CS200802B1 - Solid biocatalyst for enzymatic conversion of benzyl-penicillin into 6-amino-penicillanic acid and method for its preparing - Google Patents

Description

The present invention relates to a solid biocatalyst for the enzymatic conversion of benzylpenicillin to an acid

6-aminopenicillanic acid and a process for its preparation. By solid biocatalyst is meant a granular preparation consisting of microbial cells containing the active enzyme penicillin acylase, which are chemically bound to the surface of solid synthetic polymer particles in such a way that they are not released during the technological operations necessary for the enzymatic conversion of benzylpenicillin to 6-aminopenicillanic acid (6-APK). ).

6-APK is a starting material for the preparation of penicillin derivatives and is most preferably obtained from benzylpenicillin by cleavage of phenylacetic acid. This cleavage can be performed chemically or biocatalytically by the specific enzyme penicillin acylase (EC 3.5.1.11). Due to the chemical lability of the penicillin molecule, the chemical process for the preparation of 6-APK is very demanding (chemical protection of groups, reaction mixture temperature around -50 ° C, etc.).

The enzyme penicillin acylase is produced by various microorganisms and, by appropriate genetic manipulation, its production in cells can be increased by an order of magnitude as in the case of the strain Escherichia coli -.77, obtained according to U.S. Pat. No. 162274; strain is protected by MS. No. 188631.

For the actual conversion of benzylpenicillin, an isolated and to some degree pre-printed enzyme, preferably immobilized on a suitable carrier, or whole cells of the producing microorganism can be used. However, the use of whole cells represents considerable economic savings, since it has not been eliminated

200 802 is a process of cell disintegration, isolation and purification of an enzyme, or its immobilization to a solid support.

However, the use of a cell suspension for the actual conversion of benzylpenicillin has a number of technological and economic drawbacks. Due to their small size (ϋ, ό to 3 µm), cells are difficult to separate from the reaction mixture and cannot be reused. Therefore, not only for the process under consideration but also for other enzyme technology processes where it is advantageous to use whole cells, ways are sought to improve their separability. A method that would allow multiple use of a cell preparation would be optimal. To this end, it has been proposed in various works to use polymer particles containing physically immobilized cells. The most common method is the closure of cells into the entire volume of particles.

In particular, the method of the present invention relates to the physical enclosure of bacterial native cells containing penicillin acylase or partially disrupted, e.g. No. 113,908, into a (acrylamide) gel (T. Sato, T. Tosa, Ch Chibata, European. J. Appl. Microbiol., 2, 153, 1976) or into cellulose triacetate fibers (D. Dinelli, Proc. Eiochem., 7, 9, 1972) and the use of such closed native cells in a gelatin matrix to prepare 6-APK by cleavage of benzylpenicillin usually in a continuous manner, by circulating a strongly buffered benzylpenicillin solution and single pass or cascaded system.

These methods are not advantageous from an industrial point of view, since in this method the rate of the enzyme reaction is strongly limited by diffusion through a dual diffusion barrier (gel and cell wall), resulting in unfavorable gradient formation by local cumulation of reaction products,

6-APK and phenylacetic acid, and to inhibit the reaction, thereby reducing the reaction rate and degree of hydrolysis of benzylpenicillin, and the reaction is usually stopped at a low absolute yield (40-70%) at relatively low benzylpenicillin concentrations (1-4). Another disadvantage of utilizing the physical enclosure of native microbial cells into the gel matrix is that native cells lyse during reuse so that they release the enzyme along with the ballast proteins, thereby reducing the rate and efficiency of the enzymatic transformation, as well as product quality due to the contained proteins ( semi-synthetic penicillins from the 6-APK thus produced induce allergic reactions in humans and animals).

It is therefore imperative that not only the diffusion of the substrate to the enzyme, but especially the reaction products from the enzyme, be as fast as possible and that the enzyme is chemically stabilized in the cells. Vieth, Wang and Saini (Biotechnol. Bioeng., 15, 565, 1973) used so-called collagen complexing methods by dispersing the collagen, mixing the cell suspension and drying the film on a film. It is then treated with glutardialdehyde. Thus, they complexed Etreptomyces phaeochromogenes cells containing the enzyme glucose isomerase (Enzyme Engineering, ed. E. K. Pye and L. B. Wingard Jr., Plenum Press, N. Y., 1974). In this case, it is believed to be partially enclosed in the polymer network and partially bound to the surface of the polymer particles along with chemical stabilization of the enzyme by its covalent cross-linking within the cells.

The subject of the invention is a solid biocatalyst for the enzymatic conversion of benzylpenicillin to acid3

200 802 L-6-aminopenicillan, characterized in that it consists of a solid core of synthetic polymer of 10 to 1000 µm in size, on whose surface cells of a high-production Escherichia coli-77 strain containing a high amount of penicillin acylase are bound via glutaraldehyde. These cells are bound to the surface so tightly that they are not released during the technologically necessary operations used in the enzymatic conversion of benzylpenicillin to 6-aminopenicillanic acid and subsequent separation of the granular preparation, even during repeated production cycles. The binding of cells to a synthetic polymer bearing an amino group on its surface is mediated by glutardialdehyde, which also stabilizes the enzyme by its covalent cross-linking along with the cellular content.

The present invention further provides a process for the preparation of a solid biocatalyst, characterized in that to a granular synthetic polymer containing chemical groups capable of reacting with amines selected from the group consisting of an epoxide cycle, an aldehyde group, an azide group and activated esters of -CO-X type. - nitrophenol, 2,3,5-trichlorophenol, 8-hydroxyquinoline, N'-hydroxysuccinimide, N-hydroxyphthalimide are treated with ammonia or a substance containing at least two amino groups, at least one of which is primary at 10 to 100 ° C and pressure 0.1 to 0.3 MPa, after the reaction the excess substance and possible waste products are removed and the separated synthetic polymer bearing now on its ammonium surface is first treated with glutardialdehyde and after removing its unreacted fraction the granules are contacted with the cell suspension of Escherichia coli containing the active enzyme penicillin acylase.

Epoxy, aldehyde groups, carboxylic acid azides or activated carboxylic acid esters, ie esters -CO-X, wherein X is a residue of p-nitrophenol, 2,3,3-trichlorophenol, 8-hydroxyquinoline, N-hydroxysuccinimide, N-hydroxyphthalimide, are capable of reacting with amino-containing substances in such a way that they firmly bind the substance.

This reaction yields an intermediate consisting of a synthetic polymer bearing an amino group on the surface to which microbial cells are chemically bound by glutardialdehyde. This binding can be performed by first treating the synthetic polymer with glutardialdehyde and then contacting the cell suspension, or by mixing the cells with the synthetic polymer in a glutardialdehyde-containing medium. Contact of cells with glutardialdehyde is not a problem, but it stabilizes their enzymatic activity. The cells do not lyse and the enzyme is not released.

The method according to the invention can further be carried out in such a way that the cell suspension brought into contact with the granules already treated with glutardialdehyde contains Escherichia coli cells which have been pre-stored for 20 to 360 minutes at 0-30 ° C in a solution containing 0, 5 to 6% w / w glutardialdehyde of 5% *

In the preparation of the biocatalyst according to the invention, it is possible to introduce particles of a synthetic polymer which has already reacted with ammonia or a substance containing at least two amino groups, of which at least one is primary and therefore carries amino groups on its surface, into the cell suspension. Escherichia coli containing active penicillin acylase, wherein the liquid phase is 0.5 to 10 w / w glutardialdehyde and allowed to react at 20 to 40 ° C for 20 to 1440 minutes with gentle stirring at pH 6.5 to 8.5.

The advantage of the solid biocatalyst according to the invention is the presence of cells on the surface of particles, τη different pathway of molecules to the enzyme is not longer than in native cells. A further advantage is the broad visibility of the starting synthetic polymer, whose physical and chemical properties are determined by the requirement for permeability to the substrate and the products, as is the case with cell lines into the polymer mass. It is possible to use carriers which contain epoxy groups, for example the glycidyl methacrylate copolymer prepared and preferably according to U.S. Pat. No. 175112, aldehyde groups based on modifications of the preceding polymers according to MS. aut. No. 188372 or prepared by the polymerization of unsaturated aldehydes according to U.S. Pat. No. 174529, commercially available carriers of the Spheron, Enzacryl type, suitably modified to contain azide, active ester, and other amine-reactive groups. The advantage of the process according to the invention is that it is possible to select various molecules which introduce amino groups on the polymer surface. In this way, the cell microocyte can be adjusted and the length of the spacer link to which the polymer and the cell are connected can be regulated.

The granular biocatalyst can be used for both batch and continuous processes for converting benzylpenicillin to 6-aminopenicillanic acid when the reaction of the medium is suitably maintained. Of course, by using another microorganism (strain), a biocatalyst can be prepared according to the invention with different qualitative and quantitative representation of enzyme activities.

The invention is further illustrated by the following non-limiting examples.

EXAMPLE 1 A 5 L reaction vessel equipped with a stirrer was charged with 75 g of methcryialdehyde-divinylbenzene copolymers (1.76 mmol / g aldehyde groups), 2 L of 1,4-dioxane, 1 L of water and 530 g of hexamethylenediamine and stirred at 20 ° C. The product obtained after separation by washing with water and alcohol contained 0.96 mmol / g of bound diamine.

1.2 L of water was charged to a 2 L flask and 75 g of the previous reaction product and 75 g of E. coli-77 cell paste with penicillin acylase activity were suspended therein. While stirring at 30 ° C, 60 mL of a 50% aqueous glutardialdehyde solution was metered into the mixture and held at this temperature for 6 hours. The solid phase was filtered and washed with water to determine the captured enzyme activity, which was 70.1%. The specific activity of the preparation was 0.85 U / mg wet mass.

Penicillin acylase activity was evaluated spectrophotometrically according to MS. U.S. Patent No. 5,632,516;

116959 modified by Balasingham et al. (Biochim. Biophys. Acta, 276, 250, 1972), at 42 ° C and a pH of 7.6 in the region of zero-order reaction kinetics. The unit of enzyme activity is the amount of enzyme that catalyses the formation of one µmol of 6-APK from the benzylpenicillin Na-salt per hour.

135 g of wet immobilized cells as described were charged to a 1 L stirred glass reactor and made up to 500 ml with water. This slurry was run repeatedly to convert 7% (w / w) benzylpenicillin Na-salt to 6-APK in a batch manner at 37 ° C along with continuously maintaining the pH of the concentrated aqueous ammonia solution at 7.8. The mass of the benzylpenicillin introduced was corrected for the volume of the humid biocatalyst of the β-receptor. The preparation was capable of more than 30 repeated cycles of conversion with an average yield of β-APK it, 32% of theory in 2.5 hours. The total mass loss of solid biocatalyst between X), the repeated cycle of use was 7.8% (10.5 g). This loss was due to the loss of mannitol during filtration of the biocatalyst between cycles and losses due to the sampling required to monitor the reaction rate during each cycle. The percent ih./ic/activity between the i and 30 repeat cycles of use was 13> Z (i. Cycle> 1 X co.-r-r /; rr r. - cycla 79 Z conversion). Chromatographically determined total degradation percentage

7-penicillin and 6-APK averaged 4.7 *. In a vessel equipped with C.I. CNE-NEC ο _ .. ο .η ·· · ν ^· o yes IU xopolymeru methacrylaldehyde g divinylbenzene (1.63 moi / ioo g aidehydovýon okupiofc mt 1,4-dioxane - water (4: 1) in which was dissolved 2 g of N, N, is even Nidon stirred at 25 ° C. Washing with water and filtered alcohol and o '.' c -; _{"C, in} a .a. The n-phase contained 0.94 mmol / g of bound diamine.

g of this product were mixed with 2 g of cell paste of said strain suspended o) p. water and 1.6 mL of a 50Z aqueous solution of glutardiadehyde. After 6 hours of reaction at X., stirring was discontinued and the solid phase was filtered off, washed with water and reclaimed enzyme activity as described in Example 1 which was > The blot activity of the preparation was 0.76 J / mg wet mass.

The cells immobilized as described above were introduced into 1X ml ml of the glass reactor and made up to 50 ml with water and repeated repeatedly from 5 (w / w) Na. benzylpenicillin salts to 6-APK in a batch manner under the conditions of Example i. Three repeated conversion cycles were followed with an average yield of 7 '· 2 trays of 6-APK produced in 2 hours.

Example 3

In the same manner as in Example 1, 2 g of a methacryldehyldinylbenzene copolymer with 4 g of ethylenetriamine (1,5-diamino-3-azapentane) was modified to give a product containing 1.04 mmol / g of bound amine. Similar to Example 2, a training paste of said strain was performed to obtain a product with the entrapped activity of the enzyme 5? A and a specific activity of 0.66 J / mg wet mass.

With 5 g of wet cell-mobilized as described above, 5 repetitive conversion cycles were performed under the conditions of Example 2 with an average yield of conversion of 6-APK to 63% of theory in 2 hours.

Example. 4

The product containing bound ethylenediamine obtained according to the method of Example 2 (6 g) was mixed with 5 ml of a 50% aqueous solution of glutardialdehyde and 50 ml of water and stirred at room temperature for 1 hour. The filtered and washed water phase was suspended together with an equal amount of cell paste of said strain in 75 ml of water and the mixture was stirred at room temperature for 24 hours. The captured enzyme activity after separation was 3.2% and the specific activity was 0.09 U / mg wet.

200 802

Example 5 g of glycidyl methacrylate-ethylene dimethacrylate copolymer (3.5 mmol / g epoxy groups) was suspended at 80 ° C in 100 ml of commercial ammonia solution and allowed to react for 4 hours. After washing with water and separation, the product contained 2.0 mmol / g amino groups. 5 g of this carrier was mixed with 4 ml of a 50% aqueous solution of glutardialdehyde and 75 ml of water and 5 g of cell paste of said strain. The reaction was allowed to stir for 6 hours with stirring, after which the solid was filtered off and washed with water. The enzyme activity captured was 17%, the specific activity was 0.90 U / mg wet weight.

Enzyme activity stability tests were performed on the immobilized cell preparation described above by placing 10 g of the wet preparation in a 100 ml Erlenmeyer flask and suspending it in water to a volume of 50 ml; The flask was closed with a stopper. The suspension was shaken on a rotary shaker at 250 rpm at 38 ° C for 60 days, and enzyme activity in the filtrate and sediment samples was monitored at various time intervals. After 60 days of continuous shaking under the above conditions, there was no decrease in sediment enzyme activity; the filtrate was clear, nonalescent and its enzyme activity was zero.

Example 6 g of glycidyl methacrylate-ethylenedimethacrylate copolymer was mixed with 60 ml of ethyldiamine and kept at 40 ° C for 4 hours. After washing with water, the reaction product contained 2.4 mmol / g of bound diamine. The whole amount was mixed with the same amount of cell passage 1

of this strain and 150 ml of water and after stirring, 8 ml of a 50Λ aqueous glutardialdehyde solution were added. After a reaction time of 4 hours at 10 ° C, the solid was separated and washed with water, and the entrapped enzyme activity was determined to be 27.8 at a specific activity of 0.80 J / mg wet mass.

Enzyme activity stability assays as described in Example 5 were performed using the immobilized cell preparation described above, and after 6 days of continuous shaking, the filtrate was clear, non -alescent, and the enzyme activity therein was zero. There was no decrease in enzyme activity in the sediment.

EXAMPLE 7 g of a primary amino polymer obtained by the method of Example 5 were mixed with 5 ml of a 50 [mu] g aqueous solution of glutardialdehyde and 50 ml of water. After stirring at 20 ° C for one hour, 5 g of cell paste of the said strain was added to the mixture and stirring was continued for another 6 hours. After separation of the solid phase and washing with water, a captured enzyme activity of 5.1% and a specific activity of 0.23 J / mg wet mass were found.

Example 8

To a vessel equipped with a stirrer containing 100 ml of 1,4-dioxane-water (2; 1) was added 25 g of 1,6-diaminohexane and after dissolution 20 g of spherical (bead) copolymer of N- (hydroxypropyl) methacrylamide with p -nitrophenyl ester of methacryloylglycine crosslinked 200 Γ Γ 2 h 3-methylenepentamethylene trimethacrylate (preparation described in the author's certificate no. 173346). After stirring at room temperature for 1 hour, the polymer was washed first with 1X Wa ₂ CO ₃ solution and then with distilled water until the yellow color disappeared. The product contained 0.61 mmol / g of bound diamine. A further procedure of immobilizing the cells of said strain was carried out as in Example 1. The solid phase was filtered and washed with water. The determined entrapped enzyme activity was 52.4% and the specific activity of the preparation was 0.73 J / mg wet mass.

Enzyme activity stability tests were performed on the immobilized cell preparation described above by placing 27 g of wet mass in a 200 ml Erlenmeyer flask and suspending in distilled water to a volume of 100 ml. The procedure was as described in Examples 5 and 6. After 3 months of continuous shaking at 33 ° C, the enzyme activity found in the sediment was 37% of the original activity; the filtrate was clear, nonalescent and the enzyme activity therein was zero.

Example 9

To a 250 mL flask containing 100 mL of hexanol and 20 mL of anhydrous hydrazine was added 20 g of macroporous 2-hydroxyethyl methacrylate gel (Spheron), sealed with a reflux condenser and heated in an oil bath at 145 ° C for 8 hours. After cooling, the reaction mixture was diluted with water, the hexanol was discarded and the support washed with excess methanol and again with water. After the hydrazine had disappeared in the wash water, the support was suspended in 1000 ml of ice water and 25 ml of concentrated hydrochloric acid was added. A solution of 12.5 g of sodium nitrite in 20 ml of ice water was added with stirring at 3 ° C. After the reaction was completed, the gel was washed with ice water acidified with hydrochloric acid until the reaction to nitrites disappeared. The product was suspended in 100 ml phosphate buffer (0.2 M, pH 8.4) and 20 ml of a 50Λ solution of 1,6-diaminohexane was added under cooling. The pH of the reaction was maintained around 3.4 as necessary. The oel was dexantized and used as described in Example 1. The enzyme activity captured was 35 and the specific activity was 0.54 J / mg wet mass.

Example 10 g Enzacryl AH (Koch-Light Lab Ltd.) (was suspended in 50 ml of 2 N hydrochloric acid and cooled with ice. An ice-cold sodium nitrite solution (40 ml, 2%) was added slowly with stirring. The reaction was carried out as described in Examples 9 and 1. The captured enzyme activity was 27% and the specific activity was 0.36 J / mg wet mass.

Claims (4)

A solid biocatalyst for the enzymatic conversion of benzylpenicillin to 6-aminopenicillanic acid, characterized in that it consists of a solid core of a synthetic polymer containing chemical groups capable of reacting with amines selected from the group consisting of an epoxy cycle, an aldehyde 802 802 group, an azide group and activated -CO-X type esters, where X is a residue of p-nitrophenol, 2,3,5-trichlorophenol, ° -hydroxyquinoline, N-hydroxysuccinimide, N-hydroxyphthalimide of 10-1000 µm on the surface of which cells of high-production strain are bound by glutaraldehyde Escherichia coli-77 containing high levels of ilinacylase warblers. 2. A process for the preparation of a solid biocatalyst according to claim 1, wherein the granular synthetic polymer comprises chemical groups capable of reacting with amines selected from the group consisting of an epoxy cycle, an aldehyde group, an azide group and activated esters of the -CO-X type. p-nitrophenol, 2,3,5-trichlorophenol, 8-hydroxyquinoline, N-hydroxysuccinimide, N-hydroxyphthalimide are treated with ammonia or a substance containing at least two amino groups, at least one of which is primary at a temperature of 10 to 100 ° C and a pressure of 0, 1 to 0.3 iiPa, after the reaction, the excess substance and any reaction products are removed and the separated synthetic polymer bearing the amino group is first treated with glutardialdehyde and after removing its unreacted fraction, the granules are contacted with a suspension of Escherichia coli cells containing active enzyme penicillin acylase. 3. A process for preparing a solid biocatalyst according to claim 2, characterized in that the cell suspension brought into contact with the granules already treated with glutardialdehyde comprises Escherichia coli cells which have been pre-stored for 20 to 360 minutes at a temperature of 0 to 30 ° C in a solution containing from 0.5 to 6% by weight of glutamic acid 7. 4. A process for the preparation of a granular biocatalyst according to claim 2, characterized in that the synthetic polymer particles which have already reacted with ammonia with a substance containing at least two amino groups, at least one of which is primary and therefore already carrying amino groups on its surface, are suspended. Escherichia coli cells containing active penicillin acylase, wherein the liquid phase is 0.5-10 wt.% glutardialdehyde and allowed to heat at 20-40 ° C for 20-1440 minutes with gentle agitation at pH 6.5-8.5. Printed by Moravian Printing Works,