RU2281332C2 - Saccharopolyspora erythraea C-77 STRAIN AS PRODUCER OF ANTIBIOTIC ERYTHROMYCIN - Google Patents

Abstract

FIELD: biotechnology, in particular antibiotic production and new erythromycin producer.

SUBSTANCE: claimed strain is obtained by mutant selection from S.erythraea strain. Said strain is characterized with high specific rate of antibiotic formation (120±10 mg of erythromycin/g of dry biomass/h). Level of erythromycin formation after seven days of culturing in discontinuous process is 8250 mug/ml. Strain of present invention makes it possible to increase by 10 % level of erythromycin formation in contrast to starting strain and to decrease by 15-20 % formed biomass amount. Maximum synthesis specific rate is by 30 % higher in contrast to starting strain.

EFFECT: strain for accelerated synthesis of antibiotic erythromycin.

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Description

The invention relates to biotechnology and relates to the production of an antibiotic erythromycin with antimicrobial activity and used for the manufacture of dosage forms. The erythromycin molecule is of great interest as the basis for biological and chemical transformation in the preparation of new effective biologically active preparations that are widely used in medicine and veterinary medicine, therefore, the preparation of the erythromycin substance has a commercial prospect.

The level of synthesis of antibiotics by microbial culture depends on the characteristics of the producer strain and on the conditions under which the cultivation process is carried out.

When patenting strains producing antibiotics, the concentration of the antibiotic in the culture fluid is used as the main indicator characterizing the producer strain for a certain duration of the process, which is usually determined in laboratory conditions during fermentation in flasks. This indicator is often poorly reproduced under production conditions, especially in the case of using enriched media containing water-insoluble components such as flour, chalk and other substances.

In addition, the technology of the fermentation process, as a rule, is implemented according to the so-called controlled fermentation scheme, in which the first phase of the process is used to accumulate producer biomass, and the second is carried out with the substrates and maintaining parameters that are optimal for the antibiotic biosynthesis process itself. The second stage is crucial for the effectiveness of the whole process. The effectiveness of the second phase is determined by the antibiotic biosynthesis rate per unit time, referred to the unit mass of the producer microorganism. The high value of this producer characteristic (specific rate of antibiotic formation) makes it possible to obtain significant antibiotic accumulation at a relatively low concentration of producer biomass. This, in turn, will simplify the technology of product isolation and facilitate the resolution of issues related to the disposal of producer biomass accumulated. Thus, in the selection of the erythromycin producer, a method was used to select promising strains according to a fundamentally new, different from the previously used, indicator - the specific rate of antibiotic formation.

Erythromycin producing strains with a maximum level of erythromycin formation during the fermentation process of 4500-7500 μg / ml for 168-204 hours of fermentation are known (1,2,3). Unfortunately, the patent descriptions do not provide data on the biomass concentration of producers, which does not allow us to evaluate the value of the specific antibiotic formation rate of interest to us.

The aim of the invention is to obtain a strain with a higher specific rate of accumulation of erythromycin and a high level of productivity. This goal was achieved as a result of strain selection and selection of clones with a high rate of antibiotic formation during fermentation on complex media. The strain Saccharopolyspora erythraea C-77 was obtained from strain S.erythraea VNIIA 1658, which was characterized by activity of 7500 μg / ml by 168 hours of fermentation. For the initial strain, data were obtained on the maximum specific rate of erythromycin synthesis in the range of 0.9-1.1 μg / ml · hour.

To obtain a strain with a higher specific rate of erythromycin formation, the spore material of the initial strain was sown sequentially in 2 seed and fermentation media. After 48 hours of culture growth in a fermentation medium on a rotary shaker, the culture liquid was diluted 5 times, placed on a shaker again and incubated for 24 hours. In diluted suspensions after a day of cultivation, the concentration of erythromycin was determined and options were selected according to the maximum concentration of erythromycin after 24 hours of cultivation in a diluted medium.

In the selected variants that retained the maximum specific rate of erythromycin accumulation when they were carried out sequentially through 3 monoclonal screenings, a high specific rate of antibiotic formation was confirmed to be higher than that of the initial strain. In subsequent screenings, the properties of the selected clones were preserved. The best option was used as a new producer strain Saccharopolyspora erythraea C-77.

The spore suspension of the obtained strain was lyophilized in skim milk as a tread, which would allow such material to be stored in the refrigerator at + 5 ° C for up to 10 years.

The strain Saccharopolyspora erythraea C-77 possesses the main cultural-morphological and biochemical characters typical of the S.erythraea species and has the following characteristics.

Branched aerial mycelium, 0.4-0.6 microns in diameter, spiral hyphae, segmented in the form of bead chains remaining in the case.

Vegetative mycelium is a developed, branched, thin, protoplasmic basophilia reduced by the end of fermentation.

On Wednesday CP-15, containing soy flour, corn extract, molasses, starch and salt, on day 14, colonies reach 3-5 mm in diameter. The predominant type is colonies of a round shape with a scalloped edge and a radially folded surface, slightly deepened in agar. Aerial mycelium is light beige or gray, substrate mycelium is light brown. When plating a culture on a solid medium, cleavage usually occurs. In the general population, 7–13% of colonies with dark gray aerial mycelium are present that secrete dark brown pigment in agar and 8–15% of yellowish colonies with poor sporulation.

On oatmeal on day 14, colonies of the main type reach 2-3 mm in diameter, have a round shape with a crater in the middle and white substrate mycelium. In the general population, colonies of the main type are present in an amount of 80-86%, as well as 8-12% of colonies with dark gray aerial mycelium, which secrete dark brown pigment in agar, and 6-7% of beige colonies with poor sporulation.

On a medium containing starch, corn extract and salts, round colonies with a diameter of 2-3 mm, opaque, white, deepened in agar prevail. The colony surface is radially folded, the profile is convex, without a crater, the edge is scalloped, the consistency is dry, dense. In the population, 7-8% of colonies are light beige in color, round in shape, with a diameter of 4-5 mm, with a matte concentric-folded surface, with a scalloped edge and a flat profile. There are 3-5% of brown colonies with a diameter of 2-3 mm, not deepened in agar.

On Waxman’s environment, growth is very weak. On day 14, colonies reach a maximum size of 0.5-0.8 mm, have a round shape, white aerial mycelium, do not secrete pigment and do not go deep into agar.

There is no growth on Chapek’s environment with yeast extract, starch and salts.

The temperature for growth and fermentation is 32-34 ° C.

Ferments glucose, arabinose, maltose, fructose, sucrose, sorbitol, inositol, rhamnose. Gelatin liquefaction is observed from 10 to 20 days, peptone milk - from 11 to 20 days, hydrolysis of starch - on 4 days.

Example 1

The initial strain 1658 (VNIIA) and the Saccharopolyspora erythraea C-77 strain were sown on solid medium CP-15 containing (%): soy flour - 0.5, molasses - 0.5, corn extract - 0.5, starch - 0.5, ammonium sulfate - 0.05, sodium chloride - 0.05, magnesium sulfate - 0.07, chalk - 0.1, agar - 2, pH 8.0. Ready-made spore material from each strain was seeded with 750 ml flasks with 50 ml of inoculum No. 1 of the following composition (%): soy flour - 4.0, glucose - 0.5, glycerin - 1.0, ammonium sulfate - 0.25, ammonium phosphate - 0.06, chalk - 0.8, pH 7.0-7.2. Grown on a rotary shaker at 200-220 rpm and a temperature of 33 ± 1 ° C for 48 hours.

Sowing material in the amount of 10% of the medium volume was sown with the same flasks with 50 ml of seed medium No. 2 of the following composition (%): soy flour - 3.0, white dextrin - 2.0, starch - 3.0, ammonium sulfate - 0 , 2, potassium phosphate monosubstituted - 0.015, chalk - 1.0, pH 7.0. Incubated under the same conditions for 24 hours.

To compare the specific rate of accumulation of the antibiotic in the selected and the initial strains, laboratory fermenters with a capacity of 3 liters and 1.5 liters of nutrient fermentation medium of the following composition (%) were used: soy flour - 3.0, white dextrin - 4.0, starch - 3 , 0, ammonium sulfate - 0.2, potassium phosphate monosubstituted - 0.015, chalk - 1.0, pH 7.0. The finished vegetative inoculum in the amount of 5% was seeded with fermenters. Fermentation was carried out at a temperature of 33 ± 1 ° C, with stirring at 900 rpm and aeration: 1 volume of sterile air per 1 volume of culture medium. Starting from 48 hours, culture fluid samples were taken from fermenters every 24 hours, in which the concentration of erythromycins was determined by diffusion into agar with the Bacillus cereus var.mycoides test culture.

Due to the complex nutrient media used for the biosynthesis of erythromycin, it is impossible to determine the biomass by direct gravimetric methods; therefore, this parameter was determined indirectly by the DNA content in the producer mycelium. The dry weight of mycelium was equal to the amount of DNA in the mycelium, determined by the method with Barton's diphenylamine modification (4), multiplied by the conversion factor. The specific rate of antibiotic formation was calculated as the ratio of the concentration of erythromycin (the ratio of the increase in the concentration of erythromycin per day) to the dry weight in mycelium per unit time. Table 1 presents the indicators of the maximum specific rate of erythromycin formation for two strains.

Table 1
The maximum specific rate of erythromycin formation. No. p / p Strains mg erythromycin / g dry biomass / hour one S.erythraea 1658 (VNIIIA) 90 ± 7 2 S.erythraea C-77 120 ± 10

The strain Saccharopolyspora erythraea C-77 is characterized by an increased specific rate of antibiotic synthesis throughout fermentation compared to the original strain. Moreover, the new strain C-77 formed biomass 15-20% less than the control, which simplifies the technology of isolation of the target product and facilitates the resolution of issues related to the disposal of accumulated biomass producer.

Fermentation in a batch mode using the Saccharopolyspora erythraea C-77 strain showed an erythromycin formation level of 8250 μg / ml by 7 days of cultivation, which was 10% higher than the productivity of the control strain. When organizing controlled fermentation, even higher process outcomes are possible.

Thus, the isolated S.erythraea C-77 strain has a maximum specific synthesis rate of 30% higher, and the level of erythromycin formation is 10% higher compared to the original strain, with a 15-20% decrease in the amount of biomass formed.

A new strain of Saccharopolyspora erythraea C-77 was deposited and deposited in the collection of microorganisms LLC PKF BIGOR.

Bibliography

1. USSR author's certificate No. 1092951, priority 01.10.82, MKI C 12 P 19/62. Publ. 02/09/95 in Bull. Number 4. Strain Streptomyces erythreus 85-1 - erythromycin producer.

2. USSR author's certificate No. 1651557, priority 08/28/89, MKI C 12 P 19/62. Publ. 01/27/95 to the Bull. Number 4. The strain Streptomyces erythreus is a producer of erythromycin.

3. Patent RU No. 2142509, priority 05.19.99, MKI C 12 P 1/06. Publ. 12/10/99 to Bull. Number 34. The strain Saccharopolyspora erythraea 52 is an antibiotic producer of erythromycin.

4. Burton, K., Biochem. J., 1956, No. 62, p. 315.

Claims (1)

Strain Saccharopolyspora erythraea C-77 - erythromycin producer.