CS251457B3 - Method of cephaelosporines' derivatives preparation - Google Patents

Description

The invention "Method for the use of immobilized cells in the preparation of cephalosporin derivatives of cells in the preparation of cephalosporin derivatives" relates to a method for the enzymatic preparation of cephalosporin derivatives (CFS) of structure or formula II (see equation 1) which are intermediates in the preparation of acid 72

-aminccephalosporane (7-ACK J from compounds of structural formula Ί by immobilized cells of microorganisms containing D-aminoacid deaminating-Oα-reductase (D-aminoacid oxidase, DAAO).

equation no. 1 'Cri - i (HΌ Z'

H _or Ni

where x = -O — CO — CH3 x = —OH x = —H

R = —COOH cephalosporin C (CFS-C) desacetylcephalosporin C (DCFS-C) desacetoxycephalosporin C (DACFS-C)

The enzyme D-aminoacidoxidase produced by Trigonopsis variabilis CCY 15 - 1-2 cells can be immobilized in several ways, increasing its stability and extending its usability several times while simplifying the technology and possibly using new technological processes.

7-Aminocephalosporanoic acid can be prepared by two methods. Both utilize cephalosporins obtained as a substrate by fermentation. Chemical conversion is a multi-stage process, demanding on the purity of the raw materials used and strict adherence to technological operations at low temperatures while working with toxic substances. The method of chemical preparation is described in Japanese patent no. U.S. Patent No. 7,307,117; No. 1,445,615 et al., Differing in the manner of blocking the carboxyl and amino groups, and U.S. Pat. AO 193 181. Compared to the chemical mode, enzyme conversion is more advantageous in terms of less technological complexity, no high purity substrate is required, and the number of technological operations in the process is reduced. Enzymatic conversion is low on raw materials. This is a one-step or two-step process, wherein in the two-step reaction, compounds of structural formula I are formed of compounds of structural formula II, which are available substrates for acylases of various microorganisms to form 7-ACK. Japanese patent no. No. 74,044,895 discloses the possibility of direct enzymatic conversion of cephalosporins to 7-ACK in a fermentation pod by Fusarium sedation, Japanese patent no. 55 35 119 (1980) describes the same production strain. NSR 2,723,463 deals with the direct conversion of CFS to 7-ACK by the action of enzymes from Aspergillus and Alternaria as well as U.S. Pat. No. 3,239,393 and Japanese Patent No. 53-94,093 / 1978, which disclose strains of Brevibacterium, Achromobacter and Flavobacterium as producers.

A more advantageous method of the enzyme preparation of 7-ACK is the two-stage process in which DAAO acts on the cephalospherins of structural formula I to oxidatively deaminate the side chain at position 7 on the cephem core to give compounds of structural formula II. Japanese Patent 54,154,592 / 1979 discloses the use of Trigonopsis variabilis cells by glutaraldehyde assurance, which enhances enzyme stability and allows biological material to be used multiple times. Japanese patent no. No. 5,535,118 (1980) describes the same strain for conversion with an efficiency of 95%. CS AO no. 221,003 describes a method of using activated Trigcopsops variabilis cells with an efficacy of 80-85 percent. Activation is done with organic solvents and detergents. Brodelius in Applied Biochem. Biotechnology 6/4 / / 1981 p. 293-308 describes the use of Trigonopsis variabilis cells incorporated into an alginate gel to produce α-keto acids from D-amino acids. If cells are used for conversion in the filtrate from the fermentation broth, if appropriate in the eluate, it is recommended that the enzyme is scavenged according to Japanese patent no. No. 507,158. AO 231,458 describes methods of immobilizing cells of microorganisms using polyfunctional agents.

The invention "Method for the use of immobilized cells in the preparation of cephalosporin derivatives" employs immobilized cells of the Trigonopsis variabilis strain CCY 15-1-2 prepared by a modified procedure of the Czech Republic. AO č. The immobilized biocatalyst catalyzes the formation of cephalosporin derivatives of structural formula II from compounds of structural formula I. The compounds of structural formula II are intermediates in the preparation of 7-aminocephalosporanoic acid. The enzyme conversion substrate is an aqueous solution of cephalosporin C sodium salt or a deproteinized fermentation broth filtrate or eluates from II. purification step from the purification of CFSs at a concentration of 3-5% by weight. The concentration of immobilized biocatalyst in the reaction is 10-15% by weight and the concentration of side reaction inhibitors is 0.1-0.5%. The reaction is carried out for 2-8 hours at 32-36 ° C with vigorous stirring and aeration.

Biomass of the yeast production strain Trigonopsis variabilis CCY 15-1-2 is prepared by a two-step culture, each stage lasting 72 h at 28 ° C under aeration of 1: 1 (volume of medium: air volume per minute). 0.3% D-valine is added to the inoculum of the composition of 2% corn extract and 2% glucose as an enzyme stimulator. The pH of the soil prior to sterilization was adjusted to 5.0. For the second stage of fermentation, 8-10% of the inoculum was fermented with the fermentation broth composition: glucose 3%, peptone 1%, yeast extract 1%, KELPCU 0.1%, MgSO 3 0.09%, which contains 0.25 percent D-methionine as a stimulator. The biomass from the fermentation broth was obtained by centrifugation at a speed of 4000 min ^"1 for 30 minutes with cooling cells after washing 3 times with 0 M phosphate buffer pH 7.5 was deposited at 25 ° C. The cells thus treated retain an enzyme activity of 10-12 months.

Cross-linked Trigonopsis variabilis cells are prepared by treating the cell suspension with a 10% solids content of polyaldehyde (glutaraldehyde) with polyaldehyde reagents. With stirring, it is left to react for 3-5 hours at room temperature, then the biomass is separated by centrifugation, washed with water and deposited at -15 ° C.

Cell aggregates with enzyme activity are prepared from washed biomass by treatment with a mixture of 1% polyaldehyde (glutaraldehyde) and 2% polyfunctional reagent containing amino- or imino-clines, for example polyethyleneimine, tetramethylenediamine, 3,3-diaminodipropylamine and the like. This mixture was prepared 24 hours before use and stirred vigorously without air. After the aggregation mixture is added to the cell biomass, it is homogenized and frozen for 6-10 hours at -25 ° C.

After thawing, the resulting aggregates are separated by filtration or centrifugation, washed and deposited at +4 ° C in phosphate buffer pH 7.5.

Porous aluminosilicate materials, mineral carriers based on diatomaceous earth, porous glass, ceramic materials, clays, macroreticular resins and the like are used to immobilize the cells to a solid support. materials with high adsorption ability. Cell binding is done either by adsorption under growth, when 10% of the carrier is added to the culture medium or by binding-separated and washed biomass after fermentation. In both cases, the biomass-bound support is washed 5 times with water and the enzyme-active mobilized material is prepared by treatment with the above-mentioned mixture of poly blowing agents. 30 ml of a mixture of polyfunctional reagents are added per 5 g of adsorbed biomass carrier, frozen for 4-6 hours at -25 ° C, washed and deposited at 4-4 ° C in pH 7.5 buffer. The enzymatic activity of all of these immobilization modes is maintained for 10-12 months after preparation.

The enzyme conversion is based on CFS salt solutions, deproteinized fermentation broth filtrate, or CFS purification eluates, with an optimum CFS concentration of 10,000-12,000 μξ / ml, pH optimum ranging from 7.9 to 8.1 and temperatures from 33 to 35 ° C. If catalase is also present in the cells, it can be inactivated, otherwise the products of structural formula II, where R = -OCOOH, which are unsuitable for further enzyme processing, are formed. Inorganic azides (sodium azide 0.1-0.5%), ascorbic acid, hydrogen peroxide, boron compounds (sodium metaborate and sodium perborate) and others are used to inhibit catalase. The reaction requires aerobic conditions, optimum aeration is 1 liter of air per liter of reaction mixture per minute. The reaction time is two to six hours. Orientation monitoring of the reaction is done by thin layer chromatography on silica gel plates with UV 254 indicator, quantitative determination by high pressure chromatography.

The enzymatic conversion of compounds of structural formula II wherein R = - COOH can be used as a substrate for the enzyme preparation of 7-ACK.

Example 1

T. variabilis biomass with active DAAO, obtained by two-stage cultivation on a methionine-containing plate (0.2%), was separated by centrifugation at 4000 rpm.

. min ^-1 for 30 minutes, washed with water and stored at -25 ° C. After thawing, the biomass was diluted so that the dry weight of the resulting suspension was approximately 10%. 1% glutardialdehyde was added and the mixture was stirred at room temperature for 5 hours. The cross-linked cells were separated by centrifugation, washed and suspended in a buffer of pH 7.5 so that the solids were 10%. For the enzyme conversion, a cross-linked suspension of 0.5 ml per 10 ml reaction mixture containing sodium salt as substrate was used. CFS-C, at a concentration of 12 000 μ§ / τηϊ and sodium azide 1 mg / ml. The reaction was run at 34 ° C for 2 hours with vigorous aeration on a shaking machine. The pH was maintained at 7.9-8.0. The degree of conversion was 88%.

Example 2

Both the biomass preparation procedure and the enzyme conversion conditions are identical to Example 1, but sodium perborate NaBCkHzOa was used instead of sodium azide to inhibit catalase. 3IHO or sodium perborate NaBCH. 3 H2O.

The degree of conversion was 78%.

Example 3

The procedure is identical to that of Example 1, in the case of enzyme conversions, eluates from the purification of 12,000 µg / ml CFS-C were used instead of the CFS-C salt. The degree of conversion was 71%.

Example 4

Biomass of the production strain was obtained as described in Example 1. A reaction aggregation mixture containing 1% glutaraldehyde and 2% polyethyleneimine in distilled water was prepared 24 hours prior to use and was vigorously stirred at ambient temperature in the absence of air. Aggregates with enzyme activity were prepared by adding this mixture to biomass (which constituted 25% of the reaction mixture), which was freeze-dried at -25 ^° C for at least 8 hours after documented homogenization. The reagents produced granular or flocculated aggregates from the biomass, after which the thawed residues were removed by washing with water, the aggregates were separated by centrifugation or aspiration and deposited in pH 7.5 buffer at + 4 ° C. The enzyme conversion was followed by 0.5 g of aggregates per 10 ml reaction mixture containing 10,000-12,000 µg / ml CFS-C sodium salt. The reaction conditions are identical to those set forth in Example 1. First

The degree of conversion ranged from 75 to 83%.

Example 5

The procedure is identical to Example no. 4, 12,000 µg / ml CFS-C purification eluates were used for conversion only. The degree of conversion ranged from 70 to 75%.

Example 6

The procedure is identical to Example no. 4, but instead of polyethyleneimine, tetramethylenediamine was used in the preparation of ag251457 regimes. The conversion rate was 73%.

Example 7

The procedure as in Example no. 4, but diaminodipropylamine was used in the same concentration instead of polyethyleneimine. The degree of conversion was 75%.

Example 8

The biomass prepared as described in Example 1 was diluted to 10% solids. 15 ml of biomass were incubated with 5 g of a grain diameter aluminum silicate carrier

0.5 mm 3 hours. The support was washed with water, 30 ml of a 1% glutaraldehyde in water solution was added, and after freezing for 4 hours at -25 ° C, the support was washed with water. 5 g per 50 ml of the mixture were used for conversion. The conditions of the reaction were the same as in Example no. 1. The degree of conversion was 60%.

Example 9

The biomass was cultured in the presence of a mineral carrier added to the soil before sterilization at 5% per pod volume. After the fermentation was complete, the cell support was washed and the further procedure was as described in Example no. 8. The degree of conversion was 56%.

OBJECT OF THE INVENTION

Claims (1)

tetramethylenediamine is used. The degree of conversion was 73%. EXAMPLE 7 The procedure as in Example 4, but instead of polyethyleneimine, was used with the same concentration of diaminodipropylamine. The degree of conversion was 75%. EXAMPLE 8 The biomass prepared as described in Example 1 was diluted to 10% with dry weight. 15 ml of biomass was incubated with 5 g of an aluminosilicate silicate carrier having a grain diameter of 0.5 mm for 3 hours. The carrier was washed with water, 30 ml of a 1% solution of glutaraldehyde in water was added, and after 4 hours of freezing at -25 ° C, the carrier was washed with water. 5 g per 50 ml mixture was used for the conversion. The reaction conditions were the same as in Example 1. The conversion rate was 60%. Example 9 The biomass was cultured in the presence of a mineral carrier added to the soil by pre-sterilization at 5% per volume volume. After fermentation was complete, the cell carrier was washed and the procedure was similar to Example 8. The conversion rate was 56%. OBJECT OF THE INVENTION A method of preparing cephalosporin derivatives of the formula II. | X 'CQQH V1) wherein X = z-OCOCH3, -OH, -HR-COOH, characterized in that the aqueous solution of cephalosporin C sodium salt of a concentration of 3-5% by weight or the eluate of cephalosporins is purified with a concentration of 3 to 5% by weight, the immobilized cells of Trigonopsis variabilis CCY 15-1-1 are added immobilized by modified procedures of the MS. AO No. 231 458, wherein the catalyst concentration in the reaction is 10 to 15% by weight and the reaction concentration of the side reactions is 0.1 to 0.5% by weight and the reaction takes place for 2-8 hours at 32-36 ° C under vigorous agitation. and the obtained cephalosporin derivatives of structural formula II are isolated. Saverografia, n. P., Race 7, Most Price 2.40 KCs