CS208592B1 - Streptomyces aureofaciens microorganism strain - Google Patents

Abstract

The invention relates to a new strain of the microorganism Streptomyces aureofaciens CCM 3406 producing chlortetracycline.

Description

The invention relates to a strain of the microorganism Streptomyces aureofaciens, producing chlortetracycline. As is known, this antibiotic is obtained by aerobic cultivation of a suitable production strain in a liquid nutrient medium containing the basic necessary components. The industrial production of chlortetracycline, which has been carried out for a considerable time, has reached a stage where further increase in production by simple interventions is no longer possible, through gradual progress improving all parameters. A certain starting point is fundamental changes in production technology (reconstruction of fermentation tanks, mixers, dosing devices, etc.); however, these are very expensive matters and their possibilities are limited. Even in changing the composition of nutrient media, in using new soil components or in conducting the fermentation process, it is no longer possible to obtain economically significant reserves; one of the few possibilities is to find, select or breed a new production strain that would produce chlortetracycline in higher yields than before on soil of the usual composition, in conventional equipment and without demanding interventions in the fermentation process and also in a favorable ratio to tetracycline, which is formed at the same time. Breeding a new strain providing high yields is a lengthy and demanding matter. It is much easier and faster to apply simple mutagenic interventions to a high-production strain that is available, with the aim of trying to obtain a new strain with the desired properties.

The stated goal was achieved according to the invention with a new strain of Streptomyces aureofaciens, designated NMUXXIII/61, deposited in the Czechoslovak Collection of Microorganisms of the J. E. Purkyně University, Brno, class Obránců míru 10, under the number CCM 3406.

This strain was obtained from a production strain that was used for many years in the commercial production of chlortetracycline with an average yield of 6100 to 6300 µg/ml, under the name Streptomyces aureofaciens Gamma X/237 (internal name of the Research Institute of Antibiotics and Biotransformations in Roztoky u Prahy J. The initial strain was subjected to a series of subsequent mutagenic interventions and passive selections, primarily with ethyl methanesulfonate, nitrosomethylurea, gamma rays and ultraviolet light. The last mutagenic intervention was the treatment of nitrosomethylurea at a concentration of 0.15 M for 100 minutes in phosphate buffer with a pH of 6.8 on the spore suspension of strain Gamma X/237, obtained by washing the sporulated culture with water with 0.1% non-ionogenic surfactant. During the treatment, the mutation mixture was stirred on an electromagnetic stirrer at 25 °C. At time intervals after At 30, 45, 60 and 100 minutes of exposure to nitrosomethylurea, samples were taken and diluted in a dilution series with a factor of 10 while simultaneously diluting nitrosomethylurea to an ineffective concentration. The diluted samples were plated on Petri dishes with sporulation dextrin agar with sucrose so that there was no contact between the colonies, and after sporulation, individual colonies were re-inoculated onto agar slants. The cultures obtained on agar slants represented the starting material for the entire production evaluation process, carried out according to the following scheme:

I. vegetative generation -* II. vegetative generation -> fermentation.

The microorganism strain Streptomyces aureofaciens CCM 3406 exceeds the production of currently used strains, including production strains, for example S. aureofaciens Gamma X/237, by an average of 20 to 30%, while maintaining all suitable technological properties, including a favorable ratio of chlortetracycline to tetracycline (CTC:TCj.

The microorganism strain S. aureofaciens CCM 3406 forms colonies measuring 10.5 mm when cultivated on dextrin agar, with a marginal non-sporulating zone of 1.5 mm.

The aerial mycelium is white, but becomes brown to grayish during sporulation. Hyphae reach a size of 0.7 to 1.5 μτη depending on age, spores are mostly spherical to elliptical with a diameter of 1.97+ 40 ^m.

Details of the production evaluation of strain CCM 3406 are provided in the following examples.

Example 1

Laboratory-scale fermentation

A. Vegetative inoculum

Vegetative inoculum is prepared in two stages, i.e. I. and II. vegetative generation. 80 ml of inoculum medium (containing sucrose, soy and peanut flour, beet molasses, corn extract and nutrient salts J) is placed in 500 ml boiling flasks, which is inoculated with a loop of spores from slanted agar and incubated on a rotary shaker at 240 rpm, with an eccentricity of 25 mm, for 24 hours at a temperature of 29 °C. The II. vegetative generation is inoculated with 5% of the I. vegetative generation, the cultivation conditions are the same as for the I. vegetative generation.

B. Production evaluation

Production flasks with 40 ml of production medium (containing sucrose, soy and peanut flour, corn extract, molasses, nutrient salts and benzylrhodanide) were inoculated with 10% of the vegetative inoculum of the II. vegetative generation. Fermentation took place on rotary shakers at 240 rpm (eccentricity 25 mm) at 29 °C for 120 h. Samples for determining the production of chlortetracycline and its ratio to tetracycline were taken at the 96th and 120th hour of cultivation. The amount of antibiotic produced was determined colorimetrically with a reagent solution of 2N hydrochloric acid and the ratio of antibiotics was determined chromatographically.

Production comparison:

Production comparison:

Tribe CTC Production Ratio CTC:TC (Ug/ml % S. aureofaciens Gamma X/237 6300 100 93:7 S. aureofaciens CCM 3406 7750 123 94:6

Tribe GTC Production Ratio CTC:TC ^g/ml % S. aureofaciens Gamma X/237 4980 100 94:6 S. aureofaciens GCM 3406 6450 128 94:6

Example 3

Fermentation in 20 liter tanks

Fermentation was carried out similarly to example 1 in 20 liter tanks filled with 10 liters of production soil.

Example 2

Fermentation in 2 liter laboratory tanks

Fermentation was carried out similarly to example 1 in 2 liter tanks filled with 750 ml of soil.

Claims (1)

SUBJECT A strain of Streptomyces aureofaciens CCM 3406 producing chlortetracycline. Production comparison: Strain CTC production CTC: TC ratio µg / ml% S. aureofaciens