CS251111B3 - Method of strengthening and stabilizing biocatalyst particles for the enzymatic conversion of 7 - [(4-carboxybutanamido) -cephalosporanic acid to 7-aminocephalosporanic acid - Google Patents

Abstract

The solution concerns the consolidation and stabilization of biocatalyst particles for the enzymatic conversion of 7-/9-(4-carboxybutanamido)-cephalosporanic acid to 7-aminocephalosporanic acid, prepared according to Czechoslovak patent certificate No. 231458, in which aqueous solutions of 7-/9-(4-carboxybutanamido)-cephalosporanic acid are brought into contact with biocatalyst particles based on cells of microorganisms containing the enzyme 7-2-(4-carboxybutanamido)-cephalosporanic acid hydrolase, e.g. Pseudomonas sp. CCM 3635. The method of stabilizing the particles consists in adding, after adjusting the pH to 6.5 to 8.5, an aqueous solution of glutaraldehyde and/or an aqueous solution of technical or pure protein of animal, plant or microbial origin to the stirred mixture of biocatalyst particles with the specified enzyme activity, after which the reaction mixture is optionally finished with another addition of glutaraldehyde and allowed to freeze and/or the resulting particles are separated and dried. After grinding and swelling, the washed particles are used for the preparation of 7-aminocephalosporanic acid.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for consolidating biocatalyst particles prepared according to Basic Authentication No. 231458 by immobilized bacterial cells and stabilizing the enzyme 7-β- (4-carboxybutanamido) -cephalosporanoic acid hydrolase in these particles. Of 7-N- (4-carboxybutanamido) -cephalosporanoic acid, to form 7-aminocephalosporanoic acid and its derivatives.

Enzyme conversion products formed by the single, multiple, repeated or continuous contact of these solidified and stabilized particles with aqueous solutions of 7-aminocephene compounds, in particular solutions of 7-β- (4-carboxybutanamido) -cephalosporanoic acid to form 7-aminocephalosporic acid -ACK) can serve as sources for the production of semi-synthetic cephalosporins,

7- [4- (4-carboxybutanamido) -cephalosporanoic acid or its derivatives, can be obtained from cephalosporins C or the corresponding derivatives either chemically (Japanese Patent Nos. 77 77,078 and 78 114,891), or enzymatically, by D-amino acid oxidase, or activated cells of microbial producers of this enzyme, such as Trigonopsis variabilis and representatives of the genera Fusarium, Aspergillus, Candida and others (eg, British Patent Specification No. 1,272,769, Japanese Patent Specifications Nos. 77,444,695, 76,888,694, 77,125,696, and 80 23,966) .

A number of methods for the immobilization of cells with penicillin amidase activity, or penicillin amidase isolated from producer cells are described in the patent literature. These catalysts are useful for the enzymatic conversion of β-lactam antibiotics, particularly for the enzymatic production of 6-aminopenicillanic acid from benzylpenicillin. These are in particular the methods of immobilization described in Cs. 197101, 200802, 201621, 203607,

208931 and 209265. A method for consolidating and stabilizing biocatalyst particles for enzymatic conversion ₍ β-lactam antibiotics is described in the description of the invention for the Czech author's certificate No. 241730. Methods for using these biocatalysts are described in the descriptions of the inventions for a Czech author's certificate no. 211175 and 213099 for the production of 6-aminopenicillanic acid from benzylpenicillin and 7-aminodeacetoxycephalosporanoic acid from N-phenylacetamido deacetoxycephalosporin (cf. No. 218023).

It is known to prepare the 7-aminocephem of the compounds of formula I

HjN-, CH ₂ X ^(I) -

COOH in which X represents a hydrogen atom, -OH, COOH or a nucleogile group, from their 7-acylamino derivatives of the formula II

COOH in which R denotes, for example, the group - (CH ₂ ) 4 - COOll o X denotes the same as in formula I, hydrolase isolated from the Comamonas strain and immobilized on a porous copolymer or ion exchange resin can be used (Japanese Patent Nos. 78 59,095 and 81 85,298 ), or Pseudomonas cells ovalize ATCC 950 immobilized in polyacrylnylone (Japanese Patent Specification No. 78,940.93).

For the preparation of the 7-aminoceph of the compounds of formula I, intact or disintegrated cells of the Pseudomonas sp. CCM 3635, which was obtained according to U.S. Pat. However, native or disintegrated cells containing this specific enzyme activity can be used only once for the respective reaction.

In MS. No. 231458 discloses a largely universal method of immobilizing whole native cells, individually cross-linked or permeabilized cells, and disintegrated suspensions containing various enzymes, in particular hydrolase, lyase, oxidase, racemase, decarboxylase and other enzymes, or part or all of the sequence enzymatically active metabolic pathways, in cells of microorganisms (bacteria, yeast actinomycetes and other imperfect cells), as well as in cells of higher organisms, in particular higher fungal cells and plant cells.

By cited above, based on the mutual chemical immobilization of different cells carrying different enzymes by means of reactive water-soluble polymers (hereinafter RRP), biocatalyst particles can be formed which have high specific enzyme activity and good sedimentation properties but are soft and must be solidify, either by using adhesives dissolved in organic solvents such as acetone or postformation of the volumetric particles into granules or rollers and then drying them.

The first method of solidification results in the formation of a diffusion barrier for the substrate and products into and from the particle by reducing their porosity (partial sealing of the pores of the particles), the second method leads to the formation of large particles, of the order of 1 to 10 mm. resulting smaller particles (particle size dependence on specific surface).

Enzymes that are not extremely sensitive to the otherwise relatively aggressive effects of bifunctional crosslinking agents such as glutaraldehyde include the enzyme 7-γ- (4-carboxybutanamido) -cephalosporanic acid hydrolase. However, the use of individually cross-linked cells requires the return of material to the reactor by centrifuges, thereby leading to considerable handling losses of the catalyst upon reuse and dictating only the use of a batch reactor.

It has now been found that, depending on the method of production of immobilized cells described in U.S. Pat. No. 231458, it is possible, in combination with a mechanical method, to solidify and stabilize biocatalyst particles with 7- (4-carboxybutanamido) -cephalosporanoic acid hydrolase, the material obtained being useful for the enzymatic conversion of the 7-aminoceph of compounds, in particular 7-ύ- (4-carboxybutanamido) -cephalosporanoic acid to 7-aminocephalosporanoic acid on an industrial scale. The solidification and stabilization technology of the particles of the present invention is simple, does not require special and expensive raw materials, does not require any water-insoluble carriers, and special technological and machinery equipment.

The above drawbacks have been overcome by the development of a method of consolidating and stabilizing the biocatalyst particles for the enzymatic conversion of 7- [4- (4-carboxybutanamido) -cephalosporanoic acid to 7-aminocephalosporanoic acid, prepared according to U.S. Pat. No. 231458, in which aqueous solutions of 7-aminocephene compounds, in particular 7-β- (4-carboxybutanamido) -cephalosporanoic acid, are contacted with biocatalyst particles, which consists in introducing into a continuously stirred mixture of biocatalyst particles based on intact or cross-linked cells of microorganisms containing the enzyme 7-β- (4-carboxybutanamido) -cephalosporanoic acid hydrolase, preferably Pseudomonas sp. CCM 3635, an aqueous solution of glutaraldehyde or an aqueous solution of a technical or pure protein of animal, vegetable or microbial origin, such as a technical egg protein or a disintegrated suspension of cells of microorganisms, preferably cells containing the enzyme, is added after pH adjustment in the range of 6.5 to 8.5. 7-, 4- (4-carboxybutanamido) -cephalosporanoic acid hydrolase, after which the continuously stirred mixture is optionally added with additional glutaraldehyde and the reaction mixture is allowed to freeze, or the resulting particles are separated from the reaction liquid by filtration organic solvents, preferably in ice-cold ethanol, by filtration again and optionally dried after homogenization in the open air at room temperature for 1 to 24 hours, after which the particles are suspended in cold water or buffer, decanted several times with water or buffer and allowed to swell t at temperatures ranging from 5 to 20 ° C.

The thus obtained and stabilized biocatalyst particles are contacted with aqueous solutions of 7-aminocephene compounds, in particular 7- [4- (4-carboxybutanamido) -cephalosporanoic acid, and during the continuous pH adjustment of the reaction mixture in the range of 7.0 to 8, The alkali is fed in batchwise repeated or enzymatic hydrolysis at temperatures of 10 to 45 ° C, preferably 35 ° C, after which 7-aminocephalosporanoic acid is isolated from the filtered and cooled liquid separated from the catalyst particles. In addition to the pure forms of the substrate, a reaction mixture obtained by the amination of cephalosporin C in the presence of sodium azide or other catalase inhibitors and cells or cell-free extracts or cellular catalysts of the Trigonopsis variabilis strain with D-amino acid axidase activity may be used.

The biocatalyst particles obtained according to the present invention are solid, very rapidly sedimenting irregular size and shape formations with high stability of 7-, N- (4-carboxybutanamido) -cephalosporanoic acid hydrolase as exemplified in the following examples. Their strength and good sedimentation properties allow their discontinuous repeated or continuous use in conventional reactor types.

The lactamase-negative strain of Pseudomonas sp. CCM 3635, which was obtained according to U.S. Pat. However, the method of consolidating and stabilizing the biocatalyst particles of the present invention does not exclude the use of other strains obtained, e.g., by mutagenic intervention, selection and aggregation, or gene manipulation for industrial use in the form of immobilized biocatalysts. The biocatalyst particles can be stored at 5 ° C in an aqueous suspension or in a suctioned form in a wet state to prevent drying.

Nutrient broth and culture conditions were used to prepare native cells, as described in U.S. Pat. The cells were collected from the fermentation broth by centrifugation (5000 rpm, 30 minutes), washed with 0.1 M phosphate buffer pH 7.0 and the cell paste obtained was used to prepare a biocatalyst.

The enzymatic activity of the obtained particles was evaluated spectrophotometrically using p-dimethylaminobenzaldehyde, which reacts with 7-ACK as well as 6-APK to form a colored Schiff base, according to U.S. Pat. No. 116959 modified by Balasingham et al. (Biochim. Biophys. Acta, 276, 250 (1972)) by measuring the rate of 7-β- (4-carboxybutanamido) -cephalosporanoic acid (glutaryl-7-ACK) in 0.1 M phosphate buffer pH 7.0 and a temperature of 37 ° C with stirring in the region of zero-order enzyme kinetics.

The unit of enzyme activity is the amount of enzyme that catalyses the formation of one micromole of 7-ACK per milliliter per minute at 37 ° C and pH 7.0. The conversion of a 1% glutaryl-7-ACK solution, which was also determined by thin-layer cellulose chromatography (Polygram Cel 300) using n-propanol-water (7: 3) with ninhydrin detection (R _f 0), was considered as a biocatalyst efficiency criterion. , 5).

In the following, the invention is illustrated in more detail by the examples without limiting them.

Example 1

216 g wet cell paste Pseudomonas sp. CCM 3,635 with said hydrolase activity, obtained by centrifuging the culture after cultivation (5,000 rpm, 30 minutes) with a specific activity of 3.10 u / mg dry cell mass, was suspended in 0.05 M phosphate buffer pH 7.0 and make up to 720 ml with this buffer. To the suspension under stirring at room temperature, 30 ml of 25% glutaraldehyde was added successively and stirred continuously for 3 hours. Then, the suspension was centrifuged under the conditions above and the cells were washed twice with three times the volume of distilled water.

g of the crosslinked and washed cell paste obtained was suspended in 40 ml of distilled water and 40 ml of reactive water-soluble polymer (hereinafter RRP) was added under vigorous stirring according to the procedure of Art. RRP was obtained by previous one hour polymerization of 8% (w / v) flocculant (Sedipur CL-930, BASF-NSR) and 2% glutaraldehyde with stirring at 25 ° C. After homogenization, the mixture was further frozen in a thin layer at -20 ° C for five hours. The resulting particles were washed with water, aspirated through a textile material and ground. The particles showed an activity of 1.8.10 U / mg dry matter (dry matter content 23%).

1.2 g glutaryl-7-ACK (85% pure) was dissolved by adjusting pH 1 N NaOH to 7.0 in 90 ml distilled water and 10 g wet aggregate was added and the mixture was stirred at 35 ° C for continuous adjustment of pH 1 N NaOH to 7.0 for two hours. The degree of conversion achieved was 80% of theory. This procedure was repeated five times with an average conversion rate of 79.6% of theory.

Example 2

0.5 liter of native cell suspension of Pseudomonas sp. CCM 36 35, containing approximately

500 mg of wet cell mass per milliliter of 0.05 M phosphate buffer pH 7.0 was disintegrated by passing four times at a maximum pressure of 6.10 Pa. After each cycle, the suspension was cooled to 10-15 ° C. The degree of disintegration was evaluated microscopically. 2 g of technical egg protein was mixed into 25 ml of the disintegrated suspension.

g of cross-linked cells, prepared as described in Example 1, were suspended in 70 ml RRP, obtained by previous polymerization of 4% flocculant (Sedipur CL-930) and 2% glutaraldehyde according to the procedure of Art. No. 231458, the pH was adjusted to 7.5 with 4% NaOH under vigorous stirring. The viscous suspension was stirred for one hour and then 25 ml of the disintegrated cells and protein prepared above were added to the mixture. The suspension was stirred for 30 minutes and then 5 ml of 25% glutaraldehyde was added and stirred continuously for one hour. The material was then vacuum aspirated through the textile material, washed with water and allowed to dry at room temperature for 24 hours.

It was then suspended in water decanted several times and allowed to swell for 24 hours, then was aspirated and washed. The specific activity of the obtained particles was 0.8.10 U / mg dry matter (22% dry matter content).

1.2 g of glutaryl-7-ACK was dissolved to a volume of 80 ml in distilled water, adjusting the pH of 1 N NaOH to 7.0. Then 20 g of wet particulate matter was added and stirred at 35 ° C and continuous pH adjustment to 7.0 by addition of alkali for 2 hours. The degree of conversion to 7-ACK was 70%. The procedure was repeated a total of three times with an average yield of 69.3% of theory.

Example 3 g of cross-linked cells prepared as in Example 1 were suspended in 50 ml RRP prepared in the same manner as in Example 2. The pH of the homogeneous suspension, which was vigorously stirred, was adjusted to 7.5 with 4% NaOH, and the viscous suspension was stirred for one hour according to the procedure of US. author's certificate no. 231458.

50 ml of a disintegrated cell suspension prepared as described in Example 2 without addition of egg protein was added to the reaction mixture with stirring. The next procedure in the preparation of the biocatalyst was the same as in Example 2.

A glutaryl-7-ACK solution was prepared as described in Example 2. To 80 ml of the solution, 20 g of wet aggregate was added and the mixture was stirred under the conditions of Example 2 for one hour. The degree of conversion to 7-ACK was 72%. This same batch feed procedure was performed a total of five times with an average conversion rate of 73.2% of theory / five cycles of reuse.

Claims (1)

Process for strengthening and stabilizing particles of a biocatalyst for the enzymatic conversion of 7-3- (4-carboxybutanamido) -cephalosporanoic acid to 7-aminocephalosporic acid, prepared according to the author's certificate No. 231458, in which aqueous solutions of 7-aminocephene compounds, in particular 7- R - (4-carboxybutanamido) -cephalosporanoic acid is contacted with the biocatalyst particles, characterized in that to a continuously stirred mixture of biocatalyst particles on the basis of intact or cross-linked cells of microorganisms containing the enzyme 7-7- (4-carboxybutanamido) hydrolase - cephalosporic acid, preferably Pseudomonas sp. CCM 3635, after adjusting the pH between 6.5 and 8.5, adds an aqueous solution of glutaraldehyde and / or an aqueous solution of technical or pure protein of animal, vegetable or microbial origin, such as technical egg protein, or a disintegrated suspension of microorganisms, preferably enzyme-containing cells. 7-3- (4-carboxybutanamido) -cephalosporanoic acid hydrolase, whereupon the continuously stirred mixture is optionally added with additional glutaraldehyde and the reaction mixture is allowed to freeze, and / or the resulting particles are separated from the reaction liquid by filtration or suspended under vigorous stirring subcooled organic solvents, preferably in ice-cold acetone or ethanol, are separated by filtration again and optionally dried after homogenization in the open air at room temperature for 1 to 24 hours, and then the particles are suspended in cold water or buffer, decanted several times with water or buffer and at temperatures between 5 and 20 ° C.