CS214342B1 - Streptomyces aureofaciens CCM 3493 strain - Google Patents

Abstract

The invention relates to a new strain of Streptomyces aureofaciens CCM 3493, which produces chlortetracycline under operating conditions in higher yields than the existing strain.

Description

The invention relates to a strain of the microorganism Streptomyces aureofaciens CCM 3493 /NMU XXIV/53/, producing chlortetracycline. Chlortetracycline /CTC/ is obtained by aerobic cultivation of the production strain Streptomyces aureofaciens in liquid nutrient media under submerged conditions.

CTC production, which has been carried out in production fermentation tanks for more than two decades, has reached a stage through gradual changes in technology where further increase in chlortetracycline production is possible either through fundamental changes in the entire production technology /reconstruction of fermentation tanks, mixers, dosing equipment, etc./, which are very expensive and require the release of large amounts from the state budget, or by using new production strains whose ability to produce CTC is significantly higher.

The Streptomyces aureofaciens strain CCM-3493 NMU/XXIV/53 was obtained by a series of mutagenic interventions and passive selections, mainly by the action of ethyl methanesulfonate, nitrosomethyl urea, gamma rays and UV light. The last mutagenic intervention was the action of nitrosomethyl urea /NMU/ at a concentration of 0.15 M for 80 minutes in phosphate buffer pH 6.8 on a spore suspension of the Streptomyces aureofaciens gamma X/237 strain, obtained by washing the sporulated culture with water with 0.1% Tween 80. During the action, the mutation mixture was stirred on an electromagnetic stirrer at 25 °C. At time intervals of 30, 45, 60, 80 and 100 minutes of NMU exposure, samples were taken and diluted in a dilution series with a factor of 10 while diluting NMU to an ineffective concentration. The diluted samples were plated on Petri dishes with sporulation dextrin agar with sucrose and other ingredients so that there was no contact between the colonies after their growth and after sporulation, individual colonies were re-inoculated onto agar slants. The cultures obtained on agar slants in this way were the starting point for the entire production evaluation system, which is carried out according to the following scheme:

I. vegetative generation -> II. vegetative generation-“» fermentation

CTC production by the Streptomyces aureofaciens strain CCM 3493 /NMU XXIV/53/ was 25 to 35% and 5 to 10% higher, respectively, compared to two contemporary control strains - gamma X/237 and NMU XXIII/61.

The microorganism strain Streptomyces aureofaciens CCM 3493 /NMU XXIV/53/ forms colonies of 10 to 10.5 mm in size with a marginal non-sporulating zone of 1.3 to 1.5 mm when cultivated on dextrin agar.

The aerial mycelium is white, but becomes brown to grayish during sporulation. Hyphae reach 0.7 to 1.5 µ depending on age, spores are mostly spherical to ellipsoidal with a diameter of about 1.5 µ.

Example 1

Laboratory-scale fermentation on rotary shakers:

Vegetative inoculum:

Vegetative inoculum is prepared in two stages, i.e. I. and II. vegetative generation, in 500 ml boiling flasks with 80 ml of inoculum medium /sucrose, soybean meal, beet molasses, corn extract /CSL/ and salt mixture/. I. vegetative generation is inoculated with a loop of spores from slanted agar and incubated on rotary shakers at 240 rpm, eccentricity 25 mm

214 342 at 29 °C for 24 hours. The XI. vegetative generation is inoculated from the I. vegetative generation 5%, the cultivation conditions are identical to the I. vegetative generation.

Production rating:

Production flasks with 40 ml of production medium /sucrose, soy flour, CSL, molasses, salt mixture and benzylrhodanide/ are inoculated with 10% of vegetative inoculum /II. vegetative generation/. Fermentation is carried out on rotary shakers at 240 rpm. /eccentricity 25 mm/ at 29 °C for 120 hours. Samples for determining CTC production and the mutual ratio of produced CTC and TC /tetracycline/ are taken at the 96th and 120th hour of cultivation. The amount of produced antibiotic is determined colorimetrically with a 2 N HCl reagent solution and the CTC:TC ratio is determined chromatographically.

Production comparison:

Streptomyces strain CTC production Relative production CTC : TC aureofaciens control isolate ratio

gamma X/237 4980 100 94 : ; 6 NMU XXIII/61 6450 129 94 : : 6 NMU XXIV/53 6670 134 94 : : 6,

/CCM 3493/

Example 2

Fermentation in laboratory tanks of volume 21.

Fermentation is carried out in a similar manner to point 1 in fermenters with a volume of 2,000 ml, filled with 750 ml of soil. |

The preparation of vegetative inoculum of the I. and II. generation of the NMU XXIV/53 isolate together with two control isolates was carried out in the same way as in Example 1. From each isolate, the II. generation inoculum was prepared in 3 flasks filled with 80 ml of culture each. The actual fermentation was carried out in 9 two-liter glass fermenters. Each fermenter was equipped with one six-blade open turbine of half the diameter compared to the tank diameter and an air nozzle placed under the turbine. The fermenters were filled with 800 ml of nutrient medium with the following composition:

% sucrose, 3.5% soy flour, 0.7% CaCO^, 0.6% corn-steep, 0.6% /NH^^SO^, 0.24% NaCl, 0.22% molasses, 0.1% KH ₂ PO ₄ , 0.005% MgS0 ₄ , 0.003% MnSO ₄ , 0.002% ZnS0 ₄ , 0.0004% Co/NO ₂ / ₂ , 0.0002% benzylrhodanide. After 40 min. sterilization at 120 °C and warming to 29 °C, the soils were inoculated with 80 ml of the second generation inoculum culture. Each isolate was inoculated into 3 fermenters. The fermentation was carried out at 29 °C for 120 hours with constant stirring at 760 rpm and aeration at 700 ml air/min. During the fermentation, defoaming was carried out with soybean oil. At the end of the fermentation, CTC production and the CTC:TC ratio were evaluated. The average values are given in the following table:

214,342

Production comparison:

Streptomyces strain CTC Production % production of controls. CTC : TC ratio aureofaciens isolate gamma X/237 6300 100 93 : 7 NMU XXIII/61 7750 123 94 : 6 NMU XXIV/53 8190 130 94 : 6

/CCM 3493/

Claims (1)

A strain of Streptomyces aureofaciens CCM 3493 producing ohlortetracycline.