CS244333B1 - Method of c-cephalosporine fermentation production with utilization of fats as limitating carbonaceous substrate by means of acromonium chrysogenum strain - Google Patents

Description

The invention relates to a process for the fermentation production of cephalosporin C using fats as a limiting carbon substrate using the strain Acremonium chrysogenum.

Cephalosporin C - an intermediate for the preparation of broad-spectrum high-performance semi-synthetic antibiotics is prepared by submerged aerobic fermentation using suitable cultures and suitable sterile nutrients. The ability to produce cephalosporin C has been found in several microorganisms, e.g. Cephalosporium polyaleurum, Arachnomyces minimus, Anixiopsis peraviana, Spiroidium fuscumm (Kitano K., Kintaka K., Suzuki S., Katamoto K., Nara K., Nakao Y .: Biol. Chem. 38, 1974, p. 1761).

In production practice, they are mostly used for the biosynthetic production of cephalosporin C strains of Acremonium chrysogenum (GamsW .: Cephalosporium - artige Schimmelpilze - Hyplomycetes, Stuttgart 1971), previously classified as Cephalosporium acremonium.

Acremonium chrysogenum strains utilize spirits monosaccharides, disaccharides and polysaccharides, which are supplied to nutrients below 2-10% for their growth and production of cephalosporin C. Fats and their hydrolysates, respectively, are also suitable carbonaceous substrates for the fermentation of cephalosporin C. distillation fractions, optionally fats stabilized with antioxadants, e.g. AO - 185,858. Hubert et al. (Hubert FM, Tietz AJ: Delined measures the strategies for the biosynthesis of cephalosporin C, Biotechnology Letters, 6, 1983, pp. 385-390), compared the possibilities of fermentation of cephalosporin C on a pod where the main carbon substrate was carbohydrate-glucose with pod, where the carbon source was provided by fats - soybean oil or pork ointment distiller. In both cases, the carbon source was used as a limiting substrate. The use of fats in the fermentation of cephalosporin C has been shown to be operatively better and the fermentation process has been more stable than using dosed glycose. The fats in the fermentation of cephalosporin C also act as antifoams.

Organic nitrogen in the cephalosporin C fermentation pod can be provided by various plant or animal proteins such as soybean, peanut, cotton and fish flour, or corn extract and others.

Knowledge of cephalosporin C biosynthesis suggests the key importance of the four essential components for the construction of the cephalosporin C molecule: alpha aminoadipic acid, valine, acetate, cysteine (Loder PB, Abraham E. P., Biochem. J. 123, 1971, p. 471 to 477). The first three components are produced by the production microorganism - Acremonium chrysogenum from the previously mentioned carbon and nitrogen nutrients, while the amino acid - cysteine has always been a special problem for the fermentation of cephalosporin C.

The breeding and selection of production cultures of Acremonium chrysogenum yielded auxotrophic mutants with a genetic block on the sulphate to sulphide reduction pathway and, at the same time, increased ability to produce cephalosporin C. These strains utilized DL-methionine to feed the nutrient into the nutrient. pod in amounts of 1.0-2.2% (Demain AL, Drew SW: Antimicrob. Agents Chemother., 8, 1975, p. 5).

Substances in which the main carbon substrate is a carbohydrate and containing DL-methionine in an amount greater than 0.5% are protected in patents: U.S. Pat. No. 381,257 of Nov. 11, 1974, U.S. Pat. No. 3,082,155, issued October 19, 1963; 2 239 321 of 22 February 1973 and MS. No. 198,425 of Aug. 30, 1982. The use of DL-methionine in fermentation flasks greatly increased the raw material cost of fermentation of cephalosporin C and also caused some handling problems, e.g. when dosing DL-methionine during fermentation and complicated the purification of the final product. Therefore, mutants have been searched which are capable of utilizing sulfate for the biosynthesis of the cysteine intermediate at high yields of cephalosporin C. Such strains are also the subject of MS. AO č. 243 785.

Testing of sulphate utilizing mutants for cephalosporin C is mentioned by Komatsu et al. (Komatsu K., Iohaira R .: J. Antibiot. 30, 1977, p. 226) and Niss (Niss H. F .: Antimicrob. Agents Chemother., 4, 1973, p. 474). Martin (Martin F .: Trends Antibiot. Res. Genet. Biosynth. Actions New Subs., Proc. Int. Conf. 1982, pp. 258-268, ins. CA 100/19 / 1,535 73/1984) states the meaning of concentration phosphates for the fermentation of cephalosporin C. The optimum phosphate concentration according to this publication is 30 to 50 mM.

Our proposed method of fermentation production of cephalosporin C using fats as a limiting carbon substrate using a sulphate utilizing strain of Acremonium chrysogenum is characterized in that a fermentation feed containing soybean oil or other suitable feedable vegetable or animal fats is used for culture growth and cephalosporin C production. carbon source supplemented with a small amount of carbohydrates for the growth phase. This ensures optimal supply of C source to the cell with minimal risk of inhibiting production by catabolic repression. As the source of organic nitrogen, corn steep liquor (csl.) Or similar available raw materials, e.g. hydrolyzates of soybean, resp. peanut flour. Ammonium sulphate and ammonia water as sources of utilizable nitrogen and sulfur and other inorganic compounds CaCO3, KH2PO4, MgSCU. Throughout the cultivation, the fermentation process is controlled by feeding the substrates and adjusting the fermentation parameters (temperature, air flow, stirrer speed, and air overpressure in the fermenter) of the dissolved oxygen concentration in the ground, pH, and substrate concentration. The detailed benefits of the individual components of the soil are as follows: soybean oil as a basic carbon substrate in an amount of 1 to 2%. During the fermentation, soybean oil is metered in such a way that its concentration is maintained in the range of 0.1-1.5%. Its total consumption is 14 to 20 percent based on the base stage. The oil dosing rate is 0.5 to 2.6 liters.

. h ^_1 . m ^{3 1 of} land. An additional carbon source is the carbohydrates present in amounts below: corn starch 0.6 a, 3.5% / d, dextrin 0.35-6.0 / o, sucrose 0.2-0.7% . It is also possible to use a fermentation broth in which the above-mentioned carbohydrates are not present in the basic composition. The presence of carbohydrates, however, allows a smoother transition of the producer's metabolism from the inoculation saccharide to the production! - this will improve the adaptation of the culture and ensure a faster growth and hence the onset of the production phase. The corn liquor is added to the pod in an amount of 8 to 14%, calculated at 100%, and in addition to providing organic nitrogen to the producing microorganism, it is also a source of phosphate ions. The phosphate level is further adjusted by the addition of KH2PO4 in an amount of 0.5 to 1.0%.

The increased energy metabolism of the present process for the fermentation preparation of cephalosporin C requires, in addition to the increased phosphate concentration, also a higher level of magnesium ions, which in the bottom is secured from MgSO4.7H2O in concentrations of 0.35 to 1.1%. It is not necessary to supply methionine in pure form to the production stage. A necessary amount of methionine of 0.1 to 0.2% is provided by the corn liquor. This amount is necessary for the growth of the producer and the effect of this amino acid on the fractionation of the vultures on the arthrospores, which are indicative of maximum production activity, is also believed. The basic building block of the cephalosporin C cysteine molecule is provided from (NH4) 2SO4, which is supplied to the pod in an amount of 0.5 to 1.8%. The reduction of sulfate to sulfide and its subsequent conversion with serine to cysteine is the basis of a rational pathway for incorporating sulfur into the antibiotic molecule. Calcium carbonate is supplied to the nutrient pod in an amount of 0.6 to 1.2%. For the proposed process, it is preferable to use chemically precipitated carbonate and not natural ground - mineral.

The inoculum for fermentation of cephalosporin C is prepared by propagating a culture of Acremonium chrysogenum on an inoculum pod containing corn steep liquor, sucrose, ammonium acetate, methionine and soybean oil. The pH of the soil is adjusted to 7.0 + 0.1. It is cultivated at a temperature of 28 ° C, with aeration of 1 volume of air / 1 volume of pod and overpressure in a 70 kPa fermenter. The volume of the seed lot and the number of propagation stages are chosen according to

G of the production pod volume so that the inoculum volume to be inoculated is at least 10% of the production pod volume. Inoculum parameters suitable for inoculation at this stage are given by a sediment of at least 15% (sedimentation at 300 rpm for 10 minutes) and a pH value of 6.6 to 6.8. The duration of one inoculation stage is 48 to 72 hours.

The temperature in the production tank is maintained at 28 ° C during the start-up phase (initial biomass growth). Between 30 and 48 hours of cultivation, the cultivation temperature is reduced to 25 degrees Celsius according to the biomass growth rate characterized by the O2 consumption measured by the manometric method, which should be at least 35 μΐηοΐ. ml ¹ . h ^_1 . When the O 2 concentration in the fermentation pod falls below 6 mg / L, the temperature of the fermentation pod decreases to 24 ° C. The flow of sterile air is maintained in the range of 0.6 to 1.8 air volume / min during fermentation. per volume of fermentation broth. The pressure over the fermentation medium (overpressure in the fermenter) is maintained in the range 70 to 80 kPa. When the dissolved O2 concentration drops below 6 mg / l, it increases to 90-95 kPa.

Fermentation of cephalosporin C has high demands on mass transfer and especially oxygen transfer; the mixing of the fermentation pod largely ensures these demands by means of turbine mixers with a separating disc and 6 straight blades (ON 691021). The diameter of the fermenter diameter to the stirrer diameter D / d = 3. The mixing intensity is increased by 4 radial stops with a width b = D / 10.

The optimum number of revolutions must be determined experimentally for a given device, given the mass transfer, shear force and power input of the stirrer motor.

A suitable pH for the production charge of cephalosporin C fermentation is in the range of 5.5 to 5

6.3. Due to the stability of the fermentation process, it is necessary to maintain the pH in the range of 0.1 pH, which is maintained by adding ammonia water (20-25% NH 3 content) by automatic regulation.

Ammoniacal nitrogen content is maintained from 0.75 to 1.0 mg / ml by adding 200 mg of ammonium sulfate solution after a drop in its concentration from the initial values (2.0 to 4.5 mg / ml, depending on the initial soil composition). kg / m ³ .

The concentration of dissolved O2 after a drop from the initial concentration of 13 to 15 mg / ml (depending on the fermenter pressure) is maintained at 4 to 5 mg / ml by changing the fermentation parameters - temperature, aeration, overpressure, or stirrer speed, and mainly oil . At a dissolved oxygen concentration of 5 to 6 mg / L and an oxygen uptake of culture of at least 65 .mu.mol / ml. h, the biomass increase is optimal. The oil concentration at this stage is maintained at 0.1 to 0.2% in relation to the dissolved oxygen concentration in the fernientation pod to maintain the above-mentioned sufficient concentration of 4-5 mg / l oxygen.

Adjusted soybean oil dosing regimen controls biomass growth (avoiding excess mycelium growth and thereby reducing processable filtrate) and unwanted reduction of dissolved O2 concentration to suboptimal levels (1-3 mg / L), at which cephalosporin production rate is already significantly reduced C.

The following examples better illustrate the invention without limiting it.

Example 1

Acremonium chrysogenum CCM F-750 strain was used as production microorganism. Fermentation was done in a 30 liter tank. Vegetatively inoculum was prepared by submerged cultivation in 2-liter dishes (culture time 72 hours) at 28 ° C on sucrose 2%, corn extract (100%) 1%, ammonium acetate 0.5%, DL-methionine 0.05%, soybean oil 0.3%, with cane inoculums inoculated with 100 ml inoculum prepared on a shaking machine.

The fermentation tank - production - contained soil of the following composition: Maize leach (100%) 8.7%, maize starch 2.0 percent, white dextrin - potato 1.5%, sucrose 0.7% CaCCU precipitated 1.1% (NH4) 2SO4 1.4%, MgSO4.7H2O 0.35%, KH2PO4 0.5%, soybean oil 2.0%.

It was sterilized for 45 minutes at 120 ° C, the pH of the soil was adjusted to 6.5 before sterilization. Sterile administered, cooled to 28 ° C, was seeded with 10% v / v. inoculum. The tank was equipped with 3 turbine mixers with 6 straight blades with a separating disc and 4 stops. The stirrer speed was varied continuously from 600 to 650 rpm during the fermentation. and an air flow rate of 6 to 20 liters / min. The tray temperature was maintained at 28 ° C until the manometrically measured oxygen consumption reached 35 ° C. mh ¹ . Ir ¹ and then decreased (at 32 hr. Culture) to 25 degrees. Celsius. The dissolved oxygen concentration in the pod, decreasing from the initial value (14.4 mg / l at 70 kPa overpressure) was maintained within the range of 4 to 6 mg / l during the production phase (48 to 140 hours of cultivation) by varying the air flow in the range 6 to 20 l / min, by varying the air flow in the range from 6 to 20 l / min, by changing the overpressure in the range from 70 to 90 kPa, by changing the stirrer speed in the range from 600 to 650 rpm. and changing the soybean oil dosing rate. The oil concentration is within 92 h. fermentation was maintained by varying the dosage of oil in the range of 0.5-1.0%. Thereafter, the dosing of the oil was controlled by the dissolved oxygen concentration, and the parameters affecting its concentration hurt constantly.

Maintaining a dissolved oxygen concentration of 4-6 mg / L, the soybean oil concentration was in the range of 0.1-0.5%. The rate of oil addition during fermentation varied between 10-35 ml. h ^-1 . The pH was controlled automatically by the addition of ammonia water (25% in the range of 6.1-6.2. Ammoniacal nitrogen concentration did not fall below 0.80 mg / ml during cultivation. Cephalosporin C production activity of 23.400 was reached in 140 h fermentation ( g / ml.

Example 2

Fermentation was controlled by a method using fats as a limiting carbon substrate as described in Example 1 except that the fermentation broth contained not 8.7% but 10% corn steep liquor. Overall, however, the fermentation process was faster and some parameter changes were scored as shown in Example 1 but under the same criteria. From 72 h. the temperature was lowered to 24 ° C. In 116 h. cephalosporin C production of 25,680 µg / ml was achieved in the culture.

Example 3

Experimental conditions and fermentation regime as in Example 1 except that the changes were only in the following nutrients: corn steep liquor (100%) 10%, corn starch 0.6%, dextrin 0.35%, sucrose 0.2% . In 140 h. Fermentation produced a cephalosporin C production activity of 26.150 µg / ml.

Example 4

The fermentation was carried out in a 5000 liter fermenter equipped with mixing with one turbine with 6 straight blades with a separating disc and 2 turbines with 6 bent blades and 4 stops. The stirrer speed varied continuously within the range of 147-210 rpm, the air flow varied within the range

2 - 5 m ³ / min, the overpressure was regulated automatically. The production strain and the composition as described in Example 3 were used for the experiment. The sterile broth, cooled to 28 ° C, was inoculated with 300 L of inoculum prepared by two-stage cultivation, the first stage being carried out in two

3 liter Erlenmeyer flasks with 750 ml inoculation tray at 28 ° C for 72 hours. The second stage was carried out in a 500 liter fermenter filled with 300 liters of the inoculation tray for 72 hours at 28 ° C, with an air flow rate of 300 liters / min. and an overpressure in the fermenter of 70 kPa. During fermentation, the temperature in the production tank was maintained at 28 ° C until the manometrically measured oxygen consumption reached 35 µmol. ml ^-1 . h ^-1 and then decreased at 38 h. at 25 ° C. The concentration of dissolved oxygen in the soil after the decrease from the initial concentration of 14.8 mg / l was maintained during the production phase (48-142 h. Fermentation) in the range of 4-6 mg / l by changing the air flow in the range 2000-5000 liters / min. by varying the stirring speed of 147-210 rpm, changing the air overpressure in the range of 70-90 kPa, reducing the culture temperature to 24 ° C, and mainly changing the soybean oil dosing rate. Oil concentration up to 62 h. fermentation was maintained by varying the dosage in the range of 0.5-1.0% soybean oil. Thereafter, the oil dosing was controlled by the concentration of dissolved oxygen in the soil. Indeed, the parameters affecting the oxygen concentration of sticks are already constant at this stage. Maintaining a dissolved oxygen concentration of 4-6 mg / L, the soybean oil concentration was in the range of 0.1 to 0.5%. The rate of oil addition during the fermentation varied from 1.6 to 8 L / h, the pH was controlled automatically by adding ammonia water in the range of 0.6 to 8 L / h.

6.1 1. In 142 h. Fermentation produced a cephalosporin C production activity of 23,600 μξ / / ml.

Claims (1)

Oxygen in the post-drop from the initial concentration of 14.8 mg / L was maintained in the range of 4-6 mg / L during the production phase (48-142 h. Fermentation) by the airflow shift in the 2000 range. -5000 liters / min, by changing the stirring speed of 147-210 rpm, changing the air overpressure in the range of 70-90 kPa, lowering the cultivation temperature to 24 ° C, and mainly by changing the soybean oil feed rate . The oil concentration up to 62 h of fermentation was maintained by varying the dosage in the range of 0.5-1.0% soybean oil. Thereafter, the dosing of the oil was controlled by the dissolved oxygen concentration in the inlet. Indeed, the parameters affecting oxygen concentration were already constant at this stage. While maintaining the dissolved oxygen concentration of 4-6 mg / L, the soybean oil concentration ranged from 0.1 to 0.5%. The oil addition rate during the fermentation varied from 1.6 to 8 L / h, the pH was controlled automatically by the addition of ammonia water in the range of 0.6 to 6.1. cephalosporin C production activity 23,600 μξ // ml. PREPARATION OF THE CELLALPORIN C FERMENTING PROCESS USING FATS AS A LIMITING CARBON SUBSTANCE WITH THE ACEMONIUM CHRYSOGENUM STRAP, USING SULFUR SULFUR AND AMMONIUM NUCLEAR FOR Cefalo-Sporin C Formation On Stages Containing Vegetable or Animal Resins as Major Carbon Sources preferably soybean oil optionally supplemented with starch, dextrin and sucrose, an acodic substrate of corn liquor and sulphamic acid and, moreover, compounds KH2PO-1, MgSO4-7H2O and CaCO3 characterized in that the fermentation process is controlled by the dosing of the substrates and by adjusting the fermentation parameters temperatures in the range of 24-28 ° C, air flow in the range of 0.6-1.8 v / v to the volume of the fermentation vessel, stirrer speed according to the type of fermentor, overpressure in the fermenter in the range of 70-95 kPapodl ' and dissolved oxygen concentration, pH and substrate concentration such that the pH is maintained in the with ammonia water in the range of 5.6-6.3, preferably in the range of 6.0-6.1, that the ammoniacal nitrogen content of the populations at its initial value of 2.0-4.5 mg / ml is maintained at 0.75 1.0 mg / ml by dosing the ammonium sulfate solution so that the soybean oil is dosed during the fermentation so that its concentration is maintained in the range of 0.5-1.5% until the growth phase which it is characterized by oxygen consumption measured by a manometric method of 65 μg .. mol. ml-1. h ' 1 and lowering the dissolved oxygen concentration below 6 mg / l and then the soybean oil is dosed so that the dissolved oxygen concentration is maintained at 4 - 5 mg / l, corresponding to a soybean oil concentration of 0.1-0.2 %, while the parameters affecting the concentration of dissolved oxygen in the podium need to be regulated.