CN1022765C - Preparation method of cell variety mycomycin of new antibiotic - Google Patents

Abstract

The present invention provides cell variety mycomycin as a new antibiotic and a preparation method thereof. After flavobacterium as an antibiotic is developed, the cell variety mycomycin is another new active substance prepared by culturing streptomyces SP. SR-44. The cell variety mycomycin has specific strong growth inhibiting action on the bacteria of xanthomonas, and thus, the cell variety mycomycin can be used as a new antibiotic for agriculture.

Description

The invention relates to a new antibiotic Blebomycin (code name SR-44B) and a preparation method thereof. The antibiotic described in the present invention is obtained by separating from the culture solution of the antibiotic cytomacin-producing bacteria of the genus Streptomyces, and is a new antibiotic that has not been reported in literature. The antibiotic Cytovaricin is the same substance as Blebomycin (code name RS-44B) in the corresponding Japanese patent application of the present application.

In recent years, antibiotics are being widely used not only in medicine but also in agricultural chemicals. In order to search for new antibiotics that are more effective than before, the present inventors isolated microorganisms from various soils and cultivated antibiotics. Then these antibiotics were refined, identified and developed.

As a result, the inventors first successfully isolated and refined new antibiotic flavoparin from the culture of streptomyces SR-44 (streptomyces sp·SR-44). After further research, a new antibiotic, cytotaxin, was successfully isolated and refined from the culture of the above-mentioned SR-44 strain. Cytostatin has completely different properties from aflatarin in physicochemical and biological aspects.

The object of the present invention is to provide a new antibiotic and a method for isolating and preparing the antibiotic from the microorganisms of the genus Streptomyces.

The present invention will be described in detail below.

microorganisms used

The microorganism used to produce the antibiotic mutamycin of the present invention is a strain belonging to the genus Streptomyces, which has the ability to produce the antibiotic mutamycin.

Streptomyces SP·SR-44 (Streptomyces SP·SR-44) (collection number CGMCC-0092 of the General Microorganism Collection Center of China Microbiological Culture Collection Management Committee) (hereinafter referred to as "SR-44 strain") is one example.

The microorganism has the above-mentioned characteristics and is suitable for producing the antibiotic SR-44B of the present invention, so it can be effectively used in the preparation method of the present invention.

At the same time, the natural and artificially mutated strains of the above-mentioned "SR-44 strain" also belong to the strains of the genus Streptomyces. The method of the present invention can be applied to any microorganism that has the ability to produce the antibiotic cytastatin mentioned later.

The above "SR-44 strain" was isolated from soil in Jiangsu Province, China, and has the following properties:

1. Morphological characteristics:

This strain "SR-44 strain" was cultivated on the cell analysis medium containing glucose and yeast extract, and then the cells were separated and dried, and then heated and hydrolyzed with 6N hydrochloric acid at 110°C for 20 hours. L, L-diaminopimelic acid can be found when the amino acid composition of the cell wall is detected by paper chromatography.

In addition, the aerial hyphae grown on the agar plate showed a spiral shape, and it could be seen that the spiral hyphae was composed of dozens of spores, but no sporangia and motile spores could be seen.

It can be explained above that this bacterial strain belongs to the genus Streptomyces. The spores are cylindrical in shape and smooth in surface.

2. Growth state on various media (cultivated at 27°C for three weeks, and the color is described in the "Descriptive color names diotionary").

(1) Sucrose nitrate agar medium

grow not grow

(2) Glucose-asparagine agar medium

grow not grow

Aerial mycelium trace b gray cream (oyster white)

base b gray cream (oyster white)

Pigment No

(3) Glycerol-Asparagine Agar Medium

growth normal

Aerial mycelium trace b gray cream (oyster white)

base b gray cream (oyster white)

Pigment No

(4) Starch-inorganic salt agar medium

growing well

Aerial mycelia Large quantity 2ge Bergey

base 2ie light mustard (light mustard)

Pigment No

(5) Tyrosine agar medium

growing well

Aerial mycelium Large amount 3dc natural color (natural)

Base 2le mustard (mustard)

Pigment 2ea bright yellow (light maize)

(6) Nutrient agar medium

growing well

Aerial mycelia None

base 1 1/2 Ca cream

Pigment No

(7) Yeast-malt agar medium

growing well

Aerial mycelia Large amount 5fe gray (ash)

Base 2 lg mustard tan (mustard tan)

Pigment No

(8) Oatmeal agar medium

poor growth

Aerial mycelia None

Base Unknown

Pigment No

(9) Peptone-yeast-iron agar medium

growing well

Aerial mycelia None

base 1 1/2 ea light yellow

Pigment No

3. Utilization of carbon source (Pugo agar medium)

L-Arabinose -

D-xylose +

D-glucose +

D-Fructose -

Sucrose -

Inositol -

L-rhamnose -

Raffinose -

D-Mannitol -

+...grows -...does not grow

4. Other physiological properties

(1) Suitable temperature 25～30℃

(2) Liquefaction of gelatin Liquefaction

(glucose-peptone-gelatin medium)

(3) Starch hydrolysis Hydrolysis

(starch-inorganic salt agar medium)

(4) Coagulation of skim milk Coagulation

(5) Liquefaction of skim milk Liquefaction

(6) Production of melanin

(Tyrosine agar medium) Does not generate

(Peptone-Yeast Iron Agar Medium) Does not generate

Streptomyces species with the above properties are classified according to Bergey's Manual of Determinative Bacteriology 8 th ed. (Bergey's Manual of Determinative Bacteriology 8 th ed.), and this strain should belong to the WISIC-ISM group. Look, the closest relative is Streptomyces cocoa. It can be inferred that this strain belongs to Streptomyces cocoa or its close relatives.

Cultivation of SR-44 Strain and Preparation and Purification of Cytovaricin

Using the above-mentioned antibiotic-producing bacteria of the genus Streptomyces, the "SR-44 strain" can be cultivated by conventional antibiotic production methods to produce the antibiotic cytastatin of the present invention, and the culture method can be either liquid culture or solid nourish. However, in order to facilitate the cultivation during industrial production, it is advisable to inoculate the spore suspension or culture solution of the above-mentioned production bacteria on the medium and carry out aeration and agitation cultivation.

There is no special regulation on the nutrient source in the culture medium, and it may contain carbon source, nitrogen source and other nutrient sources commonly used in microorganism cultivation. Among them, the carbon source can be starch, dextrin, glycerin, glucose, sucrose, lactose, inositol, mannitol, etc., and the nitrogen source can be peptone, soybean powder, meat extract, rice bran, wheat bran, urea, corn steep liquor, ammonium salt, Nitrates, and other organic or inorganic nitrogen-containing compounds. For other nutrient sources, some inorganic salts, such as table salt and phosphate, can be added appropriately And potassium, calcium, zinc, manganese, iron and other metal salts, if necessary, some animals, plants, mineral oils, etc. can also be added as defoamers.

There are no strict restrictions on the culture conditions such as temperature and time during the culture, whichever is suitable for the development of the bacteria used, and it is better to choose the conditions with the highest mutamycin production. For example, the pH range of the medium can be 4-9, preferably close to neutral. The culture temperature and stirring conditions should be properly adjusted according to the type of strain used and external conditions to obtain the best results.

Starting from the culture obtained above, cytotaxin can be produced by some appropriate methods. These are some conventional methods commonly used to extract metabolites. For example, the difference in solubility, ion binding capacity, adsorption affinity and molecular weight between mutatin and impurities can be used for extraction. Specifically, mutatin is mostly present in the culture filtrate. Adjust the culture medium to be slightly acidic, such as to pH 4.0, and then extract it with an organic solvent such as ethyl acetate. After combined purification by analysis, gel chromatography, etc., the components containing mutatin and other active ingredients can be obtained. For example, the oily substance is subjected to silica gel chromatography followed by development in chloroform-methanol to obtain the active component. Then collect the active part of SR-44B by high-speed liquid chromatography [for example, Nucleosi 15C ₁₈ is selected as the filling agent, and methanol-water (8:2) is the developing solvent], and then recrystallized with methanol to obtain cytotaxin of colorless crystals.

The thus-prepared new antibiotic cytaxin has the following physicochemical and biological properties.

Physicochemical and Biological Properties of the Antibiotic Cytovaricin

Shape: colorless crystal

Melting point: 157～160℃

Elemental analysis: carbon 58.14%, hydrogen 6.77%, nitrogen 10.70%, oxygen 24.13%

Molecular weight: 895 (according to FD mass spectrum)

Specific rotation: [α] ²⁵ _D -76° (C = 0.5 methanol)

UV absorption spectrum: λ (E ^1% _1cm ) 230 (90), 265 shoulder (7.3), 268 shoulder (8.1), 274 (8.5), 282 (7.1) (see Figure 1)

Infrared absorption spectrum: ν ^kBr _max cm ^-1 3300, 2950, 1725, 1630, 1525, 1500, 1450, 1400, 1295, 1245, 1195, 1080, 1050, 1020, 825, 745, 695 (see Figure 2)

Acid hydrolysis: acid hydrolysis can obtain serine, threonine, glycine, tyrosine and other amino acids.

Color reaction: positive reaction with Dragendolf and Rydon Smith

Solubility: easily soluble in methanol, ethyl acetate, chloroform, insoluble in water, pentane, hexane, benzene.

Thin-layer analysis: (0.25mm silicon thin-layer board manufactured by Merck, USA)

Solvent Rf value

Chloroform: Methanol = 10:1 0.57

Ethyl acetate: benzene = 4: 1 0.13

Antibacterial Spectrum: Use the usual agar plate dilution method to obtain the minimum inhibitory concentration (MIC). (Potato-sucrose agar medium was used for fungi, and broth agar medium was used for bacteria.) As a result, varicosin showed a strong growth-inhibitory effect specific to bacteria of the genus Xantho-monas, whereas normal Bacteria and molds have almost no growth inhibitory effect.

Test bacteria MIC (mcg/ml)

Xanthomonas oryzae 2

(Xanthomonas oryzae)

Xanthomonas citrus 4

(Xanthomonas citri)

Escherichia coli ＞1000

(Escherichia coli)

Salmonella typhimurium ＞1000

(Salmonella typhimurium)

Staphylococcus aureus 209P ＞1000

(Staphylococcus aureus 209P)

Bacillus subtilis ＞1000

(Bacillus subtilis)

Cytovaricins belong to peptide antibiotics in terms of their physical and chemical properties. Since no peptide antibiotic has been found to have a specific and strong growth inhibitory effect on bacteria of the genus Xanthomonas before, it can be inferred that cytotaxin is a new antibiotic.

To sum up, varicosin is expected to be used as an agricultural antibiotic because it exhibits a strong growth inhibitory function against bacteria of the genus Xanthomonas.

Manufacturing example:

The above SR-44 strain (CGMCC-0092) was inoculated in this medium, and cultured with shaking at 28°C for 96 hours. Take 25 liters of its culture solution and extract it with ethyl acetate under the condition of PH=4. After the extract was concentrated under reduced pressure, 4.8g of oil was obtained, and then the oil was placed in 200ml of methanol, heated to 60°C to dissolve it, then lowered to 0°C to crystallize, filtered and washed with methanol, that is 400mg of colorless crystals can be obtained.

The above-mentioned crystals were purified by high-speed liquid chromatography. The chromatographic column is nucleoside 5C ₁₈ , with a diameter of 20 mm and a length of 250 mm. The solvent is methanol: water = 8: 2, the flow rate is 6.0ml/min, the pressure is 180Kg/cm ² , and the activity peak of cytastatin appears at the retention time of 12 minutes. The above active peaks were collected and concentrated under reduced pressure until methanol was completely removed. After filtration, the precipitate was recrystallized with methanol to obtain 150 mg of pure cytastatin in the form of colorless crystals.

Description of drawings

Fig. 1 is the ultraviolet absorption spectrogram of the antibiotic cytotaxin according to the present invention. Fig. 2 is an infrared absorption spectrum diagram of the antibiotic cytastatin.

Claims (1)

1. A method for cultivating and preparing a new antibiotic cytamycin, which has the following physical and chemical properties: Shape: colorless crystal Melting point: 157～160℃ Elemental analysis: carbon 58.14%, hydrogen 6.77%, nitrogen 10.70%, oxygen 24.13% Molecular weight: 895 (according to FD mass spectrum) Specific rotation: [α] ²⁵ _D =-76°(C=0.5methanol) UV absorption spectrum: λ ^CH30H _maxnm (E ^1% _1cm ) 230 (90), 265 shoulder (7.3),                                                                          Infrared absorption spectrum: υ ^KBr _max cm ^-1 3300, 2950, 1725, 1630, 1525,                 1500, 1450, 1400, 1295, 1245, 1195,         1080, 1050, 1020, 825, 745, 695 Acid hydrolysis: acid hydrolysis can obtain serine, threonine, glycine, tyrosine and other amino acids. Color reaction: positive reaction with Dragendolf and Rydon.Smith Solubility: easily soluble in methanol, ethyl acetate, chloroform, insoluble in water, pentane, hexane, benzene, It is characterized in that the antibiotic is prepared from metabolites cultured by streptomyces sp·SR-44 (preservation number CGMCC-0092) through acidification, organic solvent extraction and chromatography.

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