JPH0365347B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic OM-674 and a method for producing the same. In the process of searching for antibiotics produced by actinomycetes, the present inventors discovered that actinomycetes OM-674, isolated from a new species, produces a new antibiotic that is effective against anaerobic bacteria. . After identifying the production strain and isolating the substance, the present invention was completed by investigating its physicochemical and biological properties. The physicochemical properties of the antibiotic OM-674 obtained by the present invention are as follows. (1) Elemental analysis: C65.59%, H7.68%, S13.51% (2) Molecular weight: 238, molecular ion peak in high-resolution mass spectrum, m/z 238, 1023, and molecular formula C ₁₃ from the elemental analysis value H ₁₈ O ₂ S was determined. (3) Melting point is 67-70℃. (4) Specific rotation: [α] ^18.5 _D +89 (C=1, methanol) (5) Ultraviolet absorption spectrum: in methanol
It exhibits absorption maxima at 238 nm and 300 nm, with molecular extinction coefficients of 27370 and 6616, respectively (Figure 1). (6) Infrared absorption spectrum: As shown in Figure 2. (Solution method, chloroform) (7) Proton nuclear magnetic resonance spectrum: As shown in Figure 3. (8) C-13 nuclear magnetic resonance spectrum: As shown in Figure 4. (9) Solubility in solvents: soluble in methanol, acetone, ethyl acetate, and benzene;
Insoluble in water, petroleum ether and n-hexane. (10) The color reaction is positive for ferric chloride, bromcresol green, and potassium permanganate, and negative for Mauritsch and Dragendorff. (11) It is an acidic substance. The structural formula is as follows. Among known antibiotics, Thioractomycin (Gill
Antibiotics, vol. 35, pp. 391-419, 1982
year) can be mentioned. However, thiolactomycin has a molecular formula of C ₁₁ H ₁₄ O ₂ S, a molecular weight of 210, and
The proton nuclear magnetic resonance spectrum and the C-13 nuclear magnetic resonance spectrum are clearly different from those of the antibiotic OM-674. From the above, it has become clear that the present antibiotic OM-674 is a new compound. The biological properties of this substance are as follows. (1) Antibacterial properties The minimum inhibitory concentration (MIC) determined by the agar dilution method is
As shown in Table 1.

【table】

[Table] (2) Toxicity When this antibiotic was administered intraperitoneally to mice,
LD ₅₀ is 400 mg/Kg or more. As mentioned above, antibiotic OM-674 has low toxicity and shows activity mainly against Gram-negative bacteria. Therefore, this substance is expected to be used as a therapeutic or preventive agent against microbial infections in humans and animals. A practical example of the microorganism used to produce the antibiotic OM-674 of the present invention is Streptomyces sp. OM-674 strain, which was isolated by the present inventors from the soil. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession number 6510. Its mycological properties are as follows. () Morphological properties Vegetative hyphae develop well on various agar media;
Usually has no septum. Aerial hyphae are moderately or abundantly epiphytic on materials other than glycerol/calcium malate agar, and have a velvety or powdery appearance. When observed under a microscope, the sporophyte appears linear and chains of 10 or more spores are observed. The size of the spore is 0.60 x 0.88 μm and oval. The surface of the spore is smooth. No sclerotia, sporangia and zoospores are found. () Properties on various media EBShirling
and D.Gcttlieb's method (International Journal of Systematic Bacteriology, vol. 16,
The following table shows the culture properties of this producing bacterium, which were investigated by the authors (p. 313, 1966). The color tones were determined as standard colors using the Color Harmony Manual, 4th edition (Container Corporation of America, Chicago, 1958), and the code was written in parentheses along with the color chart name. The following are the results of observations on each culture medium at 27°C for 2 weeks unless otherwise specified.

【table】

【table】 () Physiological properties

【table】

[Table] () Cell wall composition Diaminopimelic acid is LL-type, and arabinose and galactose are not observed. The mycological properties of this bacterium can be summarized as follows. The cell wall composition has LL-diaminopimelic acid. Morphologically, it forms linear spore chains, and the surface of the spores is smooth. Regarding various cultural properties, vegetative mycelium exhibits an olive color or dure gold color, and aerial mycelium exhibits a light olive gray or light brownish gray color. Soluble pigments produce a slightly yellowish pigment, and melanin pigments are produced in small amounts, if at all. From these results, this bacterial strain belongs to the genus Streptomyces, and is classified according to Bridham and Tresner's classification (Burges's Manual of Determinative Bacteriology, 8th edition, pp. 748-829).
It is considered to be a bacterial species belonging to the green or gray series (1974). Mutant strains of the above-mentioned strains can also be used in the method of the invention. Other strains belonging to the genus Streptomyces that have the ability to produce the present antibiotic can also be used. As the medium, a synthetic medium or a natural medium containing appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and, if necessary, other nutrients suitable for culturing Streptomyces microorganisms can be used. The carbon source and nitrogen source used in the culture medium may be any type as long as they are available to the strain used.
That is, as a carbon source, for example, glucose,
Glycerol, fructose, maltose, mannitrate, xylose, galatose, ribose,
Various carbohydrates can be used, such as starch or its hydrolysates. Its concentration is usually 0.1 relative to the medium
~5% (in terms of glucose) is preferred. In addition, various organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, various amino acids such as glycine, glutamic acid, and alanine, alcohols such as methanol and ethanol, various non-aromatic hydrocarbons such as normal paraffin, and plants. It is also possible to use various oils and fats of natural or animal origin. Nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, urea, peptone, NZ-amine, meat extract, yeast extract, dried yeast, corn steep liquor, Nitrogen-containing organic substances such as casein hydrolyzate, fat meal or its digested product, soybean flour or its digested product, defatted soybean or its digested product, pupa hydrolysate, and various amino acids such as glycine, glutamic acid, and alanine can be used. It is. As inorganic substances, various phosphates, magnesium sulfate, common salt, etc., and trace amounts of heavy metal salts are used. Furthermore, when using a mutant strain that exhibits auxotrophy, it is naturally necessary to add substances that satisfy its nutritional requirements to the culture medium, but this kind of nutrients is especially important when using a medium containing natural substances. may not be necessary. Fermentation is carried out under aerobic conditions such as shaking culture or submerged culture with aeration and stirring. The culture temperature is usually 20-40°C. The culture period is usually 1 to 8 days, and a significant amount of the antibiotic OM-674 is produced and accumulated inside and outside the bacterial cells. After culturing, antibiotic OM-674 was added to the culture.
For example, collect it using the following method. The culture is separated into a liquid and a precipitate by centrifugation. It is extracted from the liquid by adsorbing it onto activated carbon, porous synthetic polymer resin, ion exchange resin, etc. and eluting it. The precipitate is extracted with an organic solvent such as ethyl acetate, n-butanol, or aqueous acetone. The crude substance of OM-674 is obtained by appropriately concentrating the extract to dryness. The crude substance is further purified by appropriately combining known methods commonly used in the purification of fat-soluble substances, such as concentration methods such as silica gel column chromatography. By concentrating and drying the active fraction obtained by these purification methods, powder of antibiotic OM-674 can be obtained. In the present invention, the antibiotic OM-674 was detected and quantified using silica gel thin layer chromatography (Silica gel 60F ₂₅₄ manufactured by Merck & Co., Ltd., thickness 0.2 mm, developing solvent: benzene/acetone = 7:1, antibiotic
Rf value of OM−674 0.48) and Bacteroides
Bacteroides fragilis ATCC23745
A biological assay method was used. Next, an example of the antibiotic OM-674 of the present invention will be shown, but this example is merely an example and does not limit the present invention. Example One platinum loop was obtained from a slant culture of Streptomyces sp.
100ml seed medium (glucose 1.0%, starch 1.0%,
The yeast extract was inoculated into a 500 ml Sakaguchi flask containing 0.5% yeast extract, 0.5% peptone, and 0.4% calcium carbonate, and cultured with shaking at 27°C for 2 days to obtain a seed culture. 100 ml of this seed culture was made into 15 production culture medium [Glucose
0.5%, corn steep liquor (CSL) 1.0%,
Oatmeal 1.0%, Pharmamedia 1.0%, dibasic potassium phosphate
0.5%, magnesium sulfate heptahydrate 0.5%, ferrous sulfate heptahydrate 1 mg/, manganese chloride tetrahydrate 1 mg/
, zinc sulfate heptahydrate 1 mg/, copper sulfate pentahydrate 1
mg/, cobalt chloride dihydrate 1 mg/] was added.
Inoculate a 30-volume jar and inoculate at 27℃ for 3 hours.
Daily aeration stirring culture (aeration rate 10/mm, stirring 300r.
pm). The culture solution 15 is centrifuged using a shear press type centrifuge to separate the bacterial cells and the cultured product. A 70% acetone solution 3 was added to the bacterial cells for stirring and extraction, and this was separated to obtain a hydrous acetone extract containing the antibiotic OM-674. 500ml of this extract under reduced pressure
After adjusting the pH to 4 with hydrochloric acid, add ethyl acetate to
500 ml was added and stirred to perform re-extraction. The culture supernatant 12 was adjusted to pH 4 with hydrochloric acid and extracted twice with 6 ethyl acetate. The two ethyl acetate extracts and the acetic acid extract derived from the bacterial cells were combined and concentrated to dryness under reduced pressure to obtain 4 g of an oily substance of antibiotic OM-674. Dissolve 4 g of oily substance in 10 ml benzene and add silica gel (120 g,
Merck & Co., art7734) was added to the top of the column. Column with benzene/acetone (20:
1) and fractionated into 10 ml portions. Active fraction (No.
51-74) and concentrated under reduced pressure, the antibiotic OM-674 was isolated as 180 mg of colorless needles.

[Brief explanation of drawings]

Figure 1 is the ultraviolet absorption spectrum (measured in methanol) of the antibiotic OM-674, Figure 2 is the infrared absorption spectrum (measured in chloroform), and Figure 3 is the infrared absorption spectrum (measured in chloroform).
The figure shows the proton nuclear magnetic resonance spectrum (measured in deuterated chloroform), and FIG. 4 shows the C-13 nuclear magnetic resonance spectrum (measured in deuterated chloroform).

Claims (1)

[Claims] 1. A novel antibiotic OM- represented by the following structural formula:
674. 2 A new antibiotic belonging to the genus Streptomyces
A bacterial strain capable of producing OM-674 is cultured aerobically in a medium, and antibiotics are added to the medium or cells.
A method for producing a novel antibiotic OM-674, which comprises accumulating OM-674 and collecting it.