CS218023B1 - Method of enzymatic production of the 7-aminodesacetoxycefal acid - Google Patents

Abstract

The present invention relates to an enzyme production method and isolating 7-aminodesacetoxycephalosporanic acid from desacetoxycephalosporin using specially developed, mobilized crosslinked cell catalysts and permeabilized biomass, cellular aggregates, or on the basis of each other. microbial cells containing them penicillin acylase enzyme. The obtained 7-aminodesacetoxycephalosporanic acid serves as a basic intermediate for the production of semi-synthetic desacetoxycephalosporins, especially cefalexin.

Description

The invention relates to a method for the enzymatic production of 7-aminodesacetoxycephalosporanic acid (hereinafter 7-ADCK) from N-phenylacetamidodesacetoxycephalosporin (DAOC-G) by cross-linked, aggregated or bound cells with penicillin acylase activity.

7-ADCK is a key intermediate for the synthesis of alpha-aminocephalosporin antibiotics of the cefradine type, in particular cephalexin, i.e. D - (-) - (alpha-amino-alpha-alpha-phenylacetamido) -3-methyl-3-cephem-4-carboxylic acid monohydrate , which is a highly effective, commercially produced antibiotic against some pathogenic Gram-positive and Gram-negative microorganisms, including strains insensitive to natural and semi-synthetic penicillins.

7-ADCK can be prepared in a number of ways, two of which are of industrial importance. The first method starts from the potassium salt of benzylpenicillin (penicillin Gj or phenoxymethylpenicillin (penicillin V)), the carboxyl groups of which are temporarily protected by the formation of easily saponifiable esters, e.g. p-nitrobenzyl ester,

2,2,2-trichloroethyl ester or benzylhydryl esters. These are converted in the next step by oxidation to the corresponding sulfoxide esters, which are transformed by Pummerer rearrangement from penam structures to cephem, i.e. esters of desacetoxycephalosporins G or V. In the next step, the undesired side chain needs to be removed to form 7-ADCK. still protected by a carboxyl group.

The protecting groups are removed and the free 7-ADCK is then acylated to form cephalexin, preferably according to e.g. No. 177,703. The removal of the unwanted side chain in this process is carried out exclusively by chemical means; benzylpenicillin or carboxy-protected phenoxymethylpenicillin sulfoxides are reacted with phosphorus pentachloride-type reagents to produce the chloroimine derivative which is converted to the corresponding imine derivative by alcoholysis, followed by hydrolysis to give the corresponding 7-ADCK esters (F. Huber et al., In: Cephalosporin and Penicillin Compounds, Their Chemistry and Biology, ed. EH Flynn, pp. 27-73, Academic Press, New York, 1973).

Although chemical hydrolysis proceeds in good yield, it represents a sequence of very unpleasant operations, the use of very aggressive chemicals attacking the apparatus used.

The second method of production of 7-ADCK is shorter in terms of the number of technological operations and is more economical. Phenoxymethylpenicillin (penicillin V), in particular benzylpenicillin (penicillin G), is first converted into the corresponding sulfoxide with an unprotected carboxyl group, preferably according to the procedures of the author's certificate No. 221 161 and No. 210 739. In the next step the Pummerer rearrangement is carried out. formation of desacetoxycephalosporin G.

This implies that the second process for the preparation of desacetoxycephalosporins brings numerous advantages and is therefore today used by most semi-synthetic cephalosporin manufacturers. By completely eliminating the use of protecting groups and their subsequent removal, it is considerably shorter. Even in this second, economically advantageous method of producing 7-ADCK, the problem of removing the unwanted side chain remains. The chemical process, as in the first process, uses aggressive, easily hydrolyzed phosphorus trichloride and it is necessary to operate at low temperatures in order to achieve a high yield; in large-scale production, extremely toxic waste liquors must also be treated. Thus, the chemical method of side chain removal (desacetylation) is not an advantageous solution to the problem in either of the two methods.

A method for enzymatic desacetylation of the side chain of desacetoxycephalosporins, in particular desacetoxycephalosporins G, by the enzyme penicillin acylase produced by various microorganisms, in particular Bacillus megatherium, e.g. optionally soluble penicillin acylase isolated from these cells according to U.S. Pat. AO No. 190 459. The enzymatic method of desacetylation of the side chains of desacetoxycephalosporins, although selective, eliminates many of the above-mentioned technological and economical difficulties in comparison with chemical hydrolysis (elimination of aggressive chemicals), but results in 7-ADCK products heavily contaminated with protein from autolysed cells, possibly from enzymatic and ballast proteins, leading to isolation losses during further purification, or to the risk of allergic reactions upon application of semisynthetic desacetoxycephalosporins, produced from the 7-ADCK thus obtained in clinical practice.

Another disadvantage of this procedure is that native (living) cells, or the soluble enzyme isolated therefrom, can only be used once for the enzyme reaction, resulting in a disadvantageous economic balance compared to chemical hydrolysis, since it is necessary to cultivate for each production batch enzyme active biomass in a large-capacity single-use fermenter. In the event of the application of a soluble enzyme, even if only in technical form, the cost of isolating the enzyme from the cells of the microbial producer increases to such an extent that the application of such a technology is economically unrealistic.

In an effort to economize the process of enzymatic desacetylation of the side chains of 7-phenylacetamidodesacetoxycephalospo218023 wound produced from benzylpenicillin and 7-phenoxyacetamidodesacetoxycephalosporanic acid produced from phenoxymethylpenicillin and to eliminate these undesirable effects, various forms of Bacillus megatherlum, Proteus rettgeri and Erwinia aroidea, allowing further economization of the process of preparing 7-ADCK, consisting mainly in multiple batch or continuous utilization of immobilized forms of this enzyme (BJ Abbott, In: Advances Applied Microbiology, ed. D. Perlman, Vol. 20, pp. 203-257, Academic Press, New York, San Francisco, London, 1976). These are, in particular, the work of Japanese authors (British Patent No. 1 324 159), which sorbed isolated penicillin acylase from Bacillus megatherium onto kieselguhr, activated carbon, carboxymethylcellulose, or various ion exchange resins, and thus physically, respectively. physically-chemically immobilized enzyme used to prepare 7-ADCK. Also, the Italian authors (U.S. Patent No. 190,469) physically incorporated isolated penicillin acylase from Escherichia coli cells into cellulose triacetate fibers and used this preparation both for the enzymatic preparation of 7-ADCK from DAOC-G and for the enzymatic acylation of 7-ADCK with D- The latter method of immobilizing penicillin acylase into cellulose triacetate fibers, but the enzyme was isolated from another type of microorganism (German Patent Specification No. 2,422,374), was used to prepare 7-ADCK using 7- The immobilized forms of penicillin acylase obtained by the indicated method can be used for the preparation of 7-ADČK repeatedly, biological and other toxic admixtures in the product and thus in the clinical forms of semisynthetic desacetoxycephalosporins are negligible and the whole cost of economics is controlled r, ie the cost of cultivating enzyme-active biomass and, in particular, the yield in enzyme isolation and the yield of its immobilization. It is understandable that in the economics of the process, the limiting factor is in particular the yield of enzyme isolation and the cost and availability of enzyme binding carriers and the yield of immobilization, including the operational (working) half-life of the immobilized enzyme. how long the catalyst can be reused or continuously used without significant loss of its specific enzyme activity. Last but not least, the efficiency and degree of enzyme conversion of substrate to product, recovery yield and product quality play an important role in the use of immobilized enzymes.

A number of technologies have been developed for the production of biocatalysts with penicillin acylase activity primarily intended for the enzymatic production of 6-aminopenicillanic acid (hereinafter referred to as 6-APK) by enzymatic hydrolysis of penicillin, especially benzylpenicillin. These are mainly immobilized materials with penicillin acylase activity on the principle of chemically immobilized cells of Escherichia coli and other microorganisms, both cross-linked and permeabilized cells of the production microorganism, allowing the re-use of this material for semi-continuous production of 6-APK. subject of MS. No. 201 621, on the one hand, microbial cellular aggregates with penicillin acylase activity, the production of which is the subject of MS. No. 197 101, furthermore the production of mutually chemically bound cells with peniciliacylase activity according to MS. No. 203 607, and finally the production of chemically bonded cells of prokaryotic and eukaryotic microorganisms with various enzyme activities, inter alia with penicillin acylase activity, according to US Pat. Certificate No. 209 265.

The cited processes for the production of these biocatalysts do not utilize enzyme isolation from microbial producer cells and its subsequent binding to the carrier, but use the producer cell as a natural carrier of enzyme activity and by the original immobilization procedures only enzymes are chemically (by covalent) fixed within the cells, or chemically "conserved" autolysis-resistant cells associate another chemical reaction with each other without the use of any water-insoluble carrier to improve the sedimentation properties of the formed particles, forming integral units with high penicillin acylase activity.

The properties of said cellular biocatalysts, specific activity, enzyme activity, mechanical and enzymatic stability of the particles, and other biochemical, physicochemical and physical properties of these materials allow multiple re-use (discontinuous) or continuous use for the production of 6-APK. From the economic point of view, the production costs of these types of biocatalysts are minimal, because the technology of their production requires no commercial carrier, involves a small number of operations, the raw material costs for their production are minimal.

We have now found that under the selective reaction conditions of conducting a biochemical reaction, preferably said immobilized cellular materials can be used for the present method of producing 7-ADCK from desacetoxycephalosporins, especially DAOC-G, multiple times (discontinuously or semicontinuously) or continuously, with high efficiency of enzyme conversion of substrate to product (95-100% of theory), high yield in product isolation (molar yield in isolation up to 90% of theory) and high quality (minimum active substance content for 218023 duct of 95% of theory). In conjunction with simplified and economized chemical processes for the production of desacetoxycephalosporin G, which are the subject of MS. No. 209,641, No. 210,739, the present method of enzyme production of 7-ADCK according to the present invention represents a complex, modern and economically viable chemical-enzyme technology for the production of this pharmaceutically important intermediate enabling subsequent production of semi-synthetic desacetoxycephalosporins, especially cephalexin.

The method for the enzymatic production of 7-ADCK according to the present invention is characterized in that an aqueous solution of desacetoxycephalosporins G, preferably its salts at a concentration of 0.05-10.0 is used. weight / volume, preferably 6% weight / volume, with a pH of 7.0-8.5, preferably 7.8, is stated at a temperature of 10-45 ° C, preferably 37 ° C, discontinuously, semi-continuously or continuously in contact with a biocatalyst based on cross-linked and permeabilized biomass of microbial penicillin acylase producers or on the basis of aggregated or chemically bound cells of the producer of this enzyme, after isolation of the 7-aminodesacetoxycephalosporanine from the supernatant or mother liquor after pH and temperature adjustment .

The enzyme production process of the present invention is further characterized in that the isolation of 7-ADCK is carried out in the temperature range of -1 to + 15 ° C, in the pH range of 3.5 to 5.0, preferably +2 ° C and pH 4. 2, optionally in the presence of water-immiscible organic solvents, in particular gasoline or petroleum ether, and / or the crystallized product is suspended in water-miscible solvents, in particular ethanol or acetone, or both.

The production methods of the invention are as follows

exemplified in the examples without by lita product are summarized as followed with them. Initial DAOC-G concentration (%) 1.0 3.0 6.0 8.0 10.0 reaction time required for quantitative conversion (min) 30 45 90 110 180 the amount of 7-ADCK obtained (gj 0.23 0.68 1.44 1.92 2.26 7-ADCK content in product (%) 96.83 97.7 97.57 92.65 92.24 7-ADCK yield (% of theory) 85.65 84.85 86.2 85.5 80.8

The washed aspirated catalysts were used once more as described and under the above conditions, and the same results were obtained.

Example 1 a d 2

900 ml of demineralized water was added to a 21 liter glass reactor equipped with a fused S 2 porcelain frit at the bottom of the reactor interior and an outlet valve closing the space under the frit and a duplicating apparatus for circulating water temperature maintained by the ultra thermostat, while stirring.

Example 1

Chemically-bound microbial cells with penicillin acylase activity produced according to MS. Authorization Certificate No. 203 607 with parameters: specific activity of 6.5 U / mg wet material, average particle size distribution 0.15 to 0.3 mm and average dry matter content in the material 35%, were in a quantity of 5 g gradually suspended in 45 ml of aqueous solutions of DAOC-G K salt (average active content 96.92%) over a range of initial substrate concentrations of 1.0 to 10.0% w / v. The enzyme reaction was carried out at 37 ° C with continuous stirring and continuous pH adjustment of 0.1 to 1.0 N aqueous NaOH solution using a pH-stat in the pH range of 7.6 to 7.8.

After the end of the enzymatic hydrolysis indicated by stopping the consumption of alkali, the biocatalyst particles were separated by vacuum filtration through a terylene cloth and washed with water. The clear solutions of the reaction mixtures were cooled to +4 ° C and 25% aqueous sulfuric acid solution was added with stirring while potentiometric pH control to 4.2. After adjusting the pH of the reaction mixtures, the acidified reaction mixtures were allowed to stand at this temperature for about 1 hour.

After quantitative crystallization of the product, the crystals from the individual reaction mixtures were sequentially separated by vacuum filtration on the frit and the products were suspended in 50 ml of chilled ethanol (+ 10 ° C), separated by filtration, washed with excess cold ethanol on the frit and dried to constant mass. enzymatic hydrolysis, yields and quatically placed steel anchor stirrer at 60 rpm were dissolved in the present water 57 g of DAOC-G K-salt with an active substance content of 96.92%. with pH-stat, pH-meter and dosing device for continuous pH adjustment After dissolution, the pH of the solution was adjusted to 7.9 with a single NaOH solution.

After warming the solution to 37 ° C, 110 grams of the wet mass of the biocatalyst, the parameters of which are given in Example 1, were added with continuous stirring. With the aid of the automatic continuous pH adjuster, this was continuously maintained at 7.8 with 1 N NaOH solution. continuous stirring and tempering for 90 min. enzyme reactions. After this time, the slurry was vacuum aspirated by opening the discharge valve, and the fused melt between the bottom of the reactor interior space allowed the quantitative separation of the clear reaction mixture from the biocatalyst particles remaining in the reactor.

After sucking off the reacted liquid, 900 ml of water in which the DAOC-G K-salt was previously dissolved at the same concentration as in the previous cycle was repeated to the reactor containing the wet, unscrewed sucked-off catalyst catalyst used in the previous cycle. with the same catalytic converter handle.

After aspiration of the reacted liquid from each of the cycles of repeated enzymatic hydrolysis, it was poured into a refrigerated vessel in which 330 ml of gasoline cooled to +2 ° C was pre-charged and the liquid was continuously stirred and cooled so that the resulting temperature was + 1 to +5 degrees Celsius. The pH of the cooled, stirred liquid consisting of the organic water-immiscible and aqueous phases was adjusted to 4.2 as described in Example 1. After stirring for about half an hour after adjusting the pH, the crystallization mixture was left at a temperature of about 4 ° C for about 1 hour. The product was then separated by vacuum filtration, taken up and suspended in 100 ml of ice-cold acetone, re-filtered, washed several times with excess ice water on the filter and dried to constant mass.

A total of 292 g of 7-ADCK with an average active content of 96.94% and a total yield of 89.7% of theory was obtained in 10 repeated cycles of using the same catalyst feed. The specific activity of the catalyst did not change after its 10-fold reuse. The catalyst mass loss after 2 cycles was 2% based on the particulate solids and did not affect the reaction rate and the degree of conversion between cycles 1 and 10 of its reuse.

Example 3

Chemically bound microbial cells with penicin acylase activity, obtained according to the procedure of U.S. Pat. Certificate No. 209 265 of parameters:

a specific activity of 2.5 U / mg wet material, granular particles having a size of 1-3 mm and an average dry matter content of 40%, was filled in a quantity of 55 g into a 2.6 cm diameter glass column with a tempered jacket. 0.5 L of 0.5 molar phosphate buffer pH 7.9 was pumped through the immobilized material in which 3% w / v DAOC-G K-salt was dissolved from the bottom up at a rate of 2030 ml / h at 37 ° C. Deň: 32 ° C. In this arrangement, 5 repetitive enzyme conversions of 0.5 L of substrate concentration of the indicated concentration were performed with the same catalyst feed permanently in the column, recirculating the reaction mixture while continuously adjusting the pH with dilute ammonia water (1: 1) to 7.9 in off-column. a glass stirred vessel in which a pH-electrode coupled to a pH-stat has been incorporated. The reaction time for each cycle of enzyme conversion in the recirculation reactor was 120 min.

At the end of each of the enzyme conversions, the reacted liquid was treated in the same manner as in Example 2 except that precooled petroleum ether (160 ml) was introduced instead of gasoline and the aspirated product after crystallization was suspended again in petroleum ether, aspirated and washed with ice. water and dried to constant mass.

A total of 36.5 g of 7-ADCK with an average active substance content of 97.7% and a total yield of 91.09 why were obtained in 5 repeated cycles of using the same catalyst feed in a buoyant bed recirculation reactor. theory.

Example 4

1 liter glass laboratory fermentation tanks placed in a 37 ° C water bath were each filled with 200 g of bound cells with penicillin acylase activity, the parameters of which were given in Example 1. The reactors had built-in pH electrodes coupled with pH stats and dosing device solution. alkali. 500 ml of a 6% aqueous solution of DAOC-G K-salt, pH 7.8, was charged to each of the reactors and agitators were started at 80-100 rpm. The enzyme reaction was initially carried out in a batch process with stirring, annealing and continuous pH adjustment with ammonia water to 7.8 until the substrate concentration in the first reactor dropped to 10-15%, in the second reactor to 2-0% of the starting amount. After reaching this state, continuous operation was gradually started. Substrate solution containing 6% DAOC-G solution cooled to + 5 ° C was used to pump substrate solution to the 1st reactor at 0.46 liters / hour with one pump, partially hydrolyzed substrate solution by 2nd pump from 1st reactor to the 2nd reactor at a rate of 0.47 liters / hr. (correction for the volume increase caused by increasing the volume in the first reactor by the inflow of more alkali) and by means of a third pump from the 2nd reactor to the cooled tank of the reacted conversion mixture (+5 ° Cj at a rate of 0.46 to 0.48 liters / hour. In both reactors, the pH of the reaction mixture was continually maintained by feeding 16% ammonia water using two independent pH-stats The retention of catalyst particles in the reactors was achieved by providing the reactor exhaust tubes with a filter cloth just above the stirrer.

The continuous stirred reactors connected in series were in continuous operation for 25 hours, during which time 11.7 liters of substrate were quantitatively hydrolyzed to the desired product. Isolation of 7-ADCK was performed by crystallizing the product only from the aqueous phase with suspension and washing with ethanol as described in Example 1. It was obtained

370.6 g of product having an average 7-ADCK content of 96.9%, which is 87.6% of theory. The specific activity of the catalyst did not change after 25 hours of continuous operation.

Example 5 kg of wet cell paste mass of individually covalently crosslinked permeabilized and washed E. coli cells with penicillin acylase activity (8 U / mg wet mass), obtained according to the procedure of the Czech Copyright Certificate No. 201621, were suspended in 9 liters of drinking water and pH the slurry was adjusted to 7.9 with 1N NaOH. 10 liters of the homogeneous cell suspension thus prepared were pumped into a 201 liter glass fermentation tank placed in a 37 ° C water bath, into which a glass pH electrode connected in the previous examples by the automated apparatus was built. 513 g of DAOC-G K-salt was then poured into the stirred (250 rpm) aqueous suspension and continuously maintained at pH 7.8 with a 1 N NaOH solution for 90 min. centrifuged on a Sharples centrifuge to obtain 8.4 liters of clear supernatant the one from which 7-ADCK was isolated as described below.

The thin cell paste containing a portion of the reacted reaction mixture from the previous cycle was suspended again in 9 liters of water and the whole enzymatic hydrolysis procedure of the fresh substrate solution was repeated 5 times as described, during semicontinuous use before starting the next cycle. ) compensated for weight loss caused by handling during centrifugation, replenishment of fresh stock of cross-linked and unused biomass 20 grams w / w. in the third cycle and 50 grams of the wt. in the fifth semicontinuous cycle.

7-ADCK was isolated from the semicontinuous cycles from the supernatant by the procedure described in Example 2 using the present chilled gasoline at 3 liters / cycle, treating the supernatant of the reacted liquid, suspending the product after filtration in 1 liter of iced acetone and washing with ice water.

In 5 semicontinuous cycles, a total of 1.25 kg of product was obtained with a 7-ADCK content of 96.9% and a total yield of 85.33% of theory.

Claims (2)

Process for the enzymatic production of 7-aminodesacetoxycephalosporanic acid, characterized in that an aqueous solution of 7-phenylacetamidodesacetoxycephalosporanic acid, preferably its salts, is present in a concentration of 0.05 to 10% by weight / volume, preferably 6% by weight / volume, of pH 7.0 to 8.5, preferably pH 7.8, at 10 to 45 ° C, preferably 37 ° C, contact discontinuously, semicontinuously or continuously with a biocatalyst based on a cross-linked and permeabilized biomass of a microbial penicillin acylase producer or on a biocatalyst based on aggregated or chemically bound cells of the producer of this enzyme, after which the 7-aminodesacetoxycephalosporanic acid is recovered from the mother liquor after adjusting the pH and temperature after completion of the conversion from the supernatant. 2. Process according to claim 1, characterized in that the isolation of 7-aminodesacetoxycephalosporanic acid is carried out in the temperature range from -1 to +15 [deg.] C, in the range of pH values from 3.5 to 5.0, preferably +2 [deg.] C and pH 4. 2, optionally in the presence of water-immiscible organic solvents, in particular petrol or petroleum ether, and / or the crystallized product is suspended in water-miscible organic solvents, preferably ethanol or acetone, or both.