CS209676B1 - Method of making the 6-amino-penicilane acid - Google Patents

Description

(54) Production method of 6-aminopenicillanic acid i

The present invention relates to a process for the preparation of 6-aminopenicillanic acid by the enzymatic hydrolysis of penicillin, in particular benzylic foam, by bound cells of the production microorganism Escherichia coli with a high content of foamedinacylase.

The methods used to produce 6-aminopenicillanic acid by enzymatic hydrolysis of penicillin to date include the use of soluble foam or inacylase, native cells with penicillin acylase activity, single covalently cross-linked cells or differently bound cells with foamed acylase activity and bound foamed acylase.

The use of bound cells or bound enzyme for the preparation of 6-aminopenicillanic acid (hereinafter referred to as 6-APK) has the advantage over the use of native cells or soluble enzyme that the bound materials can be reused or continuously used. Bound cells then have an economic advantage at a lower cost of preparation compared to the bound enzyme. When used for this purpose, the costs of the enzyme purification and isolation process are eliminated.

According to MS. No. 201621, individually covalently cross-linked and permeable Escherichia coli cells can be used to produce 6-APK. The disadvantage of this procedure, however, is the necessity to separate these cells because of their small size and thus poor sedimentation properties after the end of the enzyme reaction on large-capacity centrifuges, which places relatively high demands on the technical equipment of the plant.

These drawbacks are eliminated by using cells bound to the synthetic poly2 carrier materials according to U.S. Pat. No. 200802, but in addition to the high cost of carriers, their use is limited by the relatively low specific activity of the coupled preparations due to the cells being bound only to the surface of the carrier. Some disadvantages of this procedure are eliminated by the use of bound cells, the preparation of which is described in MS. No. 197101.

However, the disadvantage of the thus prepared bound cells is their relatively low specific activity and poor hydrodynamic properties, which prevent their use in fixed bed biocatalyst reactors. U.S. Pat. No. 4,113,566 discloses the use of bound cells prepared by crosslinking individual Proteus rettgeri cells with a bifunctional aldehyde and then polymerizing them into an organic matrix in a shallow bed flow reactor of about 6 cm to recirculate the penicillin solution. However, this process has the disadvantage of having to spread the biocatalyst over a large area.

We have now found that the mechanical and hydro-dynamic properties of high-endocyaninase-bound cells prepared according to CS. No. 203607, enable enzymatic cleavage of penicillin on an industrial scale in a stirred batch reactor, a fixed bed recirculation reactor and a continuous flow reactor consisting of a single fixed bed column, preferably a series of fixed bed columns of this material connected in a cascade / series /.

The process of the invention can be hydrolyzed

209 67 6 solution of penicillin by suspended particle bound particles in a stirred reactor. Since particle size reduction may occur when mixing with standard turbine type agitators, it is preferable to use agitators which allow short agitation times to be achieved with relatively low shear stresses of liquid and suspended particles.

For example, anchor stirrers and screw stirrers meet this requirement. At a bound cell activity of 6 to 9 µg / mg and a concentration of 10 to 150 mg / ml, cleavage of penicillin solutions at a concentration of 0.025 mol / l to 0.4 mol / l, preferably 0.13 to 0.25 mol / l, can be performed. . At higher concentrations, a high degree of enzyme activity inhibition and a disproportionate prolongation of the reaction time occur, and at lower concentrations there is an increased demand for product isolation. The temperature at which the reaction can be carried out is between 20 and 50 ° C, preferably between 35 and 40 ° C,

Penicillin and 6-APK are degraded at higher temperatures and the rate of hydrolysis decreases significantly at lower temperatures. The pH during the reaction is maintained in the range of 7 to 8, preferably 7.5 to 8.0, by the addition of an alkali such as ammonia water, sodium or potassium hydroxide solution or organic amines. At higher pH values the degradation products of penicillin and 6-APK accumulate, at lower values the reaction rate decreases.

The alkaline solution feed should be located in the reactor area where intensive stirring occurs, e.g. near a stirrer, thereby preventing local pH increase and degradation of penicillin and 6-APK. Under these conditions, the reaction is complete within 1 to 4 hours and product losses due to spontaneous degradation do not exceed 5 X. After completion of the reaction, the bound cells are either separated by filtration or can be decanted or washed in a suitably conditioned reactor (embedded sieve) and reused for Preparation 6-APK.

The process according to the invention can furthermore carry out the hydrolysis of the penicillin recirculation solution. The reaction mixture is pumped to the column and to which it is fed again after passing through the fixed bed column of the biocatalyst. In this case, you can. The hydrolysis can be carried out either <3 in an aqueous medium or preferably in a suitable buffer. The hydrolysis temperature is similar to that of the stirred reactor hydrolysis, preferably between 20 and 50 ° C, preferably between 35 and 40 ° C.

The pH at which the penicillin solution is maintained. The reaction mixture is between 7.5 and 8.5, preferably between 7.7 and 8.2. The efficiency of a fixed bed recirculation reactor depends on the difference between the pH at which penicillin breakdown is treated and the value at the column effluent. This value depends both on the catalyst bed height and the flow rate of the liquid through the column and on the quality and concentration of the buffering substances. The column can be filled with the bound cells alone or their mixtures with filtering aids such as kieselguhr. The optimum filling height is between 7 and 30 meters of flow rate in the range of 5 to 30 cra / min.

Under these conditions, the rate of reaction is minimally influenced by diffusion and when the pH gradient on the column of bound cells is minimal, respectively. the pH at the outflow from the fixed bed column of the biocatalyst does not fall below 7.0 (the same utilization of bound cell activity can be achieved as with a stirred reactor). The advantage of this procedure is that the bound cells are permanently placed in the column and there is no need to manipulate them between cycles of reuse.

A further process of the invention which can be used to prepare 6-APK is the continuous hydrolysis of penicillin at a flow rate of 0.025 to 0.4 mol / liter of solution through the column or preferably by a fixed bed column system of penicillin-acylase activity. The utilization of enzyme activity in a continuous fixed bed reactor is dependent on the working pH, which decreases as the penicillin solution flows through the column with increasing conversion.

For this reason, the efficiency of the reactor is dependent both on the concentration of penicillin and on the concentration of scavenging substances. At higher concentrations of penicillin, it is therefore advantageous to divide the reactor charge into a series of 2 or more columns. alkali to its original value.

It is particularly preferred to divide the bound cell material into columns of equal diameter so that the same portion of substrate is hydrolyzed after flowing through each column. A continuous enzyme reactor operating in this manner ensures a high degree of utilization of penicillin acylase activity at low demands on the concentration of buffering agents.

For a given penicillin concentration and the number of columns selected, the fill studies in individual columns can be calculated from the integrated form of penicillin acylase catalyzed hydrolysis of benzylpenicin / Warburton D., Dunnill P., Lilly M. Ď., Biotechnol. Bioeng., 1 5, 1 3, 1 973 /, which has the following shape:

SX-K in (1-X). omo 2 ./1 + - ° ₊ K _£

K K_K and fa s

O

2K

X

Κ 2K

KaΚ, Κ K _£ faf

S X + /,

2K To us it _c leduj ici importance:

S _o initial concentration of benzylic foam Inin K _in Michaelis constant X degree of conversion

Kf inhibitory constant for phenylacetic acid

K _and inhibition constant for 6-APK

Inhibθ substrate inhibition constant

E total enzyme activity in reactor V _r total reactor volume H total charge height P flow rate

If the values of all the above-mentioned constants are not known, the graphical dependence of penicillin conversion of bound cells on time in a stirred reactor may be used to approximate the relative packing height in individual columns. The times required to achieve a certain degree of conversion are then proportional to the total fill height required to achieve that degree of conversion. Of which also where the individual symbols ma

9 67 6 p

it follows that the ratio of the heights of the individual columns does not depend on the total filling height and flow rate.

However, for a given quality of bound cells and a given substrate concentration and a chosen degree of conversion, the ratio between the total column height and the flow rate is given.

Changes in capacity, respectively. The total reactor volume can be achieved while maintaining the degree of conversion, either by simultaneously increasing the incidence of the reactor by the flow rate, or by increasing the reactor diameter. The maximum reactor length is then limited by the hydrodynamic properties of the biocatalyst. A schematic of the entire 6-APK production line in a continuous manner using high-fractionated / acylase-bound bound cells is shown in Figure 1.

The scheme of the continuous cell line for the production of 6-APK using the bound cells of Fig. 1 is as follows.

Continuous preparation of demineralized water 3 7 ° C warm

Continuous preparation of benzylpentiline solution

Continuous binding of substance K-salts of benzylpenicillin or continuous inflow of concentrated penicillin solution

Dispenser Pump

Multiple cascade column continuous reactor

Regulators / pH /

Flow cooler

The evaporator pH-s t at

Container of organic solvent

1st boiler for continuous crystallization 1 2 From p ad

2nd Boiler for 14 _of 14 Controller Ice Tray Container

Wash acetone tank

Drum centrifuge

Drying

SUMMARY OF THE INVENTION Accordingly, the present invention provides a process for the preparation of 6-aminocyanic acid by enzymatic hydrolysis of penicillin, in particular benzylpenicillin, by bound cells of a high-foaming, Escherichia coli producing microorganism comprising a solution of penicillin at a concentration of 0.025-0.4 mol / liter, having a pH of 7.0 to 8.5, at 20 to 50 ° C contacts the cells of the producing microorganism Escherichia coli bound together by chemical bonding by reaction with glutardialdehyde and an organic diamine.

As a solution of penicillin at 7.0-8.5, a solution prepared by dissolving a solid substance in water or a buffer solution or obtained during the isolation of a fermentation broth filtrate, optionally diluted with water or a buffer solution, may be used.

For example, the method of the invention can be accomplished by mixing the penicillin solution with a suspension of chemically coupled Escherichia coli cells, adjusting the pH to 7.0 to 8.0 by dosing with alkali, separating the bound cell particles after completion of the reaction, and repeatedly used to break down penicillin.

This procedure can be varied so that the penicillin solution is recirculated through a fixed bed of chemically bound Escherichia coli cells at a height of 7 to 30 cm at a rate of 5 to 20 cm / min, wherein the pH of the solution is Passes the column to the value

7.5 to 8.5 by dosing the alkali, optionally so that. a penicillin solution having a pH of 7.5-8.5 flows through a column packed with chemical binding of Escherichia coli cells at a rate of 1 to 10 cm / min. or flow sequentially through at least two columns filled with a fixed bed of Escherichia coli mutually bonded by chemical bonding, at a rate of 1 to 10 cm / min, the pH of the solution being adjusted to 7.5 to 8.5 by addition of alkali prior to entering each subsequent column.

For the process of producing the 6-aminopentinic acid according to the invention, it is expedient to use, for example, bound cells with a high content of warbler and cylase, prepared from individual cells produced by the microorganism according to the procedure of U.S. Pat. Certificate No. 203 607.

In the following, the invention is illustrated in more detail, without being limited thereto. Examples

Example!

Bound E. coli cells with an activity of 6.7 µg / mg wet mass were prepared according to the procedure of US. No. 203,607. The material had the following properties: sp e citical activity 6.7 J / mg wet mass, suspension

2.5 g in 25 ml of water sedimented quantitatively in 3 minutes to a volume of 8 ml, the particle size ranged between 150-225 µm, the humidity of 60 µm, dark brown sand-like plates.

500 g of bound cells were used to hydrolyze 4.5 L of benzylpenicillin sodium salt (1600 µg / rag) at a concentration of 6.5 g / 100 ml of demineralized water. The reaction was carried out at 37 ° C with an anchor stirrer in a stainless steel vessel at the bottom of which a S 3 frit was attached. During the reaction, the pH of the reaction mixture was maintained at 7.8 by the addition of ammonia water. After completion of the reaction, the liquid was aspirated under the reactor under vacuum and further conversion started. The filtrate was cooled to 5 ° C and after addition of 1.2 L of butyl acetate the pH was adjusted to 4.3 with 1: 3 dilute sulfuric acid with stirring.

After standing for one hour, the crystalline product was filtered off and washed twice with 250 ml of ice-water and an equal amount of ice-acetone. In this way, 10 repetitive conversions were performed in 24 hours with an average hydrolysis time of 1.5 hours and no decrease in activity was observed. The total yield of 10 conversions was 1372 g of 6-aminopenicenic acid of 98.8% purity, corresponding to a yield of 79.5%; the content of insoluble residue in the product was 0.5%,

Example 2

The preparation of 6-aminopenicillanic acid was carried out in the same manner as in Example 1, except that the concentrate produced by converting penicillin from the butyl acetate extract of the fermentation broth filtrate to the aqueous phase using K 2 CO 3 was used instead of the crude benzylpenicillin sodium salt. The concentrate was diluted with demineralized water to a concentration of 100,000 µg penicillin G per ml prior to hydrolysis.

In three replicate experiments, no decrease in activity was observed and the total yield was 317 g of 6,9-lanoic acid of 97.9 Z purity, representing 63.2% of theory; the insoluble residue content of the product was 4.5.

Example 3

A column of 10 x 20 cm - (1x1 1570 ml) was filled with 977 g of bound cells described in Example 1 and washed with 3 liters of pH 7.8 buffer containing 0.048 mol KH2PO4 and 0.026 mol NagB2 O4.1G H2O. per liter.

A solution was passed through the column at 37 ° C

762 g of benzylpenicillin sodium in 11700 ml of buffer. After flowing through the column, the solution was collected in a stirred and tempered vessel in which its pH was adjusted by the addition of ammonia water to a value of 7.8 and was pumped back to the columns. After the conversion was completed, the column was washed with one liter of buffer. The converted solution and the wash solution were combined and cooled to 5 ° C. Then 3 liters of butyl acetate cooled to 5 ° C were added and the pH was adjusted to 4.3 with stirring with 1: 3 dilute sulfuric acid. After 1 hour of crystallization at 5 ° C, the 6-APK was filtered and washed 2 times 600 ml ice water and 2 times 600 ml ice acetone. The product was dried in a vacuum oven at 40 ° C. The cleavage results are shown in the following table.

batch conversion flow pressure losses on here / cm / min / s peels i / a tra /

1 92.2 15.1 0.9 2 91.9 13.6 1, 0 3 94, 1 14.8 0.9 4 95.3 14.2 1, 1 5 95,, 10.9 1, 2 . 6 94.9 11.8 1, 25 7 95.0 10.5 13 8 95.2. 10.2 13 9 92, 7 10.2 1.4 94.5 9.6 1, 6 94.3 9, 1 1, 5 1 2 93, 3 13.4 1, 1 13 93.6 11.6 1, 4 1 4 93, 7 9.4 1 »5 1 5 92.4 9.8 1, 4 / + / Mon end of batch 11 And the material was j mix L a again filled to columns.

The average yield of 6-APK was 78.3% at a purity of 98.2% and the content of insoluble residue in the product was 0.5%.

Example 4

A total of 704 g of the bound cells with penicillin acylase activity described in Example 1 were filled into four 9, 14, 33 and 34 cm high and 4 cm diameter columns with a temperature jacket and the system was washed with 2 liters of buffer containing 0.05 mol ΚΗ2 ^ θ4 ^and 0.005 mol of Na 2 B 4 O 3 · 10 2θ per liter, pH 8.4. The flow of the sodium salt solution was then started. benzylpenicillin in the above buffer at a rate of 3300 mL / hr. at 37 ° C. After passing through each column, the pH was adjusted to the value shown in the following table by dosing a 6 N NaOH solution:

pH inlet pH inlet

8.4 6.3

8.3 6.6

8.2 6.9

8.1 7.7

After effluent from the last column, the conversion of penicillin was 97.5 Z. The solution was continuously cooled to 20 ° C and vacuum evaporated to 1/3 of the original volume. The solution was then cooled to 5 ° C and the pH of the concentrate was adjusted to 4.3 with sulfuric acid diluted 2: 1 at 3-hour intervals. The pH adjustment was carried out with stirring in the presence of 600 ml of butyl acetate per liter of concentrate. After an hour of crystallization, the precipitated 6-aminopentanedioic acid was separated and washed twice with 500 ml of ice-water and an equal amount of ice-acetone. The product was dried under vacuum at 40 ° C. The average yield for operation lasting 170 hours was 89.2 7 at a purity of 98.4 7 and an insoluble residue content of 0.5 7,

No decrease in yield was observed during production.

Claims (7)

SUBJECT V A process for the production of 6-aminopenicillanic acid by enzymatic hydrolysis of penicillin, in particular benzylpenicillin, by bound cells of the production of Escherichia coli producing a high penicillinasease, characterized in that a penicillin solution having a concentration of 0.025-0.4 mol / liter having a pH value 7.0 to 8.5, at 20 to 50 ° C, contact with cells of the producing microorganism Escherichia coli bound together by chemical bonding by reaction with glutardaldehyde and an organic diamine. 2. A method according to claim 1, wherein a solution of penicillin having a pH of 7.0-8.5 prepared by dissolving the solid in water or buffer solution or obtained during isolation from the fermentation broth filtrate, optionally diluted with water, is used. or a buffer solution. 3. The method of claims 1 and 2 wherein the penicillin solution is mixed with a suspension of chemically bound Escherichia coli cells to adjust the pH to 7.0 to 8.0 by dosing alkali at a pH of 7.0 to 8.0. Y After the reaction, the bound cell particles are separated and reused to break down penicillin. 4. A method according to claim 1 or 2, wherein the solution of penicillin is recirculated through a fixed bed of chemically bound Escherichia coli cells at a height of 7 to 30 cm at a rate of 5 to 20 cm / min. adjusted to a value of 7.5 to 8.5 by dosing the alkali. 5. A method according to claim 1, wherein the pH of the penicillin solution 7.5 to 8.5 flows through a column packed with chemical binding of Escherichia coli cells at a rate of 1 to 10 cm / min. 6. The method of claim 1 wherein the penicillin solution flows sequentially through at least two columns packed with a fixed bed of Escherichia coli cells bound together by chemical bonding at a rate of 1 to 2. 10 cm / min, the pH of the solution being adjusted to 7.5 to 8.5 by the addition of alkali prior to inflow into each subsequent column.