JPS6246551B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new antibiotic and a method for producing the same, and more specifically, to a new antibiotic that is collected from the culture obtained by culturing SF-2080 substance-producing bacteria belonging to the genus Streptomyces in a medium.
This article relates to the SF-2080D material and its manufacturing method. Antibiotic SF- used in the method of the present invention
As the 2080D substance-producing bacteria, those having the ability to produce a sufficient amount of SF-2080D substance to be collected into the culture are used. An example of such a strain is Streptomyces sp. SF-2080, which was isolated by the present inventors from the soil at the bottom of the Chikuma River in Nagano City, Nagano Prefecture. The mycological properties of this strain are as follows. Morphological properties The basal hyphae are well branched and elongate in a wavy manner. The diameter of the hyphae is 0.5-0.6 microns. Subdivision of basal hyphae is usually not observed in either agar or liquid media. No aerial mycelium is formed on a conventional agar medium. Segmental spores, sporangia, flagellated spores, sclerotia, coremia, etc. have not been observed to date. Growth conditions on various media Mainly by Shirling, EB and Gottlied, D.
International Journal of Systematic
It was carried out according to the method described in Bacteriology 16 , 313-340 (1966). Observations were made after culturing at 28°C for 14 days. The growth conditions on various culture media are shown in the table below.

[Table] Physiological properties (1) Growth temperature range: 15% on yeast malt agar
It grows in a temperature range of ~45°C and grows well at 25-34°C. (2) Liquefaction of gelatin: Positive (20℃, 21 days of cultivation) (3) Hydrolysis of starch: Positive (weak, 28℃, 14 days)
(4) Effect on skim milk: No peptonization or coagulation occurs when cultured at 28℃ for 14 days. (5) Nitrate ring element: Positive (28℃, 14 days of cultivation) (6) Salt tolerance: Grows at 1.5% but not at 3.0%. (7) Production of melanin-like pigment: negative Availability of carbon source Since it does not grow on Pridham-Gottlieb agar medium, the basic composition consists of yeast extract (Difco) 0.5%, calcium carbonate 0.1%, and agar (Difco) 1.5%. The utilization of carbon sources was investigated using the culture medium. The results are as follows. Table 2 Carbon source growth D-glucose D-xylose D-fructose + L-arabinose + D-mannitol + i-inositol + L-rhamnose Sucrose + Flynose + None added + Cell wall composition Becker et al.'s method (Appl.
Microbiol. 13 , 236 (1965)) revealed that diaminopimelic acid in the cell wall composition was of the LL type. Based on the above properties, strain SF-2080 belongs to Actinomycetales (Order: Actinomycetales) and is a mesophilic bacterium.
No division of basal hyphae was observed, and this is an actinomycete that contains LL-type diaminopimelic acid in its cell wall. However, the genus of strain SF-2080 cannot be determined because the morphological characteristics such as segmental spores, flagellated spores, sporangia, sclerotia, and condensed hyphae necessary for determining the genus of actinobacteria are currently unclear. Can not. SF−
Considering that strain 2080 contains LL-type diaminopimelic acid in its cell wall, we tentatively believe that strain SF-2080 is an unidentified species of the genus Streptomyces until complete identification of the genus is possible. I decided to think about it,
Streptomyces sp. SF-2080
(Streptomyces sp. SF-2080). This strain is Streptomyces sp.
It has been deposited as SF-2080 at the Institute of Fine Technology, and its accession number is 5072. The SF-2080 strain is susceptible to changes in its properties, as seen in the case of other actinomycete strains, and can be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, radio frequencies, radiation, chemicals, etc. and
All mutant strains obtained in this way that have the ability to produce the antibiotic SF-2080D substance can be used in the method of the present invention. In the method of the present invention, the strain is cultured in a medium containing nutrients that can be utilized by common microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing actinomycetes can be used. For example, glucose, glucose, sucrose, starch, dextrin, starch syrup, molasses, soybean oil, etc. can be used as the carbon source. Further, as the nitrogen source, soybean flour, wheat germ, cottonseed meal, meat extract, peptone, yeast extract, dried yeast, cornstarch liquor, ammonium sulfate, sodium nitrate, etc. can be used. Calcium carbonate, sodium chloride, etc. as necessary.
Inorganic salts such as cobalt chloride and phosphates may be added, and organic and inorganic substances that aid the growth of bacteria and promote the production of the antibiotic SF-2080 substance may be appropriately added. The most suitable culture method is the deep culture method under aerobic conditions, similar to the method used for general antibiotic production. The appropriate temperature for culturing is 25-34°C, but in most cases it is cultured at 28-32°C. Antibiotic SF-2080
Production of the substance reaches its maximum accumulation in 2 to 7 days in both shaking culture and tank culture, and the antibiotic SF-2080 substance is accumulated in the culture solution and in the bacterial cell components (solid matter). Quantification of antibiotic SF-2080D substance is performed using Sarcina.
A combination of bioassay using Sarcina lutea and thin layer chromatography using silica gel can be carried out. Since the SF-2080D substance has the physical and chemical properties described below, it can be extracted and purified according to its properties, and the method shown below is efficient. 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, followed by stirring and extraction. The organic solvent layer obtained by removing the solid matter and the aqueous layer is concentrated under reduced pressure, a solvent such as n-hexane is added to the remaining oil to remove insoluble matter, and the solution is soaked in a suitable adsorbent such as alumina. It is adsorbed by passing it through an adsorption tower. Next, the adsorption tower is washed with a relatively highly polar organic solvent, such as ethyl acetate, methanol, or a mixture thereof, to obtain an eluted portion of the antibiotic SF-2080D substance. The residue obtained by concentrating the eluate is redissolved in an organic solvent, for example benzene, and further isolated by silica gel chromatography. The isolated SF-2080D material can be recrystallized from a suitable organic solvent, such as benzene, to obtain pure crystals. The SF-2080D material thus obtained gave a single spot in thin layer chromatography in various solvent systems, indicating its purity. The physical and chemical properties of the antibiotic SF-2080D substance obtained by the above method are as shown below. Table 3 Elemental analysis values: C: 36.41%, H: 1.10% N: 3.78%, Cl: 49.58% 2 Molecular weight: 357 (mass spectrum, Cl = 35) 3 Molecular formula: C ₁₁ H ₄ Cl ₅ NO ₂ 4 Melting point: 195-198°C 5 Specific optical rotation: [α] ²⁵ _D = 0 (C = 1.0, methanol) 6 Ultraviolet absorption spectrum: As shown in Figure 1 7 Infrared absorption spectrum: As shown in Figure 2 8 Coloration Reactions: Positive: Remieux reaction, ferric chloride reaction, Weilsch-Utaine reaction Negative: Ninhydrin reaction 9 Shape and color of substance: Yellow needle crystals 10 Distinction between neutral, acidic, and basic: Weakly acidic substances (can be mixed with aqueous sodium carbonate) 11 Rf in silica gel thin layer chromatography
Value (60FE.Merck ²⁵⁴ ): Chloroform 0.36 n-hexane/ethyl acetate/acetic acid (100/
20/1) 0.28 12 Solubility in solvents: Easily soluble in acetone and ethyl acetate, soluble in chloroform and methanol, slightly soluble in n-hexane and water Minimum resistance against various microorganisms measured by the agar dilution method of SF-2080D substance The growth-inhibiting concentrations are shown in Table 4, and in addition to showing a very strong growth-inhibiting effect on Gram-positive bacteria, it was also effective against Gram-negative bacteria and molds. Therefore, SF−
The 2080D material is useful as a pharmaceutical, veterinary drug, pesticide, disinfectant, or a material for conversion thereto.

【table】

[Table]
(Trichophyton interdigitale)
Alpergillus fumigatus 100
(Aspergillus fumigatus)
The physicochemical and biological properties of the SF-2080D substance described above were compared with those of known antibiotics, but no corresponding substances were found, and this substance is a new antibiotic. Examples of the method for producing the SF-2080D substance are shown below, but it goes without saying that many modifications and modification means not exemplified here can be used. Example 2% glucose, 0.5% peptone, 0.3 yeast extract
%, prepare a liquid medium with the composition of soybean flour 0.2%, meat extract 0.2% and ursium carbonate 0.1%,
Dispense 20ml each into a 100ml Erlenmeyer flask, sterilize it, and add Streptomyces sp.
Inoculated with −2080 strain (Feikoken Bacteria No. 5072) at 28°C.
A first seed culture solution was prepared by shaking culture for 5 days, and then the second and third seed culture solutions were prepared by sequentially increasing the volume using the same medium. Separately 2% starch syrup, 0.15% soybean oil, 1% soybean flour, 0.25% sungrain, firma media
0.5%, ferrous sulfate 0.0005%, nickel chloride
A medium 35 having a composition of 0.00005% cobalt chloride, 0.00005% cobalt chloride, and 0.1% calcium carbonate was placed in a 50-volume stainless steel fermenter, and after sterilization, 800 ml of the third type bacterial solution was inoculated and cultured with aeration at 28°C. Summer. Air flow rate is 35/min, rotation speed is 300 times/min.
It's a minute. After 120 hours, the culture was stopped and the culture solution was
Adjust the pH to 3.0 and add 36 ethyl acetate to 30
Stir for 1 minute and remove solids by filtration. The ethyl acetate layer was separated from the liquid and concentrated under reduced pressure. To the obtained dark oil was added 1.4 g of n-hexane, and insoluble matter was removed by decantation. The n-hexane supernatant is adsorbed through an alumina (Merck & Co., Art. 1097) adsorption tower filled with n-hexane in advance. When the adsorption tower was washed with n-hexane 1 and 2 and then eluted with ethyl acetate, SF-
2080D substance is eluted. The eluted fractions were collected and concentrated under reduced pressure, leaving 8.0 g of an oil. Add 60 ml of n-hexane to the residue and leave to remove the precipitate. Silica gel (C-200 manufactured by Wako Pure Chemical Industries, Ltd.) filled with n-hexane solution in advance
Adsorb onto a 400g column. After washing the silica gel column with n-hexane, elution is performed with benzene. The eluate is fractionated into 15-20 ml portions, and the components in each fraction are detected by thin layer chromatography (solvent system: n-hexane-ethyl acetate-acetic acid = 100:20:1). After the substance showing an Rf value of 0.62 is eluted, the Rf
SF-2080D substance showing a value of 0.36 was eluted. science fiction
Fractions containing -2080D material are collected and concentrated under reduced pressure. Recrystallization of the residual yellow solid from a small amount of benzene yielded pure crystals of SF-2080D material (yield 71.8 mg, mp 195-198°C).

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of SF-2080D substance.
This was measured after dissolving it at a concentration of 10 mcg/ml. Figure 2 is an infrared absorption spectrum of the SF-2080D substance, measured as a potassium bromide tablet.

Claims (1)

[Claims] 1. SF-2080D material having the following characteristics. Elemental analysis values: carbon 36.41%, hydrogen 1.10%, nitrogen
3.78%, chlorine 49.58% Molecular weight: 357 (mass spectrum, Cl = 35) Molecular formula: C ₁₁ H ₄ Cl ₅ NO ₂ Melting point: 195-198°C Specific optical rotation: [α] ²⁵ _D = 0 (C = 1.0, methanol ) Ultraviolet absorption spectrum: As shown in Figure 1 Infrared absorption spectrum: As shown in Figure 2 Color reaction: Remieux reaction, ferric chloride reaction, and Weilscheutain reaction are positive, ninhydrin reaction is negative Shape and color of substance: Yellow Needle-shaped crystals Distinction between neutral, acidic, and basic: Weakly acidic substance (soluble in aqueous sodium carbonate solution) Rf value in thin layer chromatography (silica gel): Chloroform 0.36 Hexane/ethyl acetate/acetic acid (100/20/
1) 0.28 Solubility in solvents: Easily soluble in acetone and ethyl acetate, soluble in methanol and chloroform,
Slightly soluble in water and n-hexane 2 Antibiotic SF belonging to the genus Streptomyces
- A method for producing an antibiotic SF-2080D substance, which comprises culturing a 2080D substance-producing bacterium in a medium and collecting the antibiotic SF-2080D substance from the culture. 3 Antibiotic SF belonging to the genus Streptomyces
-2080D substance producing bacterium is Streptomyces sp. SF-2080 (Feikoken Bibori No. 5072), the manufacturing method according to claim 2.