JPH0411535B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel antibiotic SF-2418 substance and a method for producing the same. Conventional techniques and problems to be solved by the invention Known substances similar to SF-2418 substance include:
Pyroquinone (Nature Vol. 199, pp. 285-286, 1963) and Haloquinone (Journal of Antibiotics, Vol. 34, 1531-
1543 pages, 1981), but it is clearly distinguished from these known substances because the elemental analysis values, molecular weight, and molecular formula are different. Therefore, SF−
2418 substances were determined to be new substances. Means for Solving the Problem The present inventors searched for a new and useful antibiotic having antibacterial activity against chicken Mycoplasma and porcine Treponema, and found that the novel antibiotic SF- The present invention was completed by discovering that 2418 substances were produced, isolating the effective substances from culture, determining their chemical structures, and examining their biological activities. That is, SF- used in the present invention
An example of a 2418 substance-producing bacterium is SF-2418, which was newly isolated from soil in Kyoto City by the present inventors.
There are stocks. The mycological properties of SF-2418 strain are as follows. Morphology The basal hyphae are well elongated and branched, and do not divide under normal conditions. It adheres well to aerial mycelia on starch agar, oatmeal agar, etc., and forms abundant spores. The branching of aerial hyphae is simple, and no axle branching is observed. The spore chain at the tip of the aerial hyphae is linear, loop-shaped, or hook-shaped, and spiral shapes are also observed. No spore motility is observed. Special structures such as sporangia and sclerotia are not observed. When observed using an electron microscope, the spores are oval or oval in shape, 0.5 to 0.7 x 0.6 to 1.0 μm in size, and have a hairy surface structure. Spores are usually 10
About 50 pieces are chained together. Growth status on various media The growth status of SF-2418 strain on various agar media is as shown in the following table. Cultivation was carried out at 28°C.
Observations were made after 14 to 21 days of culture. For the symbols shown in parentheses for the color of aerial mycelia, the Color Harmony Manual manufactured by Container Corporation of America was used.

[Table] Physiological properties (1) Growth temperature range: Grows in the temperature range of 15 to 42℃ on yeast malt agar medium, and
Grows well at 32℃. (2) Liquefaction of gelatin: Positive (3) Hydrolysis of starch: Positive (4) Reduction of nitrate: Negative (5) Effect on skim milk: Both peptonization and coagulation are negative (6) Salt-resistant product: 3% salt added Although it grows, 4
It will not grow above %. (7) Production of melanin-like pigment: negative How to use carbon sources (1) Used: D-glucose, D-fructose, D-xylose (2) Use is questionable: L-arabinose (3) Not used: D- Mannitol, i-inositol, raffinose, L-rhamnose Bacterial cell analysis Diaminopimelic acid during hydrolysis of whole bacterial cells
It was type LL. Based on the above mycological properties, strain SF-2418 is a strain belonging to the genus Streptomyces among actinomycetes. The present inventors used the SF-2418 strain as Streptomyces sp.
sp.SF-2418). This strain has been donated to the Institute of Microbial Technology.
It has been deposited as No. 8156 (FERM P-8156). The SF-2418 strain is susceptible to changes in its properties, as is the case with other actinomycetes. For example, SF
-2418 strain or a mutant strain (naturally occurring or induced) derived from this strain, whether it is a transconjugant or a genetically recombinant strain, any one that produces the SF-2418 substance can be used in the present invention. . In the method of the present invention, the above bacteria are cultured in a medium containing nutrients that can be used by common microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing actinomycetes can be used. for example,
Glucose, starch syrup, dextrin, starch, molasses, animal and vegetable oils, etc. can be used as carbon sources.
In addition, soybean flour, wheat germ, cornstarch liquor, cottonseed meal, meat extract, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used as nitrogen sources. In addition, it is effective to add, if necessary, inorganic salts capable of producing sodium, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions. Also,
Organic and inorganic substances that aid the growth of bacteria and promote the production of SF-2418 substances can be added as appropriate. The most suitable culture method is a culture method under aerobic conditions, especially a deep culture method.
The appropriate temperature for culturing is 15 to 37°C, but in most cases it is cultured at around 26 to 32°C. SF−2418
Production of the substance varies depending on the medium and culture conditions, but the accumulation usually reaches its maximum within 2 to 10 days in both shaking culture and tank culture. The SF-2418 substance produced in this way has the physical and chemical properties described below, so it can be extracted and purified from the culture according to its properties, but in particular, it can be efficiently extracted and purified by the following method. Can be purified. That is, an organic solvent that is not freely miscible with water, such as ethyl acetate, is added to the culture containing the active ingredient, and the mixture is stirred and extracted. Alternatively, a solvent that is not freely miscible with water, such as ethyl acetate, is added to the filtrate obtained by filtering solid matter from the culture, and the mixture is stirred and extracted. On the other hand, for solids, a solvent that is freely miscible with water such as acetone is added and stirred, the active ingredients are extracted from the solids, and the organic solvent is distilled off. Extract the active ingredients. The solvent of the extract containing the active ingredients thus obtained is distilled off, and n-hexane is added to the obtained oily substance to obtain a precipitate. The precipitate is subjected to silica gel chromatography to isolate SF-2418 material. The isolated SF-2418 substance is crystallized using a solvent system such as benzene and n-hexane to obtain crystals of SF-2418 substance. When testing the SF-2418 substance, Mycoplasma gallisepticum (Mycoplasma gallisepticum)
A biological assay method using M. gallisepticum as the test bacterium is used. The physical and chemical properties of SF-2418 substance are as follows. (1) Elemental analysis values: carbon 70.29%, hydrogen 5.03%,
Oxygen 24.68% (2) Molecular formula and molecular weight: C ₁₉ H ₁₆ O ₅ ; 324 (FD−
MS) (3) Melting point: 195℃ (4) Ultraviolet absorption spectra: The ultraviolet absorption spectra in methanol solution, alkaline methanol solution, and acidic methanol solution are shown in Figure 1, and the maximum absorption nm is as follows.
(E) is shown. In methanol solution: 227 nm (23,300) 255 nm shoulder (10,700) 275 nm shoulder (11,000) 283 nm (11,700) 392 nm (2,900) 502 nm (3,900) In alkaline methanol solution: 220 nm (31 ,400) 290nm shoulder (8,100) 460nm shoulder (3,900) 573nm (7,400) In acidic methanol solution: 227nm (24,800) 255nm shoulder (11,300) 275nm shoulder (12,000) 283nm ( 12,500) 395nm (3,000) 504nm (3,900) (5) Infrared absorption spectrum: As shown in Figure 2. (6) Solubility: Soluble in acetone, benzene, and chloroform, poorly soluble or insoluble in water, methanol, and n-hexane. (7) Color reaction: Appears blue in sulfuric acid, magnesium acetate, and methanol. (8) Rf value of thin layer chromatography: The Rf value when developed with the following solvent on a thin layer of silica gel 60F254 (manufactured by Merck & Co.) is as follows. Benzene: Acetone = 50:1 Rf0.33 Toluene: Ethyl acetate = 10:1 Rf0.47 Toluene: Acetone = 40:1 Rf0.40 (9) Distinction between neutral, acidic, and basic: acidic (10) Color and appearance: Brown needle-like crystals (11) Hydrogen nuclear magnetic resonance spectrum: As shown in Figure 3. (12) Chemical structural formula: Example (1) Culture Starch 2.0%, glucose 1.0% as seed medium
%, wheat embryo 0.6%, polypeptone 0.5%, powdered yeast extract 0.3%, soybean flour 0.2%, and calcium carbonate 0.1%. In addition, production media include glucose 2.5%, cottonseed oil 0.5%, wheat embryo 1.2%, corn staple liquor 1.0%, calcium carbonate 0.3%, magnesium sulfate 0.1%,
A medium containing 0.001% cobalt chloride was used. The pH was adjusted to 7.0 before sterilization. Streptomyces sp.
One platinum loop of slant culture of SF-2418 (Feikoken Bibori No. 8156) was inoculated and cultured with shaking at 28°C for 4 days.
This was used as the first type culture solution. Next, four 500 ml Erlenmeyer flasks containing 80 ml of the seed medium were heated at 120°C.
After sterilization for 30 minutes, the above-mentioned first type culture solution was inoculated in four batches, and cultured with shaking at 28°C for 2 days to obtain a second type culture solution. Next, four 5-volume Erlenmeyer flasks into which 1 ml of the seed medium was dispensed were sterilized at 120°C for 30 minutes, and 80 ml each of the above second type culture was inoculated, cultured with shaking at 28°C for 1 day, and this was used as the third type. It was used as a culture solution. Four 50-volume jar fermenters containing 30 production media previously sterilized at 120°C for 30 minutes were inoculated with one third type culture solution each, and incubated at 28°C for 5 days with aeration (35/min) and stirring ( Initial 200rpm, 41
300 rpm). (2) Isolation After completion of the culture, diatomaceous earth was added as a filter aid and filtered, 50% acetone water 72 was added to the obtained bacterial cells to extract the active ingredient, and the bacterial cells were separated by filtration. Next, the bacterial cell extract was concentrated under reduced pressure to remove acetone, and ethyl acetate was added to the resulting concentrated solution (8.5%).
8.5 was added and shaken to extract the active ingredient.
The obtained ethyl acetate extract (8.0%) was concentrated under reduced pressure to obtain an oily substance. Add n-hexane to this oily substance, collect the resulting precipitate by filtration, and obtain the crude substance.
2.8g was obtained. Dissolve this crude material in toluene,
Silica gel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.) 300ml
column and fractionated with a toluene-ethyl acetate mixture. SF-2418 substance for active fraction dispensing was prepared using a thin layer of silica gel (Kieselgel 60F254,
571; Merck & Co.) chromatography was performed,
After immersing in phosphomolybdic acid and heating, it was detected as Rf0.47 (purple red). The above active fractions were combined and concentrated to dryness under reduced pressure to yield 220 mg.
A coarse powder was obtained. This crude powder was dissolved in benzene, and n-hexane was added to crystallize the SF-
190 mg of brown needle-like crystals of substance 2418 were obtained. Effects of the Invention The minimum inhibitory concentration of the SF-2148 substance against various microorganisms is shown in Table 1, and it is particularly effective against Mycoplasma and Treponema.

【table】

[Table] Micrococcus luteus * 〓0.025
(Micrococcus luteus)
Batius brevis* 0.05
(Bacillus brevis)

Claims (1)

[Claims] 1. SF-2418 substance having the following chemical structural formula. 2 Antibiotic SF- belonging to the genus Streptomyces
A method for producing a new antibiotic SF-2418 substance, which comprises culturing a 2418 substance-producing bacterium in a medium and isolating an SF-2418 substance having the chemical structural formula shown below from the culture.