SK35894A3 - Method of fermentating preparation of l-treonine by cultivation of production strain of escherichia coli 472 t-23 - Google Patents

Abstract

This invention involves improving the manner of controlling the fermentation of L-threonine, an essential amino acid used in the feed industry and also for pharmaceutical (therapeutic) purposes, by cultivating the production strain Escherichia coli 472 T-23 in a liquid production medium containing common nutriments and additives. The principle of the new procedure, by which a high growth and production speed and a yield of L-threonine of about 80 g/l is achieved with a conversion of saccharose of about 42%, consists of controlling the agitation and aeration performed according to the value of the pH (6.5 to 7.0) and the content of dissolved oxygen (0.7 to 0.9 mg/l) in the medium, and in the continual addition of a solution of saccharose and ammonia. The novelty of the invention consists of controlling the fermentation of L-threonine using a concentration of dissolved oxygen which is kept at 0.7 to 0.9 mg/l.

Description

FIELD OF INVENTION .......

The invention relates to a method of fermentative production; L-threonaria by culturing the Escherichia coli 472 T-23 production strain in a liquid nutrient medium containing assimilated carbon and nitrogen sources, mineral nutrient salts and / or trace elements and / or biofactors under aerobic conditions.

Pote paradise. wider ....... state ....... techniques

L-threonine is essential and is used both in animal production for feed compound enrichment and in medicine. For economic and technical reasons, it has therefore been necessary to have a technological process which can be used to obtain this amino acid in a simple and acceptable manner. Biosynthetic processes, in particular the cultivation of suitable microorganisms, have been found to be cost-effective and further developed in which their ability to accumulate L-threonine in the culture medium in relative amounts has been exploited. M i k roorgan i z m y ,. Suitable for this purpose, Escherichia coli, synthesize, however, those which are particularly required for proteosynthesis or as an isoleucine biosynthesis intermediate. It was only through a combination of mutation, selection and genetic engineering methods that the strain Escherichia coli 472 T-23 (U.S. Pat.

no. No. 4,278,765), which was able to accumulate up to 40 g / L of L-threonine in sucrose medium at 40% sucrose conversion in 40 hours in culture with sucrose as a carbon source. Other media interventions appropriate

G, s and o r up to 73 g / l, especially at high levels in quantities,

L-threonine in 1 and 1, and penicillin

L-threonine at 66 40%.

A number of further developments in the production of L-threonine has been to find a method of fermentation control that would allow the isoleucine concentrations to gradually increase the sucrose

(b) influencing the metabolic activity of the production strain, which would not only increase production but also create the prerequisites for the automatic control of the newly developed L-threonine fermentation production process.

Summary of the invention .......

The fermentation process of L-threoriine by culturing the production strain of Escherichia coli 472 T - 23 according to the invention in a liquid nutrient medium containing assimilated carbon and nitrogen sources, mineral nutrient salts and / or trace elements and / or biofactors under aerobic conditions satisfies these requirements. ; The principle of the invention is that after the start of the growth of the production microorganism with the simultaneous production of L-threonine, the pH of the nutrient medium is maintained at 6.5 to 7.0, and the oxygen content is 20 to 25% saturated by adding a solution of sucrose and ammonia. , and after stopping the growth, which results in a decrease in the rate to zero and after the sucrose concentration in the nutrient medium falls below 0.5 g / l, the cultivation is terminated.

The high growth and production rate of the microorganism used is possible in the realization! of the method according to the invention maintained throughout the fermentation by continuous dosing of the sucrose solution and the ammonia solution based on a pH measurement that decreases due to increased metabolic activity of the microorganism in the exponential growth phase and manifested by increased respiration, decreased dissolved oxygen concentration, oxygen limit and increased consumption sucrose. By measuring dissolved oxygen concentration and / or pH, a sucrose solution is dosed to restore respiration and further increase production.

In a very simple manner, the process according to the invention was able to increase the yields of L-threonine up to 80 g / l, with a sucrose conversion of about 42%.

in an embodiment of the invention

In a 30 liter fermentation tank, with stirrer and sterile air supply, 15 liters of culture medium containing 450 g of affinity sucrose was charged.

g of yeast autolysate, 75 g of ammonium sulfate, 6 g of magnesium sulfate, 30 g of potassium dihydrogen phosphate, 0.3 g of iron sulfate, 0.3 g of manganese sulfate and 9 g of soybean oil. After sterilization (120 ° C / 30 min), the culture medium was cooled to 37 ° C, 2 g of penicillin G was added and seeded with 1 ml of cell suspension obtained by culturing the production microorganism Escherichia coli 472 T-23 under the same conditions as the production. Fermentation was carried out at a constant temperature of 37 ° C, an overpressure of 0.5 barr and an air flow rate of 0.9 m / s, at a stirring speed of 500 to 900 rpm, maintained automatically so that the dissolved oxygen concentration (initial state 100 % saturation) did not drop below 20% saturation. Dissolved oxygen concentration (initial 100% saturation), pH, temperature, pressure and air flow were continuously measured during the process. The optical density (growth rate) of the L-threonine concentration (production rate), the sucrose concentration and the ammonia and nitrogen concentration were continuously monitored.

After 8 hours, the production organism was increasing as the process was growing and producing at a rate and thereby decreasing the dissolved oxygen concentration. To the 20 th culture hour, adding a 7.0% aqueous solution of 7.0. Consumption of carbon source (probably indicated by decreasing the dissolved concentration concentration of the dissolved solution dosed a liter of 400 so long).

into the medium in one latent phase ammonia, began L-threoriine. As a result of respiration, the pH started to fall and at the same time.

The pH automatically adjusted ammonia to 6.5 up to the 20 th culture (hour) of respiration, resulting in an increase in oxygen in the medium. With an oxygen increase of 25% (to 25% saturation) of affine sucrose containing g sugar and 25% of a 25% aqueous solution, the dissolved oxygen concentration stabilized at 20% saturation.

After the dissolved oxygen dropped below 20% saturation (with a maximum stirring of 900 rpm), a 70% sucrose solution was dosed at a rate of 200 ml / h, again automatically as the concentration increased. dissolved oxygen by 25%; The pH was automatically regulated to 6.5 to 7.0 by the addition of 25% aqueous ammonia solution.

When the growth stopped and the growth rate dropped to zero, the dosing of sucrose was stopped, its concentration was allowed to drop below 0.5 g / l and the fermentation was stopped. After 42 hours of culture, 80 g / L of L-threonine was produced, with a sucrose conversion of 42%.

L-threonine can be isolated from the culture medium either as a technical product or as a pure crystalline substance.

For usability

The L-threonine prepared by the process of the invention can be used both in the feed industry for the production of compound feed and for pharmaceutical (therapeutic) purposes.

Claims (1)

PATENT Ο V E Process for fermentative production of L-threonium by culturing the production strain Escherichia coli 472 T-23 in a liquid nutrient medium containing sources of about a m. Other carbon and nitrogen, mineral nutrient salts and / or trace elements and / or biofactors, under aerobic conditions, characterized by the fact that, following the start of the growth of the production micro-organism, the production of L-threoriine is maintained in the nutrient medium at pH 6.5 to 7.0 and an oxygen content of 20 to 25% saturation by the addition of sucrose solution and ammonia solution and after the growth has stopped and the growth rate of the microorganism has dropped to zero and the concentration in the nutrient medium has fallen below 0.5 g / l the cultivation is terminated.