RU2420581C1 - Method of producing biocatalyst exhibiting cephalosporin acid synthesis activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method provides cultivation of Escherichia coli cells producing cephalosporin acid synthetase. As selection criteria of the moment of enzyme biosynthesis truncation point, the pH value of a cultural fluid and a enzyme distribution constant C_dist=A_cbm/A_p-p, where A_cbm is enzymatic adaptation of a cell biomass (Me/g humid) and A_p.p-is enzymatic adaptation of a supernatant (ME/ml). The cephalosporin acid synthetase biosynthesis is truncated at C_dist being not less than 350 and pH value 7.9÷8.2. The produced cell biomass in the concentration of 100÷150 mg dry/g of a polymerisation mixture is suspended in a solution of acrylamide and N, N'-methylene-bis-acrylamide in the weight relation 20:1. During 5÷30 min, the cells are modified by glutaric aldehyde taken in the relative concentration of 1·10^-3÷9·10^-3 mmol/mg of protein. The polymerisation process is initiated by persulfuric acid salt. Synthetase activity of the biocatalyst is 750-950 MU/g dry.

EFFECT: method allows producing the biocatalyst which exhibits high target activity and stability and does not contain proteins washed out in operation of proteins contaminating the biocatalytic transformation end-product.

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Description

The claimed invention relates to the production of heterogeneous biocatalysts (CD) based on bacterial cells, namely, a biocatalyst based on Esherichia coli cells having activity in relation to the synthesis of cephalosporins acids.

There is a method of immobilization of E. coli cells with penicillin acylase activity, which consists in incorporating stitched with glutaraldehyde cells with specially increased permeability of cell walls into porous spherical balls of polyacrylamide gel [1]. Upon receipt of the gel beads, polymerization is initiated by a redox pair in which N, N, N ', N'-tetramethylethylenediamine serves as a catalyst, and ammonium persulfate is the initiator [2]. Get biocatalyst intended for enzymatic hydrolytic cleavage of benzylpenicillin (BP) with the aim of producing 6-aminopenicillanic acid (6-APC). Its enzymatic activity for BP hydrolysis is very low - less than 8 IU / g ¹ ( ¹ The international unit (ME) of the enzymatic activity of the drug in relation to any biocatalytic transformation is taken as an amount of this drug that catalyzes the conversion of 1 μmol of substrate (or the formation of 1 μmol product) in the selected conditions for 1 minute).

A known method of immobilization of microbial cells, including E. coli cells, by covalently joining them to active groups of crosslinked polymers (gels) obtained by copolymerization of various monomers, mainly acrylate nature [3]. In this case, the cells are brought into contact with either the finished polymer or with monomers, which are then polymerized. Before contact with the polymer or monomers, the cells can be treated with a polyfunctional crosslinking agent, including glutaraldehyde. The polymerization of monomers is initiated by a pair of beta-dimethylaminopropionitrile - ammonium persulfate. This patent does not contain examples of the preparation of a biocatalyst based on E. coli cells, however, methods are described for immobilizing Proteus rettgeri cells containing penicillin acylase in order to obtain biocatalysts for the production of 6-APC by enzymatic hydrolysis of BP. Even the best of the described biocatalysts has a very low operational stability, characterized by a twofold drop in activity during 20 cycles of use in the process of BP hydrolysis, which corresponds to 60 hours.

Closest to the claimed method for producing a biocatalyst based on immobilized E. coli cells is a method for producing a biocatalyst for hydrolytic cleavage of BP, carried out by incorporating E. coli cells containing penicillin amidase (penicillin amidohydrolase) into the polyacrylamide gel [4]. According to the closest analogue, the procedure for obtaining a biocatalyst is as follows. The deep cultivation of E. coli strain ATCC 9637 is carried out at 30 ° C in a medium containing organic sources of carbon and nitrogen, as well as inorganic salts. The biosynthesis process is carried out within 24 hours; the use of any special criteria for choosing the moment of stopping biosynthesis is not described. The grown biomass of the cells is suspended in a solution of polyacrylamide gel monomers (acrylamide and N, N'-methylenebisacrylamide) and the gelation process is carried out by adding a polymerization catalyst (beta-dimethylaminopropionitrile) and its initiator (potassium persulfate) to the reaction mixture. The biomass of cells is used in the amount necessary to create its concentration in the polymerization mixture (p.cm) With _BMK = 33 mg dry / g p.s. The resulting gel is ground and washed.

The main disadvantages of the method according to the closest analogue are the lack of covalent fixation of cells during immobilization, which entails the possibility of contamination of the target product of enzymatic transformation with impurities of an organic nature, primarily proteins washed from the biocatalyst, as well as low activity of cells immobilized by this method. So, the biocatalyst obtained according to the closest analogue with respect to the hydrolysis of BP has an activity of only 22 IU / g; its activity with respect to the synthesis of beta-lactam antibiotics, in particular with respect to the synthesis of cephalosporins, was not controlled.

Reproduction of the method of cell immobilization according to the closest analogue (Example 3) using the E. coli strain VKPM B-10182 producing cephalosporin acid synthetase made it possible to obtain a biocatalyst having a cefazolin synthesis activity of about 40 IU / g wet. (300 IU / g dry) when the activity of the immobilization process is 50%. The low activity of such a biocatalyst does not allow its use for the synthesis of cephalosporins acids in a batch reactor with stirring. So, for the synthesis process of cefazolin in such a reactor, a biocatalyst with an activity of at least 60 IU / g wet., 330 IU / g dry. The stability of the biocatalyst obtained according to the prototype is also low. Under conditions of accelerated inactivation (40 ° C, pH 6.5), the half-inactivation period is only 20 hours, which is the result of not only the thermal inactivation of the enzyme, but also its physical leaching from the gel matrix. Loss of synthetase activity during a single washing of the biocatalyst with 0.1 M phosphate buffer with a pH of 7.5 under mild conditions (1 g of wet BC in 3 ml of buffer, 20 ° C, 1 hour) is about 30%. Protein leaching into the supernatant in an amount of 5 mg with 1 g of CD is observed. The inevitable contamination of the target product of the biocatalytic transformation process with substances of protein nature makes it impossible to use such a biocatalyst for the production of antibiotics.

The objective of the invention is to develop a method for producing a biocatalyst based on Escherichia coli cells immobilized in a polyacrylamide gel, which is active in the synthesis of cephalosporin acids, characterized by high activity and stability and does not contain weakly bound proteins that can contaminate the target product of biocatalytic transformation.

The problem is solved by the combined use in the process of obtaining a biocatalyst of a number of techniques carried out in the selected optimized conditions.

According to the claimed method, the starting material for the preparation of the biocatalyst is E. coli strain producing the enzyme cephalosporin acid synthetase. The cultivation of this producer is carried out on a liquid fermentation medium containing organic sources of carbon and nitrogen. As a criterion for stopping the process, the distribution constant of the target enzyme between the cell biomass (BMC) and the external solution K _ras , which characterizes the binding strength of the enzyme to the cell structures, is used. The grown cell biomass is suspended in a solution of polyacrylamide gel monomers, modified with glutaraldehyde, and then the polymerization initiator, a salt of sulfuric acid, is added. The introduction of a polymerization catalyst into the reaction mixture is not required. The resulting gel is ground and washed from substances that are not bound during the polymerization process (proteins, monomers).

The process of biosynthesis of cephalosporin acid synthetase is stopped in the phase of stationary growth of E. coli cells with a distribution constant of cephalosporin acid synthetase K _ra ≥ 350 and a pH of the culture fluid of 7.9 ÷ 8.2. The time frame for the onset of the selected optimal state for immobilization of the cells may shift depending on the E. coli strain used, which affects the duration of biosynthesis (see Table 1 in Example 4). The cells obtained under the selected conditions combine high activity on the target enzyme (at least 1600 IU / g dry) and strong binding of cephalosporin acid synthetase to cell structures that remain intact in the stationary phase. Preservation of the natural cellular microenvironment of the target enzyme protects it from the adverse effects of the immobilization processes and subsequent operation of the biocatalyst, therefore, the immobilization of E. coli cells with _Kpas ≥ 350 provides a biocatalyst with high activity for the synthesis of cephalosporins acids and good stability. At a culture fluid pH below 7.9, cephalosporin acid synthetase is strongly bound to cell structures (K _distribution ≥ 350), however, the use of such cells for immobilization is impractical due to the insufficiently high activity of the obtained cell biomass. The use of immobilized cells with a weakly bound target enzyme (K _dis <350, pH> 8.2) that are in a state of limited cytolysis (destruction of the cytoplasm of cells) or subsequent autolysis (destruction of the cytoplasm and cell membranes) leads to a decrease in the stability of the biocatalyst , as well as loss of activity during immobilization (Example 5).

The use of high concentrations of cell biomass (cell suspension density) C _BMK = 100 ÷ 150 mg dry / g p.s. (3-5 times higher than in the method according to the closest analogue) allows to obtain a highly active biocatalyst, due to the introduction of a large number of active cells into the polymerization mixture. Subsequent modification with glutaraldehyde prevents them from being washed out of the gel during the preparation and washing of the biocatalyst. The upper density limit of the cell suspension (150 mg dry / g bcm) is limited by the possibility of introducing wet BMK into the polymerization mixture with a dry matter content of 20-23% in an amount of not more than 70% of its total weight (W _pc.cm ). The use of the density of the suspension is less than 100 mg dry / g p.s. does not provide a biocatalyst with a sufficiently high activity (see Table 2 in Example 6).

Modification of cells with glutaraldehyde in the implementation of the proposed method has two positive consequences. Firstly, this leads to irreversible fixation of the protein content of cells, including the target enzyme, in the biocatalyst. The result is an increase in the activity and stability of immobilized cells, as well as the absence of leaching of proteins from the finished biocatalyst. Secondly, intermediate products of the interaction of glutaraldehyde with proteins act as catalysts for the polymerization of polyacrylamide gel monomers, which makes it unnecessary to add to the reaction mixture the substances commonly used for this purpose (beta-dimethylaminopropionitrile or N, N, N ', N'-tetramethylethylenediamine). The possibility of such a replacement was previously demonstrated by immobilization of free proteins in acrylate gels [5]. According to the claimed method, glutaraldehyde is used in a relative concentration With _GA = 1 · 10 ^-3 ÷ 9 · 10 ^-3 mmol / mg of protein, carrying out the modification for τ _{mod =} 5 ÷ 30 min. The use of a modifier at a concentration above 9 · 10 ^-3 mmol / mg of protein leads to an insufficiently active biocatalyst, and when the concentration of glutaraldehyde is below 1 · 10 ^-3 mmol / mg of protein and / or a modification time of less than 5 min, the stability of the biocatalyst decreases and the possibility leaching of proteins from it. When the time of modification with glutaraldehyde for more than 30 min, the concentration of intermediate products of the interaction of proteins with glutaraldehyde, which are catalysts for the polymerization of acrylate monomers, decreases in the reaction mixture, so the subsequent gelation process proceeds slowly, and the mechanical properties of the obtained biocatalyst deteriorate (see Table 3 in Example 7).

The method in General

The biosynthesis of cephalosporin acid synthetase is carried out by culturing an E. coli strain producing the corresponding enzyme in a liquid fermentation medium of the following composition (wt.%): Corn extract (2% by dry weight) and thiophene-2-acetic acid (0.1% ) The fermentation is stopped at the binding constant of the target enzyme K _{ra ≥} 350 and the pH of the culture fluid in the range of 7.9 ÷ 8.2. The grown cell biomass is separated by centrifugation (5-8 thousand rpm), washed with 0.1 M phosphate buffer with a pH of 7.5 and again separated by centrifugation under the same conditions. The sediment, which is the biomass of cells (BMC), is used for immobilization by incorporating modified cells in a polyacrylamide gel obtained by copolymerization of acrylamide (AA) and N.N'-methylene-bis-acrylamide (BIS).

The cell biomass, taken from the calculation of _BMK = 100 ÷ 150 mg dry / g bcm, is suspended in phosphate buffer, pH 7.5, containing monomers of polyacrylamide gel in a weight ratio of AA: BIS = 20: 1 at a total concentration of in the polymerization mixture C _mon = 9%. With constant stirring and cooling of the cell suspension in an ice bath, cells are modified with glutaraldehyde taken in a relative concentration of C _GA = 1 · 10 ^-3 ÷ 9 · 10 ^-3 mmol / mg of protein contained in the cells. Modification time with glutaraldehyde τ _mod = 5 ÷ 30 min. At the end of the modification, a polymerization initiator — a salt of supra sulfuric acid, for example ammonium persulfate or potassium persulfate — is added to the reaction mixture in the concentration necessary for gelation for 20-120 sec. The resulting block of gel is kept in the cold for another 20-30 minutes, and then crushed, twice forcing through a metal sieve with a mesh size of 0.5 mm, and washed from substances not bound during the polymerization. Immobilized cells are separated from the washings by vacuum filtration on a porous glass filter. A biocatalyst is obtained with cefazolin synthesis activity of at least 100 IU / g wet., 600 IU / g dry., Half-inactivation period (τ _1/2 ) of at least 800 hours (40 ° C and pH 6.5). The biocatalyst is stable against leaching of protein substances from it and can be used in the synthesis of cephalosporins acids in batch reactors with stirring.

The inventive method for producing a biocatalyst based on cells of E. coli strains immobilized in a polyacrylamide gel producing cephalosporin acid synthetase (Examples 1, 2, 6, 7) has a number of advantages compared to the control — a method performed according to the closest analogue and carried out using E. coli cultures, VKPM strain B-10182 (Example 3):

- increased activity of the obtained biocatalyst in relation to the synthesis of cefazolin by 2-3 times (600 ÷ 950 IU / g dry. by the present method and 300 IU / g dry. according to the control);

- increasing the stability of the obtained biocatalyst in the conditions of accelerated inactivation of not less than 40 times (τ _1/2 = 800-2000 hours according to the present method and τ _1/2 = 20 hours according to the control);

- the lack of leaching of the target enzyme and other proteins from the biocatalyst obtained according to the claimed method, in contrast to the biocatalyst obtained according to the closest analogue.

The invention is illustrated by the following examples of a method for producing a biocatalyst using two E. coli strains producing cephalosporin acid synthetase, namely VKPM B-10182 from the All-Russian Collection of Industrial Microorganisms (VKPM) and CGMCC No.3508 from the Chinese Collection of Microorganism Cultures (CGMCC).

In all examples cited, the activity of enzyme preparations for the synthesis of cephalosporin-acid cefazolin from 3-methyl-mercapto-5-methyl-1,3,4-thiadiazol-2-yl-7-aminocephalosporanic acid (MMTD-7-ACC) and methyl 1 (H) -therazolylacetic acid ester (METZUK). It is determined by the initial rate of cefazolin formation under the following conditions: pH 7.5, temperature (30 ± 1) ° C, substrate concentrations - (0.059 ± 0.002) mol / L MMTD-7-ACC and (0.23 6 ± 0.004) mol / l MATZUK. The cefazolin content in the reaction mixture is determined by high performance liquid chromatography (HPLC).

The solids content in the cell biomass and biocatalysts is determined by the gravimetric method after the preparation is dried at (105 ± 1) ° C to constant weight.

Constant distribution synthetase cephalosporin acids (K _dis) between the biomass of the cell and the outer solution is determined by analyzing the enzymatic activity of cell biomass (A _BMC, lU / g wet.) And supernatant (A _rr, IU / ml) after slurrying 1 g wet BMK in 5 ml of 0.1 M phosphate buffer, pH 6.5 and calculated by the formula: To _rasp = A _BMK / A _rr .

The protein content in the biomass of cells and solutions is determined by the Lowry method [6]. The dynamics of inactivation of biocatalysts is monitored by monitoring their residual activity after incubation for various times under conditions of accelerated inactivation at 40 ° C and pH 6.5 in 0.1 M phosphate buffer. The stability of the samples is characterized by the time of their half-inactivation under these conditions (τ _1/2 , hour).

The loss of proteins and the target enzyme during washing of the biocatalysts is controlled by determining the protein content and determining the synthetase activity in the supernatant after suspending 1 g of wet biocatalyst in 3 ml of 0.1 M phosphate buffer at pH 7.5 for 1 hour at 20 ° C.

Example 1. The implementation of the proposed method for producing a biocatalyst using a culture of E. coli, strain VKPM B-10182

The biosynthesis of cephalosporin acid synthetase by E. coli VKPM B-10182 culture is carried out in 750 ml Erlenmeyer flasks containing 100 ml of fermentation medium of the following composition:

- corn extract - 2% (on dry weight)

- thiophene-2-acetic acid - 0.1%

- tap water - to the desired volume.

pH after sterilization of 6.9-7.1.

Cultivation of the enzyme producer is carried out at a temperature of 25-26 ° C on a circular rocking chair with a rotation speed of 220-240 rpm. The process of biosynthesis of cephalosporin acid synthetase is carried out for 18 hours and stopped at pH 8.1, K _rasp = 380. The grown biomass of the cells is separated by centrifugation at 6000 rpm for 20 minutes in a centrifuge with cooling, washed with 0.1 M phosphate buffer, pH 7.5 and re-separated by centrifugation under the same conditions.

With 3.70 L of culture fluid, 46.6 g of wet biomass of E. coli cells containing cephalosporin acid synthetase and characterized by the following quality indicators are obtained:

- synthetase activity (A _BMK ) - 460 IU / g wet .; 2009 IU / g dry .;

- solids content (β _BMK ) - 22.9%;

- protein content - 114 mg / g wet.

The resulting biomass of E. coli cells, strain VKPM B-10182, is used for immobilization by incorporation into a polyacrylamide gel.

Immobilization of cell biomass is carried out under the following conditions:

W _{P. cm} = 7.0 g; With _BMK = 37 mg dry / g p.cm; With _GA = 1.7 · 10 ^-3 mmol / mg protein; τ _mod = 15 min; With _mon = 9%; the initiator of the polymerization of ammonium persulfate is 0.2%.

The procedure is carried out in a glass beaker placed on a magnetic stirrer in an ice bath. 4.2 g of wet cell biomass are suspended in 1.4 ml of 0.3 M phosphate buffer at pH 7.5, then 1.05 ml of a 60% aqueous solution of acrylamide gel monomers is added (AA: BIS = 20: 1). After obtaining a homogeneous suspension, 0.32 ml of a 25% aqueous solution of glutaraldehyde is added to it and the proteins are modified with a cross-linking agent for 15 minutes with constant stirring. Then, 0.06 ml (60 μl) of a 25% aqueous solution of ammonium persulfate was added to the polymerization mixture with vigorous stirring, and the stirrer was stopped. After 30 seconds, a gel is formed, which is kept in an ice bath for 20 minutes, crushed by successive twisting through a metal sieve with a mesh size of 0.50 mm and washed with distilled water and a 1 M aqueous solution of sodium chloride. The biocatalyst is separated from the washings by vacuum filtration.

Get 7.01 g of wet biocatalyst, characterized by the following quality indicators:

- synthetase activity (A _BC ) - 165 IU / g wet .; 900 IU / g dry .;

- solids content (β _BC ) - 18.3%;

- half-inactivation time at 40 ° C, pH 6.5 (τ _1/2 ) - 1200 hours.

The output of the activity of the immobilization process is 59.9%.

Losses when washing the finished biocatalyst buffer:

- synthetase activity - not detected

- protein - not detected.

Example 2. The implementation of the proposed method for producing a biocatalyst using a culture of E. coli, strain CGMCC No.3508

The biosynthesis of cephalosporin acid synthetase by E. coli culture, strain CGMCC No.3508, is carried out as described in example 1 for strain E. coli VKPM B-10182, with the only difference that the enzyme biosynthesis is carried out for 16 hours and stopped at pH 8.05; To _dec = 370. The grown biomass of the cells is separated and washed in accordance with the description given in example 1.

With 1.2 l of culture fluid, 13.1 g of wet biomass of E. coli cells containing cephalosporin acid synthetase and characterized by the following quality indicators are obtained:

- synthetase activity (A _BMK ) - 380 ME / g wet .; 1820 IU / g dry .;

- solids content (β _BMK ) - 23.0%;

- protein content - 90 mg / g wet.

The resulting biomass of E. coli cells, strain CGMCC No.3508, is used for immobilization by incorporation into a polyacrylamide gel.

Immobilization is carried out under the following conditions:

W _{P. cm} = 7.0 g; With _BMK = 145 mg dry / g p.cm; With _GA = 5.0 · 10 ^-3 mmol / mg protein; τ _mod = 10 min; With _mon = 9%; the initiator of the polymerization of ammonium persulfate is 0.2%.

The procedure is carried out in a glass beaker placed on a magnetic stirrer in an ice bath. 4.4 g of wet cell biomass is suspended in 0.7 ml of 0.3 M phosphate buffer at pH 7.5, then 1.05 ml of a 60% aqueous solution of acrylamide gel monomers is added (AA: BIS = 20: 1). After obtaining a homogeneous suspension, 0.79 ml of a 25% aqueous solution of glutaraldehyde is added to it and the proteins are modified with a cross-linking agent for 10 minutes with constant stirring. Then, 0.06 ml (60 μl) of a 25% aqueous solution of ammonium persulfate was added to the polymerization mixture with vigorous stirring, and the stirrer was stopped. After 20 seconds, a gel forms, which is kept in an ice bath for 20 minutes, crushed by successive twisting through a metal sieve with a mesh size of 0.50 mm and washed with distilled water and a 1 M aqueous solution of sodium chloride. The biocatalyst is separated from the washings by vacuum filtration.

Get 6.95 g of wet biocatalyst, characterized by the following quality indicators:

- synthetase activity (A _BC ) - 150 IU / g wet .; 840 IU / g dry .;

- solids content (β _BC ) - 17.8%;

- half-inactivation time at 40 ° C, pH 6.5 (τ _1/2 ) - 1250 hours.

The output of the activity of the immobilization process is 62.4%.

Losses when washing the finished biocatalyst buffer:

- synthetase activity - not detected

- protein - not detected.

Example 3. The implementation of the method for producing a biocatalyst according to the closest analogue using E.coli culture, strain VKPM B-10182, (control)

The biosynthesis of cephalosporin acid synthetase by E. coli culture, VKPM strain B-10182, separation and washing of the cell biomass is carried out according to example 1. The obtained cell biomass, described in example 1, is used for immobilization by incorporation into a polyacrylamide gel according to the procedure described in the closest analogue .

Immobilization of cell biomass is carried out under the following conditions:

W _{P. cm} = 7.0 g; With _BMK = 33 mg dry / g p.cm; With _mon = 12.7%; beta-dimethylaminopropionitrile polymerization catalyst - 0.36%; the initiator of the polymerization of potassium persulfate is 0.07%.

1.0 g of wet cell biomass is suspended in 4.0 ml of a 0.9% sodium chloride solution, then 0.75 g of acrylamide and 0.40 g of N, N'-methylene-bis-acrylamide are dissolved in this suspension (AA: BIS = 18: 1). 0.5 ml of a 5% solution of beta-dimethylaminopropionitrile and a 1% solution of potassium persulfate are added successively. The resulting gel was incubated for 20 minutes, then crushed by successive two-time punching through a metal sieve with a mesh size of 0.50 mm and washed with 0.9% sodium chloride solution.

Get 6.20 g of wet biocatalyst, characterized by the following quality indicators:

- synthetase activity (A _BC ) - 38 IU / g wet .; 300 IU / g dry .;

- solids content (β _BC ) - 12.7%;

- half-inactivation time at 40 ° C, pH 6.5 (τ _1/2 ) - 20 hours.

The output of the activity of the immobilization process is 51.4%.

Losses when washing the finished biocatalyst buffer:

- flush of synthetase activity - 11 ME with 1 g of CD (29% of the initial activity of CD)

- protein washout - 5 mg of protein with 1 g of CD.

Example 4. Dynamics of the biosynthesis of cephalosporin acid synthetase by E. coli culture

Cultivation of the producer E. coli, strain VKPM B-10182 or CGMCC No.3508, is carried out in Erlenmeyer flasks under the conditions according to example 1. During the process, two flasks are removed from the experiment, the culture fluid contained in them is combined, the pH value is controlled in it, and then the cell biomass is separated by centrifugation and washed with 0.1 M phosphate buffer with a pH of 7.5, used in an amount of 5 ml per 1 g BMK. Wash water and the resulting cell biomass are analyzed and the distribution constant of the cephalosporin acid synthetase K _{rasp is calculated.} The obtained data on the relationship between the pH of the culture fluid, K _ras and the activity of the resulting cell biomass (A _BMK , IU / g dry.) _Are presented in Table 1.

Table 1 Dynamics of the cephalosporin acid synthetase biosynthesis Time hour E.coli VKPM B-10182 E.coli CGMCC No.3508 pH A _BMK , ME / g dry. To _rasp pH A _BMK , ME / g dry. To _rasp 10 7.61 1490 350 7.55 1250 390 12 7.75 1640 360 7.65 1360 380 fourteen 7.80 1770 370 7.95 1620 410 16 7.92 2140 350 8.05 1840 370 17 8.04 2180 360 8.10 1670 360 eighteen 8.11 2000 380 8.18 1700 400 19 8.13 2060 420 8.28 1650 340 twenty 8.17 1980 420 8.38 1590 320 21 8.25 1990 340 22 8.32 1980 330 23 8.46 1855 300 24 8.58 1666 290 25 8.70 1600 270 26 8.95 1540 190

Example 5. Use for immobilization of cells with loosely coupled target enzyme

Cultivation of the producer of cephalosporin acid synthetase, separation and washing of the cell biomass is carried out according to example 1, with the difference that the enzyme biosynthesis is carried out for 22.5 hours and stopped at pH 8.38, K _pacp = 320.

With 0.70 l of culture fluid, 8.1 g of wet biomass of E. coli VKPM B-10182 cells containing cephalosporin acid synthetase and characterized by the following quality indicators are obtained:

- synthetase activity - 440 IU / g wet .; 1900 IU / g dry .;

- solids content - 23.2%;

- protein content - 95 mg / g wet.

The obtained cell biomass is used for immobilization in a polyacrylamide gel, carried out under the conditions according to Example 1.

Get 6.91 g of wet biocatalyst, characterized by the following quality indicators:

- synthetase activity (A _BC ) - 113 IU / g wet .; 750 IU / g dry .;

- solids content (β _BC ) - 15.1%;

- half-inactivation time at 40 ° C, pH 6.5 (τ _1/2 ) - 750 hours.

The output of the activity of the immobilization process is 42.2%.

Losses when washing the finished biocatalyst buffer:

- synthetase activity - not detected

- protein - not detected.

Example 6. The effect of the density of the cell suspension on the properties of the resulting biocatalyst

For immobilization, the biomass of E. coli VKPM B-10182 cells obtained according to Example 1 is used. The immobilization procedure is carried out under the conditions of Example 1, varying the content of cell biomass in the polymerization mixture (cell suspension density) while maintaining the total volume of the polymerization mixture due to a corresponding change in the introduced buffer volume. To initiate the polymerization, various salts of sulfuric acid are used. The properties of the obtained biocatalysts are presented in Table 2.

table 2 The effect of cell suspension density on the properties of the resulting biocatalyst No. With _BMK , mg dry / g p.s. Polymerization initiator β _BC ,% A _BC τ _1/2 , hour IU / g wet IU / g dry one 75 (NH ₄ ) ₂ S ₂ O ₈ 13.0 63 485 - 2 one hundred K ₂ S ₂ O ₈ 16.1 108 670 1100 3 (Example 1) 137 (NH ₄ ) ₂ S ₂ O ₈ 18.3 165 900 1200 four 150 Na ₂ S ₂ O ₈ 19,4 185 950 1340

Example 7. The effect of the concentration of glutaraldehyde and modification time on the properties of the resulting biocatalyst

For immobilization, the biomass of E. coli VKPM B-10182 cells obtained according to Example 1 is used. The immobilization procedure is carried out under the conditions according to Example 1, varying the cell modification time with glutaraldehyde and its relative concentration (while maintaining the total volume of the polymerization mixture due to a corresponding change insertion volume of the buffer). The gelation times (polymerization, τ _polym , sec) and the properties of the obtained biocatalysts are presented in Table 3.

Table 3 Effect of glutaraldehyde concentration and modification time on the properties of the resulting biocatalyst No. With _GA , mmol / mg protein τ _mod , min τ _polym , sec β _BC ,% A _BC τ _1/2 , hour Flushing with 1 g of BK IU / g wet IU / g dry ME mg protein one 0.5 · 10 ^-3 fifteen 60 14.1 121 870 500 0.26 0.15 2 1,0 · 10 ^-3 thirty 120 17.0 150 880 810 no no 3 1.7 · 10 ^-3 one 45 13.8 130 950 620 0.2 0.1 4 (Example 1) 1.7 · 10 ^-3 fifteen thirty 18.3 165 900 1200 no no 5 1.7 · 10 ^-3 45 600 (weak gel) 6.8 52 760 - - - 6 9.0 · 10 ^-3 5 90 19.1 118 620 2000 no no 7 14.0 · 10 ^-3 fifteen 60 18.7 95 510 2200 no no

Thus, a method has been developed for the preparation of a biocatalyst having activity with respect to the synthesis of cephalosporins acids based on E. coli cells immobilized in a polyacrylamide gel. The method allows to obtain a stable biocatalyst with an activity of at least 600 IU / g dry, not containing loosely coupled proteins that can contaminate the target product of biocatalytic transformation.

The features of the method are:

- the use of E. coli culture strains producing the enzyme cephalosporin acid synthetase;

- the choice of the moment of stopping the cultivation of the producer at K _rasp not less than 350 and a pH value in the range of 7.9 ÷ 8.2;

- modification of cells suspended in a solution of polyacrylamide gel monomers with glutaraldehyde under optimized conditions that ensure fixation of cellular proteins in the biocatalyst, as well as the participation of intermediate products of the interaction of glutaraldehyde with proteins during gelation as a catalyst for the polymerization of monomers.

Information sources

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3. US3957580 "Immobilized microbial cells"; SU608495 "Method for the immobilization of microbial cells."

4. Sato T., Tosa T., Chibata I .: Continous production of 6-Aminopenicillanic acid from Penicillin by immobilized microbial cells. European J Appi Microbiol 2: 153-160 (1976).

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Claims (1)

A method for producing a biocatalyst based on bacterial cells of Escherichia coli, which is active in the synthesis of cephalosporin acids, comprising cultivating the producer and immobilizing the grown biomass of cells containing the target intracellular enzyme by suspending it in a solution of polyacrylamide gel monomers followed by gelation under the action of a catalyst and a polymer initiator , characterized in that they use a culture of Escherichia coli, producing the enzyme synthetase of cephalosporin acids, in As the criteria for choosing the moment of termination of the enzyme biosynthesis process, the distribution constant of the target enzyme is used K _rasp = A _bmk / A _rr ,
where A _BMK is the enzymatic activity of cell biomass (IU / g wet.);
And _rr - the enzymatic activity of the supernatant (IU / ml);
and the pH of the culture fluid, stopping the process of biosynthesis of cephalosporin acid synthetase at a _{Kp of} not less than 350 and a pH value of 7.9 ÷ 8.2, the cell biomass at a concentration of 100 ÷ 150 mg dry / g polymerization mixture is suspended in a solution of acrylamide and N , N'-methylene-bis-acrylamide at a weight ratio of 20: 1, for 5 ÷ 30 min, cells are modified with glutaraldehyde taken in a relative concentration of 1 · 10 ^-3 ÷ 9 · 10 ^-3 mmol / mg protein, as the initiator of the polymerization process using a salt of sulfuric acid.