CS218023B1 - A method for the enzymatic production of 7-aminodesacetoxycephalosporanic acid - Google Patents

Abstract

The invention relates to a method for enzymatic production and isolation of 7-aminodesacetoxycephalosporanic acid from desacetoxycephalosporin using specially developed, mobilized, cell catalysts based on cross-linked and permeabilized biomass, cell aggregates, or based on chemically bound microbial cells containing the enzyme penicillin acylase. The obtained 7-aminodesacetoxycephalosporanic acid serves as a basic intermediate for the production of semi-synthetic desacetoxycephalosporins, especially cephalexin.

Description

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

7-ADCK is a key intermediate in the synthesis of alpha-aminocephalosporin antibiotics of the cephradine type, but in particular cephalexin, i.e. D-(—)-(alpha-amino-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 by a number of methods, of which two are the most important from an industrial point of view. The first method is based on the potassium salt of benzylpenicillin (penicillin G) 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 ester. These are then converted by oxidation into the corresponding sulfoxide esters, which are transformed from penam structures into cephem structures by the Pummerer rearrangement, i.e. into esters of desacetoxycephalosporins G or V. In the next reaction step, the unwanted side chain must be removed to form 7-ADCK with the carboxyl group still protected.

The protecting groups are removed and the free 7-ADCK is then acylated to form cephalexin, preferably e.g. according to Czechoslovak Patent No. 177 703. Removal of the unwanted side chain in this method is carried out exclusively by chemical means; sulfoxides of benzylpenicillin or phenoxymethylpenicillin with a protected carboxyl group are reacted with reagents of the phosphorus pentachloride type, an intermediate chlorimine derivative is formed, which is converted into the corresponding imino derivative by alcoholysis, subsequent hydrolysis leads to the corresponding 7-ADCK esters (F. Huber et al., In: Cephalosporin and Penicillin Compounds, Their Chemistry and Biology, ed. E. H. Flynn, pp. 27—73, Academie Press, New York, 1973).

Although chemical hydrolysis proceeds with good yield, it is a sequence of very unpleasant operations, using very aggressive chemicals that attack the equipment used.

The second method of producing 7-ADCK is shorter in terms of the number of technological operations and more economically advantageous. Phenoxymethylpenicillin (penicillin V), but especially benzylpenicillin (penicillin G] is first converted into the corresponding sulfoxide with an unprotected carboxyl group, preferably using procedures according to Czechoslovak Patent No. 221 161 and No. 210 739. In the next stage, the Pummerer rearrangement is carried out to form desacetoxycephalosporin G.

It follows from the above that the second method of preparing desacetoxycephalosporins brings numerous advantages and is therefore used today by most manufacturers of semi-synthetic cephalosporins. 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 method, similar to the first method, uses aggressive, easily hydrolyzed phosphorus pentachloride and, if a high yield is to be achieved, it is necessary to work at low temperatures; in larger-scale production, it is also necessary to process extremely toxic waste liquors. The chemical method of removing the side chain (deacetylation) therefore does not represent an advantageous solution to the problem in either of the two methods mentioned.

The technical and patent literature also describes a method of enzymatic deacetylation of the side chain of desacetoxycephalosporins, especially desacetoxycephalosporins G, with the enzyme penicillin acylase, produced by various microorganisms, especially Bacillus megatherium, e.g. according to GDR patent specification No. 97,213, and native (living) Escherichia coli cells, or with soluble penicillin acylase isolated from these cells according to MS. AO No. 190 459. The mentioned enzymatic method of deacetylation of the side chains of desacetoxycephalosporins is selective, eliminates a number of the mentioned technological and partly also economic difficulties in comparison with chemical hydrolysis (elimination of aggressive chemicals), however, it leads to the formation of 7-ADCK products heavily contaminated with protein impurities resulting from autolyzed cells, or from enzyme and ballast proteins, which leads to isolation losses during further purification, or to the risk of allergic reactions when applying semi-synthetic desacetoxycephalosporins produced from 7-ADCK obtained in this way in clinical practice.

Another disadvantage of this procedure is that native (living) cells, or soluble enzyme isolated from them, can only be used once for the enzyme reaction, which in its consequences means an unfavorable economic balance compared to chemical hydrolysis, because for each production batch it is necessary to cultivate enzyme-active biomass in a large-capacity fermenter for single use. In the eventual application of soluble enzyme, used even only in technical form, the costs of isolating the enzyme from the cells of the microbial producer will increase so much that the application of such technology is economically unrealistic.

In an effort to economize the process of enzymatic deacetylation of the side chains of 7-phenylacetamidodesacetoxycephalosporanic acid produced from benzylpenicillin and 7-phenoxyacetamidodesacetoxycephalosporanic acid produced from phenoxymethylpenicillin and to eliminate the aforementioned undesirable effects, various forms have been developed, primarily immobilized preparations of the penicillin acylase enzyme, isolated from the cells of Escherichia coli, Bacillus megatherlum, Proteus rettgeri and Erwinia aroidea, enabling further economization of the 7-ADCK preparation process, consisting primarily in multiple batch or continuous use of immobilized forms of this enzyme (B. J. Abbott, In: Advances Applied Microbiology, ed. D. Perlman, Vol. 20, pp. 203— —257, Academic Press, New York, San Francisco, London, 1976). These are mainly the works of Japanese authors (British patent document No. 1,324,159), who sorbed isolated penicillin acylase from Bacillus megatherium onto diatomaceous earth, activated carbon, carboxymethylcellulose, or various ion exchange resins, and used the thus physically or physico-chemically immobilized enzyme for the preparation of 7-ADCK. Italian authors (Czech Patent No. 190,469) also 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-(—)-phenylglycine methyl ester to form cephalexin. The latter method of immobilizing penicillin acylase into cellulose triacetate fibers, however, the enzyme was isolated from a different type of microorganism (German Patent No. 2,422,374), was used for the preparation of 7-ADCK using 7-phenoxyacetamidodesacetoxycephalosporanic acid or its salts as a substrate. The immobilized forms of penicillin acylase obtained in the indicated method can be used for the preparation of 7-ADCK repeatedly, biological and other toxic impurities in the product and thus in the clinical forms of semi-synthetic desacetoxycephalosporins are negligible and the entire process economy is driven primarily by the cost of the catalyst, i.e. the cost of cultivating enzyme-active biomass and primarily by the yield in enzyme isolation and the yield of its immobilization. It is understandable that in the economics of the entire process, the limiting factor is mainly the yield of enzyme isolation and the price and availability of carriers for enzyme binding and the yield of immobilization, including the operational (working) half-life of the immobilized enzyme, i.e. in practice, the value of how many times, or how long, the catalyst can be repeatedly or continuously used without significant loss of its specific enzyme efficiency. Last but not least, the efficiency and degree of enzyme conversion of the substrate to the product, the yield in isolation and the quality of the product play a significant role in the use of immobilized enzymes.

A number of technologies have been developed for the production of biocatalysts with penicillin acylase activity intended primarily for the purposes of enzymatic production of 6-aminopenicillanic acid (hereinafter referred to as 6-APK) by enzymatic hydrolysis of penicillin, especially benzylpenicillin. These are primarily immobilized materials with penicillin acylase activity based on the principle of chemically immobilized cells of Escherichia coli and other microorganisms, namely cross-linked and permeabilized cells of the production microorganism, allowing repeated use of this material for semi-continuous production of 6-APK, their production and use for the stated purpose is the subject of Czechoslovak patent No. 201 621, and microbial cell aggregates with penicillin acylase activity, the production of which is the subject of Czechoslovak patent No. 197 101, as well as the production of chemically bonded cells with penicillin acylase activity according to Czechoslovak patent No. of the author's certificate No. 203 607, and finally the production of chemically bound cells of prokaryotic and eukaryotic microorganisms with various enzyme activities, including penicillin acylase activity, according to the Czechoslovak author's certificate No. 209 265.

The cited production processes of these biocatalysts do not use the isolation of the enzyme from the cells of the microbial producer and its subsequent binding to the carrier, but rather use the producer cell as a natural carrier of the enzyme activity and, using the original immobilization processes, only the enzyme is chemically (by covalent bonding) fixed inside the production cells, or the thus chemically "preserved" cells, resistant to autolysis, are connected to each other by a further chemical reaction without the use of any water-insoluble carrier with the aim of improving the sedimentation properties of the resulting particles, which form integral units with high penicillin acylase activity.

The properties of the aforementioned cellular biocatalysts, specific activity, enzyme efficiency, mechanical and enzyme stability of particles and other biochemical, physico-chemical and physical properties of these materials allow for multiple repeated (discontinuous) or continuous use for the purposes of 6-APK production. From an economic point of view, the costs of producing the aforementioned types of biocatalysts are minimal, since the technology of their production does not require any commercial carrier, includes a small number of operations, and the raw material costs for their production are minimal.

We have now found that under selective reaction conditions for conducting the biochemical reaction, the aforementioned immobilized cell materials can be advantageously used for the subject method of producing 7-ADCK from desacetoxycephalosporins, especially from DAOC-G, both repeatedly (discontinuously or semi-continuously) and continuously, with high efficiency of enzymatic 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 content of active substance in the product 95% of theory). In conjunction with simplified and economized chemical processes for producing desacetoxycephalosporin G, which are the subject of the MS. of copyright certificates No. 209,641, No. 210,739, the subject method of enzymatic production of 7-ADCK according to the invention represents a complex, modern and economically feasible chemical-enzyme technology for the production of this pharmaceutically important intermediate, enabling the subsequent production of semi-synthetic desacetoxycephalosporins, especially cephalexin.

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

The method of enzyme production according to 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 methods are combined.

The production methods according to the invention are further

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

The washed, suctioned catalysts were used once more in the described manner and under the stated conditions, with the same results being achieved.

Example 1 and 2

Into a 21-liter glass reactor equipped with a fused porcelain frit S 2 at the bottom of the inner space of the reactor and a drain valve closing the space under the frit and a duplication device for circulating water temperature control of the reaction mixture maintained by an ultrathermostat, 900 ml of demineralized water was introduced and, with stirring, verExamples of embodiments.

Example 1

Chemically bound microbial cells with penicillin acylase activity produced according to Czechoslovak author's certificate No. 203 607 with parameters: specific activity 6.5 U/mg of wet mass of material, average particle size distribution 0.15 to 0.3 mm and average dry matter content in the material 35%, were gradually suspended in 5 g each in 45 ml each of aqueous solutions of K-salt DAOC-G (average active substance content 96.92%) in the range of initial substrate concentrations 1.0 to 10.0% mass/volume. The enzyme reaction took place at a temperature of 37 °C with continuous stirring and continuous pH adjustment with 0.1 to 1.0 N aqueous NaOH solution using a pH-stat in the range of pH values 7.6 to 7.8.

After the end of the enzymatic hydrolysis, which is indicated by the cessation of alkali consumption, the biocatalyst particles were separated by vacuum filtration through a terylene sheet and washed with water. The clear solutions of the reaction mixtures were cooled to a temperature of +4 °C and 25% aqueous sulfuric acid solution was added with constant stirring while potentiometrically controlling the pH to a value of 4.2. After adjusting the pH of the reaction mixtures, these acidified reaction mixtures were left undisturbed for about 1 hour at the specified temperature.

After quantitative crystallization of the product, the crystals from the individual reaction mixtures were gradually separated on a frit by vacuum filtration and the products were gradually suspended in 50 milliliters of cooled ethanol (+10 °C), separated again by filtration, washed on the frit with an excess of cold ethanol and dried to a constant mass. The achieved enzyme hydrolysis parameters, yields and 57 g of DAOC-G K-salt with an active substance content of 96.92% were dissolved in the water provided using a vertically placed steel anchor stirrer with a speed of 60/min. A glass pH electrode connected to a pH-stat, pH-meter and dosing device for continuous pH adjustment was attached to the reactor. After dissolution, the pH of the solution was adjusted to 7.9 with a one-normal NaOH solution.

After the solution was brought to a temperature of 37 °C, 110 grams of wet mass of the biocatalyst, the parameters of which are given in Example 1, were added with continuous stirring. Using the aforementioned automatic device for continuous pH adjustment, this was continuously maintained at a value of 7.8 with a 1 N NaOH solution during continuous stirring and tempering for the entire 90 min. of the enzyme reaction. After this time, the suspension was vacuum-sucked off by opening the outlet valve, while the fused frit between the bottom of the inner space of the reactor allowed for quantitative separation of the clear reaction mixture from the biocatalyst particles that remained in the reactor.

After suctioning off the reacted liquid, 900 ml of water, in which the K-salt of DAOC-G had been previously dissolved in the same concentration as in the previous cycle, was again introduced into the reactor, which contained the wet, unwashed, suctioned catalyst batch used in the previous cycle, and the entire procedure was repeated 10 times with the same catalyst batch.

After suctioning off the reacted liquid from each of the repeated enzyme hydrolysis cycles, it was poured into a refrigerated container in which 330 ml of gasoline cooled to +2 °C had been previously introduced and the liquid was continuously stirred and cooled so that the resulting temperature ranged from +1 to +5 degrees Celsius. The pH of the cooled, stirred liquid consisting of an organic water-immiscible and aqueous phase was adjusted to 4.2 in the manner described in Example 1. After approximately half an hour of stirring after pH adjustment, the crystallization mixture was left to stand at a temperature of about 4 °C for approximately 1 hour. The product was then separated by vacuum filtration, removed and suspended in 100 ml of ice-cold acetone, separated again by filtration, washed several more times on the filter with excess ice-cold water and dried to constant mass.

In 10 repeated cycles of using the same catalyst batch, a total of 292 g of 7-ADCK was obtained with an average active substance content of 96.94% and a total yield of 89.7% of theory. The specific activity of the catalyst did not change after its 10-fold repeated use. The loss of catalyst mass was 2% after 10 cycles based on the dry matter of the particles and did not affect the reaction rate and the degree of conversion between the 1st and 10th cycle of its repeated use.

Example 3

Chemically bound microbial cells with penicillin acylase activity, obtained by the procedure according to Czechoslovak patent certificate No. 209 265 with the following parameters:

specific activity 2.5 U/mg wet mass of the material, particles in the form of granules with a size of 1—3 mm and an average dry matter content of 40%, were filled in an amount of 55 g into a glass column with a diameter of 2.6 cm with a tempered jacket. 0.5 l of 0.5-molar phosphate buffer pH 7.9, in which K-salt DAOC-G was dissolved at a concentration of 3% mass/volume, was pumped through the immobilized material from the bottom up at a rate of 2030 ml/hour, at a temperature of 37 °C. In this arrangement, 5 repeated enzyme conversions of 0.5 l of the substrate solution of the indicated concentration were carried out with a continuous catalyst feed placed permanently in the column, recirculation of the reaction mixture with continuous pH adjustment with diluted ammonia water (1: : 1) to a value of 7.9 in a glass stirred vessel located outside the column, in which a pH electrode coupled to a pH-stat was built in. The reaction time for each enzyme conversion cycle in the recirculation reactor was 120 min.

After completion of each of the enzyme conversions, the reacted liquid was processed in the same manner as in Example 2, with the difference that instead of gasoline, pre-cooled petroleum ether in an amount of 160 ml was introduced and the aspirated product after crystallization was suspended again in petroleum ether, aspirated again and washed with ice water and dried to constant mass.

In 5 repeated cycles of using the same catalyst batch in a recirculating reactor with its buoyant bed, a total of 36.5 g of 7-ADCK was obtained with an average active substance content of 97.7% and a total yield of 91.09% of theory.

Example 4

1-liter glass laboratory fermentation tanks placed in a water bath heated to 37 °C 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 an alkali solution dosing device. 500 ml of a 6% aqueous solution of K-salt DAOC-G with a pH of 7.8 was added to each of the reactors and the stirrers were started, the intensity of which was 80 to 100 revolutions/min. The enzyme reaction initially proceeded in a batch manner with stirring, tempering and continuous adjustment of the pH with ammonia water to a value of 7.8, until the substrate concentration dropped in the first reactor to 10—15%, in the second reactor to 2—0% of the initial amount. After reaching this state, continuous operation was gradually started. From the tank containing a 6% DAOC-G solution cooled to +5 °C, the substrate solution was pumped into the 1st reactor by one pump at a rate of 0.46 liters/hour, the partially hydrolyzed substrate solution was pumped from the 1st reactor to the 2nd reactor by a 2nd pump at a rate of 0.47 liters/hour (correction for the increase in volume caused by the increase in volume in the first reactor due to the inflow of a larger amount of alkali) and from the 2nd reactor to the cooled tank of the reacted conversion mixture (+5 °C) by a third pump at a rate in the range of 0.46 to 0.48 liters/hour. In both reactors, the pH of the reaction mixture was maintained by continuously dosing 16% ammonia water using two independent pH-stats. Retention of catalyst particles in the reactors was achieved by providing the tubes sucking the reaction mixture from the reactors with a filter cloth, which was placed just above the stirrer.

Continuous stirred reactors connected in series were operated continuously for 25 hours, during which time 11.7 liters of substrate were quantitatively hydrolyzed to the desired product. Isolation of 7-ADCK was carried out by crystallization of the product from the aqueous phase only with suspension and ethanol washing according to the procedure given in Example 1. The product was obtained

370.6 g of product with 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 mass of cell paste of individually covalently crosslinked permeabilized and washed E. coli cells with penicillin acylase activity (8 U/mg of wet mass), obtained by the procedure according to the Czechoslovak Patent No. 201621, was suspended in 9 liters of drinking water and the pH of the suspension was adjusted to 7.9 with 1N NaOH solution. 10 liters of the homogeneous cell suspension thus prepared were pumped into a 201 liter glass fermentation tank placed in a water bath tempered at 37 °C, into which a glass pH electrode connected to the automatic device mentioned in the previous examples and continuous pH adjustment was built in. Then, 513 g of K-salt DAOC-G was poured into the stirred (250 rpm) aqueous suspension and the pH was continuously maintained at 7.8 with 1N NaOH solution after for 90 min. After this time, the cross-linked cells were centrifuged in a Sharples centrifuge. 8.4 liters of clear supernatant were obtained, from which 7-ADCK was isolated as described below.

The thin cell paste containing part of the reacted reaction mixture from the previous cycle was suspended again in 9 liters of water and the entire procedure of enzymatic hydrolysis of the fresh substrate solution was repeated 5 times in the described manner, while during the semi-continuous use, before starting the next cycle (after checking the amount of biomass by determining its dry matter), weight losses caused by manipulation during centrifugation were compensated by adding fresh stock of cross-linked and unused biomass of 20 grams of wet weight in the third cycle and 50 grams of wet weight in the fifth semi-continuous cycle.

From the individual semi-continuous cycles, 7-ADCK was isolated from the supernatant by the procedure given in Example 2 using the presented cooled gasoline in an amount of 3 liters/cycle, processing the supernatant of the reacted liquid, suspending the product after filtration in 1 liter of ice-cold acetone and washing with ice-cold water.

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

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.