The strain streptomyces cinnamonensis ac-1638-producer and monensin

 

(57) Abstract:

The invention relates to the microbiological industry (biotechnology). The strain Streptomyces cinnamonensis AC-1638 producer of veterinary antibiotic coxcidiostatics monensin obtained from a strain of S. cinnamonensis S1009 by processing nitrosoguanidine and selection on agar medium containing the antibiotic chloramphenicol, followed by isolation of mutants resistant to 50 μg/ml of chloramphenicol, selection among these options with the highest production of antibiotic monensin. Strain VKPM as-1638 productivity has more than 35 g/l at a regulated fermentation process with the contents of monensin And from monensin not less than 85%, which allows the use of a new strain for cost-effective industrial production of monensin.

The invention relates to the microbiological industry, in particular to biotechnological production of veterinary antibiotic - coxcidiostatics monensin.

First coccidiostatic and antibiotic properties poliarnogo of antibiotic monensin (trade name “rumensin”) were described in 1967 in the study of complex A3 823 synthesized by a strain of Streptomyces cinnamonensis ATCC 15413 [1, 2]. Further Audacia only by the presence of radicals in the molecule of the antibiotic [3]. In the complex of monensin strains industrial producers dominate monensin and In more than 99%. Monensin In has about two times smaller antibiotic activity than monensin A. Therefore, in veterinary practice, has found application preparation containing not less than 85% of monensin And from the amount of monensin.

The final product is the sodium salt of monensin And found greatest application in the manufacture of various forms of veterinary drugs. Monensin And is used in the form of crystalline powder or dry mycelial culture of S. cinnamonensis with a high content of monensin depending on the forms of manufactured drugs (injectable, premixes and so on).

Monensin represent the mechanism of action of polyester ionophores that can change the permeability of membranes specific for sodium ions. Monensin used with curative purpose, as protivokariosnoe substance (in the form of a premix containing 10% or 20% of monensin sodium) in all species of animals and birds, in addition to the horses. With the preventive purpose used throughout the rearing period birds and other animals. The most important property is the ability to prevent the development of kriesa) for industrial production in the United States, Bulgaria and Czechoslovakia [2, 4, 5]. Known producing strains of monensin S. cinnamonensis VNIIBT-5, which received a patent in Russia [6]. However, industrial production monensin in Russia is not present.

The purpose of the invention is a strain of S. cinnamonensis for the industrial production of monensin.

Imagine the strain of S. cinnamonensis AC-1638 (NICB 109) derived from the original strain of S. cinnamonensis 1009, registered in the all-Russian collection of industrial microorganisms, state research Institute of Genetics and selection of industrial microorganisms under the number ACIM-S1009. The level of activity of the parent strain under standard conditions of fermentation in flasks is about 12 g/l of the complex monensin, the percentage of monensin And from the complex is about 70%, which does not meet modern industrial production monensin, and does not meet the requirements for the quality of medicines (content component And not less than 85%). Describes how the selection of strains of different producers polyketide antibiotics, based on receiving pleiotropic mutants that are resistant to antibiotics, including chloramphenicol [7-9]. One of the producers of macrolides found that this is due to regulatory mutation, activerules monensin is what spore culture of strain, treated with N-methyl-N-nitro-N-nitrosoguanidine (NG), plated on standard agar medium ISP containing chloramphenicol and 50 are selected spontaneous resistant to antibiotic mutants (Cmlr), which modified the regulation of the biosynthesis of monensin. Selected Cmlrmutants with increased production of monensin (a and b) check on the stability of the save the resulting properties. The result is a strain of S. cinnamonensis, NICB 109 having an enhanced ability to produce monensin containing monensin And a minimum of 85% and resistant to 50 μg/ml of the antibiotic chloramphenicol. Productivity monensin in strain S. cinnamonensis, NICB 109 when a specific mode controlled fermentation for 300 hours is more than 35 g/L. Strain of S. cinnamonensis, NICB 109 deposited in culture collections of industrial microorganisms PMBC (,Moscow) # AU-1638.

The main morphological and biochemical characteristics of S. cinnamonensis AC-1638 (NICB 109) is not significantly different from the typical producers of this type and has the following characteristics:

Cultural morphological traits

Shporonosni not spiral, often collected into bundles, sitting on short legs. SP who="ptx2">On a solid nutrient medium (ISP) on the 6th day of growth at a temperature of 28°With the strain forms a white rounded radially folded, wrinkled colonies with a grayish tinge, scalloped edge and a convex dark center. The diameter of the colonies to 2-5 mm. On the 9th day of incubation, the sizes of colonies reach 5-9 mm in diameter, configuration and folding colonies is not changed, the color becomes more intense, formed aerial mycelium with spores beige-gray color. On Wednesday highlighted brown pigment.

On solid nutrient maltose-yeast medium (YEME) on the 6th day of growth at a temperature of 28°With the strain forms a greyish-white rounded radially wrinkled colonies with scalloped edge and a convex darker center. The diameter of the colonies to 2-5 mm. On the 9th day of incubation, the sizes of colonies reach 4-8 mm in diameter, configuration and folding colonies is not changed, the color becomes more intense, formed aerial mycelium with spores gray-white in color.

Physiological and biochemical characteristics

Sprayway glucose, sucrose, galactose, mannose, maltose, fructose, rhamnose, mannitol, glycerol, acetate, but not sprayway lactose. Gelatino thins slightly, only peptonized, Brahma is.

Resistance to antibiotics: chloramphenicol - 50 µg/ml erythromycin - 100 µg/ml, tetracycline - 20 mcg/ml

Example 1. Obtaining a strain of Streptomyces cinnamonensis AC-1638 (NICB 109).

To obtain mutants that are resistant to chloramphenicol (Cmlr), the original strain was subjected to the processing of N-methyl-N-nitro-N-nitrosoguanidine (NG). The spore suspension is prepared by flushing with a “jamb” two weeks of age the liquid medium used for growing seed. The resulting suspension (3 ml) is transferred into a sterile tube, which add a pre-prepared in buffer (pH 8.8-9) solution of NG at a final concentration of 30 μg/ml of Cultivation carried out on a shaker at 220 rpm at 28°C for 20-25 hours until seedlings (control is carried out microscopically). The thus treated suspension of spores plated on agar medium ISP containing chloramphenicol at a concentration of 50 μg/ml Cups are grown in an incubator at 28°C for 10-14 days. Formed in the cups mutant colonies (mlr) carry on “weeds” with agar medium ISP and grown for 14 days. Selected mlrmutants in the amount of not less than 500 checks on the ability to biosynthesis of monensin in liquid form ml at 260 rpm The two-stage fermentation process. The first stage of the growing seed for 24 hours in the medium of the following composition, %: glucose (glucose syrup in an equivalent amount) and 1.5 - 2.5%; soy flour - 1.3 to 1.7%; sodium chloride - 0,3%; chalk chemical - 0,3%.

The second stage is the process of biosynthesis of monensin carried out within 240±12 hours in the medium of the following composition:

glucose (glucose syrup in an equivalent amount) - 3,0-4,0%;

the soy flour (coarse) - 3,0-4,0%; soybean oil (or other vegetable or animal fats in the equivalent amount of 5-8%;

manganese chloride 4-water - 0,33%; chalk chemical besieged - 0,25%; ammonium sulfate 0,03%; aluminum sulfate is 0.07%.

From trusted so mlrmutants were selected variants with activity higher than the original strain. The level of antibiotic activity was determined by high performance liquid chromatography, as described in [10]. In the described procedure was selected strain of S. cinnamonensis, NICB 109 (AU-1638) resistant to 50 μg/ml of chloramphenicol and are able to produce with a particular method of cultivation in a laboratory fermenter (see example 2) more than 35 g/l monensin. Determination of cu CLASS="ptx2">Thus, an isolated strain of S. cinnamonensis AC-1638 (NICB 109) has the biosynthetic capacity and quality (component composition) of the final product is considerably higher than that of the original strain.

Example 2. Fermentation of a strain of Streptomyces cinnamonensis AC-1638 in laboratory fermenter

The fermentation is carried out in two stages. The first stage in the flasks 750 ml receive, as described in example 1, vegetative planting material. Ready seed must meet the following requirements:

- appearance is a dense mass that fills the entire volume of liquid;

- color - gray or light brownish;

- when using the microscope stained with methylene blue fixed smears in the field of view should be dense mesh long thin basophilic hyphae;

- pH of 6.6 to 7.0.

Sowing the fermenter is vegetative seeds at the rate of 5% of the volume of the fermentation medium in the fermenter. The composition of the fermentation medium described in example 1.

20 minutes after the inoculation of the fermentor and thereafter every 24 hours or, if necessary, select a sample of the culture fluid in which contras; wet biomass; starting with 72 hours; the accumulation of monensin; starting with 120 hours, the fat content; the content of dry substances. The process of biosynthesis is conducted at the following parameters:

Temperature- 34±0,5°With

Aeration:

from 0 up to 7 hours - 0.20 m3/m3/min

from 7 to 12 hours - 0.40 m3/m3/min

from 12 to 16 hours - 0.85 m3/m3/min

16 hours before the end of fermentation - 0.90 m3/m3/min

Pressure of 0.3 kg/cm2

Mixing is continuous.

During fermentation the pH has a tendency to a slight increase followed by a decline. PH adjusted 25% ammonia water. The first 24-36 hours of culture characterized by severe foaming. As antifoam serves propanol. After 72 hours of growth 3 times per day (8 hours) sterile filed a number of soybean oil. The quantity of oil depends on the results of the analysis of the fat contained in the culture fluid. The oil content in the culture fluid should be in the range of 0.3-0.8%. In the period from 40 to 100 hours is not acceptable lipid-lowering below 0,3-0,35%.

With 100 hours begin to apply sterile in the process and to prevent the increase of wet biomass in the culture fluid of more than 50%. The beginning of the synthesis of the antibiotic was observed after 36 hours of fermentation. The content of monensin determine daily after 48 hours of fermentation.

In the normal course of fermentation level synthesis monensin 35-40 g/l, the content of monensin And is at 85%.

Thus, the strain Streptomyces cinnamonensis AC-1638 (NICB 109) has a high ability to produce monensin wapishana biosynthesis of more than 35 g/l high content (85%) monensin A.

Given the above, this strain can be used for the industrial production of monensin.

References

1. W. M. Stark, N. Knox. Antimicrobial agents and Chemotherapy, 196-8, p.353.

2. Patent US No. 3501568, 1970.

3. A. W. Birck, G. A. Robinson. Biotechnology 1995, 28, p.443.

4. Patent US № RE 34698, 1994.

5. Patent Czechoslovakia No. 272657, 1991.

6. RF patent №2057810, 1996.

7. RF patent №2142509, 1999.

8. Nastasia I. N., Zavorotnaja S. A., Fedorenko, C. A., Danilenko Century. N. Fabrication and characterization of mutants Sacharopolyspora erythraea, resistant to chloramphenicol. Antibiotics and chemotherapy, 1999, so 4, no 3, page 5.

9. Elizarov S. M., Mironov, C. A., Danilenko Century. N. The change in the activity of serine/treoninove protein kinases in the growth of CmlrHaya strain of Streptomyces cinnamonensis, NICB 109.

The strain Streptomyces cinnamonensis PMBC-1638 - producer of monensin A.



 

Same patents:

The invention relates to agriculture and can be used in the manufacture of various types of granulated mineral fertilizers (ammonium nitrate, required for plant growth, urea, ammofoska and others) for crop production
The invention relates to the field of biotechnology and relates to a method of biosynthesis of cephalosporin C in liquid nutrient medium with the use of culture Acremonium chrysogenum

The invention relates to biotechnology and is a method of obtaining gamma-aminobutyric acid (GABA) using auxotrophic for L-isoleucine bacteria Escherichia coli

The invention relates to applied Microbiology and can be used for cleaning the surface of the water from spilled oil and petroleum products

The invention relates to applied Microbiology and can be used for cleaning the surface of the water from spilled oil and petroleum products

The invention relates to the field of biotechnology, and more particularly to a strain of Shigella sonnei No. 262 and can be used to produce vaccine and diagnostic products

The invention relates to the mushroom industry and can be used in biotechnology for the production of fruit bodies of mushrooms

The invention relates to the field of biotechnology, hydrolysis industry and winemaking, and is intended for the production of purified pectolytic enzyme preparation
The invention relates to the processing of the fruit before putting it into storage
The invention relates to biotechnology, namely, construction of Escherichia coli - producer cholera toxin, and can be used to create a diagnostic test systems, allowing to detect toxigenic clones of Vibrio cholerae and Escherichia coli, as well as for the design of live vaccine strains against diarrhoeal diseases caused by pathogenic strains of V. cholerae and E.

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the strain Lactobacillus buchneri 600 is isolated from barley grains at milkwax stage of ripeness. The strain Lactobacillus buchneri 600 is registered in 05. 08. 2002 year in the All-Russian State collection of microorganism strains used in veterinary science and animal husbandry at number Lactobacillus buchneri VGNKI 02.08.04-DEP and deposited in the collection 000 "Biotrof". Invention provides the more effective multiplication of the strain in maize ensilaging green mass and preserving flattened grains with enhanced formation of lactic acid, inhibition of putrid microflora that allows preparing fodder from vegetable raw with enhanced quality. Invention can be used in fodder production for ensilage maize green mass and preserving flattened grains.

EFFECT: valuable properties of strain.

3 tbl, 2 ex

FIELD: biotechnology, agriculture, microbiology.

SUBSTANCE: invention relates to a new isolated strain of Lactobacillus acidophilus 1660/08 as a producer of the protein fodder. The strain Lactobacillus acidophilus 1660/08 is obtained by the selection method and selected by its ability to form significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.10.-DEP. Invention provides eliminating the environment pollution in producing the protein fodder, to enhance the specific protein yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process in its preparing, to simplify equipment fitting out and to utilize waste in manufactures using natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the strain Lactobacillus plantarum 578/25 is obtained by method of step-by-step selection and selected by its ability to produce significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.09.-DEP. Invention provides eliminating the pollution of environment in producing the protein fodder, to elevate the protein specific yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process of its preparing, to simplify apparatus equipment, to utilize waste in manufacturing using the natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: biotechnology, microbiology, dairy industry.

SUBSTANCE: the strain of microorganism Lactobacillus lactis VKPM B-8354 is prepared without using mutagens and genetic methods and shows resistance against broad spectrum of lactophages. The strain ferments effectively milk from different trading sorts, with broad range of fatness and different methods of thermal treatment. Individual specific properties of the strain allows its applying as a monostrain ferment. Curd obtained with applying the strain L. lactis VKPM B-8354 shows good organoleptic qualities, nonacid taste and homogenous consistence. The strain is suitable especially for plants with small volume of manufacture but with varied assortment.

EFFECT: valuable properties of strain.

3 tbl, 2 dwg, 5 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for preparing inosine and 5'-inosinic acid that are prepared by using microorganism Escherichia coli. Production of inosine by indicated microorganisms is elevated due to the enhanced activity of protein encoded by gene yijE. Invention provides increasing yield of inosine and 5'-inosinic acid.

EFFECT: improved preparing method, valuable properties of strain.

8 cl, 3 dwg, 2 tbl, 3 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: inosine and 5'-inosinic acid are obtained by using microorganism Escherichia coli. Production of inosine by indicated microorganism is elevated due to enhancing activity of protein encoding by gene ydeD. Invention provides elevating yield of inosine and 5'-inosinic acid.

EFFECT: improved preparing method.

8 cl, 3 dwg, 2 tbl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: amino acid L-threonine is prepared by culturing microorganisms belonging to genus Escherichia eliciting ability for producing L-threonine. Indicated microorganism is modified with aim to elevate activity of aspartate aminotransferase. Invention provides preparing amino acid L-threonine with high degree of effectiveness.

EFFECT: improved preparing method.

12 cl, 1 tbl, 2 ex

FIELD: plant production, in particular method for fruit treatment before dispatch for storage.

SUBSTANCE: method includes purifying of fruit surface from mechanical impurities and application of essence from the same fruits in combination with cuticular-layer epiphytic substances of vegetable raw material by using condensation from supercritical medium as carrier. Micelle obtained by extraction from biomass of Mortierella hydrophila micromycete with non-polar extractant under supercritical extractant parameters is used as supercritical medium.

EFFECT: improved safety of fruit protection from bacterial damage; increased fruit keeping time.

3 ex

FIELD: plant production in particular method for fruit treatment before dispatch for storage.

SUBSTANCE: method includes purifying of fruit surface from mechanical impurities and application of essence from the same fruits in combination with cuticular-layer epiphytic substances of vegetable raw material by using condensation from supercritical medium as carrier. Micelle obtained by extraction from biomass of Mortierella reticulata micromycete with non-polar extractant under supercritical extractant parameters is used as supercritical medium.

EFFECT: improved safety of fruit protection from bacterial damage; increased fruit keeping time.

3 ex

Up!