CS246954B1 - Production method of the l-lysine by biosynthesis - Google Patents

Abstract

The invention addresses L-lysine aerobic biosynthesis, wherein the culture medium is inoculated with the production inoculum microorganisms grown in inoculation the soil of the same composition as the cultivation soil and taken from the cultivation soil to beginning of the stationary phase of the growth curve, with a decrease in glutamic acid concentration in inoculum to 50% vol., acid aspartic to 60% by volume and isoleucine to 30 4 vol. Defaults, with permissible deviation - 10%.

Description

The invention relates to a process for the fermentative production of L-lysine by culturing auxotrophic mutants of microorganisms of the genera Microooccus, Brevibaoterium or Corynebaoterium, requiring homoseYin for growth in liquid nutrient media containing sources of assimilable carbon and nitrogen and mineral nutrient under aerobic conditions.

Known processes for the biosynthetic production of L-lysine using auxotrophic mutants of microorganisms, using in particular nutrient media as essential components mainly of amino acids, supplied in the form of variously modified natural nitrogen sources / British Pat. No. 851,396, U.S. Pat. No. 2,979,439, Japanese Pat. No. 26,943 (63), generally produce less than 55 g / L-lysine per liter of nutrient bore in the 80th hour of fermentation, which has hitherto been considered an upper limit, according to known data, due to the occurrence of reverse production microorganism to the original prototrophic strain. According to Japanese Pat. 55 g / liter production is reported to be achievable only by suppressing the reverse auxotrophic mutant of the antibiotic additive to which the original prototrophic strain is much more sensitive.

It has now been discovered that in L.lysine biosynthesis with homotinin-dependent auxotrophic mutants, production ability is significantly influenced by intervention in the Krebs citrate cycle, which essentially potentiates the metabolic pathway to L-lysine. By regulating the phosphorus concentration on the one hand, the respective phosphorylation processes leading to the formation of the puruvate can be potentiated, but on the other hand, limiting the magnesium concentration associated with decreased carboxylase activity and suppressing unwanted side-processes through acety1-coenzyme Λ. to oxaloacetate and thus 1 complete citric acid cycle.

Under these conditions, the cycle of citric acid can then be initiated by the controlled addition of some biosynthesis intermediates, particularly citric acid. By this intervention, the production rate can be unexpectedly increased including the degree of conversion of the carbohydrate component to L-lysine, with the possibility of achieving a much higher final L-lysine production. Balancing the content of the basic components in the broth plays a very important role in achieving these maximum yields.

At the start of the process, the production capacity of biomass is basically conditioned by the relative ratio of the concentrations of amino nitrogen and ammonia nitrogen. If this system is potentiated by phosphate additions while limiting the magnesium to a certain maximum concentration, a very rapid production acceleration can be achieved which allows additional sugar additions during the fermentation and thus a higher degree of conversion of the sugar component to L-lysine.

Based on the above findings, a process for the fermentative production of Lvlysin according to the invention has been developed, which is characterized in that the cultivation is carried out in a nutrient medium which initially contains 1: 6 amine nitrogen and ammoniacal nitrogen at a minimum amine nitrogen concentration of 1.5 mg / ml, phosphate and magnesium ions in a ratio of at least 10: 1, at a minimum phosphate ion concentration of 1 mg / ml, respectively. citric acid or a salt thereof, preferably calcium, at a concentration of 0.1-2% by weight.

If necessary, the minimum ratio of phosphate ions to magnesium ions can be maintained during the biosynthesis by adding phosphate in one or more portions.

Under these conditions, a high production rate exceeding 1 mg / 1 ml of L-lysine per hour is achieved with a high final fermentation yield. The results obtained by the process according to the invention are at least 30% higher in terms of production rate than those previously known, for example, in Japanese Patent No. 26,943 / 63.

Examples

He did

In a 250-liter fermentation tank, 150 liters of non-suspension broth were prepared containing neutralized and filtered hydrolyzate from 7 kg of peanut flour, 2.25 kg of corn liquor (60%), 30 kg of sucrose, 300 g of potassium phosphate, 450 g soybean oil. After 40 min. sterilization at 120 ° C, cooling to 29 ° C and adjusting the pH of the aqueous ammonia solution to 7.0, sterile broth containing 2 mg / ml of amino nitrogen and 12 mg / ml of ammoniacal nitrogen was inoculated with 15 liters of inoculum of Corynebacterium sp. VÚA 9366/454 / collection designation of an unspecified strain grown in the Research Institute of Antibiotics in Roztoky u Prahy /.

The cultivation was carried out at 29 ° C under constant aeration. At 20 and 40 hours, 50 g. Of potassium phosphate was added. 4.5 kg of sucrose was also added at 40 o'clock. During the entire fermentation, the pH was maintained at 6.0-7.0 by addition of ammonia. The fermentation was terminated at 60 hours. The content of L-lysine in the fermented soil was 60 g / liter with a final soil volume of 170 liters. The yield of 10.2 kg of L-lysine hydrochloride from 34.5 kg of sucrose corresponds to a conversion rate of 29.6%.

Example 2

Procedure and Nutrient broth as in Example 1 except that Micrococcus glutamicus strain ATCC 13287 was used as the production microorganism, and 1.5 kg of calcium citrate was added to the broth at the start of fermentation and after 3 and 30 hours at 30 and 45 hours. 75 kg of sucrose. Phosphate was not added. The content of L-lysine in the soil after fermentation at 60 hours was 72 g / liter with a final soil volume of 170 liters. The yield of 12.22 kg of L-lysine hydrochloride from 37.5 kg of sucrose corresponds to a conversion rate of 32.6%.

Example 3

The procedure and the culture medium were the same as in Example 1 and the production organism as in Example 2.

0.5 kg of citric acid was added to the soil before sterilization and 3.75 kg of sucrose at 30 and 45 hours. Phosphate was not added. The content of L-lysine in the soil after fermentation at 72 hours was 67 g / liter with a final soil volume of 170 liters. The yield of 11.38 kg of L-lysine hydrochloride from 37.5 kg of sucrose corresponds to a conversion rate of 30.4%.

Claims (2)

Process for the fermentative production of L-lysine by culturing auxotrophic mutants of a microorganism requiring homoserine for growth in liquid nutrient media containing assimilable carbon and nitrogen sources and mineral nutrient salt under aerobic conditions, characterized in that the cultivation is carried out in nutrient medium , which at the beginning of the fermentation contains amine nitrogen and ammoniacal nitrogen in a ratio of 1: 6, at a minimum concentration of amine nitrogen of 1.5 mg / ml phosphate and magnesium ions in a ratio of at least 10: 1, at a minimum concentration of phosphate ion 1 mg / ml, . citric acid or a salt thereof, preferably calcium, at a concentration of 0.1-2.0% by weight. 2. The method of claim 1, wherein the minimum phosphate ion to magnesium ion ratio is maintained during the biosynthesis by adding phosphate in one or more portions.