SU539538A3 - Method for producing metabolite 2776 - Google Patents

Description

The invention relates to methods for producing biologically active substances by microbiological synthesis.

A known method of obtaining biologically active substances, including the cultivation of microorganisms of the species Streptomyces candidus in an aqueous nutrient medium containing sources of carbon, nitrogen and mineral substances, during its aeration.

The aim of the invention is to obtain a new biologically active substance with reduced toxicity, effectively acting against coccidia, as well as increasing the digestibility of feed ruminants.

For this, Streptomyces candidus NRRL 5449 is taken as a culture of the microorganism, and glucose and monensin produced by the organism Streptomyces cinnamonensis are additionally introduced into the nutrient medium. At the same time, sterptized growth medium of Streptomices cinnamoneusis is used as a source of monensin. The nutrient medium can also be contacted with either the cultured and lyophilized broth of the growth medium separated from the cells upon receipt of a culture of Streptomyces candidus NRRL 5449, or with the separated cellular material of this microorganism purified by sonication, centrifugation and dialysis.

The accompanying drawing shows an infrared absorption spectrum of metabolite A 27106 in chloroform.

The biologically active substance obtained by the proposed method in the form of a sodium salt is designated as the metabolite A 27106. The starting material for the preparation of the substance A 27106 is monensin, which is obtained either from culture culture or from the mycelium of the culture Streptomyces cinnamonensis ATCC 15413. Substance A 27106 is obtained from monensin the presence of glucose under the influence of enzymes secreted by the new strain of Streptomyces candidus, present in the collection of permanent cultures in the North of the separate research and experimental works of the agricultural research service vany Ministry agriculturally US-keeping under the accession number NRRL 5449.

A new strain of Streptomyces candidus NRRL 5449 was isolated from a soil sample on Mount Ararat in Turkey. Streptomycetes NRRL 5449 are characterized by the presence of sporophore. having a shape from straight to wave; the spores have a smooth surface and the shape is from oval to weakly cylindrical; the size of the spores is on average 1.165 x 10.5 microns; the size of the epor chains ranges from 10 to 50 microns. Aerial mycelium is usually white, at 26 ° C only vegetative growth is observed, with 45 ° C growth does not occur, and maximum growth and sporulation occur at 30-37 ° C. Characteristics of the culture medium: ICP 1. Satisfactory growth; light yellow reversible color (1IC1); the absence of a raised mycelium or epor; soluble pygmept is absent. ICP 2. Abundant growth; light yellow reversible color (1013); abundant aerial mycelium and spores; (W) white a; soluble pigment is missing. ICP 3. Good growth; light yellow, reversible green color (10С1); good aerial mycelium and spores; (W) white a; soluble pigment is missing. ICP 4. Growth good to abundant; reversible moderate yellow color (10H4); good to abundant mycelium and spores; (W) White brown soluble pigment. ICP 5. Abundant digs; reversible, moderate orange-yellow color (1016); plentiful aerial mycelium and eiory, (Y) light yellow 2db; slightly brown soluble pigment. ICP 7. Good growth; reversible light yellow, green color (10В1); good aerial mycelium and spores; (W) white a; soluble pygmyite is absent. Glycepp-glycine. Abundant digs; medium intensity brown reversible color (1114); plentiful formation of spores and aerial mycelium, (W) white a and (Y) light yellow 2 db; weak, light brown soluble pigment. Emerson Wednesday. Abundant growth; greyish yellow reversible color (12NZ); aerial mycelium and spores are absent; soluble pigment is missing. Wednesday Bennett. Good height; light yellow reversible color (1112); poor aerial mycelium and spores; (W) white soluble pigment is missing. Wednesday Chacek. Good height; moderate orange-yellow color (1117); abundant aerial mycelium and spores; (W) white a; no soluble pigment. Glucose-asparagine. Growth is satisfactory to good; light yellow, reversible 6 green color (10H1); aerial mycelium and spores are absent; soluble pigment is missing. Calcium salt malic acid. Abundant growth; reversible grayish-yellow color (11E4), abundant aerial mycelium and supports; (Y) light yellow 2 La; slightly brown soluble pigment. Nutrient agar. Good height; reversible light yellow, green color (10 C1); no air - targets or dispute; soluble pigment is missing. In the study of the physiological properties of Streptomyces eandidus NRRL 5449 in accordance with standard techniques, the following was found. Action on skim milk. Enlightenment; Texturing after 14 days. Nitrate reduction. Positive. Formation of melanin from yeast extract of tryptone broth. Minor. Agar-tyrosine. Absence Gelatin liquefied. Absence for 21 days. Temperature requirements. 26 ° C - only vegetative growth; 36-37 ° С - good vegetative digs, good aerial mycelium and spores; at 43-53 ° C growth is absent. The results of testing the disposal of carbon with the aid of Streptomyces eandidus RRL 5449 are given below. The symbols used to indicate the growth response are as follows: -f utilization - good growth; -f-: scrapping likely to occur - poor growth; - utilization is doubtful - weak or no ost; - no disposal - no growth. Carbon source Raffinose + D-Fructose + Cellobiose- (L-Arabinose- | /) -Mannitol + Rhamnose-} Pellulose- Dextrose + to - D-Xylose 4- Inositol-Jс (no carbohydrate - to + Medium for extracting Streptomyces eandis NRRL 5449 can be any approach for this microorganism.However, predtchitelnymi sources of carbohydrates in the ovedenii fermentation in large quantities can be invert sugar or corn ron, but you can use glucose, frukzu, maltose, starch, inositol, and the like. If you want to use cult S. eandidus NRRL 5449 to obtain biogich if active substance A 27106, then the food must contain a source of glucose

to ensure efficient conversion. If S. candidus NRRL 5449 is grown to obtain an enzyme system characteristic of it for later use to obtain substance A 27106, the presence of glucose during fermentation is arbitrary.

Preferred sources of nitrogen are peptones, soy flour, mixtures of amino acids and other similar substances.

As mineral salts, ordinary soluble salts containing nonons of iron, sodium, potassium, ammonium, calcium phosphate, chloride, carbonate, etc. can be incorporated into the medium. The medium must also contain the necessary amount of trace elements.

The initial pH of the culture medium can be varied, but it is desirable to select and maintain the pH of the medium when cultured in the range of 5.7-7.5.

Like other Streptomyces species, S. candidus NRRL 5449 requires aerobic conditions for cultivation. The inoculation of the aqueous medium can be carried out with a suspension containing spores or with a vegetative graft material. The growing temperature is chosen in the range of 26-40 ° C, however, the maximum growth and sporulation occur at 32-37 ° C. The supply of sterile air to the culture medium to ensure effective growth of the microbial culture and to obtain a sufficiently high yield of substance A 27106 is carried out at a rate of at least 0.1 volume of air per minute per unit volume of culture medium, but it is preferable if the volume of air is from 1/3 to 1/2 volume of air in 1 min per volume of culture medium.

The presence of a suitable source of glucose is a prerequisite for the conversion of monensin or factor A of the antibiotic complex A 3823 to the biologically active substance A 27106.

When glucose is present in the medium in the required amount, and monensin in an amount of 0.1-1.0 g / l of the medium, the cultivation generally ends in about 36-72 hours. The optimal degree of conversion is achieved when monensin is present in an amount of 0.5-0.7 g / l, and glucose is 2-2.5 wt. % by amount of medium.

If the content of glucose in the medium is less than 2%, then it must be added, keeping the concentration at an optimal level.

If the enzyme system of S. candidus NRRL 5449 is used to obtain the active substance A 27106, it is necessary that the enzyme preparation has an increased degree of purification. The active enzyme system of S. candidus NRRL 5449 is present in the filtered broth obtained by fermentation and in the cellular material. For example, the filtered broth can be lyophilized and stored for at least two weeks until re-dissolving.

buffer solution in the amount of 1/6 of the volume of the initial broth. Effective conversion is achieved in about 72 hours when 2.5 g of such a lyophilized preparation and 25 mg of monensin are taken.

The enzyme preparation from S. candidus NRRL 5449 cellular material, separated from the broth after fermentation, can be obtained as follows.

Cell material can be frozen and stored for at least three months. After thawing, the cells are suspended in a buffer solution, further purified by sonication and centrifugation, the separated residues of cellular material are suspended in a buffer solution and dialyzed. From 200 g of cellular material, a quantity of dialysate is obtained, sufficient to convert glucose and 25 mg of monensin.

in the biologically active substance A 27106.

Substance A 27106 is also contained in the culture broth in the mycelium of Streptomyces candidus NRRL 5449.

In accordance with this technique

for selection, it is selected with the intent of extracting the product from one or both sources. For example, the fermentation medium is filtered, after which the filtrate and dense mycelium mass obtained

after filtration, it is extracted with the use of suitable solvents to obtain substance A 27106. The product is extracted from the solvents using conventional techniques in the industry.

However, the isolation of the substance A 27106 can be made from the culture medium and mycelium, without being extracted, but preferably by removing water from them. For example, the culture medium can be dried.

lyophilization, and then mixed with the material received for processing. In addition, water can be completely removed from the solids and a suspension suitable for adding mash (wort) to the wet mass or to similar feedstock can be prepared.

One of the satisfactory methods for isolating A 27106 may be the following. Wednesday in which it was made

cultivation of the microorganism, filtered, dense mass, pressed on the filter, extracted with a solid solvent, such as methanol. The methanol extract is concentrated and then added to the filtrate. The diluted solution is extracted twice by adding half the volume of chloroform. Chloroform extracts are evaporated in a vacuum, an oily product of dark amber color is formed, which discolor, passes over activated charcoal in a column using chloroform.

The extract obtained after washing out the adsorbent is again concentrated in vacuo, which ensures the production of an oil from light yellow to colorless. This oil is dissolved in a minimum amount of chloroform and chromatographed on a silica gel column using ethyl acetate as the solvent. Elution is desirable to produce by thin layer chromatography. The impurities are washed from the adsorbent with ethyl acetate, and washing the adsorbent with a mixture of ethyl acetate and methanol allows the isolation of the biologically active substance A 27106.

The biologically active substance A 27106 (sodium salt) is a white solid crystalline substance melting at a temperature of about 170-175 ° C. It tends to easily form hydrate and other solvates, and its melting point changes (usually a few degrees lower than indicated).

The elemental composition of substance A 27106 is the following (%): C 58.78, H 8.51, O 27.85, and Na 3.63, which corresponds to the empirical formula C42H7iO, 6Na.

Substance A 27106 shows practically no ultraviolet absorption in the region higher than 235 microns.

Its infrared absorption spectrum in chloroform is shown in the accompanying drawing. The achievable absorption maxima at the spectrum are: 3.1; 3.36; 3.69; 6.82; 7.1; 7.25; 7.9 (plateau); 8.1; 8.3; 8.67; 8.8 (plateau); 9.04; 9.22; 9.51; 9.66; 10.03; 10.26; 10.66; 11.23; 11.47; And, 83 and 12.15.

The mass spectrum of substance A 27106 shows the presence of a molecular-ion peak at gp / e S54.44630 and characteristic peaks at m / e836.44I29, 779.44690, 761.44740

These resulting drops coincide with an empirical formula and a molecular weight value of 854 for the sodium salt of substance A 27106.

Typically, substance A 27106 is easily soluble in 3 high-polarity solvents, insoluble in non-polar solvents, and exhibits varying degrees of solubility in intermediate polar solvents. For example, substance A 27106 is soluble in lower aliphatic alcohols, partly in | enol, diethyl ether and acetone, and is comparatively insoluble in liquid lower ones.

Electrometric titration of the substance 27106 in acid form at the initial pH value of 8 showed the presence of a grouping: a benefit titrated with pKa 7.2.

The monopotric salt described is the natural form of substance A 27106. Isho1 from the sodium salt is easily obtained sourly; ammonium and other salts derived from alkali metal lithium, ka, rubidium, cesium are obtained from the acid). All of them have biological activity.

The exact structure of substance A 27106 is unknown: tna. It is known that the conversion of monensin O of substance A 27106 requires the presence of lucose, which is consumed even in the absence of S. candidus cells undergoing metabolism. The molecular weight of substance A 27106 corresponds to the molecular weight of glucosyl monenzip.

Substance A 27106 is less toxic than monensin. For example, when monensin was injected into groups of mice with 6 heads in each intraperitoneally at a dosage of 50 mg / kg, one out of six died, at a dosage of 100 mg / kg, three of

six died. At the same time, under similar conditions of the experiment, at 10 mg / kg dosage, one of six mice died, and three of six mice, at a dosage of 20 mg / kg, under the same experimental conditions.

Example 1. S. candidus NRRL 5449 was grown on agar on Bennett medium before obtaining well-defined colonies. The grown culture was removed and prepared from

it is suspended in sterile, deionized water (10 ml). This suspension was poured into four glass shaking vessels, which contained 100 ml of vegetative medium of the following composition (g per 1.1 l): soluble products obtained from the grain after the distillery (Nadrizol, National Distillers Products Company), 25, 0; lactose 10.0; maltose 10.0, FeSO4-7H2O 0.01; MgSO4-7H2O 2.0; KH2P04 2.0; CAS02; deionized

water. Incubation was carried out at 30 ° C on a rotating rotary shaker at 250 rpm for 24 hours.

This culture medium was used to inoculate 15 glass vessels with a volume of 500 ml, each containing 100 ml of sterile fermentation medium of the following composition (g per liter): meat extract 5.0; pancreatic hydrolyzate of casein 5.0; sodium chloride 5.0; glycerin 15.0; calcium carbonate 2.0; deionized water up to 1 l. pH of 7.2. Inoculated medium was incubated for 72 hours.

In a 40 L fermenter, 24 liters of a production medium of the following composition was prepared,

g: anti-foaming agent in the form of oily polysiloxane 5.0, glycerin 375, dextrose 625, pancreatic casein hydrolyzate 125, meat extract 125, sodium chloride 125, calcium carbonate 50; deionized water.

The initial pH value of 7.0. The medium was sterilized in an autoclave at 120 ° C for 30 minutes under a pressure of 1.05-1.41 kg / cm (15-20 LB per square inch). After sterilization, the pH of the medium is 7.6.

To a sterile medium, 25 g of purified monensin, ω-dissolved in 200 ml of ethanol were added and 700 ml of vegetative graft material prepared as described were added.

The fermentation media was aerated, passing sterile air at a speed of about 1 liter (0.35 cubic feet), and stirred with a stirrer at 420 rpm.

Incubated at 30 ° C for about 9

about 114.5 hours The fermentation process was followed up with a thin layer chromatography on silica gel. Initially, only monensin was present in the fermentation medium. A second spot gradually appeared, indicating the presence of substance A 27106. At the end of the fermentation, only a spot was present, indicating the presence of substance A 27106.

Example 2. The culture of the microorganism Streptomyces cinnamonensis ATSS 15413 was grown in 55 ml of medium placed in a 250 ml volume for 47 hours. This vegetative graft was added to 220 ml of medium in a flask and incubated with shaking for 21 hours. - The prepared graft material was used for planting in a 40 L fermentor containing 24 liters of sterile medium of the following composition, g: glucose 750.0; soy flour 625.0; soybean oil 500.0; methyl oleate 500; anti-foaming agent in the form of an oily polysiloxane 5.0; potassium chloride 2.5; disubstituted potassium phosphate 2,5; manganese chloride, tetrahydrate 15.0; iron sulfate, hydrated 7.5; calcium carbonate 25.0; water is deionized to a volume of 24 liters. pH was adjusted to 8.0 by adding 16 ml of 10 n. potassium hydroxide solution.

The inoculated medium was incubated at 32 ° C for 234 hours. 72 hours after the start of incubation, the amount of blown air was increased from 0.01 to 0.023. (0.55 to 0.8 cubic feet per minute) and a stirring speed of 500 to 700 rpm. Obtaining factor A of antibiotic A 3823 or monensin generally ended after 210. hour.

After 234 hours, the contents of the pasteurized fermenter were used to deactivate S. cinnamonensis, after which the following nutrients were added to the fermentor, g: dextrose 750; soya flour 625; manganese chloride, tetrahydrate 155; calcium carbonate 12.5; iron sulfate, hexahydrate 7.5, potassium chloride 2.5; disubstituted phosphate cal-s 2.5; methyl oleate 250 ml; soybean oil 250 ml; water is deionized to a volume of 24 liters.

The pH value was adjusted to 8.0 by adding 215 ml of 5N. solution of latry oxide hydrate, after which the medium was sterilized. Cpe.Tv was inoculated with a vegetative culture. Streptomyces candidus NRRL 5449. Incubation was carried out for 137 hours at 30 ° C with aeration at a rate of 0.01 (0.35 cubic feet iB 1 min), first stirring at 120 rpm, at 16 rpm at 420 rpm; and after 40 hours, the stirring speed increased to 500 rpm. Dextrose (400 g) was added to the following incubation periods, hours: 44, 66.5, 89, 97, 113, and 127.

Calcium carbonate (175 g) was added to the beginning, at the 72nd and 99th hour of incubation.

ten

The incubation controls were performed using thin layer chromatography. After 137 hours, the production of substance A 27106 was essentially pumped up.

Example 3. The culture of S. candidus NRRL 5449 microorganism was grown as described in Example 1. The cell material was separated from the fermentation medium by filtration under vacuum and divided into equal parts of 200 g each, which was stored in a frozen state. Two of the parts obtained were thawed at room temperature and suspended in 0.05 M phosphate buffer (pH 5.8) to a volume of 600 ml. This cell suspension was sonicated for 30 minutes, then centrifuged at 10,000 rpm for 30 minutes and the residue from the fragments of cellular material was suspended in 100 ml of the indicated buffer. This suspension was dialyzed for 18 h against the indicated buffer. 120 mg of glucose and 25 mg of monensin in 2 ml of ethanol were added to 50 ml of dialysate. The reaction mixture was moved 72 hours at 30 ° C, then filtered; the filtrate was extracted with chloroform (100 ml). The obtained chloroform extract was concentrated in vacuo, chromatographed on a column of silica gel (10 g). The washing of the adsorbent with ethyl acetate indicated the presence of only traces of factor A of the antibiotic A 3823. When the adsorbent was washed with a mixture of ethyl acetate and methanol (19: 1), substance A 27106 (17 mg) was released.

The resulting preparation A 27106 is an effective agent for the treatment and prevention of coccidiosis in poultry. The amount of drug administered to the feed for poultry should be 0.005-0.05% by weight of the feed, preferably 0.01-0.04%.

Substance A 27106 also improves feed utilization by ruminants, primarily adults. It can be administered to ruminants with a iB dose of 0.5-2.5 mg / kg per day. The best results are obtained when giving it to animals in an amount of 0.1-1.5 mg / kg per day.

Claims (4)

1. Method for the production of metabolite A 27106, including the cultivation of microorganisms of the type Streptomyces candidus with aeration in an aqueous nutrient medium containing sources of carbon, nitrogen and mineral substances, characterized in that, in order to obtain a substance with reduced toxicity, iB as a microorganism take Streptomvces candidus MRRL 5449, and glucose and monensin produced by Streptomyces cinnamonensis are added to the nutrient medium. 2. A method according to claim 1, wherein a sterilized liquid growing medium of Streptomyces cinnamonensis is used as a source of monensin. 3. Method according to paragraphs. 1 and 2, characterized in that the nutrient medium is contacted with the culture medium broth of Streptomyces Candidas NRRL 5449 pre-grown medium separated from the cells and lyophilized. 4. Method according to pi. 1 and 2, characterized in that the nutrient medium is contacted with cell material separated from the medium prior to Streptomyces sapdidus NRRL 5449 cultivation, purified by sonication, centrifugation and dialysis.