CS211175B1 - Process for the preparation of 6-penicillane acid - Google Patents

Abstract

The invention protects the process of using the filtrate fermentation liquids and intermediate products benzylpenicillin (penicillin O), respectively. intermediates from different phases of its isolation as starting raw substrates for the enzyme preparing 6-aminopenicillinic acid (6-APK) using crosslinked, aggregated and bound cells with penicillin acylase activity, which leads to further economization production of this acid and thus semi-synthetic penicillins.

Description

The invention relates to a process for the preparation of 6-aminopenicillinic acid (hereinafter 6-APK) by enzymatic cleavage of benzylpenicillin by cross-linked or aggregated or bound cells of microorganisms having penicillin acylase activity.

6-APK is a key intermediate for the production of semisynthetic penicillins; a number of processes for its production are known, essentially by two basic routes, by chemical or enzymatic hydrolysis of the basic substance, i.e., isolated pure benzylpenicillin potassium or sodium salt. The first way, chemical hydrolysis, has essentially no choice but to proceed from the pure dry substance benzylpenicillin, since the principle of this hydrolysis itself dictates the anhydrous environment and the protection of chemically labile groups (TE Carrington, Proc. R. Soc., Lond., B 179, 321). (1971).

The other way, classical enzyme hydrolysis by native microorganism cells or soluble enzyme (NSR Pat. No. 1,114,766), must also be based on the substance potassium or sodium benzylpenicillin dissolved in water or buffer, as the native (soluble) penicillin acylase enzyme would was inactivated when using crude benzylpenicillin isolation intermediates, for example proteolytic non-digestion when using fermented soil, treatment with organic solvents, changing ionic strength, or various toxic impurities from other phases of benzylpenicillin isolation, which would adversely affect isolated 6-APK recovery.

Enzymatic hydrolysis of penicillin, especially benzylpenicillin, is a process that is increasingly used, since it is less expensive to use raw materials and equipment, especially when using bound enzymes or bound cells. Another advantage of bound penicillin acylase or bound cells with this activity is the possibility of their multiple or continuous use for the preparation of 6-APK, the quality and economy of the prooes being controlled not only by the cost of producing the biocatalyst but also by its operational (working) stability; in practice this means a value that indicates how many times the bound biocatalyst can be reused, or how many hours of continuous operation the biocatalyst is able to operate without a significant decrease in the rate of reaction and the degree of conversion of benzylpenicillin to 6-APK. It is understood that the operational stability of most bound biocatalysts will be higher when using pure benzylpenicillin substance (in the form of potassium or sodium salt) dissolved in water or buffer than when using benzylpenicillin intermediates.

Several types of chemically coupled cell-based biocatalysts with penicillin acylase activity have been developed to produce δ-APK by enzymatic hydrolysis of benzylpenicillin (US Patent Nos. 201,621, 197,101, 203,607 and 209,265), which allow the use of various intermediates in the production of benzylpenicillin. obtained, for example, directly from the fermented soil filtrate or at various stages of isolation without significantly affecting their operational stability, the method of covalent cross-linking of the enzyme in producer cells according to the cited inventions contributes significantly to the stability of bound cells even under various physical, physicochemical, chemical and biochemical effects .

It has been found that longer significant technical and, in particular, economic progress can be achieved through a particularly inexpensive method of separating and concentrating benzylpenicillin from fermented soil without conventional extraction in conjunction with cleavage of the obtained concentrate by the subject of the invention and will be explained in more detail below. By applying this process, the production cost per unit of benzylpenicillin in the filtrate of fermented soil can be reduced several times compared to the same amount of benzylpenicillin in the form of a solid potassium salt.

Furthermore, the advantageous fact that the enzyme digestion by the biocatalysts of the present invention is so substrate-specific that the addition of foreign matter in the crude benzylpenicillin concentrate obtained from the fermented soil filtrate does not interfere with the enzyme process and isolation of 6-APK from the reaction is utilized.

211175,

Accordingly, the present invention provides a process for the preparation of 6-aminopenicilenic acid by enzymatic cleavage of bezylpenicillin by cross-linked or aggregated or bound cells of microorganisms having penicillinase activity; The essence of this process consists in using as the starting material for the cleavage an aqueous phase having a pH of 7.0 to 8.0 obtained in some of the stages of the fermentation of the soils into benzylpenicillin containing a soluble salt of benzylpenicillin, in particular sodium, potassium or ammonium, at a concentration of 2-96% by weight, after which the 6-aminopenicillanic acid is isolated and separated, after the acidification of the reaction mixture at 0-10 ° C in the presence of an organic, water-immiscible solvent such as butyl acetate. the phase is worked up to phenylacetic acid.

According to the invention, the aqueous phase having a pH of 7.0 to 8.0 of the benzylpenicillin ammonium salt solution formed by neutralization with ammonia water from the free benzylpenicillin acid obtained from the filtrate of the fermented soil by acidification to pH 1.5 to 2.5 and separation, preferably by centrifugation at -5 to 0 ° C.

Another preferred option is to use a solution of the sodium or potassium salt of benzylpenicillin obtained from the rich butyl acetate extract containing free benzylpenicillin acid by reextraction with an aqueous solution of sodium or potassium hydroxide or carbonate as the aqueous phase having a pH of 7.0 to 8.0.

As an aqueous phase having a pH of 7.0 to 8.0, for example, the same aqueous solution of crude benzylpenicillin potassium salt obtained from the rich butyl acetate extract is salted out with a saturated potassium acetate solution and separated in solid form.

It goes without saying that, in addition to the illustrative examples, other aqueous phase events as starting materials are not excluded as long as they meet the conditions.

An essential advantage of the process according to the invention is that it is not necessary to isolate benzylpenicillin in the form of a pure substance, usually in the form of the potassium salt as the final product, for the production of δ-APK, thus eliminating the most costly isolation step, ie extracting the acidified filtrate In a non-extractive process for the preparation of an aqueous benzylpenicillin concentrate, the filtrate of the fermented soil is expediently cooled to a temperature of -5 to -3 ° C, preferably around -2 ° C, whereby the cryoscopic the effect of the soluble salts allows the filtrate to be supercooled to temperatures below 0 ° C with constant agitation, without the formation of icing.

The filtrate of fermented soil can be cooled both in the tank and through-flow during the processing of the whole batch. The supercooled filtrate is then acidified, again preferably by flow, with an inflow of an inorganic acid, for example 30% sulfuric acid, so that the resulting pH of the supercooled solution is 1.8 to 2.0. Under these conditions, the benzylpenicillin in the free acid form is certain, has been relatively stable for a short time, and its solubility is very low.

Benzylpenicillin in the form of the free acid does not crystallize out of solution, but precipitates as an amorphous turbidity, the particles of which form oily droplets, which agglomerate and solidify due to hypothermia. They are specifically heavier than water and can be separated by centrifugal force. In a flow-through method for treating the filtrate of fermented soil, one of the alternatives of the invention is to remove benzylpenicillin on continuously operating self-emptying centrifuges, where the material to be treated is only slightly warmed at a relatively fast flow rate. The heavy phase of benzylpenicillin blasted off in a relatively small volume of several percent is immediately neutralized with an alkali or buffer solution after being shot from the centrifuge drum.

The benzylpenicillin salt concentrate obtained is relatively pure; calculated on the dry matter, it contains more than 92% by weight. pure salts. Depending on the concentration of the alkali solution or buffer used, the concentrate obtained has a dry matter content of 10 to 30 wt%, and has a sufficient concentration to be used as a substrate for subsequent cleavage of benzylpenicillin by bound penicillin acylase in crosslinked or aggregated or bound cells of this enzyme.

Both the concentration and the purity of the concentrate depend on the quality of the fermented soil, the degree of autolysis of the benzylpenicillin producer, secondary contamination, etc. In cases where the fermented soil contains a large amount of proteins and other macromolecular substances of biological origin, it is advisable to partially deproteinize it in advance or pH range 4.0 to 6.0, preferably in the presence of flocculating agents, de-filter most of the macromolecular substances and cellular detritus by filtration or centrifugation before eliminating the free benzylpenicillin acid by lowering the pH of the fermented soil filtrate, separating, neutralizing and dissolving into an aqueous concentrate in the manner described.

In the case of completely anomalous batches of fermentation, for example in highly contaminated or poorly separable fermented soils, in addition to the previous partial deproteinization mentioned above, it is expedient to load the filtrate with a small amount of non-polar solvent of greater specific gravity than water, for example chloroform, trichlorethylene or the like; , and then eliminate the free benzylpenicillin acid by the above procedure. The amount of non-polar solvent used is either within the solubility limits or the emulsifiability of the solvent in the fermented soil, or exceeds this limit a few.

The amount of surfactant is in the range of 0.5 to 2.0% by volume, preferably nonionic surfactants, for example of various types based on alkylphenols, may be used. If the obtained aqueous neutralized benzylpenicillin concentrate is too colored, this coloring can be reduced by adsorption of the dyes to the activated carbon or ion exchange resins.

For enzyme cleavage, the benzylpenicillin concentrate is diluted with demineralized water so that the final benzylpenicillin concentration is equivalent to a 1-7% solution of benzylpenicillin potassium or sodium, depending on the type of bound biocatalyst and the choice of cleavage processes by cross-linked or aggregated or bound penicillin acylase cells.

The process according to the invention makes it possible, as already mentioned, to use as starting substrate for the enzymatic cleavage of various intermediates for the production of the substance benzylpenicillin and intermediates from different stages of isolation, in particular an aqueous concentrate obtained from butylacetate extract of fermented soil or crude benzylpenicillin salts. organic solvents, resulting in further economization of 6-APK production by eliminating losses in the isolation and purification of benzylpenicillin.

The enzymatic hydrolysis of the neutral diluted benzylpenicillin conoentate obtained by one of the described processes, depending on the type of bound cellular catalyst used, is carried out according to Art. 20, 621 and 209 676. In a batch re-use of the biocatalyst, cleavage takes 90 to 120 minutes and the amount of 6-APK generated exceeds 95% of theory.

It is obtained from the 6-APK conversion mixture by changing the pH to an isoelectric point, having previously been mixed with a water-immiscible nonpolar organic solvent, such as butyl acetate, chloroform, and the like, and precooling the mixture to a temperature in the range 10 to -2 ° C. Under these conditions, the crystallized 6-APK is then washed with water and acetone and dried. The 6-APK obtained is generally of such high quality that no further cleaning operations are required; its content in the product is between 97 and 100% by weight. and the insoluble residue is 0.5 to 5.0 wt.

Another advantageous consequence of the process of the invention is that after the enzyme hydrolysis process and the recovery of 6-APK, mother liquors can be evaluated to obtain phenylacetic acid in purity suitable for use as a precursor in benzylpenicillin biosynthesis and as an inducer of bio211175 synthesis of penicillin acylase enzyme in cell culture for the production of bound cell biocatalysts.

The process according to the invention is further illustrated by the following examples which illustrate some of the possible uses of suitable starting materials, but are not limited thereto.

Example 1

000 l of fermented broth containing 26,300 U penicillin G per ml filtrate was gradually cooled and at the same time stripped of the mycelium of the producing microorganism on a rotary vacuum filter. Combined with the wash water, 42,000 liters of a yellow-brown, almost clear filtrate having an activity of 18,930 U / ml was obtained, which was further cooled down to an average temperature of -2 ° C and fed to a mixing pump.

The pumped filtrate was acidified to pH 1.8-2.0 by the inflow of precooled 30% sulfuric acid. Acidification slightly increased the temperature of the filtrate, which was immediately after the mixing pump led to self-evacuating centrifuges of the Leval BRPX type with a gravitational field of about 6000 O on the drum periphery. The flow through the centrifuges was controlled to about 6000 l / h and the automatic partial emptying was ordered for one minute interval.

About 1 liter of free penicillin G acid was separated with each partial shot of the sediment, which fell out of the centrifuge into a cooled neutralizing cauldron equipped with a stirrer. During centrifugation, the temperature of the treated material increased and the apparatus arrangement therefore had the shortest possible joints. The filtrate flow through the centrifuge was selected as quickly as possible, and also after the shortest partial blast, both depending on the workability of the fermented soil and thus the ability to completely separate free penicillin acid G from the filtrate in the centrifugal field.

Penicillin acid G was precipitated in the neutralization boiler with stirring _; immediately neutralized by the addition of dilute ammonia water so that the resulting pH of the concentrate was in the range of 6.2 to 7.2. After the neutralization boiler was filled, the inlet from the centrifuge was switched to the second one and the concentrate from the first one was pumped into a central stainless steel tank where the obtained concentrate was maintained at a temperature of 3 to 8 ° C.

A total of 2,430 liters of a concentrate with a biological activity of 291,000 U / ml, ie.

% yield based on the fermented soil filtrate. The concentrate was mixed with 30 kg of Karbovent, after stirring for 15 min filtered through a filter press with an aluminous kieselguhr layer. The bleached concentrate was stored again at a temperature between 3 and 8 ° C and further used for the enzyme preparation of 6-APK in two repeated batches.

To a 5000 L reaction stainless steel boiler equipped with heating coils, stirrer and opaque 2 above the bottom outlet of a 1 m filter screen covered with a filter cloth, 1,200 L of bleached concentrate was pumped and not diluted to 3000 L with demineralized water. To the penicillin solution was added 500 L of an aqueous suspension of bound cells of the penicillin acylase product obtained by the procedure of US. Certificate No. 203 607.

The resulting dilute cell bound cells for cleavage of the penicillin G concentrate were 14% based on wet cell sediment and 2.15% based on dry matter, and then proceeded according to U.S. Pat. Certificate No. (PV 2533-79). The end of the solution cleavage process was 125 minutes at pH 7.8 and the resulting 6-APK concentration was 32,800 µg / ml reaction mixture. The cooled contents of the boiler were discharged immediately through the bottom outlet through a filter cloth and pumped through a Seitz 40x40 type safety filter with 20 inserts into the flow cooler.

Through the condenser, the solution was cooled to 2 ° C and then collected in a crystallization kettle where 350 L of butyl acetate was cooled, cooled to ° C. At the same time as the cold reaction mixture was fed, the contents of the boiler were vigorously stirred and the pH of the solution was adjusted to pH 4.2 with 30% sulfuric acid while maintaining a temperature of 2 ° C. After adjusting the entire volume of the solution in this manner, it was stirred for a further 90 minutes and further, after determining the residual content in the mother liquors after crystallization, the 6-ACK was filtered on a drum centrifuge, washed with cold water and then with acetone. A total of 80.1% of the theory of 6-APK with an active substance content of 98.9% and a moisture content of 0.4% was obtained in two repeated batches.

Example 2

36.5 np of fermented broth containing 18,400 U penicillin G / ml filtrate was filtered successively through a rotary filter while cooling. Since the fermented soil was contaminated during the last 48 hours of cultivation, the filtrate was not clear. To the 48 filtrate and wash water from the filter was added 1.5 vol% trichlorethylene and 0.2% surfactant (Slovafol 9095 with stirring and cooling) and the mixture was further cooled with stirring and passed through a flow cooler at -2 ° C to a directing pump, wherein it was continuously acidified with 20% sulfuric acid to pH values between 1.8 and 2.1 and further immediately processed on a self-evacuating centrifuge as described in Example 1.

The separated heavy phase was collected in a receiver and immediately neutralized with dilute ammonia water to pH 6.8 with vigorous stirring and cooling. After processing the entire batch of filtrate, the separated heavy solvent phase was drained from the aqueous concentrate in the receiver, and 0.2% activated carbon and 0.5% diatomaceous earth were added to the remaining concentrate. The aqueous concentrate was filtered and treated by a continuous process of enzyme cleavage of a 2% solution (based on dry matter of penicillin G ammonium salt) with the addition of phosphates (0.06 M phosphate buffer, pH 7.8) on a 3 column fixed bed bed system as described. Ss. No. 209 676.

The reaction mixture of the last stage of the cleavage cascade was continuously cooled to + 5 ° C by passing through an exchanger and continuously extracted on a rotary horizontal extractor adjusting the pH with 20% sulfuric acid to 1.9 with 6: 1 butyl acetate. The extracted aqueous phase was continuously collected in a boiler, where it was continuously neutralized with dilute potassium hydroxide solution to pH 6.8 under intensive rinsing and cooling to 2 ° C. Dilute 6-APK solution was continuously pumped from the neutralization boiler to a film evaporator and the solution was concentrated at vapor vapor temperature of 26 ° C 5 to 7 times and collected into the crystallization boiler. In this boiler, the concentrated solution was continuously cooled to +2 ° C and acidified to pH 4.3.

After adjusting and crystallizing the entire volume of the solution under continuous cooling, it was stirred for a further 2 hours. The crystallized 6-APK was centrifuged on a drum centrifuge, washed with ice water and acetone, and dried under vacuum. 163.2 kg of 6-APK were obtained with an active substance content of 97.6%.

The organic butyl acetate phase obtained in the continuous extraction contained, in addition to the small residues of penicillin and decomposition products, virtually all equivalent amounts of phenylacetic acid and was worked up as follows.

In the extraction boiler, the rich butyl acetate was neutralized with ammonia water to a pH of 7.8 to 8.0 and the aqueous phase obtained was separated. The heavy aqueous phase was separated and another extraction of the remaining butyl acetate phase with water was carried out, the aqueous phases combined to give a total of 360 L of a 32% ammonium phenylacetate solution, suitable for direct use in the precursor for penicillin G biosynthesis.

2,1175

Example 3 m ^ of fermented broth containing 16,900 µl penicillin G / M 1 filtrate was filtered successively by cooling through a rotary filter. Since the fermented broth was strongly opalescent due to partial autolysis of penicillin G producer cells, 0.25 vol% flocculant (Sedipur KA) was added to the 90 m ^ filter and wash the water from the filter with stirring and cooling. Then the filtrate was acidified with 3016 sulfuric acid at pH 4.6; After stirring and cooling to +5 ° C, the resulting precipitate was separated through a rotary filter and the clear filtrate was passed through a flow cooler at -2 ° C to a routing pump where it was continuously acidified with 20% sulfuric acid to a pH between 1.8 to 2.1 and the resulting heavy phase was immediately treated as described in Example 1, including immediate continuous neutralization with dilute ammonia water while stirring and cooling and decolorizing the neutral concentrate with a mixture of activated carbon and diatomaceous earth.

The cooled neutral penicillin G concentrate was successively diluted in portions to a volume of 2000 L with demineralized water to a concentration of 6.2% (w / v) benzylpenicillin; The cleavage was carried out in 6 semi-continuous cycles of hydrolysis of the screened and permeabilized cells according to the procedure described in U.S. Pat. No. 201 621 and 318 kg of 6-APK were produced having an active substance content of 98.6% insoluble residue 4.25%. Phenylacetic acid was regenerated as a 40% ammonium salt solution as described in Example 2.

Example 4

760 liters of rich butyl acetate extract, with an efficiency of 68,500 U of penicillin G, obtained by treating the filtrate of fermented soil, was re-extracted to a rotary extractor to

070 1 2,75% potassium carbonate solution in demineralised water. The obtained aqueous concentrate having an activity of 93,800 U / ml and a pH value of 7.5 was divided into two equal volumes, which were processed in a semi-continuous manner by means of the stitched cells according to the procedure of U.S. Pat. 201 621. From the first cycle, 100.30 kg of 6-APK of 98.6% purity was obtained, and from the second cycle, 111.20 kg of 6-APK of 97.8% purity.

Example5

The rich butyl acetate extract was treated into the crude benzylpenicillin K-salt with a saturated solution of potassium acetate in water. The crude K-salt, salted with excess potassium acetate from the aqueous phase, was separated on a high speed drum centrifuge, and after separation of the mother liquors, the crude non-washed benzylpenicillin salt was dissolved into a 6% solution and subjected to enzymatic hydrolysis by sieved and permeabilized cells. The obtained 6-APK had a purity of 98.8% and a yield of 80.2% of theory.

Claims (4)

SUBJECT OF THE INVENTION A process for the preparation of 6-aminopenicillanic acid by enzymatic cleavage of benzylpenicillin by cross-linked or aggregated or bound cells of microorganisms having penicillinoylase activity, characterized in that the starting material for the cleavage is an aqueous phase having a pH of 7.0 to 8.0 obtained in any of processing steps of the fermented soil into henzylpenicillin containing a soluble salt of benzylpenicillin, in particular sodium, potassium or ammonium salt, at a concentration of 2 to 7% by weight, after which it is separated by acidification of the reaction mixture at 0 to 10 ° C and in the presence of organic, a water-immiscible solvent such as butyl acetate, 6-aminopenicillanic acid, which is isolated and the separated organic phase is processed to phenylacetic acid. 251175 2. A process according to claim 1, wherein the aqueous phase having a pH of 7.0 to 8.0 is a solution of the ammonium salt of benzylplenicillin formed by neutralization with ammonia water of free benzylpenicillin acid obtained from the filtrate of fermented soil by acidification to pH 1. 5 to 2.5 and separation, preferably by centrifugation, at a temperature of -5 to 0 ° C. 3. The process of claim 1, wherein the aqueous phase having a pH of 7.0 to 8.0 is a benzylpenicillin sodium or potassium salt solution obtained from a rich butylacetate extract containing benzylpenicillin free acid by reextracting with an aqueous hydroxide solution; sodium or potassium carbonate. 4. A process according to claim 1, wherein the aqueous phase having a pH of 7.0 to 8.0 is an aqueous solution of the crude potassium salt of benzylpenicillin obtained from the rich butyl acetate extract by salting out with a saturated potassium acetate solution and separating it in solid form. .