RU2381273C2 - Method for production of heterogeneous biocatalyst, biocatalyst based on hydrolase of alpha-aminoacid ethers and method for synthesis of aminobeta-lactam antibiotic under action of this biocatalyst - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method for production of heterogeneous biocatalyst for fermentative synthesis of aminobeta-lactam antibiotic based on intracellular hydrolase of alpha-aminoacid ethers from bacteria of Xanthomonas type, including damage of biomass in cells of Xanthomonas rubrilineans, extraction of ferment from it into water phase and its further immobilisation. Besides, damage of cells is carried out in stages, first damaging their integrity by cryogenic action, and then by treatment of water suspension of thawed cells by butyl acetate. Extraction of ferment into water phase is carried out in process of treated cell suspension heating first in conditions of spontaneously established gradient of pH, and then at constant value of pH. Produced cell-free extract is used for procedure of ferment immobilisation by means of its deposition by polyethylene glycol in the form of ferment aggregates and their further cross linkage in presence of glutaric dialdehyde. Thus obtained heterogeneous biocatalyst contains up to 0.95 g of protein/g of dry biocatalyst and has synthetase activity of up to 2200 ME/g of dry biocatalyst.

EFFECT: invention makes it possible to improve efficiency of biocatalyst production procedure, to increase activeness and stability of produced biocatalyst.

6 cl, 2 dwg, 4 tbl, 9 ex

Description

Technical field

The claimed group of inventions relates to the production of heterogeneous biocatalysts for the enzymatic synthesis of aminobeta-lactam antibiotic.

State of the art

Alpha-amino acid ester (AEG) hydrolases are called enzymes that catalyze the hydrolysis of alpha-amino acid esters, hydrolysis of the acylamide bonds of various cephalosporins and penicillins, as well as N-acylation of 7-aminocepheme and 6-aminopenam with alpha-amino acid derivatives (esters or amides). Such substrate specificity allows the use of these enzymes as biocatalysts for the synthesis of cephalosporins and penicicillins containing in the side chain an amino group in the alpha position of aminocephalosporins and aminopenicillins that make up the aminobeta-lactam antibiotic group.

Studies of the structure of AEG showed that these enzymes belong to the class of α / β hydrolases and contain the Ser-His-Asp catalytic triad in the active center. The structure, structure of the active center, substrate specificity, and catalytic properties of AEG are fundamentally different from such acylases of beta-lactams such as penicillin G acylase, also used for the synthesis of beta-lactam antibiotics. These enzymes structurally belong to the class of N-terminal hydrolases containing in the active center an N-terminal β-chain residue that acts as a nucleophile in a catalytic event.

In relation to the synthesis of aminobeta-lactam antibiotics, enzymes from bacteria of the Pseudomonadaceae family, which are alpha-amino acid ester hydrolases, are known [GB 1382255, Jpn. J. Antibiot Suppl. 30S: 230 (1977), CA 1044627, RU 2136759, KR 20010069097]. The biochemical, physicochemical and catalytic properties of AEG from Acetobacter turbidans [Enzyme Microb. Technol. 9: 339 (1987)], Xanthomonas citri [Agric. Biol. Chem. 44: 1069 (1980); Agric. Biol. Chem. 44: 1663 (1980)], Xanthomonas rubrilineans [Biochemistry 55 (12): 2226 (1990); Enzyme Microb. Technol. 15: 965 (1993)], Pseudomonas melanogenum [BBA - Protein Struct. & Molecul. Enzymol. 1040 (1): 12 (1990)]. It has been shown that these enzymes have a subunit structure. The enzyme from Pseudomonas melanogenum consists of two subunits with a molecular weight of 70,000-72,000 each, the enzymes from Acetobacter turbidans, Xanthomonas citri and Xanthomonas rubrilineans consist of four subunits of the same molecular weight. The biochemical and catalytic properties of AEG from various sources are close. The crystal structure was established, the composition of the active center was determined, and genes for enzymes from Xanthomonas citri were cloned [WO 02086111; J. Biol. Chem. 278: 23076 (2003); WO 02086127] and Acetobacter turbidans [WO 02086111; J. Biol. Chem. 277: 28474 (2002)].

The development of enzymatic synthesis of antibiotics using AEG requires the creation of technological heterogeneous biocatalysts, including by immobilizing enzymes isolated from biomass cells. The procedures for obtaining AEG-based biocatalysts consist of three main steps: (1) isolation of the enzyme from the biomass of cells by its destruction and extraction of the enzyme into the aqueous phase (obtaining cell-free extract); (2) obtaining an enzyme preparation of one degree or another degree of purification; (3) immobilization of the enzyme on any carrier.

Known methods of destroying the biomass of bacterial cells containing AEG under pressure (French Press) [Enzyme Microb. Technol. 15: 965 (1993); Enzyme Microb. Technol. 25: 336 (1999)] or by homogenization [BBA - Protein Struct. & Molecul. Enzymol. 1040 (1): 12 (1990)]. These methods lead to the total destruction of cellular structures and allow almost completely extracting the target enzyme from the destroyed cellular biomass, providing a high activity yield at the stage of obtaining cell-free extract (up to 100%). However, proteins and other water-soluble substances also easily pass into the aqueous phase from completely destroyed cells; therefore, the obtained cell-free extract contains many ballast substances and has a low specific activity. A method is described for destroying the biomass of bacterial cells containing AEG using ultrasound in the presence of surfactants [Biotechnol. Bioeng. 23 (2): 361 (1981)]. When using this relatively mild method, the cells are not completely destroyed, which leads to a decrease in the yield of activity during the extraction of the enzyme, however, it provides a higher purity of the extracellular extract. The method of destroying the biomass of bacterial cells containing AEG under the action of an organic solvent of butyl acetate immiscible with water [Biochemistry 55 (12): 2226 (1990); Zaslavskaya P.L.,. Biotechnol. Appl. Biochem. 18: 299 (1993)] acts more selectively than those discussed above, but its effectiveness is not characterized by a sufficiently high yield of activity.

Table 1
Comparison of different methods of destruction of the biomass of cells of bacteria of the genus Xanthomonas - producers of AEG Microorganism The method of destruction of cells The output of the destruction procedure,% Specific activity of cell-free extract, IU / mg protein Link Xanthomonas citri Ultrasound in the presence of surfactants 80 2 Choi W.G. et al. Biotechnol. Bioeng. 23 (2): 361 (1981) - Closest equivalent Xanthomonas rubrilineans Under pressure (French Press) one hundred Less than 1 Blinkovsky A.M. et al. Enzyme Microb. Technol. 15: 965 (1993) Xanthomonas rubrilineans Butyl acetate (10 vol.%), 40 ° C, pH 8. 70-90 Less than 1 Krestyanova I.N. et al. Biochemistry 55 (12): 2226 (1990); Zaslavskaya P.L. et al. Biotechnol. Appl. Biochem. 18: 299 (1993) Xanthomonas rubrilineans Butyl acetate (4.5 vol.%), 30 ° С, pH gradient one hundred 2.5-4.5 The inventive method (see example 1 and 3)

Table 1 compares various methods of destroying the biomass of cells of bacteria of the genus Xanthomonas with the aim of extracting the AEG enzyme from them. The values of the specific activity of the cell-free extract are given in international units (ME) * (* - The amount of this drug that catalyzes the conversion of 1 μmol of the substrate (or the formation of 1 μmol of the product) is taken as the international unit (ME) of the enzymatic activity of the drug for any biocatalytic transformation ) in the selected conditions for 1 minute), referred to a milligram of protein, and in all cases are reduced to the activity of the synthesis of the cephalexin antibiotic using experimental ratios between g idrolase and synthetase activity.

Known methods for producing AEG from various bacterial sources in water-soluble form, aimed at obtaining highly purified enzyme preparations, up to obtaining homogeneous proteins [Enzyme Microb. Technol. 9: 339 (1987); BBA - Protein Struct. & Molecul. Enzymol. 1040 (1): 12 (1990); Biochemistry 55 (12): 2226 (1990); Enzyme Microb. Technol. 15: 965 (1993)]. These methods necessarily include several successive stages of chromatographic purification of the enzyme on various sorbents and are unsuitable for use in the production of technological biocatalysts due to the complexity and duration of the procedure, as well as low activity yields.

Known methods for producing heterogeneous AEG-based biocatalysts from Acetobacter turbidans by isolating an enzyme from cell biomass, partially purifying it, and then immobilizing it. A method for isolating AEG, consisting in the mechanical destruction of cell biomass under pressure, extraction of the enzyme into the aqueous phase and partial purification of the AEG by treating the aqueous extract with streptomycin followed by dialysis [Enzyme Microb.Technol. 25: 336 (1999)] is not characterized in terms of yield of activity of the procedure. The resulting crude AEG preparation has a very low specific activity, determined with respect to the hydrolysis of benzylpenicillin - 0.06 IU / mg protein. A method for covalent immobilization of a crude enzyme obtained in this way on glutaraldehyde-modified agarose [Enzyme Microb.Technol. 25: 336 (1999)], as well as an improved method for its immobilization, including an additional modification of the enzyme immobilized on agarose with aldehyde dextran [J. Mol. Catal. In: Enzym. 11: 633 (2001)], time-consuming and require special training media. These methods are not characterized in terms of the yield of activity of the immobilization procedure and the absolute activity of the biocatalyst in relation to the synthesis of any antibiotic (synthetase activity). There is also known a method for deeper purification of AEG from Acetobacter turbidans and its covalent immobilization on agarose followed by aldehyde dextran modification [Biomacromolecules 2 (1): 95 (2001)]. The crude aqueous extract with a specific activity of 0.1 IU / mg protein, determined with respect to the hydrolysis of D-phenylglycine methyl ester (MEFG), is subjected to two-step chromatographic purification on CM-52 and phenyl-sepharose. The yield of activity of the purification procedure is 62% with respect to the crude extract, however, the total yield with respect to the biomass of the cells is not known. The AEG is purified 420 times as compared to a crude aqueous extract, reaching a specific activity of 42 IU / mg protein. The procedure for immobilizing a purified AEG was not characterized in terms of yield activity and absolute synthetase activity of the product. This method of obtaining a biocatalyst is very laborious and ineffective due to the application of the deep purification of the enzyme before immobilization and the complex multi-stage immobilization procedure.

Closest to the claimed method for producing a heterogeneous biocatalyst based on intracellular hydrolase of alpha-amino acid esters from bacteria of the genus Xanthomonas is a method for producing a biocatalyst based on an enzyme from a microorganism of the same genus, namely, Xanthomonas citri [Biotechnol. Bioeng. 23 (2): 361 (1981)]. Like the inventive method, the closest analogue reflects the complete procedure for obtaining a heterogeneous biocatalyst based on the biomass of cells (figure 1).

As the closest analogue for the claimed heterogeneous biocatalyst based on intracellular hydrolase of alpha-amino acid esters from bacteria of the genus Xanthomonas, a heterogeneous AEG based biocatalyst selected from Xanthomonas citri immobilized on kaolin obtained by the method of [Biotechnol. Bioeng. 23 (2): 361 (1981)], containing not more than 0.08 g of protein / g of dry biocatalyst and characterized by a synthesis activity of 510 IU / g of dry biocatalyst.

As the closest analogue to the proposed method for the enzymatic synthesis of aminobeta-lactam antibiotic by acylating a key beta-lactam compound with the corresponding derivatives of D-aminophenylacetic acid esters, a method for synthesizing the aminofephalosporin antibiotic cephalexin by acylating 7-aminodetacetoxycephalosuccinicamino-aminomethyl-dimethyl-aminosuccinic acid-7-aminomethyl under the action of a heterogeneous biocatalyst based on AEG from Xanthomonas citri [Biotechnol. Bioeng. 23 (2): 361 (1981)].

A method of obtaining a heterogeneous biocatalyst according to the closest analogue is as follows. Cell biomass is subjected to ultrasonic disintegration in the presence of surfactants and the enzyme is extracted into the aqueous phase. A cell-free extract with a specific activity of 2 IU / mg protein, determined by the synthesis of cephalexin, is obtained. The output of activity at the stage of obtaining cell-free extract (η _extract ) reaches 79%. AEG is concentrated from a cell-free extract by fractional precipitation with ammonium sulfate. After dissolution of the enzyme precipitate, the preparation is desalted by dialysis and subjected to two-stage chromatographic purification first on DEAE-cellulose and then on 4-B Sepharose. After each of the chromatographic steps, the enzyme preparation is freeze-dried. The yield of synthetase activity at the stages of concentration and purification of AEG (η _Pts ) is 28%, while the degree of purification of the drug reaches 30 times. The output of the procedure for obtaining an enzyme preparation suitable for immobilization (η _FP ) is determined by the outputs of the stages of extraction and purification of the enzyme and is 22%: (η _FP = η _extract · η _Pts ).

The resulting partially purified AEG preparation with specific activity for cephalexin synthesis of 60 IU / mg protein is immobilized by adsorption on bentonite or kaolin. The output of the activity of the immobilization procedure (η _them ) is 71 ÷ 92% depending on the media used. The total output of the process of obtaining a heterogeneous biocatalyst from cell biomass (η _total ) according to the closest analogue is 16-20%, the duration of the procedure is at least 70 hours.

The best of the biocatalysts obtained by the method according to the closest analogue is AEG immobilized on kaolin. This heterogeneous biocatalyst contains not more than 0.08 g of protein / g of dry biocatalyst and has synthetase activity (in relation to the synthesis of cephalexin), referred to the mass of the dry carrier, 510 IU / g dry. Operational tests of it in the process of cephalexin synthesis at 30 ° C using a low (4 mg / ml) initial concentration of 7-ADCA substrate show that after 10 cycles the residual activity of the biocatalyst is 85% of the original. The half-inactivation period of the biocatalyst calculated according to these data is τ _1/2 = 42 cycles. However, the processing of the experimental data presented in the closest analogue on the dependence of the biocatalyst operating stability on the number of cycles shows that the biocatalyst inactivation does not obey the first-order kinetic laws and proceeds 4-5 times faster in the first five cycles than in the next five. This may indicate a partial washing off of the enzyme from the carrier during the synthesis, even at very low substrate concentrations. Raising the temperature to 35 ° C reduces the operational stability of the AEG immobilized on kaolin by 8 times (τ _1/2 = 5 cycles).

The method for synthesizing the cephalexin aminocephalosporin antibiotic according to the closest analogue consists in acylating 7-ADCA, taken at a concentration of 4 mg / ml, with D-phenylglycine methyl ester at a molar ratio of acylating agent (MEFG) and 7-ADCA in the initial reaction mixture from 1 to 3. Reaction synthesis is carried out for 2 ÷ 3 hours at a pH of 6.2 and a temperature of 35 ° C in an aqueous medium under the action of a heterogeneous biocatalyst based on AEG from Xanthomonas citri immobilized on kaolin. The best yield of cephalexin (85% by 7-ADCA) was achieved using a molar ratio of substrates of 3: 1. There are no data on the synthesis of other aminocephalosporins or aminopenicillins in the closest analogue.

The main disadvantages of the heterogeneous biocatalyst, as well as the methods for its preparation and use according to the closest analogue, are as follows:

1. The complexity and multi-stage procedure used for the isolation and purification of the enzyme; associated with this large loss of enzymatic activity, causing a low yield of activity of the procedure for obtaining the biocatalyst as a whole.

2. Obtaining a biocatalyst with a low protein content (not more than 0.08 g protein / g dry biocatalyst) due to the use of the method of immobilization of the enzyme on a carrier that is ballast.

3. The low level of activity of the final product is a heterogeneous biocatalyst.

4. The low level of operational stability of the biocatalyst due to the use of the adsorption immobilization method, which does not provide multi-point, strong and irreversible binding of the enzyme to the carrier; the consequence of this is the low thermal stability of the immobilized enzyme, as well as the possible desorption of the enzyme and other proteins by a solution of substrates (especially at high concentrations of substrates).

5. The need to use low concentrations of substrates in the synthesis of antibiotics to reduce the risk of contamination of the target product with protein impurities.

Disclosure of invention

The claimed group of inventions is aimed at solving the following problems:

- increasing the efficiency of the method for producing a heterogeneous biocatalyst based on a hydrolase of alpha-amino acid esters by simplifying the procedure for producing a biocatalyst and increasing the yield of enzymatic (synthetase) activity of this procedure;

- increasing the activity and stability of the resulting biocatalyst;

- development of a method for the enzymatic synthesis of aminobeta-lactam antibiotic using the resulting heterogeneous biocatalyst.

The tasks are solved by the combined use in the process of obtaining a heterogeneous biocatalyst of a number of techniques carried out in the selected optimized conditions. To isolate AEG, the biomass of Xanthomonas rubrilineans bacteria cells is used. Cells containing the enzyme are destroyed in stages under mild conditions, first violating their integrity by cryogenic exposure, and then treating the aqueous suspension of thawed cells with butyl acetate. The enzyme is extracted from the destroyed cellular biomass by extraction into the aqueous phase. The cell-free extract obtained after separation of the solid phase, without any purification, is used to immobilize the enzyme. To obtain such a cell-free extract that is directly suitable for immobilization, the procedure for destroying cell biomass and extracting the enzyme is carried out under mild conditions optimized for the concentration of cell biomass in suspension, the concentration of butyl acetate, temperature, pH, and the moment the process is stopped. Extraction is carried out first under conditions of a spontaneously established pH gradient, and then at a constant pH value maintained by adding a titrant, the flow rate of which is recorded.

The criterion for choosing the moment the extraction is stopped is the change in the rate of titrant consumption. Obtaining a cell-free extract of the required purity upon reaching (100 ± 5)% of the activity yield in the process of enzyme extraction is provided by stopping the procedure at a time when the rate of titrant consumption decreases a certain number of times compared to the initial value. The degree of reduction in titrant consumption, providing optimal results (K _opt ), depends on the Xanthomonas rubrilineans strain used, as well as on the conditions of biosynthesis of the culture and is established in a series of preliminary experiments (see table 2 in Example 3).

Immobilization is carried out by deposition of AEG in the form of enzyme aggregates from a cell-free extract under the action of polyethylene glycol with a selected molecular weight, taken in the amount necessary for complete precipitation of the target enzyme, and subsequent cross-linking of these aggregates with glutaraldehyde, taken in an optimized concentration calculated with respect to the content protein in a cell-free extract (see Example 4, No. 1-4 in table 3). Obtaining a cell-free extract under conditions optimized for each strain provides a standardized starting material for the immobilization procedure, as can be seen when comparing No. 2, 3 and 5 of table 3 in Example 4.

The inventive method for producing a heterogeneous biocatalyst is schematically represented in figure 1 and is as follows. The biomass of Xanthomonas rubrilineans bacteria cells containing the AEG enzyme is placed in a freezer with a temperature of -25 ° C or lower, preferably from -25 to -30 ° C, and incubated for the time required to freeze the entire volume of biomass to a predetermined temperature. Then the cell biomass is thawed, keeping the required time at room temperature, and suspended in Tris-HCl buffer, 0.05 mol / L, pH 7.8. The concentration of cell biomass in suspension is 20 ÷ 100 mg dry / ml, preferably 40 ÷ 50 mg dry / ml. Butyl acetate is added to the resulting suspension at a concentration of 2 ÷ 6 vol.%, Preferably 4.5 vol.%. The procedure for the destruction of cell biomass and extraction of the enzyme into the aqueous phase is carried out with vigorous stirring at a temperature of 25 ÷ 35 ° C, preferably 30 ° C. The extraction is carried out first under conditions of a spontaneously established pH gradient, and then at a constant pH value maintained by adding alkaline titrant, and its consumption is recorded. As the titrant, an aqueous solution of ammonia or other titrants, such as an aqueous solution of sodium hydroxide or potassium hydroxide, are used.

The pH range that provides optimal results depends on the Xanthomonas rubrilineans strain used, as well as on the biosynthesis conditions of the culture and is set in a series of preliminary experiments. The enzyme extraction procedure is stopped when the flow rate of the ammonia solution is reduced by the experimentally determined number of times K _opt . The time to reach K _opt , that is, the duration of the process of destruction of cell biomass and extraction of the enzyme is not standardized and ranges from 3 to 7 hours, depending on the biochemical parameters of the initial cell biomass, as well as on the selected process conditions in terms of temperature and concentration of cell biomass and butyl acetate.

The destroyed cellular biomass is separated from the cell-free extract by any available effective method, for example by centrifugation, and washed with sodium acetate buffer, 0.1 mol / L, pH 6.5, containing 0.005 mol / L calcium acetate. The buffer is used in an amount of 20% of the initial volume of the cell suspension. Wash water is separated from the destroyed cellular biomass and attached to the acellular extract. Get a clear cell-free extract with a specific activity of 2.5 ÷ 4.5 IU / mg protein, determined by the synthesis of cephalexin. The activity yield at the enzyme extraction stage with respect to the activity of the initial cell biomass is (100 ± 5)%, that is, it is quantitative within the experimental error.

The obtained cell-free extract is used without any purification for the immobilization of the AEG enzyme by obtaining enzyme aggregates and their cross-linking. Thus, the output of the procedure for obtaining an enzyme preparation suitable for immobilization is identical to the output of the enzyme extraction procedure:

η _FP = η _extract = (100 ± 5)%.

In order to precipitate the enzyme aggregates, polyethylene glycol with a molecular weight of 4000 ÷ 10000, preferably 6000 ÷ 8000, taken in an amount necessary for complete precipitation of the target enzyme, namely 0.25 ÷ 0.30 g / ml of cell-free extract, is added to the cell-free extract, depending from the molecular weight of polyethylene glycol. The procedure is carried out at a temperature of 2 ÷ 4 ° C with stirring for the time required to completely dissolve the polyethylene glycol (15 ÷ 30 min). Then, for crosslinking of the enzyme aggregates, glutaraldehyde is added to the reaction mixture in an amount necessary to create its relative concentration of 1 · 10 ^-2 ÷ 3 · 10 ^-2 mol / mg of protein, and the proteins are modified and crosslinked for 2 hours with gentle stirring at a temperature of 2 ÷ 4 ° C. A heterogeneous biocatalyst in the form of cross-linked enzyme aggregates is separated from the reaction mixture by vacuum filtration and washed on a porous glass filter to colorless washings with sodium acetate buffer, 0.1 mol / L, pH 6.5, containing 0.005 mol / L calcium acetate.

The activity output of the AEG immobilization procedure is 48–70%. Since the isolation of the enzyme is carried out without loss of activity, the total yield of the process of obtaining a heterogeneous biocatalyst (from the biomass of cells) by the present method is 48–70%, the duration of the procedure is 12–15 hours.

The characteristics of the inventive biocatalyst. The heterogeneous biocatalyst immobilized by AEG does not contain a ballast carrier and consists mainly of protein (up to 0.95 g protein / g dry). Immobilized AEG has activity (in relation to the synthesis of cephalexin), referred to the mass of dry biocatalyst, 1300 ÷ 2200 IU / g dry. The range of the relative concentration of glutaraldehyde in the reaction mixture during cross-linking of enzyme aggregates 1 · 10 ^-2 ÷ 5 · 10 ^-2 mol / mg protein provides an immobilized enzyme that combines the indicated level of synthetase activity with operational stability, characterized by maintaining 95% of the initial activity after the use of a biocatalyst in 10 cycles of cephalexin synthesis (see Example 4, table 3).

Longer operational tests of the biocatalyst during the synthesis of cephalexin at 30 ° C and an initial substrate concentration of 7-ADCC 20 mg / ml show that the inactivation of the immobilized enzyme obeys first-order kinetic laws. This indicates the absence of AEG washing out of the biocatalyst even when relatively high substrate concentrations are used (5 times higher than that described in the closest analogue). The half-inactivation period of the biocatalyst, determined by 35 cycles of use in the process of cephalexin synthesis, is τ _1/2 = 200 cycles (see Example 10 and figure 2).

The inventive method for the synthesis of aminobeta-lactam antibiotic in General is as follows. Using the obtained biocatalyst, enzymatic synthesis of various aminopenicillins, for example, ampicillin, amoxicillin, and aminocephalosporins, for example, cephalexin, cefadroxyl, cefradine, cefaclor, cephaloglycine, is carried out by acylation of the key beta-lactam compound taken in ml with a concentration of 5 mg to 40 mg -aminophenylacetic acid, for example, methyl esters of D-phenylglycine, D-para-hydroxyphenylglycine, D-2,5-dihydrophenylglycine taken in a molar excess of 1.5 ÷ 4.0. The synthesis reaction is carried out at a pH of 6.0 ÷ 6.2 and a temperature of 1 ÷ 40 ° C in an aqueous medium or in mixtures of water with a polyhydric alcohol ethylene glycol with a content of the latter in the initial mixture of 30 ÷ 50%. The content of the heterogeneous biocatalyst in the reaction mixture varies from 5 to 50 IU / ml, depending on the selected synthesis reaction conditions so that the duration of the process does not exceed three hours.

The advantages of the claimed group of inventions:

1. Obtaining a heterogeneous biocatalyst according to the simplest possible scheme (see Fig. 1), which includes only the steps of isolating an enzyme from biomass of cells and immobilizing it (directly from a cell-free extract), in contrast to the method according to the closest analogue, which also includes a multistage step for obtaining purified enzyme preparation; a corresponding reduction in the total time of the procedure to 12 ÷ 15 hours, instead of at least 70 hours of the prototype.

2. The increase in the total yield of activity of the procedure for obtaining a biocatalyst is more than 3 times (48 ÷ 70% according to the claimed method; 16 ÷ 20% according to the closest analogue).

3. Obtaining a biocatalyst that does not contain a ballast carrier and consisting mainly of protein (in the inventive biocatalyst up to 0.95 g protein / g dry; in the closest analogue, not more than 0.08 g protein / g dry.).

4. An increase in the synthetase activity of the biocatalyst (in relation to the synthesis of cephalexin) by 3.3–4.3 times (in the inventive biocatalyst 1300–2200 IU / g dry; in the closest analogue - 510 IU / g dry).

5. Increasing the operational stability of the biocatalyst at least 5 times (at 30 ° C: τ _1/2 = 200 cycles for the inventive biocatalyst; not more than τ _1/2 = 42 cycles according to the closest analogue).

6. The absence of leaching of the enzyme from the biocatalyst during its operation even at initial substrate concentrations 5 times higher than the concentrations used in operational tests.

7. The possibility of using a biocatalyst for the enzymatic synthesis of aminobeta-lactam antibiotic in a wide range of temperatures and concentrations of the starting compounds in an aqueous medium or in mixtures of water with polyhydric alcohol ethylene glycol, which allows to achieve higher yields of the target antibiotic (up to 98%, see Example 6) compared with 85% according to the closest analogue.

The claimed group of inventions is illustrated by the following figures of the graphic image:

Figure 1 Scheme of methods for producing a biocatalyst based on AEG.

Figure 2 The operational stability of the biocatalyst in the synthesis of cephalexin (according to table 4).

The implementation of the invention

The invention is illustrated by the following examples of a method for producing a biocatalyst and a method for synthesizing an aminobeta-lactam antibiotic under the action of the obtained heterogeneous biocatalyst. In all the examples described, the activity of enzyme preparations for the synthesis of cephalexin is given. It is determined by the initial rate of formation of the target product under the following conditions: pH 6.0 ÷ 6.2, temperature (40 ± 1) ° C, substrate concentrations - (0.040 ± 0.002) mol / L 7-ADCA and (0.080 ± 0.004) mol / l MEFG. The cephalexin content in the reaction mixture was determined by high performance liquid chromatography (HPLC). The protein content in the preparations is determined by traditional methods [Biotechnol. Appl. Biochem. 29: 99 (1999)], and the solids content - by the gravimetric method after drying the drug at (105 ± 1) ° С to constant weight.

Example 1. The implementation of the proposed method for producing a biocatalyst using a strain of Xanthomonas rubrilineans VKPM B-9915.

As a starting material for obtaining a heterogeneous biocatalyst, the biomass of cells of the strain Xanthomonas rubrilineans VKPM B-9915 from the All-Russian Collection of Industrial Microorganisms (VKPM) was used. Cultivation of the producer was carried out for 22 hours in Erlenmeyer flasks on a medium consisting of corn extract (2% dry.), Peptone (1%) and glucose (1%) at 28 ° C on a circular shaker at 240-260 rpm . Cell biomass is separated by centrifugation at 7000 rpm.

Taken 273 g of wet cell biomass with a solids content of 18.5%, synthetase activity of 83.3 IU / g wet, 450 IU / g dry.

To carry out cryogenic exposure, the biomass of the cells is placed in a plastic vessel and frozen in a freezer at a temperature of - 28 ° C. After 3 hours, the vessel is removed from the freezer, the biomass is thawed at room temperature and transferred to a 2.5 L thermostatic reactor equipped with a mechanical stirrer and a pH control and maintenance system.

The procedure for treating cells with butyl acetate and extracting the enzyme into the aqueous phase is carried out in this reactor under the following conditions: concentration of cell biomass in a suspension of 40 mg dry / ml; butyl acetate concentration 4.5 vol%; temperature 30 ° C; pH gradient, constant stirring. Thawed cell biomass is suspended in Tris-HCl buffer, 0.05 mol / L, pH 7.8, taken in the amount necessary to obtain a cell suspension of the desired concentration, taking into account the moisture contained in the cell biomass. During this procedure, 1040 ml of buffer is used to obtain 1260 ml of suspension. Thawed biomass is suspended in a buffer at a temperature not exceeding 20 ° C, 150 μl of mercaptoethanol are added to stabilize the enzyme, and 57 ml of butyl acetate are added. The moment of adding butyl acetate is considered the beginning of the procedure. At the initial time, the reactor is heated to maintain a temperature of 30 ° C and the initial pH value is controlled, the value of which should not exceed 8.0. During the process, the pH decreases and the procedure proceeds in a spontaneously established gradient of its values, up to a pH of 6.8. Upon reaching a pH of 6.8, an automatic tinting system is activated and pH 6.8 ± 0.1 is maintained by adding an aqueous solution of ammonia with a concentration of 12.5% to the reaction mixture, fixing the flow rate of this solution during the process. The enzyme extraction procedure is stopped after 4 hours 30 minutes, when the flow rate of the ammonia solution is reduced by 2.5 times.

The reaction mixture is quickly cooled to a temperature of 15 ÷ 20 ° C and treated with a flocculant to facilitate the separation of the destroyed biomass of cells. As a flocculant, a water-soluble polymer anion exchange resin containing pyridinium or quaternary ammonium bases, for example Flocaton PPS-400, is used. The destroyed biomass of cells is separated from the cell-free extract by centrifugation and washed with 250 ml of sodium acetate buffer, 0.1 mol / l, pH 6.5, containing 0.005 mol / l of calcium acetate. The washings separated by centrifugation are added to the acellular extract. In the resulting solution, control the pH and set the pH value to 6.8 ÷ 7.2.

1440 ml of a clear, acellular extract with synthetase activity of 16.0 IU / ml, protein content of 4.2 mg / ml, specific activity of 3.8 IU / mg of protein are obtained. The output of the activity of the process of extracting the enzyme from the biomass of cells is η _extract = 101%.

The enzyme immobilization procedure is carried out in a 2 L reactor equipped with a mechanical stirrer and placed in an ice bath. 1440 ml of cell-free extract is placed in this reactor, cooled to 2-4 ° C, and polyethylene glycol with a molecular weight of 6000 taken in an amount of 403 g (0.28 g / ml of cell-free extract) is added to it with constant stirring, which ensures complete precipitation of the target enzyme. Stir the reaction mixture for 15 min (until the precipitator is completely dissolved) and add 76.7 ml of a 25% aqueous solution of glutaraldehyde (3.5 · 10 ^-2 mmol / mg protein). The reaction mixture is incubated with gentle stirring at a temperature of 2 ÷ 4 ° C for 2 hours. The resulting solids were separated by vacuum filtration and washed on a porous glass filter to colorless washings with sodium acetate buffer, 0.1 mol / L, pH 6.5, containing 0.005 mol / L calcium acetate. 1,500 ml of this buffer are used for washing.

38.6 g of wet heterogeneous biocatalyst are obtained in the form of cross-linked AEG enzyme aggregates with a solids content of 20.4%. The synthetic activity of the product is 370 IU / g wet or 1810 IU / g dry biocatalyst. The protein content in the product is 0.93 g protein / g dry. The activity yield of the AEG immobilization procedure is η _im = 59.1%, the total activity yield of the process of obtaining a heterogeneous biocatalyst (from cell biomass) η _total = 59.8%. The half-inactivation period of the biocatalyst, determined by 35 cycles of its use in the process of cephalexin synthesis, is τ _1/2 = 200 cycles (see Example 10).

Example 2. The implementation of the proposed method for producing a biocatalyst using a strain of Xanthomonas rubrilineans CGMCC No. 2339

As a starting material for obtaining a heterogeneous biocatalyst, the biomass of cells of the strain Xanthomonas rubrilineans CGMCC No. was used. 2339 from the Main Chinese Microorganism Culture Collection (CGMCC). Biomass obtained under the conditions described in example 1.

91.2 g of wet cell biomass with a solids content of 19.5%, synthetase activity of 70.2 IU / g wet, 360 IU / g dry were taken.

Cryogenic effect on the biomass of cells is carried out according to the method described in example 1, but at a temperature of -30 ° C and for 5 hours.

The procedure for treating cells with butyl acetate and extracting the enzyme into the aqueous phase is carried out according to example 1 under the following conditions: concentration of cell biomass in suspension 50 mg dry / ml; butyl acetate concentration 4.0 vol%; temperature 28 ° C; pH gradient, constant stirring. The following amounts of reagents are used: 280 ml of buffer, 50 μl of mercaptoethanol, 14 ml of butyl acetate. The volume of the suspension is 355 ml. The procedure for extracting the enzyme is carried out for 6 hours 10 minutes and stopped at the time of achieving a decrease in the rate of titrant consumption by 2.8 times compared to its initial value.

Processing the reaction mixture with a flocculant and obtaining a cell-free extract is carried out according to example 1. Receive 420 ml of a clear cell-free extract with synthetase activity of 15.7 IU / ml, protein content of 3.5 mg / ml, specific activity of 4.5 IU / mg of protein. The output of the activity of the process of extracting the enzyme from the biomass of the cells is η _extract = 103%.

The enzyme immobilization procedure is carried out according to the method described in example 1, with the difference that they use 420 ml of cell-free extract, 105 g of polyethylene glycol with a molecular weight of 8000 (0.25 g / ml of cell-free extract) and 8.8 ml of a 25% aqueous solution of glutaric aldehyde (1.5 · 10 ^-2 mmol / mg protein).

Obtain 11.1 g of wet heterogeneous biocatalyst in the form of cross-linked AEG enzyme aggregates with a solids content of 20.2%. The synthetic activity of the product is 380 IU / g wet or 1880 IU / g dry biocatalyst. The protein content in the product is 0.92 g protein / g dry. The activity yield of the AEG immobilization procedure is η _im = 64.0%, the total activity yield of the process of obtaining a heterogeneous biocatalyst (from cell biomass) η _total = 65.9%. The relative residual activity of the biocatalyst after 10 cycles of cephalexin synthesis (see Example 10) is 96.3%.

Example 3. The implementation of the proposed method for producing a biocatalyst when varying the moment of stopping the process of extracting the enzyme

Table 2 (No. 1-3) shows the data obtained using the culture of the strain Xanthomonas rubrilineans VKPM B-9915. The cryogenic treatment of the cell biomass, its processing with butyl acetate and the extraction of the enzyme into the aqueous phase is carried out according to Example 1, with the difference that the stopping time of the enzyme extraction is varied, correlating it with the flow rate of the ammonia solution. Cell-free extracts are used to immobilize the enzyme according to the procedure described in example 1.

The presented results reflect the effect of the moment the process was stopped on the properties of cell-free extracts and biocatalysts, as well as on the efficiency of the processes of extraction and immobilization of the enzyme. Table 2 (No. 4) also shows the data of Example 2 on the preparation of a biocatalyst based on the biomass of cells of the strain Xanthomonas rubrilineans CGMCC No. 2339, showing the dependence of the optimal stopping time of the enzyme extraction process on the strain used.

table 2
The effect of stopping the enzyme extraction process on the properties of the cell-free extract and heterogeneous biocatalyst, as well as on the efficiency of their preparation No. Strain Xanthomonas rubrilineans Process time Cell-free extract Heterogeneous biocatalyst Total output act., η _total ,% Act., ME / ml Protein, mg / ml Specific act., IU / mg protein Exit act., Η _extract ,% Act., ME / g dry Output Act.,
η _them ,% one VKPM
B-9915 3 hours 20 minutes 14.1 3,1 4,5 90 1450 49.0 44.1 2
Example 1 VKPM
B-9915 4 hours 30 minutes 16,0 4.2 3.8 101 1810 59.1 59.8 3 VKPM
B-9915 6 hours 10 minutes 16,2 7.5 2.2 102 1220 65.9 67.2 four
Example 2 CGMCC No. 2,339 6 hours 10 minutes 15.7 3,5 4,5 103 1880 64.0 65.9

Example 4. The implementation of the proposed method for producing a biocatalyst at various concentrations of glutaraldehyde

Table 3 (No. 1-4) shows the data obtained using the culture of the strain Xanthomonas rubrilineans VKPM B-9915. The procedures for cryogenic exposure of the cell biomass, its processing with butyl acetate and extraction of the enzyme into the aqueous phase are carried out according to example 1, that is, under conditions optimized for this strain. The obtained cell-free extracts are used to immobilize the enzyme according to the procedure described in example 1, with the difference that the relative concentration of glutaraldehyde in the reaction mixture is varied by cross-linking the enzyme aggregates.

As a criterion for comparing the stability of immobilized enzymes obtained under various conditions, the residual enzymatic activity of the biocatalyst after its operation in 10 cycles of cephalexin synthesis carried out according to Example 10 is used. The presented results reflect the effect of glutaraldehyde concentration on the properties of biocatalysts and the efficiency of the immobilization process.

Table 3
The effect of the relative concentration of glutaraldehyde in the reaction mixture on the properties of a heterogeneous biocatalyst and the efficiency of the enzyme immobilization process No. Strain Xanthomonas rubrilineans Relative concentration of glutaraldehyde, mmol / mg protein Heterogeneous biocatalyst Activity, IU / g dry. The output of activity η _them ,% Relative residual activity after 10 cycles of synthesis,% one VKPM B-9915 0.2 · 10 ^-2 2200 58.3 89.7 2 VKPM B-9915 1,0 · 10 ^-2 2210 70,2 96.3 3
Example 1 VKPM B-9915 3.5 · 10 ^-2 1810 59.1 96.7 four VKPM B-9915 5.0 · 10 ^-2 1320 48.1 98.1 5
Example 2 Cgmcc
No. 2,339 1.5 · 10 ^-2 1880 64.0 96.3

Table 3 (No. 5) also shows the characteristics of the biocatalyst obtained on the basis of the biomass of cells of the strain Xanthomonas rubrilineans CGMCC No. 2339. According to Example 2, a cell-free extract is obtained under conditions optimized for a given strain, then enzyme aggregates are precipitated and crosslinked under conditions falling into the concentration range of glutaraldehyde that is optimal for VKPM B-9915, while achieving similar characteristics of the final biocatalyst.

Example 5. Synthesis of cephalexin in an aqueous medium

The process of enzymatic synthesis of cephalexin is carried out in a thermostatic glass reactor with a capacity of 100 ml, equipped with a mechanical stirrer and a device for measuring and maintaining pH, under the following conditions: aqueous medium; temperature 2 ° С; pH 6.0 ÷ 6.2; enzyme content (25 ± 1) IU / ml; initial concentration of 7-ADCA 25 mg / ml; the molar ratio of MEFG and 7-ADCA in the initial reaction mixture is 3.0: 1. The calculation of the concentrations of the reagents in the initial solution is carried out taking into account the amount of moisture introduced with a portion of the biocatalyst.

To prepare a substrate solution, 1.25 g (5.84 mmol) of 7-ADCA was placed in a beaker with a capacity of 100 ml, 40 ml of distilled water was added. While stirring on a magnetic stirrer and controlling the pH, a 12.5% ammonia solution is added in portions to the suspension until the 7-ADCA sample is dissolved. In this case, the pH should not exceed 8.2. Then, 3.53 g (17.52 mmol) of MEGH hydrochloride are added portionwise to the solution with stirring, without allowing the pH of the solution to drop below 6.0 by adding a 12.5% ammonia solution. After dissolution of MEFG, the solution is quantitatively transferred to a graduated cylinder and the volume of the solution is adjusted with water to 50 ml.

The prepared substrate solution is transferred to the reactor, the temperature is set at 2 ° C and 3.4 g of the biocatalyst obtained in Example 1 are added with vigorous stirring with an activity of 370 IU / g of wet biocatalyst and a solids content of 20.4%. The moment of addition of the biocatalyst is considered the beginning of the synthesis reaction. The process is carried out for 70 minutes at a temperature of 2 ° C and a pH value of 6.0 ÷ 6.2, which is supported by adding a 12.5% ammonia solution in automatic mode. At the end of the process, the biocatalyst is separated from the reaction mixture by vacuum filtration, washed with 10 ml of water, and the cephalexin content is determined by HPLC in the filtrate combined with washing. The yield of cephalexin at 7-ADCA is 85.7%.

Example 6. Synthesis of cephalexin in a mixture of water with ethylene glycol

The process of enzymatic synthesis of cephalexin is carried out in the reactor described in example 5 under the following conditions: the content of ethylene glycol in the initial reaction mixture is 50 vol.%; temperature 5 ° С; pH 6.0 ÷ 6.2; enzyme content (40 ± 1) IU / ml; initial concentration of 7-ADCA 30 mg / ml; the molar ratio of MEFG and 7-ADCA in the initial reaction mixture is 2.5: 1.

The preparation of a solution of substrates of 1.50 g (7.01 mmol) of 7-ADCA and 3.53 g (17.52 mmol) of MEGH hydrochloride is carried out according to the procedure described in example 5, with the difference that instead of 40 ml of water for dissolution Samples 7-ADC use a mixture of 15 ml of distilled water and 25 ml of ethylene glycol.

The synthesis of cephalexin is carried out according to example 5 with the difference that the process is carried out for 180 minutes at a temperature of 5 ° C, using 5.41 g of the biocatalyst obtained in example 1, with an activity of 370 IU / g of wet biocatalyst and a solids content of 20.4 % The yield of cephalexin at 7-ADCA is 98.3%.

Example 7. Synthesis of cefaclor

The process of enzymatic synthesis of cefaclor is carried out in the reactor described in example 5, in the following conditions: the content of ethylene glycol in the initial reaction mixture is 45 vol.%; temperature 10 ° С; pH 6.0-6.2; enzyme content (40 ± 1) IU / ml; initial concentration of 3-chloro-7-aminocephalosporanic acid (7-AHCC) 15 mg / ml; the molar ratio of MEFG and 7-AHCC in the initial reaction mixture is 4: 1.

The preparation of a solution of substrates of 0.75 g (3.20 mmol) of 7-AHCC and 2.58 g (12.80 mmol) of MEFG hydrochloride is carried out according to the procedure described in example 5, with the difference that instead of 40 ml of water for dissolution Samples 7-AHCC use 17.5 ml of distilled water and 22.5 ml of ethylene glycol.

The synthesis of cefaclor is carried out according to example 5 with the difference that the process is carried out for 100 minutes at a temperature of 10 ° C, using 5.41 g of the biocatalyst obtained in example 1, with an activity of 370 IU / g of wet biocatalyst and a solids content of 20.4 % The yield of cefaclor by 7-AHCC is 92.4%.

Example 8. Synthesis of ampicillin

The process of enzymatic synthesis of ampicillin is carried out in the reactor described in example 5 under the following conditions: the content of ethylene glycol in the initial reaction mixture is 30 vol.%; temperature 20 ° С; pH 6.0 ÷ 6.2; enzyme content (30 ± 1) IU / ml; initial concentration of 6-aminopenicillanic acid (6-APA) 40 mg / ml; the molar ratio of MEFG and 6-APC in the initial reaction mixture is 3: 1.

The preparation of a solution of substrates of 2.00 g (9.26 mmol) of 6-APA and 5.60 g (27.7 mmol) of MEFG hydrochloride is carried out according to the procedure described in example 5, with the difference that instead of 40 ml of water for dissolution 6-APA samples are used with 25 ml of distilled water and 15 ml of ethylene glycol, and the pH of the solution when dissolving 6-APA does not exceed 7.5.

The synthesis of ampicillin is carried out according to example 5 with the difference that the process is carried out for 75 minutes at a temperature of 20 ° C, using 3.97 g of the biocatalyst obtained in example 1, with an activity of 370 IU / g of wet biocatalyst and a solids content of 20.4 % The output of ampicillin for 6-APA is 88.6%.

Example 9. Synthesis of amoxicillin

The process of enzymatic synthesis of amoxicillin is carried out in the reactor described in example 5, under the following conditions: aqueous medium; temperature 40 ° C; pH 6.0 ÷ 6.2; enzyme content (6.5 ± 0.5) IU / ml; initial concentration of 6-APC 10 mg / ml; the molar ratio of methyl ester of D-para-hydroxyphenylglycine (MEGPH) and 6-APA in the initial reaction mixture is 1.7: 1.

To prepare a solution of substrates, 0.720 g (3.97 mmol) of MEGFG was placed in a beaker with a capacity of 100 ml, 40 ml of distilled water was added. While stirring on a magnetic stirrer and controlling the pH, a 20% hydrochloric acid solution is added in portions to the suspension until the MEGPH sample is dissolved. Then the pH of the solution was adjusted to 6 by adding a 12.5% ammonia solution and immediately introduced into the solution of 0.500 g (2.34 mmol) of 6-APA. With stirring, a 12.5% ammonia solution is added in portions to the suspension until the 6-APA sample is dissolved, ensuring that the pH does not exceed 6.5. After dissolution of the 6-APA, the solution is quantitatively transferred to a graduated cylinder and the volume of the solution is adjusted with water to 50 ml.

The synthesis of amoxicillin is carried out according to example 5 with the difference that the process is carried out for 60 minutes at a temperature of 40 ° C, using 0.86 g of the biocatalyst obtained in example 2, with an activity of 380 IU / g of wet biocatalyst and a dry matter content of 20.2 % The 6-APA yield of amoxicillin is 72.8%.

Example 10. The operational stability of the biocatalyst in the synthesis of cephalexin

To study the operational stability of the biocatalyst, a series of successive cycles of cephalexin synthesis are carried out using the same portion of the biocatalyst under the following conditions: the ethylene glycol content in the initial reaction mixture is 40 vol.%, Temperature 30 ° C, pH 6.0 ÷ 6.2, the enzyme content (15 ± 1) IU / ml, initial concentration of 7-ADCA 20 mg / ml; the molar ratio of MEFG and 7-ADCA in the initial reaction mixture is 2.5: 1.

The first cycle of cephalexin synthesis is carried out according to the procedure described in example 5, but at a process temperature of 30 ° C and 100 ml in the volume of the reaction mixture. At the end of the reaction, the biocatalyst is separated from the reaction mixture by vacuum filtration, washed with 10 ml of water and used in the next cycle of cephalexin synthesis. In the combined filtrate, the cephalexin content was determined by HPLC and the yield of cephalexin was calculated by 7-ADC.

Subsequent synthesis cycles are carried out in a similar manner. After every five cycles, the residual activity of the biocatalyst and its dry matter content are determined. To create the selected standard process conditions for the concentration of the biocatalyst, the volume of the reaction mass of the corresponding cycle is adjusted taking into account the decrease in the activity of the biocatalyst and the decrease in its mass due to the consumption of analytical procedures. The required number of consecutive cycles of cephalexin synthesis is carried out. The results of experiments conducted using the biocatalyst obtained in example 1 are presented in table 4.

Processing the experimental data of table 4 shows that the inactivation of the biocatalyst in the synthesis of cephalexin obeys first-order kinetic laws with a half-inactivation period τ _1/2 = 200 cycles (see figure 2).

Table 4
The operational stability of the biocatalyst obtained in example 1 during the synthesis of cephalexin at a temperature of 30 ° C The number of synthesis cycles The product yield at 7-ADCC,% Biocatalyst Activity Residual activity, A _N , IU / g dry Relative residual activity, Aotn,% lg _rel 0 95.7 1810 one hundred 2 5 93.9 1780 98.3 1,993 10 96.1 1750 96.7 1,985 fifteen 94.5 1721 95.1 1,978 twenty 95.6 1692 93.5 1,970 25 93.6 1663 91.9 1,963 thirty 94.9 1636 90,4 1,956 35 95.2 1609 88.9 1,949

Information sources

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10. Blinkovsky A.M., Markaryan A.N., Synthesis of β-lactam antibiotics containing α-aminophenylacetyl group in the acyl moiety catalyzed by D - (-) - phenylglycyl-β-lactamide amidohydrolase. Enzyme Microb. Technol. 15: 965-973 (1993)

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13. Barends T.R.M., Polderman-Tijmes J.J. et al., The sequence and crystal structure of the α-amino acid ester hydrolase from Xanthomonas citri define a new family of β-lactam antibiotic acylases. J. Biol. Chem. 278: 23076-23084 (2003)

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Claims (6)

1. A method of producing a heterogeneous biocatalyst for enzymatic synthesis of an aminobet-lactam antibiotic based on an intracellular hydrolase of alpha-amino acid esters from bacteria of the genus Xanthomonas, including the destruction of cell biomass, extraction of the enzyme from it into the aqueous phase and its subsequent immobilization, characterized in that cell biomass is used bacteria Xanthomonas rubrilineans; cell destruction is carried out in stages, first violating their integrity by cryogenic exposure, freezing the biomass to a temperature of -25 ° C or lower, preferably from -25 ° C to -30 ° C, and then treating an aqueous suspension of thawed cells with a biomass concentration of from 20 to 100 mg dry / ml butyl acetate at a concentration of 2 ÷ 6 vol.%; the extraction of the enzyme into the aqueous phase is carried out by heating the treated cell suspension, first under conditions of a spontaneously establishing pH gradient, and then at a constant pH maintained by adding a titrant solution, until the extraction procedure is terminated, using the degree of solution flow rate reduction as a criterion for choosing the stopping moment titrant; cell-free extract obtained after separation of the destroyed cellular biomass is used for the immobilization of the enzyme by precipitation with polyethylene glycol in the form of enzyme aggregates and their subsequent crosslinking in the presence of glutaraldehyde. 2. The method according to claim 1, characterized in that the extraction of the enzyme is carried out at a temperature of 25 ÷ 35 ° C. 3. The method according to claim 1, characterized in that for the deposition of enzyme aggregates from the cell-free extract using polyethylene glycol with a molecular weight of 4000 ÷ 10,000 in the amount necessary for complete precipitation of the enzyme. 4. The method according to claim 1, characterized in that for the crosslinking of enzyme aggregates use glutaraldehyde in a relative concentration
1 · 10 ^-2 ÷ 5 · 10 ^-2 mmol / mg protein. 5. A heterogeneous biocatalyst for the enzymatic synthesis of aminobeta-lactam antibiotic based on intracellular hydrolase of alpha-amino acid esters from bacteria of the genus Xanthomonas, characterized in that it is obtained by the method according to claim 1, contains up to 0.95 g protein / g dry biocatalyst and has a synthetase activity up to 2200 IU / g of dry biocatalyst. 6. A method for the enzymatic synthesis of an aminobeta-lactam antibiotic by acylating a key beta-lactam compound with the corresponding derivatives of D-aminophenylacetic acid esters, characterized in that the enzymatic synthesis reaction is carried out under the action of the heterogeneous biocatalyst according to claim 5.

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