RU2689699C1 - Amycolatopsis umgeniensis strain - producer of uremycin antibiotic - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and can be used in producing a valuable antibiotic of eremomycin sulphate. Strain Amycolatopsis umgeniensis possessing the ability to produce eremomycin is deposited in the Russian National Collection of Industrial Microorganisms under registration number VKPM As-1978.EFFECT: strain enables to obtain high-output Eremomycin.1 cl, 2 tbl, 1 ex

Description

Strain Amycolatopsis umgeniensis - producing antibiotic eremomycin

The technical field to which the invention relates.

The invention relates to medicine and the biotechnology industry and relates to a new strain of actinomycete forming the antibacterial antibiotic eremomycin.

The chemical structure of the antibiotic eremomycin belongs to the polycyclic antibiotics of the vancomycin-ristomycin group (dal-peptides), which are life-saving drugs in the treatment of very serious infections.

Structure of antibiotic eremomycin sulfate

Eremomycin is effective in the treatment of diseases caused by gram-positive bacteria, including methicillin-resistant strains of Staphylococcus aureus (MRSA) and the anaerobic pathogen of pseudomembranous colitis Clostridium difficile. Eremomycin is also used as an initial substance in obtaining semi-synthetic derivatives of a natural structure for improving chemotherapeutic parameters (1), including the weakening of the allergenic effect characteristic of glycopeptide antibiotics.

Prior art

A known strain of Amycolatopsis orientalis subsp. eremomycini VKPM Ac 807 - producing the antibiotic eremomycin [2], adopted as a prototype. The disadvantage of this strain is low antibiotic activity - 2300 μg in 1 ml of fermentation liquid.

DISCLOSURE OF INVENTION

The essence of the invention is that a new producing strain of eremomycin has been obtained by selectively transforming the properties of the Amycolatopsis umgeniensis soil culture as a result of sequential multi-step exposure to N-methylnitrosacrea and gamma rays ⁶⁰ Co to dispute the original culture and select the original mutant strain No. 1472 with increased antibiotic activity among the mutagenized population of actinomycete colonies grown on a selective background of resistance to the glycopeptide antibiotics vancomycin and bleomycin.

The resulting new strain Amycolatopsis umgeniensis No. 1472 is deposited in the culture collection of the State Research Institute of Genetics under the strain VKPM Ac-1978 number.

The strain Amycolatopsis umgeniensis VKPM Ac-1978 has a high antibiotic activity of eremomycin formation (3300 mcg in 1 ml of fermentation liquid) and is characterized by the following taxonomic, cultural-morphological and physiological-chemical characteristics.

Taxonomic, cultural-morphological and physiological signs of the strain Amycolatopsis umgeniensis VKPM Ac-1978

Taxonomic features. Duration of cultivation for the study of taxonomic characters (7-21) days at a temperature of 28 ° C. For seeding diagnostic media, spore seed of a ten-day culture of a strain grown on glucose-aspartic agar or Lindenbein medium is used. The color is determined by the table Bondartseva and Prauzera. The data are given in table №1.

Morphological signs. Hyphae of aerial mycelium are slightly sinuous, breaking into fragments of various lengths; spore surface is smooth.

Physiological and biochemical properties. Aerobe. Grows at (24-37) ° С, growth optimum is 28 ° С and in the range of pH values (5.0-10.0) with growth optimum at pH 7.0; thins gelatin, milk peptonized. Saccharose inverts. Starch hydrolyzes. Fiber is not growing. Restores nitrates to nitrites. Melanoid pigments do not form when grown on Tresner's provocative medium with citric acid iron.

Use of carbon sources. Well utilizes glucose, fructose, lactose, rhamnose, xylose, ribose, arabinose, mannitol, maltose; does not utilize sucrose, raffinose, sorbitol, mannose, cellulose.

Antagonistic properties. The strain Amycolatopsis umgeniensis VKPM Ac-1978 with growth on agar media moderately inhibits the growth of gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis, Bacillus mycoides, Micrococcus luteus, and does not act on Gram-negative bacteria Escherichia coli, Comamonas-iurea-nauri-hygiene, and the anthropology of the anthracidae, irius, and the anthracididae, which doesn’t affect the gram-negative bacteria Escherichia coli, Micrococcus luteus.

Storage of strain Amycolatopsis umgeniensis VKPM Ac-1978. The strain is stored in the form of spores on sterile soil with quartz sand or on millet grains, as well as in the form of a mature culture on agar media under vaseline oil. The antibiotic activity of Amycolatopsis umgeniensis VKPM Ac-1978 strain during deep cultivation is determined by the well method of diffusion into agar (Gauze medium No. 2) using Bacillus subtilis ATCC 6633 as a test microorganism and by comparing growth suppression zones of fermentation liquid samples with the calibration standard of eremomycin sulfate ( 100 µg, 50 µg, 25 µg, 12.5 µg). The biological activity of the resulting biosynthesis and chemically purified antibiotic substance is determined by the three-dose variant of the method (GFH, p. 949).

The strain Amycolatopsis umgeniensis VKPM Ac-1978 is illustrated by the following example.

Example 1. Spore material with mycelium culture Amycolatopsis umgeniensis VKPM-Ac-1978, grown for (10-14) days at 28 ° C in Lindenbein medium, seeded with culture medium (150 ml in Erlenmeyer flasks with a volume of 0.75 l), and grown at 28 ° C for (24-36) hours on a circular rocking chair at a rotation speed of 220-240 rev / min.

The composition of the sowing medium,%:

The medium is sterilized for 30 minutes. at a pressure of 0.6 atm. at 120 ° C.

The grown mycelium in the amount of (5-10)% seeded nutrient medium (100 ml in Erlenmeyer flasks with a volume of 0.75 l) of the following composition,%:

Sterilize the medium for 30 min. at a pressure of 0.6 atm. at 120 ° C.

Fermentation cultivation is carried out at 28 ° C for 120 hours on a rocking chair at a rotation speed of 220-240 rev / min. Eremomycin accumulates in the culture fluid of the producer. Antibiotic activity was determined by diffusion into agar using the Bacillus subtilis ATCC 6633 test culture. The content of eremomycin in the culture fluid of the Amycolatopsis umgeniensis strain VKPM Ac-1978 by the end of fermentation is 3300 μg / ml.

To isolate eremomycin (3), it is sorbed from the native culture solution (filtered from the mycelium) on the carboxyl cation exchanger of CB-2 in salt form, eluted with ammonia solution, the eluate is neutralized, desalted, passed through Amberlite XAD-2 sorbent, the column is washed with water, the antibiotic is eluted with a 4% solution of isopropyl alcohol in 0.2 M acetic acid, the eluate is concentrated in vacuo to contain eremomycin (120-140) mg / ml and crystallized in the form of sulfate.

Eremomycin sulfate, obtained by biosynthesis with a new strain-producer of Amycolatopsis umgeniensis VKPM Ac-1978, is a white crystalline powder, readily soluble in water and insoluble in organic solvents. The indicators characterizing eremomycin sulfate (physicochemical properties, antibacterial activity and toxicity) fully correspond to the indicators that are defined for eremomycin sulfate protected by the RF patent №2110578 (gross formula: C ₇₃ H ₈₉ N ₁₀ ClO ₂₆ XH ₂ SO _4. Mol weight is 1656 amu

UV spectrum: λ _max (0.01 N HCl) nm (E ^1% _{1 cm} ): 280 (42); λ _max (0.01 N. NaOH) nm (E ^1% _{1 cm} ): 300 (60);

IR Spectrum (KBr), ν _max , cm ^-1 : 3350, 1650, 1500, 1200-1000; [α] ²⁰ _D = -100 ° (c 1.0, H ₂ O).

The yield of the target product - purified (97% purity) of the crystalline preparation of eremomycin sulfate from the content in the native solution is 65.0%, i.e. 2.1 g with 1 liter of native solution (filtered fermentation liquid).

Thus, a new mutant strain of Amycolatopsis umgeniensis VKPM Ac-1978 was obtained, which differs from a well-known producer in its antibiotic formation capacity, which is more than 40% higher than the activity of the prototype producer Amycolatopsis orientalis subsp. eremomycini VKPM-807 (Table 2). The use of a new strain of Amycolatopsis umgeniensis VKPM Ac-1978 allows you to increase the yield of the target product. The yield of eremomycin is 2.1 g from 1 liter of the native solution.

Information sources.

1. M.N. Preobrazhenskaya, E.N. Olsufyeva, O.V. Miroshnikova et al., G. Antib. 60 (4): 235-244, 2007

2. Patent RU 2352631 dated 10.2008

3. Patent RU 2110578 dated 10.1998

* / +++ - good; ++ - moderate; + - weak.

Claims (1)

Strain Amycolatopsis umgeniensis VKPM Ac-1978 - producer of the antibiotic eremomycin.