CS201621B1 - Method for semi-continuous production of 6-amino penicillanic acid - Google Patents

Description

The invention relates to a process for the semi-continuous production of 6-aminopenicillanic acid, a basic intermediate for the production of a number of clinically used semisynthetic penicillins. As is known, 6-aminopenicillanic acid (hereinafter, for brevity 6-APK) is obtained by chemical or enzymatic cleavage of penicillin, in particular benzylipenicillin, by the action of penieilinacylase produced by various microorganisms, most commonly Escherichia coli (German Pat. No. 1,114). 766). The prior art is almost exclusively a single use of native cells in a batch method of cleavage of penicillin, which is a significant disadvantage. In native cells, the possibility of reuse is very limited. Native cells undergo autolysis, the active enzyme penicillin acylase - it is washed out of them and with it the content of cells, especially intracellular soluble proteins, which in part accompany the resulting 6-APK throughout the isolation process and contaminate the final product. Semi-synthetic penicillins, prepared from the 6-APK thus obtained, cause allergic reactions in humans and animals. Decomposition of native cells is accelerated by the high ionic strength of the cleaved penicillin solution, respectively. the relatively high pH values at which the enzyme hydrolysis proceeds (7.6 to 8.2). The washed penicillin acylase is then lost as a result of the repetitive enzyme process. In practice, this means that the native cells can only be used once, that they must always be prepared by re-cultivation in a culture medium whose residues cannot be completely removed during cell separation. These residues contribute to the contamination of the final product or complicate the choice of a suitable 6-APK isolation process.

Some progress has been made in preparing 6-APK by cleaving penicillin with bound penicillin acylase. For the binding of this enzyme to a wide variety of inorganic and artificial organic materials, purified preparations obtained from microorganism cells are used by relatively expensive, complex and mainly loss-making processes. For example, in MS. No. 145849 discloses a method for purifying penicillin acylase and its binding to a carrier by culturing E. coli cells and separating biomass, mechanically crushing and repeatedly extracting the enzyme, fractional precipitation of the concentrate, dialysis thereof, and optionally lyophilizing the enzyme. This multi-step preparation results in a yield of about 30% of the original amount of enzyme, with a crude preparation purity of about 10% based on pure penicillin acylase. The enzyme preparation thus obtained is then used to bind to a carrier of the type of carbohydrate derivatives, after its prior chemical activation with cyanogen bromide.

However, carriers of this and similar types are very expensive formulations that increase the cost of the bound enzyme to the extent that only about 200-fold use of the bound enzyme is more efficient than the single use of native cells. The possibility of multiple use of the bound enzyme is not always provided, especially when batch operation is carried out in the reactor under agitation, which exerts a strong mechanical influence on the bound enzyme. Artificial materials with higher mechanical strength tend to have the disadvantage of limited diffusion speed and the process of penicillin hydrolysis slows down. The bound enzyme is denatured sooner or later in frequent reuse, especially when cheaper solutions of crude penicillin obtained from the fermented culture liquid are used for hydrolysis.

It has been found that the method of semi-continuous production of aminopenicillanic acid 6 by the cleavage of penicillins, in particular benzylpenicillin, by microbial cells containing penicillin acylase, in particular cells of the microorganism Escherichia coli according to the invention, has been found to be particularly high. penicillin acylase-containing microbial cells pre-chemically stabilized by treatment with a bifunctional agent such as dialdehydes, preferably glutardialdehyde, and using a solution of penicillin at a concentration of 1 to 10 wt. (volume, preferably 4 to 7 wt.%) volume, at pH 7.2 to 8.2, after the cleavage is complete and the reaction mixture is cooled to 0 to 5 ° C, the microbial cell suspension is separated and used to cleave the fresh batch solution. After adjusting the pH and temperature, 6-aminopenicillanic acid is recovered from the supernatant or mother liquor, and the cleavage of penicillin and isolation of 6-aminopenicillanic acid is repeated at least twice.

For enzyme cleavage according to the invention, a solution of the sodium or potassium salt of benzylpenicillin or a crude solution of benzylpenicillin in the form of an acid obtained in the treatment of fermented soil after the cultivation is expediently used as the penicillin solution.

The stabilization of cells of a microorganism, in particular Escherichia coli, is essential for the optimal course of the enzymatic cleavage of penicillin by the method according to the invention; the use of high penicillin acylase strains is particularly preferred. Stabilization of cells, resp. their intracellular content, together with the penicillin acylase, is achieved by covalent cross-linking with suitable bifunctional agents, preferably glutardialdehyde. This stabilization, which is an easily feasible reaction, provides a cell's resistance to autolysis and penicillin acylase fixation in cells without significantly losing their hydrolytic activity. The stabilized cell suspensions may be reused to break down penicillin, with a decrease in potency being mild and slow. The activity of the cross-linked cells can be further enhanced by treatment with a suitable organic solvent and a suitable wetting agent, which results in the leaching of unreacted cellular components, especially lipids that do not participate in the glutardialdehyde reaction and thereby wash the cells and improve their diffusion properties. If a high penicillin acylase strain is available, a more concentrated penicillin solution may be used under appropriate conditions than before, with very rapid conversion within 2-3 hours, with a high yield of very pure 6-aminopenicillanic acid.

In the process of semi-continuous production of 6-aminopenicillanic acid, after the first cleavage of the penicillin solution, usually at a concentration of 5-7% by weight. (The volume, which is carried out according to the usual process of vigorous stirring, pH and temperature control, separates the chemically stabilized cells, preferably by centrifugation, pouring fresh penicillin solution over them without any further treatment and running the next enzyme hydrolysis cycle under the same conditions. Handling losses of the cell suspension, which arise primarily during centrifugation, are usually replaced after five cleavage cycles by replenishing the suspensions of the stock stabilized cells.

Apart from the advantageous possibility of re-use of chemically stabilized cells, the advantage of the method according to the invention is its speed, since it is not necessary to ensure complete separation of the cell mass into the paste form and there is no need to wash the stabilized cells. The residual volume of the hydrolysis reaction mixture remaining in the sediment is immediately returned to the process at the start of the second and subsequent hydrolysis cycles. The former one-off procedure is thus changed to a repeated semi-continuous process, with a cycle repeated every 3 hours. The supernatant resulting from separation of the stabilized cell suspension is immediately processed to crystalline 6-APK, either by direct adjustment of pH and temperature or after concentration. A significant amount of the product can also be obtained from the concentrated mother liquors after phenylacetic acid removal in the usual manner.

Significantly, the 6-APK obtained by the process of the present invention is substantially cleaner than the products obtained by the one-time process using native unstabilized cells which partially autolyse during cleavage and contaminate the 6-APK with their breakdown products, especially proteins which accompany the 6-APK throughout the entire insulation, which is extremely difficult and costly to remove. The products obtained by cleavage with chemically stabilized cells do not need to be crystallized for further use and their solubility is better.

From this is evident the technical and economic effect in the production of the basic intermediate for the preparation of a number of therapeutically important semisynthetic penicillins.

The process according to the invention is described in detail in the following examples.

Example 1

A culture of a high-production mutant of the strain Escherichia coli CCM 2843, grown in a 5t fermentation tank, was concentrated by centrifugation to a volume of about 300 liters. The thickened native cell suspension contained about 400 mg wet weight / ml. This suspension was homogenized by stirring to adjust the pH to 7.5 with 50% sodium hydroxide solution. A 50% aqueous glutardialdehyde solution was then added with stirring to a final concentration in the reaction mixture of 2% (w / v). The reaction was carried out at 20-25 ° C for 3 hours. The reaction mixture was then diluted with 3000 liters of drinking water and the suspension was again concentrated to 300 liters by centrifugation.

The thus prepared chemically stabilized cell suspension was used to hydrolyze penicillin to 6-APK by transferring it to the reactor and diluting with stirring 2100 liters of deionized water to contain about 80 mg wet cell mass / ml, i.e. about 8% volume of sediment. The suspension temperature was set at 37 ° C and benzylpenicillin potassium was added to a final concentration of 7% (w / v).

The enzymatic hydrolysis proceeded with stirring at 37 ° C, while continuously maintaining the pH at 7.6-8.2 by the addition of a dilute aqueous ammonia solution. After 2 hours, the enzyme hydrolysis was terminated, as evidenced by both the cessation of ammonia solution consumption and the analytically determined conversion value of benzylpenicillin to 6-APK (95% of 6-APK theory in 2 hours). The reaction rate and the yield of 6-APK were monitored spectrophotometrically according to U.S. Pat. No. 116959.

Upon completion of hydrolysis, the reaction mixture was immediately cooled to 0-5 ° C and the cell suspension was centrifuged to 1/9 of the original volume and separated from the reaction mixture. The supernatant at about 2 ° C was acidified with concentrated hydrochloric acid to pH 4.2, thereby precipitating crystalline 6-APK, which was collected by filtration. The filtrate was acidified with concentrated hydrochloric acid to pH 2.0 and extracted with 30% (v / v) carbon tetrachloride. The aqueous phase was neutralized to pH 7.0 with concentrated ammonia solution and concentrated azeotropically in vacuo at 35 ^° to 1/3 volume. The pH was then adjusted to 4.2 with concentrated hydrochloric acid while cooling to 0 ° C. The crystallized 6-APK was filtered off. Phenylacetic acid was recovered from the extract.

The concentrated cell suspension was immediately transferred to another reactor, diluted again to a volume of 2400 liters and a repeated cycle of benzylpenicillin enzyme hydrolysis was performed under the conditions described above. In this way, the semi-continuous cleavage of benzylpenicillin was repeated with the same cell suspension of 12 kral while being reduced to yield of hydrolysis and isolation of 6-APK,

Example 2

Combined thickened suspensions of native E. coli cells from two 5-liter fermentation tanks, 600 liters, were centrifuged into a mold. cell paste, thereby separating the remains of the broth. The cell paste was suspended in 1000 liters of deionized water and homogenized with stirring to contain 300 mg wet mass of native cells / ml. The pH of this suspension was adjusted to 7.0 with stirring. by adding 50% (w / v) sodium hydroxide solution, and then adding a 25% aqueous solution of glutardialdehyde in an amount such that its final concentration in the reaction mixture is 1% (w / v). The reaction was carried out at 25 ° C for 3 hours. After this time, the reaction mixture was diluted with 4000 L of deionized water and concentrated to a volume of 400 L.

The thus prepared suspension of chemically stabilized cells was pumped into the reactor, diluted with 3600 liters of deionized water with stirring and homogenized to contain approximately 60 mg of wet cell mass / ml, i.e. 6% by volume of sediment. With this cell suspension, 25 cycles of a 4% (w / v) benzylpenicillin sodium salt solution were converted to 6-APK semicontinuously for 125 hours under the same conditions as in Example 1. After every fifth cycle of use, the reaction mixture was needed supplemented with a stock suspension of chemically stabilized cells to compensate for the loss of handling.

After cell separation, the supernatant was processed to 6-APK such that after acidification to pH 2.0 it was extracted with butyl acetate, the aqueous phase was basified to pH 7.5 and concentrated in vacuo to 1/2 of the original volume at 40 ° C. After cooling to 0 ° C and acidifying to pH 4.2, crystalline 6-APK precipitated, which was filtered off and washed with ice water and acetone. Phenylacetic acid was recovered from the extract in the usual manner.

The average analytical value of the conversion of benzylpenicillin to 6-APK was 90% of the theory in 2.5 hours, the average yield of dry 6-APK was 84.5% of the theory.

Example 3

A 000 liter culture of Escherichia coli CCM 2843 strain grown in a 15 t fermentation tank containing approximately 500 kg wet cell mass (120 kg dry cell mass) was concentrated to a volume of 1000 liters using sedimentation centrifuges. The concentrated cell suspension was homogenized by adjusting the pH to 7.0 with 25% sodium hydroxide. While stirring at 25 ° C, 40 L of a 25% aqueous glutardialdehyde solution was added. The reaction was allowed to stir at 25 ° C for 3 hours. The mixture was diluted with dilute water to a volume of 3500 liters and further stirred for 15 minutes. The reaction mixture was again concentrated on sedimentation centrifuges.

400 liters of such cross-linked cellular biomass were diluted with drinking water to a total volume of 500 liters and 50 liters of butyl acetate were added. The suspension was stirred for 5 hours at 25 ° C. After this time a solution of 10 kg of Slovafol 909 was added and further stirred for 15 minutes. After this time, the suspension was diluted with drinking water to a total volume of 4000 liters and separated on sedimentation centrifuges. The washed cell paste was suspended in a tank with vigorous stirring in 3500 liters of drinking water and, after homogenization, further stirred for 30 minutes. After this time, the suspension was concentrated by sedimentation centrifuges to a total volume of 400 liters. The concentrated suspension was again pumped into a stirred vessel, diluted to a total volume of 3500 liters with drinking water, and the concentration of cross-linked and washed cells from the stirred suspension was repeated once again to 400 liters as described above.

From a total of 500 kg of wet mass (120 kg of dry mass) of native E. coli cells, 350 kg of wet mass (84 kg of dry mass) of chemically stabilized cells was obtained by the described cross-linking, washing and washing process, which is 70 % yield.

300 liters of the thus prepared thickened suspension of chemically stabilized cells were pumped into the reactor, diluted to a total volume of 4000 liters with deionized water, and homogenized by stirring. After mixing, the cross-linked cell concentration was 60 mg wet mass / ml, i.e. 17-20 mg dry mass / ml. The suspension temperature was set at 37 ° C and benzylpenicillin potassium was added to a final concentration of 6.5% (w / v), ie 414 billion units (268 kg). The enzyme hydrolysis was carried out for 2 hours under the conditions of Example 1.

After the hydrolysis was complete, the chemically stabilized cells were concentrated to a total volume of 400 liters by sedimentation centrifuges and the concentrated cell suspension was cooled to 5 ° C. The clear reaction mixture after separation of the stabilized cells was pumped into tanks, diluted with 800 liters of methyl isobutyl ketone, stirred, cooled to 2 ° C and acidified to pH 4.2 with 40% w / v sulfuric acid solution. The mixture was then allowed to crystallize for 3-4 hours, then filtered, the filter cake covered with 10 liters of ice-cold deionized water, the crystals were removed and suspended in 120 liters of ice-cold deionized water and filtered again. The product was then covered with 10 liters of cooled acetone, removed and suspended in 120 liters of cooled acetone and filtered again. Phenylacetic acid and methyl isobutyl ketone were recovered from the filtrate.

400 liters of concentrated cell suspension in the remainder of the reaction mixture from the previous enzyme hydrolysis cycle was pumped back to the reactor after 4 hours at 5 ° C, diluted to 4000 liters with deionized water and a repeated benzylpenicillin enzyme hydrolysis cycle was performed.

In this manner, semi-continuous cleavage of benzylpenicillin to 6-APK was repeated with the same suspension of chemically stabilized cells 5 times with a time lag between the start of one and the end of the second cycle of 6 hours, achieving an average degree of conversion APK in isolation of 80% of theory without processing mother liquors. The quality of the product was assessed in terms of 4 criteria, namely purity (98.8%), insoluble triethylamine salt residue in diehlormethane (2.18%), humidity (0.9%) and tintometric color measurement (0.1 / 0). , 1).

Example 4

Concentration of E. coli cells after cultivation in a 151 fermenter and their chemical stabilization was performed as described in Example 3. Washing and washing of the stabilized cells was also performed in the same manner as described in Example 3 except that toluene was used instead of butyl acetate and instead of Slovafol 909, Slovafol 910 in the same concentrations.

The semi-continuous hydrolysis of the 6.5% benzylpenicillin potassium salt was also carried out in the same manner as in Example 3, except that 15 repetitive hydrolysis cycles were carried out, each time before the start of the fifth, tenth and fourteenth cycles. liters of thickened stock suspension of chemically stabilized cells to compensate for the loss of handling.

The 6-APK isolation procedure was also the same as in Example 3 except that 1,500 liters of butyl acetate was used instead of methyl isobutyl ketone.

The average conversion rate of 15 semicontinuous digestions was 92%, the average yield of 6-APK in isolation was 81.6% of the theory without processing of mother liquors. The quality of the 6-APK was similar to that shown in Example 3 except that the average value of the insoluble residue was 4.8%.

Claims (3)

1. A process for the semi-continuous production of 6-aminopenilic acid by digesting penicillins, in particular benzylpenicillin, with penicillin acylase-containing microbial cells, in particular cells of the microorganism Escherjchia coli, characterized in that the digestion is carried out using a suspension of microbial cells such as dialdehydes, preferably with glutardialdehyde, and using a solution of penicillin at a concentration of 1 to 10% w / v, preferably 4 to 7% w / v, at a pH of 7.2 to 8.2 po. After complete cleavage and cooling of the reaction mixture to 0-5 ° C, the microbial cell suspension is separated, used to cleave a fresh penicillin solution batch and 6-aminopenicillanic acid is isolated from the supernatant or mother liquor after adjusting the pH and temperature, the cleavage of penicillin and the isolation of 6-aminopenicillanic acid is repeated at least twice. 2. A process according to claim 1, wherein the solution of penicillin is a solution of sodium or potassium salt of benzylpenicillin. 3. The process of claim 1 wherein the penicillin solution is a crude acid solution of benzylpenicillin obtained by treating fermented soil after cultivation.