SK145794A3 - Method of submerged cultivation of cells producing g-penicillin amidase - Google Patents

Abstract

This method for the submerged cultivation of cells producing G-penicillin amidase, in particular cells of Escherichia coli containing recombined plasmids in a mineral cultivating medium containing fermentable saccharide as a source of available carbon and inorganic salt as a source of nitrogen and mineral nutrient salts, is based on the fact that at the beginning of cultivating the productive micro-organism in the cultivating medium containing soluble salts of calcium at a concentration of Ca = 2.10<-5> to 0.1 % by weight, and/or calcium carbonate at a concentration of 0.05 to 1% by weight, and soluble salts of magnesium at a concentration of Mg = 0.001 to 0.05% by weight, the pH value is adjusted to 7.3 to 8.0, and then during the cultivation the pH is kept at a value of 6.0 to 7.5 while adding fermentable saccharide in doses at a concentration of 0.01 to 3% by weight and an inorganic source of nitrogen in a quantity corresponding to the concentration in the medium of 0.01 to 3% by weight, in particular ammonium hydroxide.

Description

A method for submerged cultivation of G-penicillin amidase producing cells

FIELD OF INVENTION .......

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.

The state of the art

G-penicillin amidase (PAG only) 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-aminopenicillanic acid. (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 batches during fermentation for strains with PAG activity is reported in CS 151 240. however, even with chemically defined soils, the rate of PAG synthesis is greatly increased by the addition of phenylacetic acid iriducer, especially when phenylacetic acid is used as the sole carbon source (Vojtíšek and Slezák, 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, Foil Microbiol. 20, 298-306, 1975) have been prepared in the past. various regulatory 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) (Vojtišek 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 Escherichia coli strains carrying various recombinant plasmids are disclosed in CS 278 516 and CS (PV 1269-92).

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a method for submerged cultivation of G-penicillin amidase (PAG) producing cells in a mineral nutrient medium containing an fermentable carbohydrate as a source of assimilable carbon and an inorganic salt and mineral nutrient salts as a nitrogen source. , in particular Escherichia coli containing recombinant plasmids, adds the fermentable carbohydrate at doses corresponding to a concentration of 0.01 to 3.0%, or continuously depending on the carbohydrate consumption, while adjusting the pH in the range of 6.0 to 7.5, with 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 cultivation. The nutrient medium may advantageously contain calcium in the form of a soluble salt, at a concentration of Ca 2.10 " ^with up to 0.1% and / or in the form of CaCO 3 at a concentration of 0.05 to 1 iron as soluble salts in a concentration of 1.10" ⁶ to 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 cultivation involves the use of 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 high cell concentration with high activity PAG. 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 regulation may adversely affect enzyme production, it is preferable to increase the starting pH of the medium or to add CaCO 3, which increases the buffering capacity of the medium. An appropriate concentration of Mg ²⁺ ions and a low concentration of Fe ²⁺ resp. Fe ³⁺ 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 the arrest of cell culture growth from a lack of carbon or nitrogen source. After adding the next dose of the necessary source and its continued utilization, the p02 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 according to CS (PV 1269-92). Likewise, the culture process can be used for various PAG producers without recombinant plasmids, particularly with reduced catabolic repression against the carbon source used and without the need for an inducer. Preferably, a strain of Escharichia coli CCM 4228 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 ^-1 dry cell mass.

The obtained highly active cell suspension can 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 a paste resp. 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 with the following composition was prepared: 2% glycerol, 0.4% (NhU 2 SO 4, 1.36% KH 2 PO 4, 0.3% NaOH, 0.001% FeSO 3 · 1 H 2 O, 0.005% CaCl 2 · 6 H 2 O, 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, to aseptic conditions kanamycin at a concentration of 35 µg.rnl ^-1 .

The glycerol can strain stored at -20 ° C was inoculated 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 with the following composition: 2% glycerol, 0.2% (NH4) 2SO <t, 1.36% KH 2 PO 4, 0.3% NaOH, 0.5 CaCO 3, 0.001% FeSO 4 .7H ₂ O, 0.005% CaCl ₂ .6H ₂ O, 0.2% MgSO 4 .7H 2 O, 0.015% silicone defoamer SAG 471, pH 7.2-7.3; sterilization for 40 minutes at 1.2 atm. Solutions MgSO4, FeSO4, CaCl ₂ and CaCO are sterilized separately.

ml of inoculum from a flask prepared as described above was aseptically poured into sterile broth in a fermentation tank and cultured 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% (NH 4) 2 SO 4 was added in three portions during the culture. Cultivation was carried out for 17 hours. 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 ^-1 dry cell mass.

Example 2

Prepared culture on solid agar and the inocula 10 cells of Escherichia coli CCM 4228 (pKA19), as described in Example 1, except that the liquid medium for the inoculum was enriched reprecipitation corn steep liquor ⁽¹ 250 ml.l 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 (diameter 348 mm, height 810 mm) equipped with four stops, three radial mixers with six blades of 122 mm diameter, placed on one shaft (stirrer spacing 170 mm, bottom stirrer from bottom 160 mm) ) and aeration rims with a diameter of 158 mm, 40 mm from the bottom, were prepared with 50 L of nutrient broth with the following composition: 1% glycerol, 0.4% (NM2SO4, 1.36% KH2PO4, 0.3% NaOH, 0, 5 CaCO 3, 0.001% FeSO 4 .7H 2 O, 0.003% CaCl 2 · 6 H 2 O, 0.2% MgSO 4 · 7H 2 O, 0.015% silicone defoamer SAG 471. After sterilization (40 min, 1.2 atm), MgSO solutions were added aseptically. , FeSO 4, CaCl 2 and CaCO 2 (sterilization separately) 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 (PO2) was measured and samples were taken to determine optical density, aminiacal nitrogen, dry cell mass, and PAG activity. As the pO2 decreased to zero (from about 10 hours of culture), the stirring frequency gradually increased. Since 17 hours of culture, glycerol additions corresponding to 0.15% of the culture medium 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 ^{-1 of} cell dry matter.

Example 3

A culture of Escherichia coli strain CCM 4228 (pKA18) on a slant agar was prepared as described in Example 1. 10 inoculum flasks were prepared as described 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% for this purpose with corn extract). sucrose) but without kanamycin. To prepare 12.5 liters pre-precipitated as described in Example 2. under aseptic conditions, carried out a liter of inoculum obtained by pouring the contents of 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.

In a 300 liter stainless steel tank (diameter 490 mm, height 1460 mm) equipped with 4 stops, three radial stirrers with six 190 mm diameter vanes, placed on 1 (250 mm spacing, 85 mm bottom bottom) and aeration rim, a distance of 50 mm prepared 180 l of soil with the following composition: 1 sucrose, 0.4% (NH-OzSCU, 1.36% KH2PO4, 0.4% NaOH, 0.1 stirrer shafts from below,

CaCl2.6H2O, 0.2% temperature 25 ° C oxygen (pO2)

CaCO 3, 0.001% FeSO 4 .7H 2 O, 0.003

MgSO 4 .7H 2 O, 0.015% SAG 471 silicone defoamer, pH 7.6 to 7.8, sterilization for 40 minutes, 1.2 atm. In addition, sterile stock solutions were prepared: 15 L of a 50% sucrose solution, 0.25

15% NH 4 Cl solution, 6 L of 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. Cultivation was carried out at pH. During the cultivation, pH, parcillants were measured and samples were taken for determination of optical density, ammoniacal nitrogen, dry cell mass and PAG activity. At 17 hours of culture, NH-oCl was added at a concentration corresponding to 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 pC> 2, with doses corresponding to the final sucrose concentration in the nutrient. soil 0.15%. From 19 hours of culture, the pH was regulated in the range of 6.4 to 6.6. The cultivation takes place 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 U. g ^-1 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-batches, automatically controlled according to pO2 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 ^{-1 of} dry cell mass.

Industrial applicability ......

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

Claims (3)

A method of submerged cultivation of G-penicillin amidase producing cells in a mineral nutrient medium containing an fermentable carbohydrate as an assimilable carbon source and an inorganic salt and a mineral nutrient source as a nitrogen source. 6.0 to 7.5, with an increased pH by adding a fermentable carbohydrate at a concentration of 0.01 to 3%, or a carbohydrate consumption, during the cultivation of Escherichia coli containing, with a concomitant 6.4 to 7.3 to 8.0 characterized by a strain of the microorganism, in particular recombinant plasmids, at dosages correspondingly to a pH adjustment in the range of 7.0 by adding alkali at the start of the cultivation. 2. Method below! Claim 1, characterized in that the inorganic nitrogen source is simultaneously metered in an amount corresponding to a concentration of 0.01 to 3% in the medium, the ammonium hydroxide being preferably used as the nitrogen source, which simultaneously serves to control the pH in said range. 3. The method according to claims 2, characterized in that the nutrient medium contains calcium in the form of soluble salts in a concentration ^of Ca ~ 2.10 to 0.1%, and / or in the form of CaC03 at a concentration of 0.05 to 1%, the iron in the form of soluble salts in a concentration of 1.10 " ⁶ to 2.10" ³ % and magnesium in the form of soluble salts in a concentration of Mg corresponding to 0.001 to 0.05%.