CS206946B1 - Strain brevibacterium sp.ao 6/79 - Google Patents

Description

The invention relates to a strain of Brevibacterium sp. Both AO and 6/79, producing L-lysine when cultivated on liquid nutrient media containing carbon sources such as sucrose or molasses, nitrogen sources such as corn steeping water, hydrolyzate; peanut or soy flour and mineral nutrient and salts, under single and in particular continuous cultivation conditions.

I The strain of Brevibacterium sp. AO 6/79 was prepared for breeding aimed at preparing mutants that were not phenotypically different from the wild strain but ·; which differs from the strain of initial nutritional requirements. ; j Starting strain of Brevibacterium sp. CB (Research Institute for Antibiotics and Biotransformation in Roztoky u Prahy) is homoserine dependent and resistant to the lysine (SB-7 aminoethyl) -cysteine (hom ^- , AEC ^r ) analogue. In obtaining the above strain, the work focused on the trait of homoserine. The AEC character ^r remained unchanged. The result was a strain of Brevibacterium sp. AO 6/79 phenotypically hom ^- genetic mutation suppressor-type, among other things, when compared with the starting strain with unchanged production capacity. The strain was isolated from a mutagenesis experiment in which nitrosomethylurea at a concentration of 0.1 M was used as a mutagen for 60 minutes. Solution

Both the mutagen and the cell suspension were prepared in citrate to phosphate buffer pH 6.0. After the experiment, both control and test suspensions were embroidered in Petri dishes (10 mm diameter) with complete agar broth to determine the proportion of surviving cells. The test suspension was also inoculated into liquid complete soil and cultured on a shaker (240 rpm, 25 mm deflection) at 28 ° C for 48 hours in 10 ml Erlenmeyer flasks containing 20 ml of soil inoculated with 1 ml of test suspension. At the end of the culture, the suspension was embroidered in petri dishes with minimal agar medium containing glucose and sodium citrate, ammonium sulfate and trace elements. The grown colonies were tested for lysine production by a simple test in which they were monitored by the growth zones of the lysine (lys ^- ) dependent Escherichia coli indicator strain around the rollers of agar soil with a culture of tested isolates at the top. Brevibacterium sp. AO 6/79 was isolated in this manner. Its production was verified by a shake culture test and in laboratory tanks on complete soil and found to be consistent with that of the parent strain of Brevibacterium sp. CB. ____________

Brevibacterium sp. AO 6/79 does not differ morphologically from the parent strain, but is distinguished by its physiological properties, which are a manifestation of genetic change. These are nutritional requirements, growth kinetics and production properties.

ί The difference in Brevibacterium sp. CB is an AO 6/79 strain capable of growing on minimal soil and the difference in demand or stimulation of growth by homoserine is also significant. Under standard conditions (optimal homoserine level, baseline culture cell concentration, etc.), the highest specific growth rate of 0.48 hours ^-1 was measured for the CB strain and 0.27 hours ^-1 for the strain of the invention. When the soil content of homoserine was reduced to 1/10 of the optimal level, the specific growth rate of the CB strain decreased to 38% of the baseline; The different enzymatic equipment of both strains is also evidenced by growth kinetics. The saturation constant of growth on sucrose is 105 mg / liter for the CB strain, while 259 mg / liter for sucrose for the strain according to the invention.

There were no significant differences in flask culture levels when tested for production capability under standard conditions (CB strain 31.1 g lysine / liter, with a sucrose to lysine conversion coefficient of 0.174, strain according to the invention 30.5 g lysine / liter, with a coefficient of 0.170). Similar results were obtained in two-liter fermenters (CB strain 71.3 g / liter and 0.214 strain according to the invention).

78.1 g / liter and 0.22). However, at the level of twenty-liter fermenters, again under standard conditions, the CB strain accumulated 65 g of lysine / liter, with a conversion coefficient of 0.22, while the strain of the invention produced 60 g / liter with a conversion coefficient of 0.26. ------------------- Strain of Brevibacterium sp. AO 6/79 was grown for both single and mainly for the continuous process of lysine biosynthesis. Auxotrophic strains are more or less unstable and therefore suppressor-type mutants, with their physiological and production stability, are genetically based on one of the few solutions for the preparation of a stable strain for continuous cultivation.

A new strain of Brevibacterium sp. was deposited in the type culture collection at IHE, Šrobárova 48, 100 42 Praha 10, under number AO 6/79.

Further details can be seen from the examples.

Examples *

Example 1

By culture of Brevibacterium sp. AO 6/79, grown on sloping agar of complete soil (J. Lederberg, Methods in Med. Res. 3,5-22,1950), inoculated with 60 ml of inoculum broth in 500 ml flasks. Soil · has the following composition (in% by weight):

sucrose 2.5 corn steeping water 3.0 tap water to 100.0 pH before sterilization 7.0, after sterilization for 30 minutes at 120 ° C 6.8 to 7.0. Cultivation on a rotary shaker (240 rpm, 25 mm deflection) at 28 ° C lasted 24 hours.

The resulting vegetative inoculum is seeded in an amount of 10% by volume with 20 ml of production soil in 500 ml boiling flasks of the following composition (% by weight):

sucrose 18.0 maize steeping water 1.0 potassium phosphate primary 0.1 magnesium sulfate cry. 0,015 calcium carbonate 3,0 peanut flour acid hydrolyzate 25,0 ammonium sulphate 1,0 tap water up to 100,0

Sterilization at 120 ° C for 30 minutes; The pH adjusted to 7.8 to 8.0 with 25% ammonia solution ranged from 6.7 to 7.0 after sterilization. Cultivation at 28 ° C lasted 48 to 96 hours on a rotary shaker. The average lysine production was 96 hours

30.5 g / liter.

Example 2

The vegetative inoculum prepared as in Example 1 was inoculated at 7.5% by volume with 800 ml of production soil of the same composition as in Example 1, placed in 2-liter laboratory tanks. Sterilization at 120 ° C for 30 minutes. Stirring 770 ipm, aeration 0.8 liter / min. The average lysine production at 72 hours of fermentation was 78 g / liter. According to consumption and fermentation, sucrose was added during the cultivation.

Example 3

Cultivation in 20-liter laboratory fermenters (10-liter working volume) was carried out in three stages: flask inoculum - inoculation feeder - production fermenter. The inoculum in the flasks was prepared as in Example 1. The inoculation fermenter was filled with soil of the following composition (in% by weight):

sucrose 2.5 i corn steep water) 3.0 technical biotin (1%) 0.0006 thiamine 0.00002 soybean oil 0.3 tap water up to 100.0 pH was adjusted to 6.8 to 7.0, sterilization at 122 ° C for 60 minutes. The fermentor was inoculated with 2% by volume flask inoculum, propagation was continued for 14 hours, with stirring at 350 rpm.

and aeration 10 liters / min., at 28 ° C.

The third stage, ie the actual production process, took place in the soil of the following composition (in% by weight): sucrose 18.0 maize steeping water 1.0 peanut flour hydrolyzate 25.0% by volume potassium phosphate primary 0.2 magnesium sulfate crystals. 0.03 technical biotin (1%) 0.0005 thiamine 0.00002 polypropylene glycol 0.02% v / v

tap water up to 10 liters

The pH was adjusted and maintained at 6.8 to 7.0 during fermentation. The inoculum was 10% by volume of the grown second stage culture. Additional sucrose was added from the stock solution, depending on consumption, culture, and pH. Culture conditions: stirring 470 rpm, aeration 10 liters / min, temperature 28 ° C. Depending on the conditions used and the quality of the production micro-organism

Claims (3)

SUBJECT Brevibacterium sp. AO 6/79 producing L-lysine. 3 ......... '................... was achieved at 72 hours of production '63 to 65 g of lysine / liter, with a conversion factor of sucrose to lysine of 0.29 to 0.32.