JPH0354104B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic OM-704-A and a method for producing the same. The present invention is based on the finding that certain actinomycetes have the ability to produce novel antibiotics. The purpose of the present invention is to provide the OM-704
An object of the present invention is to provide a novel antibiotic named -A and a method for producing the same. According to the present invention, an antibiotic OM-
704-A is provided. The physicochemical properties of this antibiotic are as follows. (1) Elemental analysis value (%): O H N Actual value 68.19 6.24 7.93 Theoretical value 67.78 6.26 7.91 (2) Molecular weight: Mass spectrum (EI mass spectrum and FD
In mass spectrum analysis, an ion peak was observed at mass number 354. From the above results and elemental analysis values, the molecular formula C ₂₆ H ₂₂ N ₂ O ₄ was given. (3) Melting point: 293±2℃ (4) Specific optical rotation: This substance is poorly soluble, so it does not give a constant measured value. (5) Ultraviolet absorption spectrum: (Measured in methanol) As shown in Figure 1. The absorption maximum (nm) is as follows (molecular extinction coefficient is in the box): 250 (11800, shoulder), 260 (13600, shoulder), 278 (20100, shoulder), 286 (21700), 309 (9760), 321 ( 8950), 367 (4130), 490 (1150) (6) Infrared absorption spectrum: (KBr method) as shown in Figure 2. cm ^-1 2960, 1670, 1625, 1535, 1470, 1350,
1300 (7) Proton nuclear magnetic resonance spectrum: (measured in deuterium chloroform/deuterium methanol) as shown in Figure 3. (8) Solubility in solvents: Slightly soluble in chloroform, dioxane, methanol, and dimethylformamide, and insoluble in water. (9) Classification of basic, neutral, and acidic: Neutral substances. (10) Color of substance: red-orange. (11) Rf value: Silica gel thin layer chromatography (Merck, TLC aluminum sheet, silica gel
60F ₂₅₄ , thickness 0.2mm) by the usual method, the Rf value is as follows. (i) Chloroform/methanol (9:1) 0.45 (ii) Benzene/methanol (4:1) 0.58 Based on the above physical and chemical properties and the properties of the methyl form, this antibiotic has the structure represented by the above formula. It is recognized that This compound is an antibiotic and its biological properties are as follows. (1) Antibacterial properties The minimum inhibitory concentration (MIC) determined by the agar dilution method is
As shown in Table 1.

[Table] As described above, this antibiotic OM-704-A shows activity against Gram-positive bacteria. The LD ₅₀ of this antibiotic is 100 mg/Kg (intraperitoneal administration in mice). Therefore, the present antibiotic is expected to be used as an antibacterial agent for the prevention or treatment of human, animal, or plant diseases caused by these bacteria. As a result of comparing the above physicochemical and biological properties with known antibiotics, there were no known antibiotics, and Streptococcus faecium
Antibiotic OM-704-A was found to be a novel antibiotic since the antibacterial activity against IFO3181 was restored by the addition of thymidine, leucovorin or dihydrofolic acid. The method for producing antibiotic OM-704-A according to the present invention is directed to the production of antibiotic OM-704-A, which belongs to the genus Streptomyces.
-704-A-producing microorganisms are aerobically cultured in a medium, the desired compound is accumulated in the culture, and the antibiotic compound OM-704-A is recovered from the culture. . Belongs to the genus Streptomyces and is an antibiotic OM
Any microorganism can be used for the purpose of the present invention as long as it has the ability to produce -704-A. The bacterial strain described in the Examples is Streptomyces sp. OM-704-KA333 strain, which was newly isolated from soil by the present inventor. Mutant strains produced from this strain by natural or artificial induction can also be used for the purpose of the present invention, as long as they have production ability. The above strains Streptomyces sp. OM
Strain -704-KA333 has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 6520. Its mycological properties are as follows. () Morphological properties Vegetative hyphae develop well in both natural and synthetic media and usually do not have septa. Aerial mycelium grows moderately or abundantly on media other than peptone yeast iron agar, and is velvety and white to pale pink in color. When observed under a microscope, the sporophyte appears linear and chains of 10 or more spores are observed. The size of the spore is 0.57 x 1.0 μm, cylindrical, and the surface of the spore is smooth. No sclerotia, sporangia and zoospores are found. () Properties on various media E.B. Shearing (Int.J.Syst.
Bacteriol. Vol. 16, p. 313, 1966), and the culture medium and test method used are known.
The results are shown in Table 2. The color tones were determined using the Color Harmony Manual, 4th edition (Container Corporation of America, Chicago, 1958) as standard colors, and the code was written in parentheses along with the color table name. Unless otherwise specified below, observations were made on each medium at 27°C for 2 weeks.

【table】

【table】

[Table] Media selected by Seth Project
() Physiological properties (1) Formation of melanin pigment (a) Tyrosine agar negative (b) Peptone/yeast iron agar negative (c) Glucose/peptone/gelatin medium (puncture at 21-23°C) positive (d) Tryptone・Yeast solution negative (2) Tyrosinase reaction negative (3) Nitrate reduction negative (4) Hydrogen sulfide production negative (5) Gelatin liquefaction (glucose, peptone,
Gelatin medium) Positive (6) Hydrolysis of starch Positive (7) Coagulation of skim milk (37℃) Positive (8) Peptonization of skim milk (37℃) Positive (9) Growth temperature range 15℃~40℃ (10 ) Availability of carbon sources (Pridham-Godelib agar medium) Used: D-glucose, D-xylose, D-mannitol, D-fructose Slightly used: L-arabinose, i-inositol Not used: ruffinose, sucrose, melibiose ( 11) Decomposition of cellulose Negative () Cell wall composition Diaminopimelic acid is LL- type, and arabinose and galactose are not observed. The mycological properties of this bacterium are summarized as follows. The cell wall composition has LL-diaminopimelic acid. In terms of morphology, it forms linear spore chains, and the surface of the spores is smooth. Regarding the culture characteristics, the vegetative hyphae are pale pink or pale yellow;
Aerial mycelia are white or pale pink in color.
Soluble pigments produce slightly yellow-brown pigments,
Melanin pigment is produced only in glucose-peptone-gelatin medium. Based on these results, this bacterial strain belongs to Streptomyces and is classified according to Pridham and Tresner's classification (Burgess Manual of Determinative Bacteriology, 8th edition, 1974).
It is considered to be a species belonging to the White or Red series, according to the authors (2010, pp. 748-829). In the method of the present invention, antibiotic OM-704-
As a medium for culturing the microorganism that produces A, various media containing carbon sources, nitrogen sources, and inorganic substances that are commonly used for culturing Streptomyces microorganisms can be used. Examples of carbon sources for the culture medium are glucose, maltose, lactose, sucrose, starch, dextrin,
Examples of nitrogen sources are soybean flour, corn steep liquor, cottonseed flour, peptone, meat extract, dried yeast, casein hydrolyzate, etc.
Ammonium salts, nitrates, etc. Examples of inorganic substances are salts such as phosphate, magnesium, potassium, calcium, sodium, iron, manganese, etc. Cultivation is usually carried out aerobically, and aerated agitation culture is preferred. The culture temperature can be changed as appropriate within a range that allows microorganisms to grow and produce antibiotic OM-704-A, but is preferably 25 to 30°C. PH is 6-7
is preferred. Although the culture time varies depending on various conditions, the antibiotic OM-704-A accumulated in the culture usually reaches its maximum titer in about 30 to 100 hours. After completion of culture, antibiotic OM-704 from culture
-A can be recovered by known means for separating and purifying antibiotics having the above-mentioned physicochemical properties from a culture of microorganisms, either alone or in combination.
An example of a practical separation and purification method is as follows. The culture is separated into bacterial cells and filtrate. The bacterial cells are extracted with acetone or methanol, the extract is concentrated, and then extracted with an organic solvent such as ethyl acetate or chloroform. The filtrate is separated from water such as ethyl acetate and benzene, extracted with an organic solvent that dissolves antibiotic OM-704-A, and then extracted with antibiotic OM-704-A by a known method commonly used in the purification of fat-soluble substances. Collect 704-A. For example, the extract (ethyl acetate layer) is concentrated under reduced pressure, the resulting precipitate is separated, washed with n-hexane, and then subjected to silica gel column chromatography using a mixed solvent of chloroform and methanol as the elution solvent. is used to isolate antibiotic OM-704-A. The produced antibiotic OM-704-A was detected and quantified using silica gel thin layer chromatography (manufactured by Merck & Co., Ltd., silica gel 60F ₂₅₄ , thickness 0.2 mm,
Developing solvent; chloroform/methanol = 9:1,
Rf value of antibiotic OM-704-A (0.45) and Streptococcus faecium (Rf value 0.45) and Streptococcus faecium
faecium) IFO3181 as the test bacterium. Next, an example of the antibiotic OM-704-A according to the present invention will be shown, but this example is merely an example and does not limit the present invention. Example Streptomyces sp.
(Streptomyces.sp) strain OM-704-KA333, Microtechnical Research Institute No. 6520) was inoculated into a seed medium, and after culturing at 27°C for 2 days, it was placed in a 30-volume jar fermenter. Inoculate 20 media at a rate of 1% and incubate at 27℃ for 48 hours with aeration rate of 10/min and agitation.
Aerated agitation culture was performed at 250 rpm. The composition of the seed medium is glucose 1.0%, starch 2.0%
%, yeast extract 0.5%, peptone 0.5%, calcium carbonate 0.4%, the composition of this medium is glucose 0.5%,
Con steep liquor 1.0%, oatmeal 1.0
%, Pharmamedia 1.0%, K ₂ HPO ₄ 0.5%,
_MgSO4・_7H2O0.05 %, _FeSO4・_7H2O0.001 %,
_MnCl2・_4H2O0.001 %, _ZnSO4・_7H2O0.001 %,
_CuSO4・_5H2O0.001 %, _CoCl2・_2H2O0.001 %,
15 at 121℃ after adjusting to PH7.0 during use.
Sterilized for minutes. Culture solution 20 is centrifuged using a shear press (10,000 rpm) to separate bacterial cells and culture supernatant. To the obtained bacterial cells, 1.5 g of 60% acetone solution was added and extracted with stirring, and this was filtered to obtain a 60% acetone extract. This 60% acetone extract was concentrated to 500 ml under reduced pressure, 400 ml of ethyl acetate was added, and the mixture was thoroughly stirred and re-extracted. The acetic acid extract obtained here was concentrated to 20 ml under reduced pressure, left overnight at room temperature,
By filtering and drying the resulting precipitate, 130
mg of crude material was obtained. Next, ethyl acetate 12 was added to the culture supernatant, and after stirring well, the ethyl acetate layer was separated by centrifugation. This ethyl acetate extract was concentrated under reduced pressure to 500 ml, left overnight at room temperature, and the resulting precipitate was filtered off to obtain 510 mg of a pink-red crude material. Antibiotic OM obtained from these bacterial cells and culture supernatant
640 mg of the crude material containing -704-A was dissolved in chloroform/methanol (4:1) and added to the top of a silica gel column (Kieselgel 60, 20 g, manufactured by Merck & Co.) that had been previously filled with chloroform. Fill the column with chloroform (160 ml) and chloroform/
Elute with methanol (20:1, 400ml),
It was fractionated into ml portions. Here, the active fractions (fraction Nos. 4 to 14, 15 ml/fraction tube) obtained by elution with a mixed solvent of chloroform/methanol (20:1) are concentrated under reduced pressure and left overnight at room temperature. As a result, 120 mg of crude crystals were obtained. By crystallizing this with a mixed solvent of chloroform/methanol, red-orange needle crystals of antibiotic OM-704-A were obtained.
I got mg. The properties of this substance were consistent with the physicochemical and biological properties described above.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum (measured in methanol) of the antibiotic OM-704-A, Figure 2 shows the infrared absorption spectrum (KBr method), and Figure 3 shows the nuclear magnetic resonance spectrum (measured in deuterium chloroform/dew methanol). measurement).

Claims (1)

[Claims] Antibiotic OM-704-A represented by the following formula: 2 A microorganism belonging to the genus Streptomyces and capable of producing the antibiotic OM-704-A expressed by the following formula is cultured aerobically in a medium, the desired compound is accumulated in the culture, and the desired compound is extracted from the culture. A method for producing antibiotic compound OM-704-A, which comprises the step of recovering OM-704-A. 3 The microorganism is Streptomyces sp.
(Streptomyces sp.) OM-704-KA333 (Feikoken Bibori No. 6520).