SK279771B6 - Method of a submerged cultivation of cells producing g-penicillin amidase - Google Patents

Abstract

A method of a submerged cultivation of cells, especially Escherichia coli strain which contains recombinant plasmides, producing G-penicillin amidase in a mineral, cultivating medium containing fermentable saccharide-as a source of an assimilable carbon-and inorganic salt and mineral nutrient salts-as a source of nitrogen-resides in admixing fermentable saccharide, during the microorganism strain cultivation, in amounts corresponding with the concentration from 0.01 to 3 percents or, continually, depending on the saccharide consumption; at a simultaneously regulated pH ranging from 6.0 to 7.5, preferably 6.4 to 7.0, by alkali addition, at increased 7.3 to 8.0 pH, in the beginning of cultivation.

Description

The invention relates to a method for submerged cultivation of cells with G-penicillin amidase activity, in particular cells containing plasmids carrying a gene for the synthesis of G-penicillin amidase.

BACKGROUND OF THE INVENTION

G-penicillin amidase (hereinafter PAG) hydrolyzes the amidic bond of G-penicillin (benzyplpenicillin) to 6-aminopenicillanic acid (6-APK), or the amidic bond of deacetoxycephalosporin G (N-phenyl-7-aminodeacetoxycephalosporanoic acid) to 7-aminocecein 7-ADCA).

Methods for culturing bacteria with PAG activity using complex nutrient components such as yeast and corn extracts, various protein hydrolysates in combination with an enzyme inducer-phenylacetic acid are known (U.S. Pat. No. 1,015,554). The use of a complex medium in combination with glucose and phenylacetic acid has also been reported for the cultivation of strains carrying the PAG gene on recombinant plasmids (Robas et al., Biotechnol. Bioeng. 41, 14-24, 1993; Lee and Chang, Biotechnol. Lett. 10,787-792,1988).

The use of a synthetic, chemically defined nutrient medium containing an organic nitrogen source and a fermentable carbohydrate (e.g. sucrose) as a carbon source that can be added in multiple portions during fermentation for strains with PAG activity is reported in CS 151240. It is known that however, the use of chemically defined soils significantly increases the rate of PAG synthesis by the addition of a phenylacetic acid inducer, particularly when using phenylacetic acid as the sole carbon source (Vojtíšek and Slezák, Foil Microbiol. 20, 289-297, 1975; Babu and Panda, Bioprocess Eng. 6, 71-74 (1991).

Since PAG synthesis is subject to catabolic glucose (or other carbohydrate) repression and partial acetate repression, also produced by glucose utilization (Vojtíšek and Slezák, Microbiol Foil 20, 298-306, 1975), various regulatory regimes have been prepared in the past. mutant strains less sensitive to this repression (Robas et al., Biotechnol. Bioeng., 41, 14-24, 1993; CS 188 631). However, the specific activity of PAG without phenylacetic acid did not reach high values (40 µg * 1 dry cell mass) (Vojtíšek and Slezák, Foil Microbiol. 20, 289-297, 1975). Recombinant strains prepared from these mutant strains are also less sensitive to catabolic repression and many of them can synthesize PAG constitutively in the absence of phenylacetic acid. The properties of strains of Escherichia coli carrying various non-combined plasmids are disclosed in CS 278 516aCS (PV 1269-92).

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a method for submerged cultivation of cells of a strain of a microorganism, in particular Escherichia coli, containing recombinant plasmids producing G-penicillin amidase (PAG) in a mineral nutrient medium containing an fermentable carbohydrate as an assimilable carbon source. in that during fermentation of the microorganism strain a fermentable carbohydrate is added in doses corresponding to a concentration of 0.01 to 3.0%, or continuously depending on the carbohydrate consumption, while simultaneously adjusting the pH in the range of 6.0 to 7.5, preferably 6 4 to 7.0 by addition of alkali at an elevated pH of 7.3 to 8.0 at the beginning of the culture. The nutrient medium may advantageously contain calcium in the form of a soluble salt in a concentration of Ca 2.10 * 5 to 0.1% and / or in the form of CaCC> 3 in a concentration of 0.05 to 1%, iron in the form of soluble salts in a concentration of 1.10 * 5 to 5%. 2.10 * 3% _and magnesium in the form of soluble salts at a concentration of Mg corresponding to 0.001 to 0.05%. In particular, the invention relates to the cultivation of cells containing recombinant plasmids carrying the PAG gene. These recombinant strains usually produce PAG even without the addition of phenylacetic acid, while reducing the repression of PAG synthesis by carbon sources (e.g. glucose, sucrose, glycerol). These properties favor the present invention by suitably controlled cultivation to achieve maximum PAG production. The method of submerged culture consists in using synthetic, chemically defined soils containing a utilizable carbon source, preferably a carbohydrate, and an inorganic nitrogen source, both dosed during cultivation, due to eventual and partial repression, to achieve a high cell concentration with high PAG activity . Since organic acids (e.g. acetic acid) are formed during utilization of the carbon source, which lower the pH below the optimum value and thereby negatively affect growth, simultaneous pH control is desirable. Since early pH control may negatively affect enzyme production, it is preferable to increase the start pH of the medium or to add CaCC> 3, which increases the buffering capacity of the medium. An appropriate concentration of Mg2 + ions and a low concentration of Fe2 +, respectively, are important for PAG production. Fe3 + ions. It is advantageous to feed the nitrogen source in the form of ammonium hydroxide, thereby simultaneously maintaining an optimal pH and the necessary nitrogen level. The whole culture process can be combined by monitoring the oxygen partial pressure (pO2) regulated so that a rapid and sudden increase in pO2 signals a halt in cell culture growth due to a lack of carbon or nitrogen source. After adding the next dose of the necessary resource and its continued utilization, pC> 2 will decrease again. In this way, it is possible to achieve a high concentration of cells with a high PAG activity (i.e., a high total and specific PAG activity) and thus greatly increase the yield of the enzyme and obtain a quality cellular material.

The culture method is particularly useful for strains of Escherichia coli containing recombinant plasmids carrying a PAG gene, such as e.g. strains containing plasmids according to CS 278 516 and CS (PV 1269-92). Also, the culture procedure can be used for various PAG producers lacking recombinant plasmids, particularly with reduced catabolic repression against the carbon source used and without the inducer need. Preferably an Escherichia coli CCM 4228 strain containing the recombinant plasmid pKA18 can be used. With the described culture method, significant PAG production can be achieved in the absence of phenylacetic acid, eliminating the risk of intermittent lysis (disintegration) of the cell population in the pharmaceutical industry's scale of induction of PAG with phenylacetic acid (probably genetic association of prophage release with intensive gene transcription) .

The amount of PAG was determined in cells washed with 0.1 M phosphate buffer pH 8.0 by measuring the initial rate of hydrolysis of penicillin G in 0.05 M phosphate buffer pH 8.0 at 37 ° C in the zero order kinetics . The product of the 6-APK reaction was determined spectrophotometrically using p-dimethylaminobenzaldehyde (Balasingham et al., Biochim. Biophys. Acta 276, 250-260, 1972). One unit is the amount of enzyme that corresponds to the formation of one micromole of 6-APK per minute. Specific activity is defined as Ug 'dry cell mass.

The obtained highly active cell suspension may be used or in the form of a separate cell paste, or in the form of a thickened suspension as a starting material for the production of immobilized whole cells or parts thereof after disintegration, or it is possible to paste respectively. The thickened slurry can be used to isolate the crude enzyme and then immobilize it to obtain catalysts with high PAG activity and high G-penicillin cleavage efficiency.

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

DETAILED DESCRIPTION OF THE INVENTION

Example 1

A solid and liquid broth was prepared with the following composition: 2% glycerol, 0.4% (NH4) ₂ SC> 4, 1.36% KH2PO4, 0.3% NaOH, 0.001% FeSO4.7H2O, 0.005% CaCl2.6H2O 0.2% MgSO 4 .7H 2 O, pH 7.2-7.3; sterilization for 30 minutes at 1.2 atm. In the case of solid broth, this medium was supplemented with 1.5% agar. Under aseptic conditions, kanamycin was added to the liquid and solid soils at a concentration of 35 µg / ml.

The glycerol can strain stored at -20 ° C was seeded on solid soil in Petri dishes and the plates were incubated at 30 ° C for 48 hours. The grown strain was inoculated with 100 ml of liquid soil in a 500 ml beaker and incubated on a rotary shaker (240 rpm, 25 mm eccentric) for 24 hours at 30 ° C.

In a 5 liter glass fermentation tank (158 mm ID, 250 mm height) equipped with a radial mixer with six 70 mm blades, 2 liters of soil were prepared: 2% glycerol, 0.2% (NH4) ₂ SO 4, 1.36% KH ₂ PO _4, 0.3% NaOH, 0.5 CaCOj, 0.001% FeSO4.7H2O, 0.005% CaC12.6H2O, 0.2% MgSO4-7H2O, 0.015% silicone antifoam SAG 471 pH 7.2-7.3; sterilization for 40 minutes at 1.2 atm. The MgSO4, FeSO4, CaCl2 and CaCO3 solutions were sterilized separately.

ml of inoculum from the flask prepared as described above was aseptically added to sterile broth in a fermentation tank and cultivated at 25 ° C, aerating 1 volume of air per broth volume at a stirring frequency of 400 rpm. During the cultivation, the stirring speed was gradually increased up to 600 rpm. and samples were taken to determine the optical density of the culture, ammoniacal nitrogen, dry cell mass, and PAG activity. A total of 0.4% (NH4) 2SO4 was added in three batches during the cultivation. The pH of the culture at the end of the fermentation was 6.0. PAG production of 4700 U and cell growth of 8.3 g dry matter were achieved per liter of culture liquid. The specific activity was 560 µg / l of dry cell mass.

Example 2

A solid agar culture and 10 cells of Escherichia coli CCM 4228 (pKA19) inoculum as described in Example 1 were prepared except that the liquid medium for the inoculum was enriched with pre-precipitated corn extract (250 ml. Of culture medium).

The precipated corn liquor was prepared as follows:

g of corn extract (60% dry matter) was suspended in demineralised water, the pH was adjusted to 7.6 with 40% NaOH, the solution was made up to 1 liter with water and heated at 100 ° C for 15 minutes. The resulting precipitates were removed by filtration. In a 75-liter stainless steel tank (348 mm diameter, 810 mm high) equipped with four stops, three radial mixers with six 122 mm blades mounted on one shaft (stirrer spacing 170 mm, bottom stirrer from bottom 160 mm) ) and aeration wreaths with a diameter of 158 mm, 40 mm from the bottom, were prepared 50 l of nutrient broth with the following composition: 1% glycerol, 0.4% (NH4) ₂ SO4, 1.36% KH2PO4, 0.3% NaOH 0.5 CaCO3, 0.001% FeSO4.7H ₂ O, 0.003% CaC12.6H ₂ O, 0.2% MgSO4.7H2O, 0.015% silicone antifoam SAG 471. After sterilization (40 minutes, 1.2 atm) was aseptically MgSO4, FeSO4, CaCl2 and CaCO3 solutions (sterilization separately) were added and the pH was adjusted to 7.6 with a 40% NaOH under aseptic conditions.

In addition, sterile stock solutions were prepared: 2.8 liters of 50% (w / v) glycerol (sterilization 2x30 min., 1.2 atm) and 1 liter of 1: 1 ammonia water.

After cooling of the soil, inoculation of sterile broth with 1 liter of inoculum was obtained, which was obtained by pouring the contents of 10 flasks under aseptic conditions. The cultivation was carried out at 25 ° C. The initial aeration was 0.3 volumes of air per volume of culture medium at a stirring frequency of 400 rpm. During cultivation, pH, oxygen partial pressure (pO ₂ ) was measured and samples were taken to determine optical density, ammoniacal nitrogen, cell dry matter, and PAG activity. As the pO ₂ decreased to zero (from about 10 hours of culture), the stirring frequency gradually increased. Since 17 hours of cultivation, glycerol additions corresponding to a concentration in the culture medium of 0.15% were dosed. When the pH dropped to 6.6, pH control was started by the addition of ammonia water in the pH range of 6.3 to 6.6. Cultivation was carried out for 21 hours.

In one liter of culture liquid, PAG 5050 U production was achieved with an increase of 7.2 g dry cell mass. The specific activity was 701 µg of dry cell mass.

Example 3

A culture of Escherichia coli CCM 4228 (pKA18) on slanted agar was prepared as described in Example 1. 10 inoculum flasks were prepared as in Example 2, except that the soil contained 1% sucrose instead of glycerol. .

In a 75 liter stainless steel tank with the same equipment as in Example 2, 50 liters of soil were prepared with the same composition as in the inoculum preparation (i.e. carbon source 1% sucrose) but without kanamycin. For this purpose, 12.5 liters of pre-precipitated corn extract were prepared in the manner described in Example 2. After cooling the soil, the broth was seeded with 1 liter of inoculum under aseptic conditions, obtained by pouring 10 flasks under aseptic conditions. Cultivation in the seed tank was carried out under aeration conditions of 0.3 volume of air per volume of soil at a stirring frequency of 300 rpm. and a temperature of 28 ° C. During culture, the growth of the culture was monitored by measuring the optical density. At the transition to the stationary phase of the culture (5 pm culture), the culture grown in the seed tank was used to inoculate the production tank.

To a 300 liter stainless steel tank (diameter 490 mm, height 1460 mm), equipped with 4 stops, three radial stirrers with six 190 mm diameter blades mounted on one shaft (250 mm apart, bottom stirrer from bottom 85 mm), and aeration rims, 50 mm from the bottom, prepared 180 l of soil with the following composition: 1% sucrose, 0.4% (NH ₄ ) ₂ SO ₄ , 1.36% KH ₂ PO ₄ , 0.4% NaOH, 0, 1 CaCO ₃ , 0.001% FeSO ₄ .7H ₂ O, 0.003% CaCl ₂ .6H ₂ O, 0.2 MgSO ₄ .7H ₂ O, 0.015% SAG 471 silicone defoamer, pH 7.6 to 7.8, sterilization 40 minutes, 1.2 atm. In addition, sterile stock solutions were prepared: 15 L of 50% sucrose solution, 0.25 L of 15% NH 4 Cl solution, 61 ammonia water (diluted 1: 1).

After cooling the soil, aseptic conditions were inoculated with 4 liters of culture from an inoculation tank. The initial aeration was 0.33 volume of air per volume of nutrient medium at a stirring frequency of 150 rpm. The cultivation was carried out at 25 ° C. During cultivation, pH, oxygen partial pressure (pO ₂ ) was measured and samples were taken to determine optical density, ammoniacal nitrogen, cell dry matter, and PAG activity. At 17 hours of culture, NH ₄ Cl was added at a concentration of 0.02% in the broth and dosing of sterile sucrose solution from the storage bottle was started in the event of a sudden increase in pO ₂ , with doses corresponding to a final sucrose concentration of broth 0, 15%. Since 19 hours of culture, the pH has been regulated in the range of 6.4 to 6.6. Cultivation was performed for 34 hours.

In one liter of culture liquid, PAG 14,050 U production and 15 g dry cell mass were achieved. The specific activity was 937 µg of dry cell mass.

Example 4

Cultivation of Escherichia coli CCM 4228 (pKA18) was performed in the same manner as in Example 3, except that sucrose was dosed continuously in micro doses, automatically controlled by pO ₂ changes (dosing at pO ₂ above 7%).

Cultivation was carried out for 42 hours. In one liter of culture liquid, PAG 21,800 U production and 22.4 g dry cell mass were achieved. The specific activity was 973 µg of dry cell mass.

Industrial usability

The fermentation process according to the invention can be used for the preparation of intermediates in the production of antibiotics, the so-called. semi-synthetic penicillins, respectively. cephalosporins.

PATENT CLAIMS

Claims (3)

A method for submerged cultivation of cells of a strain of a microorganism, in particular Escherichia coli, comprising recombinant plasmids producing G-penicillin amidase, in a mineral nutrient medium containing an fermentable carbohydrate as an assimilable carbon source and an inorganic salt and mineral nutrient salts as a nitrogen source. m that during fermentation, the fermentable carbohydrate is added at doses corresponding to a concentration of 0.01 to 3%, or continuously depending on the consumption of carbohydrate, an inorganic nitrogen source corresponding to a concentration of 0.01 to 3% in the medium, calcium, iron and magnesium; while simultaneously adjusting the pH in the range from 6.0 to 7.5, preferably 6.4 to 7.0, by adding alkali, at an elevated pH of 7.3 to 8.0 at the beginning of the cultivation. The process according to claim 1, characterized in that ammonium hydroxide is used as the inorganic nitrogen source and at the same time serves to control the pH in said range. Method according to claims 1 and 2, characterized in that the calcium is used in the form of a soluble salt in a concentration of Ca 2.10 -5 to 0.1% and / or in the form of CaCO ₃ in a concentration of 0.05 to 1%, iron in the form of soluble salts at a concentration of 1.10-5% 2.10-3% _and magnesium in the form of soluble salts at a concentration of Mg corresponding to 0.001 to 0.05%.