally by subsequently suspending in cooled organic solvents unrestrictedly miscible with water and / or a mixture thereof with water, re-separating and optionally further drying, and finally washing it with water or buffer.

263 016. The present invention relates to a granular biocatalyst based on immobilized 7β 3- (4-carboxybutanamido) cephalosporanoic acid hydrolase enzyme for the enzymatic production of 7-aminocephalosporanoic acid and a process for its preparation according to U.S. Pat. Certificate No. 231 656.

7 / 3- (4-carboxibutanamido) cephalosporic acid (glutaryl-7-ACK) is an intermediate of the two-step preparation of 7-aminocephalosporanoic acid (7-ACK) from cephalosporin C. 7-ACK serves as a pharmaceutically important intermediate for the production of semi-synthetic cephalosporins such as cephalotin and cefazoline.

A number of methods of immobilizing cells or enzymes for industrial biotransformations using a variety of carriers and methods of immobilization are described in the patent literature. There are also immobilization methods that do not require the use of a carrier at all, such as universal procedures using chemically reactive water-soluble polymers to immobilize various prokaryotic and eukaryotic cells with different enzyme activity (cf.

231 458). Using chemically reactive water-soluble polymers, a simple and versatile process for the production of immobilized enzymes such as hydrolase, isomerase, racemase, lyase and oxydoreductase according to U.S. Pat. By the above process, water-insoluble, water-insoluble enzymes of various purity and quality can be prepared using reactive soluble polymers (RRP), preferably obtainable by reacting commercially available flocculants containing a polyethyleneimine fraction with a bifunctional crosslinking agent such as glutaraldehyde and soluble enzyme preparations of different purity and quality. enzyme, in the form of biocatalyst particles with good sedimentation properties, high filterability and specific activity of the desired enzyme.

For the enzymatic hydrolysis of glutaryl-7-ACK to 7-ACK, in addition to the native cells with the activity of--β- (4-carboxybutanami-2263 016 do) cephalosporic acid hydrolase (hereinafter glutaryl-7-ACK hydrolase), the use of biocatalysts for a base of immobilized cells with this activity, such as cells of the genus Comamonas or Pseudomonas incorporated in cellulose acetate capsules (Japanese Patent Publication No. 76 70 884), or Pseudomonas sp. immobilized with reactive soluble polymers (Author's Certificate No. 251 111 dependent on Author's Certificate No. 231 458).

The use of biocatalysts based on immobilized technical or isolated enzymes of varying degrees of purity is, in principle, more advantageous for the conversion of glutaryl-7-ACK, since the production strain cells may contain other enzymatic activities undesirable for 7-ACK preparation. , such as esterases, catalysing the formation of deacetylated derivatives of glutaryl-7-ACK and 7-ACK or 3-lactamase.

Several methods of using glutaryl-7-ACK hydrolase isolated from a strain of the genus Comamonas sp. and Pseudomonas, especially Pseudomonas oval, immobilized with glutaraldehyde to various carriers such as Amberlit Ira-940 basic ion exchanger, or acrylonitrile methacrylate copolymer (Japanese Patent Nos. 78 59 095, 81 85 298 and 78 139 791). All of these methods, however, require immobilization to particles of water-insoluble carriers or methods of immobilization to the polymer-forming network, whereby the cost of producing immobilized enzyme preparations is known to be significantly influenced by the cost and availability of the carriers used. Another disadvantage of these processes is that the specific activity of the obtained immobilized materials based on this immobilized enzyme is very low and does not allow a realistic assumption of industrial realization.

The process of the present invention eliminates these disadvantages since it requires no organic, inorganic or natural water-insoluble carriers.

Crosslinking of glutaryl-7-ACK hydrolase enzyme molecules to form a covalently cross-linked network using conventional cross-linking agents (glutaraldehyde, toluene diisocyanate, etc.) is not possible as this enzyme is highly sensitive to the relatively aggressive effects of these agents and occurs at very low concentrations to irreversibly inactivate this enzyme. Therefore, in order not to inactivate this enzyme during immobilization, it is necessary to work with reactive water-soluble polymers formed, for example, by reacting cationic flocculants containing the polyethyleneimine fraction with glutaraldehyde separately, after which the resulting particles can be cross-linked

3263 016 and solidify by pulse addition of low concentrations of free glutaraldehyde, or add a water-soluble substance that reacts with glutaraldehyde, such as polyethyleneimine or a protein solution, and crosslink the resulting mixture of volumetric particles together with the reacting solutes. In the present process, the decrease in glutaryl-7-ACK hydrolase activity is substantially lower, the particles are more solid and exhibit high enzyme activity stability.

Accordingly, the present invention provides a granular biocatalyst based on the immobilized enzyme hydrolase 7 (i- (4-carboxybutanamido) cephalosporanoic acid hydrolase enzyme for the enzymatic production of 7-amicephalosporanoic acid according to U.S. Patent 231,656) obtainable from the biocatalyst particles based on crosslinked hydrolase 7 {3- (4). -carboxybutanamido) cephalosporanoic acid by subsequent treatment with an aqueous solution of glutaraldehyde and / or polyethylenimine or proteins of microbial, vegetable or animal origin and an aqueous solution of glutaraldehyde, separation and mechanical homogenization of the resulting product and its partial drying or subsequent suspension in non-cooled organic solvents water and / or mixtures thereof with water, re-separation and partial drying, and finally washing it with water or buffer.

According to the invention, the biocatalyst is produced by adding an aqueous solution of glutaraldehyde in the concentration range of 0.01 to 1 to a continuously stirred reaction mixture of the particles after adjusting the pH in the range 6.0 to 8.5, preferably 7.5. 0% v / v, or an aqueous solution of polyethhylenimine or an aqueous solution of proteins, followed by an aqueous solution of glutaraldehyde, re-adjust the pH of the reaction mixture within the stated values and stir continuously for 10 minutes to 5 hours, or leave to stand, optionally allowed to freeze, whereupon the particles are separated, mechanically homogenized and partially dried, or treated with cooled acetone or ethanol and / or mixtures thereof with water, with separation, re-separated and partially dried for 1 to 24 hours at 10 to 40 ° c, then the biocatalyst is suspended in water or buffer, decanted several times and washed, allowed to swell and separate.

The biocatalyst particles obtained according to the present process have a high sedimentation rate, good filterability and high glutary-1-7-ACK hydrolase activity. These properties, and in particular the complete absence of undesirable enzyme activities, allow a realistic assumption of its use in the production of 7-ACK with the following advantages:

a) higher degree of theoretical conversion of substrate to product,

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b) higher yield of 7-ACK in isolation,

c) higher quality 7-ACK,

d) absence of degradation products in isolated 7-ACK.

Glutaryl-7-ACK hydrolase activity was determined by measuring the rate of hydrolysis of glutaryl-7-ACK to 7-ACK in 0.1 M phosphate buffer pH 7.0 at 37 ° C in the region of zero order enzyme kinetics. 7-ACK was determined spectrophotometrically using p-dimethylaminobenzaldehyde, which reacted with 7-ACK to form a colored Schiff base according to U.S. Pat. No. 111,959, modified by Balasingham et al. (Biochim. Biophys. Acta, 276, 250 (1972)).

One unit of enzyme activity (U) is the amount of enzyme that corresponds to the formation of one µmole of 7-ACK per minute under the above reaction conditions. The specific activity of the starting and immobilized material is expressed as U. g ” ¹ proteins respectively. dry matter.

The 7-ACK was further monitored in the reaction mixture using a thin layer of cellulose (Polygram cell-300) in a n-pro panol-water (7: 3) system with ninhydrin detection (R ^ 0.5) or high pressure liquid chromatography (HPLC). ).

The invention is further illustrated in the examples without limiting them.

Examples

Example 1

One liter of non-specific esterase glutaryl-7-ACK technical hydrolase solution containing 3 g of protein. liter and a total of 800 U. liter was added slowly and in portions to the continuously stirred reaction mixture of the reactive soluble polymer (RRP) formed by the reaction of the cationic flocculants containing the polyethyleneimine and glutaraldehyde fraction according to U.S. Pat. Certificate No. 231 656.

After all of the enzyme solution had been incorporated, the pH of the reaction mixture was adjusted to 7.5 with 20% aqueous sodium hydroxide while stirring continuously. A 25% aqueous solution of glutaraldehyde was added gradually and slowly to the resulting suspension of the voluminous particles so that its final concentration in the reaction mixture was 0.5% (v / v). Thereafter, the pH of the reaction mixture was adjusted to 7.5 with the solution and method, and the mixture was stirred for one hour.

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Then the particles were separated by filtration, and the wet voluminous cake was introduced into a food processor into a grated bread roll, the drum of which was provided with cutting holes of 0.5 to 1 mm. The homogenized granular material was suspended in ethanol: water (1: 2) at -5 ° C, stirred briefly with a high-speed propeller stirrer, immediately and sharply vacuum-dried, and dried in a vacuum oven at 30 ° C for 2 hours. hours. Subsequently, the material was suspended in drinking water, decanted several times with water, the last decanting being carried out with distilled water. The washed particles were separated by vacuum filtration.

29.5 g wet mass of dry matter biocatalyst was obtained

22.5% and a specific activity of glutaryl-7-ACK hydrolase 60.2 U. g ” ¹ .

g glutaryl-7-ACK (95.4% purity) was dissolved by adjusting the pH with 1 N NaOH to pH 7.0 in 90 mL of 0.05 M phosphate buffer pH 7.0 and adding 10 g wet mass of the obtained biocatalyst and the mixture was stirred at 35 ° C while continuously adjusting the pH with an alkaline solution to 7.0 for 60 minutes. 90% of the theoretical conversion of the substrate to 7-ACK was achieved. The described procedure was repeated a total of 5 times with the same batch of particles of the same biocatalyst and the average conversion rate of the substrate to 7-ACK / 5 cycles of reuse was 89.3%.

Example 2

One liter of the technical enzyme solution was mixed with 0.8 liters of RRP in the same manner as in Example 1. The technical enzyme solution showed the same activity and protein concentration. 10 ml of a 10% aqueous polyethyleneimine solution having a molecular weight of 30,000 to 40,000 were added while stirring continuously, the pH was adjusted to 7.5 with a 20% aqueous NaOH solution, and an aqueous solution of glutaraldehyde was added so that the final concentration in the reaction mixture was 0.5% and the pH was again adjusted as described. The next procedure for the preparation of the biocatalyst was the same as in Example 1.

32.2 g of wet mass of the biocatalyst with a dry matter content of 23.8% and a specific activity of glutaryl-7-ACK hydrolase of 53.3 U were obtained. g ¹ .

Further, the enzyme conversion of the substrate to the product was carried out under the conditions and method set forth in Example 1 except that distilled water was used instead of the phosphate buffer. 87.3% of the theoretical conversion of glutaryl-7-ACK was achieved, with an average conversion rate of 5 repetitive cycles using the same biocatalyst feed of 86.3%.

-6Example 3

263 016

One liter of technical enzyme solution of the same quality and in the same manner as in Example 1 was mixed with RRP. In addition, an aqueous protein solution (technical egg protein) was added to the reaction mixture to give a total protein concentration of 50 g. After incorporation of the protein solution, the pH was adjusted as described during continuous mixing, and a 25% aqueous glutaraldehyde solution was added to the resulting suspension of particles to bring the actual concentration in the reaction mixture to 1% (v / v). Furthermore, the procedure for preparing the biocatalyst was the same as in Example 1.

70.3 g of wet dry matter biocatalyst was obtained

24.3% and a specific activity of glutaryl-7-ACK hydrolase of 29.8 U. G

Example 4

A biocatalyst was prepared in the same manner as in Example 2 except that a glutaryl-7-ACK technical hydrolase solution without non-specific esterase activity obtained by concentration on a rotary evaporator at 38 ° C containing 30 g was used as the starting enzyme material. protein. liter ^-1 and a total amount of this enzyme of 7 100 U. liter ¹ . It was obtained

56.4 g of wet biocatalyst with a dry matter content of 24% and a specific activity of glutaryl-7-ACK hydrolase 314.7 U. G

Example 5

One liter of the technical enzyme solution, which was concentrated as described in Example 4, was mixed with RRP as in Example 1; The pH of the reaction mixture was adjusted to 7.8 during continuous stirring as described, the mixture was thoroughly homogenized and then frozen in a thin layer at -20 ° C for 5 hours. After the mixture was thawed, the particles were suspended in a 0.1% aqueous glutaraldehyde solution and after stirring at room temperature for 30 minutes, the suspension was diluted with distilled water and the particles separated by vacuum filtration. The wet voluminous cake was mechanically homogenized and dried at 30 ° C for 3 hours under vacuum. Then, the particles were suspended in distilled water, decanted, washed and separated as in Example 1.

55.3 g of wet mass of dry matter biocatalyst were obtained

24.6% and a specific activity of glutaryl-7-ACK hydrolase 307 U. g ¹ .

-ΊExample 6 _{263 β1θ}

One liter of an aqueous hydrolase solution, which was concentrated as described in Example 4, containing the protein concentration and activity also shown in Example 4, was mixed with the RRP as described in Example 1, including the pH adjustment method.

A 25% aqueous solution of glutaraldehyde was gradually added to the resulting suspension of the voluminous particles to give a final concentration in the reaction mixture of 0.25% (v / v). The immobilization was carried out as described in Example 5.

55.5 g of wet mass of biocatalyst with a dry matter content of 24.6% and a specific activity of glutaryl-7-ACK 312 U hydrolase were obtained. g ^- l.

Example 7

950 ml of a technical enzyme solution of the same quality and in the same manner were mixed with RRP as in Example 1. The immobilization was carried out in the same manner as in Example 1, except that the pH of the reaction mixture was adjusted to 7.0, the actual concentration of the added solution. glutaraldehyde was 0.05% (v / v). Further, immobilization was continued by freezing the reaction mixture as described in Example 5, including decanting, washing and separating.

26.4 g of wet mass of the biocatalyst with a dry matter content of 22.8% and a specific activity of glutaryl-7-ACK 70 U hydrolase were obtained. G

Claims (2)

A granular biocatalyst based on immobilized 7β- (4-carboxybutanamido) cephalosporanoic acid hydrolase enzyme for the enzymatic production of 7-aminocephalosporanoic acid according to c. No. 231,656, obtainable from the particles of a biocatalyst based on cross-linked 7 / 3- (4-carbopoxybutanamido) cephalosporic acid hydrolase by subsequent treatment with an aqueous solution of glutaraldehyde and / or polyethyleneimine or proteins of microbial, vegetable or animal origin and an aqueous solution of glutaraldehyde, separation and mechanical treatment. by homogenizing the resulting product and partially drying it, optionally by subsequently suspending it in cooled organic solvents unrestrictedly miscible with water and / or mixtures thereof with water, re-separating and optionally further partial drying and finally washing it with water or buffer. 2. A process according to claim 1, characterized in that an aqueous solution of glutaraldehyde is added to the continuously stirred particulate reaction mixture after adjusting the pH in the range of 6.0 to 8.5, preferably 7.5, in a concentration range of 0.01. up to 1.0% v / v, or an aqueous polyethyleneimine solution or an aqueous protein solution, followed by an aqueous glutaraldehyde solution, re-adjusting the pH of the reaction mixture within the range indicated and stirring continuously for 10 minutes to 5 hours, or The mixture is left to stand, optionally allowed to freeze, whereupon the particles are separated, mechanically homogenized and optionally dried, or treated with cooling acetone or ethanol and / or mixtures thereof with water, separated again and optionally partially dried for one to 24 hours. at 10 to 40 ° C, then the biocatalyst is suspended in water or buffer, decanted several times and washed, swollen and separated .