JPH0254074B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic SF-2197A and a method for producing the same. More specifically, it relates to a novel antibiotic SF-2197A substance and a method for producing the substance, which comprises culturing SF-2197A substance-producing bacteria belonging to the genus Microbispora and collecting the SF-2197A substance from the culture. . As a result of our search for new and useful antibiotics that have antibacterial activity against various Gram-positive and -negative bacteria, the present inventors discovered that a novel antibiotic substance SF-2197A was produced in a culture of a strain belonging to the genus Microbispora. The present invention was completed by discovering that the active substance was isolated from the culture, and determining its physicochemical and biological properties. That is, the present invention provides SF-2197A substance having the following physical and chemical properties: 1. Elemental analysis values: carbon 60.43%, hydrogen 8.64%, nitrogen 10.32%, oxygen 19.12% 2. Molecular weight: m/m by mass spectrometry (FDMS)
Since a peak of (M ⁺ +1) is observed at z438, the molecular weight is thought to be 437. 3 Melting point: Melts gradually from around 61℃, but does not show a clear melting point. 4 Specific rotation: [α] ²⁵ _D = -132° (C1, methanol) 5 Ultraviolet absorption spectrum: Does not show characteristic absorption. 6 Infrared absorption spectrum: As shown in FIG. 7 Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. 8 Color reaction: Remieux and iodine reagent positive. Ninhydrin and sulfuric acid reagents negative. 9 Rf value of thin layer chromatography: Silica gel 60F-254 (manufactured by Merck & Co., Ltd.) Chloroform-methanol (5:1) 0.65 Benzene-acetone (1:1) 0.63 10 Distinction between neutrality, acidity, and basicity: Neutral 11 Color of substance: White 12 ¹ H-NMR spectrum: As shown in Figure 2. 13 ¹³ C-NMR spectrum: As shown in Figure 3. Further, the present invention cultivates antibiotic SF-2197A-producing bacteria belonging to the genus Microbispora in a medium,
SF with the above physicochemical properties from culture
This is a method for producing the new antibiotic SF-2197A, which is characterized by isolating the 2197A substance. An example of the SF-2197A substance-producing bacterium used in the present invention is the actinomycete SF-2197 strain isolated from soil in Yaizu City, Shizuoka Prefecture. The mycological properties of SF-2197 strain are as follows. Morphological properties: It adheres well to simply branched aerial hyphae on glucose-asparagine agar, Bennetts agar, etc., and forms numerous short, alternating spore chains on the aerial hyphae. When observed using an electron microscope, there are often two chains of spores, but chains of three spores are also frequently seen, and there are cases of four. The surface of the spore is smooth, the shape of the spore is oval to oval, and the size is 0.4 to 0.6 x 0.6 to 1.2 μm.
It is. No motility observed. The basal hyphae are well elongated and branched, with no division observed. Other special structures such as sporangia, sclerotia, and binding threads were not observed.

[Table] The growth status of SF-2197 strain on various media is shown in this table. Culture at 28℃, observation at 14
This was done after ~21 days of culture. Physiological properties (1) Growth temperature range: 15% on yeast malt agar
Grows in a temperature range of ~42°C and grows well at 25-30°C. (2) Liquefaction of gelatin: Negative (3) Hydrolysis of starch: Negative (4) Peptonization of skim milk: Negative Coagulation of skim milk: Negative (5) Reduction of nitrate: Negative (6) Salt tolerance: 5% salt However, it will not grow at 7% or more. (7) Production of melanin-like pigments: doubtful Utilization of carbon source (Pridham-Gotzlieb agar medium) (1) Sugars used: D-glucose, D-fructose, D-xylose, glycerol, L-arabinose, D-mannitol , raffinose, L-rhamnose, sucrose (2) Unused sugar: i-inositol Cell wall composition Becker et al.'s method [Appl. Micro-
biol., 12 , 421 (1964) and the method of Lechevalier [J.Lab.Clin.Med., 71 ,
934 (1968)], the diaminopimelic acid in the whole cell hydrolyzate was of the DL type, and xylose and arabinose could not be detected. Based on the above mycological properties, strain SF-2197 is considered to be an intermediate strain between the genera Microbispora and Microtetraspora among actinomycetes, but it is mainly due to the fact that it has two spore chains. We decided to place it in the Microbispora genus. Therefore, the present inventors used the SF-2197 strain as Microbispora sp.
SF-2197). Furthermore, this strain has been deposited with the FIKEN under accession number 7213 (FERM P-7213). The SF-2197 strain is susceptible to changes in its properties, as is the case with other actinomycete strains, and can be mutated by artificial mutagenesis methods using, for example, ultraviolet rays, X-rays, radiation, and chemicals. Any mutant strain capable of producing the SF-2197A 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, any known nutrient source that has been conventionally used for culturing actinomycetes can be used. For example, glucose, glycerol, sucrose,
Starch, starch syrup, dextrin, molasses, oatmeal, cooking oil, etc. can be used. Nitrogen sources include soybean flour, wheat germ, cotton seed cake, tomato ketchup, cornstarch liquor, meat extract,
Peptone, yeast extract, ammonium sulfate, sodium nitrate, etc. can be used. In addition to adding other inorganic salts such as calcium carbonate, sodium chloride, cobalt chloride, phosphates, and magnesium sulfate as necessary, organic and inorganic substances that support the growth of bacteria and promote the production of SF-2197A substances are added. It can be added appropriately. The most suitable culture method is a liquid culture method under aerobic conditions, especially a deep culture method, similar to known methods for producing general antibiotics. The appropriate temperature for culturing is 20°C to 35°C, but in most cases it is preferable to culture at a temperature in the range of 25°C to 30°C. The production of SF-2197A substance varies depending on the medium and culture method used, but in the shaking culture method or the deep culture method using a culture tank, the accumulation usually reaches its maximum within 2 to 7 days. The following method is used to test SF-2197A substances. Gum agar medium (Nissui Pharmaceutical Co., Ltd.) is used as the assay medium. Bacteroides fragilis is used as the test bacterium. The SF-2197A substance shows a linear relationship between the logarithm of the concentration and the inhibition circle diameter at 10 μg/ml to 100 μg/ml, giving inhibition circle diameters of 13 mm to 27 mm, respectively (paper disk plate method). The SF-2197A substance obtained by the present invention is a fat-soluble, neutral substance. When collecting this from the culture, Amberlite is used for its extraction and purification.
Synthetic adsorbents such as XAD-2 (manufactured by Rohm and Haas), Diaion HP-20 (manufactured by Mitsubishi Kasei), gelling agents such as Cephadex LH-20, precipitation with hexane, solvents such as ethyl acetate Extraction methods, column chromatography using silica gel, etc. are effective, but the following method is more efficient. That is, bacterial cells and other solid substances are separated from the culture solution using a super-aiding agent such as diatomaceous earth, and then,
The active ingredients in the liquid are adsorbed by Diaion HP-20. After washing the resin part with water and pre-washing with 50% methanol water, the active ingredient is eluted with 80% acetone water. The active fraction is concentrated under reduced pressure, and the active ingredient is extracted with ethyl acetate from the concentrated solution after distilling off the acetone. After concentrating this extract under reduced pressure, n-hexane is added to precipitate the active ingredients. A pure substance of SF-2197A can be obtained by appropriately combining the precipitate with Sephadex LH-20, column chromatography such as silica gel, high performance liquid chromatography, etc. The physical and chemical properties of SF-2197A substance are shown below. (1) Elemental analysis values: carbon 60.43%, hydrogen 8.64%, nitrogen 10.32%, oxygen 19.12% (2) Molecular weight: m/m by mass spectrometry (FDMS)
Since a peak of z438 (M ⁺ +1) is observed, the molecular weight is thought to be 437. (3) Melting point: It melts gradually from around 61℃, but does not show a clear melting point. (4) Specific rotation: [α] ²⁵ _D = -132° (C1, methanol) (5) Ultraviolet absorption spectrum: Shows no characteristic absorption. (6) Infrared absorption spectrum: The spectrum measured in the potassium bromide tablet is shown in Figure 1. (7) Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. (8) Color reaction: Remieux and iodine reagent positive. Ninhydrin and sulfuric acid reagents negative. (9) Rf value for thin layer chromatography: Silica gel 60F-254 (manufactured by Merck) Chloroform-methanol (5:1) 0.65 Benzene-acetone (1:1) 0.63 (10) Neutral, acidic, basic Distinction: Neutral (11) Color of substance: White (12) ¹ H-NMR spectrum: in deuterated chloroform
Figure 2 shows the spectrum measured at 400MHz. (13) ¹³ C-NMR spectrum: in deuterated chloroform
Figure 3 shows the spectrum measured at 100MHz. Next, the minimum inhibitory concentration of the SF-2197A substance against various microorganisms is shown in the table below, and it is used as a medicine, agricultural or livestock drug, or an intermediate for conversion thereof.

【table】

[Table] As a result of testing the acute toxicity of this substance using mice, all mice survived after intraperitoneal administration of 100 mg/Kg. It has the above biological and physical and chemical properties.
Although there is no corresponding SF-2197A substance in the literature, Cerevioccidin (K. Anzai) is a product of actinomycetes with a molecular weight of 437.
et al., J. Antibiotics A 8 , 42 (1955)] and O-
611B substance [JP-A-55-83797] is known. However, since they all have different physical and chemical properties, SF-2197A was determined to be a new antibiotic. Examples of the present invention are shown below, but these are merely examples and do not limit the present invention. Of course, many variations or modifications not exemplified here may be used. Example 1 Inoculum culture medium (glucose 1.0%, starch 1.0%)
%, peptone 0.5%, yeast extract 0.3%, soy flour 0.2%, meat extract 0.2%, calcium carbonate 0.1
%) into a 100ml Erlenmeyer flask.After sterilization,
Microbispora SB SF-2197 strain (FERM
P-7213) was inoculated by 3 to 4 platinum loops from yeast starch agar slant culture and cultured at 28°C for 3 days. 50 ml of the obtained inoculum was inoculated into two sterilized 5-volume Erlenmeyer flasks containing the above medium 1, and incubated at 28°C.
It was cultured with shaking for 24 hours. Next, this inoculum 1 was sterilized by putting the above medium 35 into two 50-volume jar fermenters, and then inoculating it.
Cultured at 28°C for 24 hours. Production medium in which this seed culture solution 30 was sterilized (6.0% starch syrup, 0.3% soybean oil, 1.0% Sungrain (manufactured by Suntory), 1.5% cottonseed oil,
Gluten meal 1.2%, wheat germ 0.8%, calcium carbonate 0.2%) were inoculated into two 300 volume stainless steel tanks and cultured at 28℃ for 4 days with aeration and stirring (aeration rate 200/min, initial stirring 100 revolutions/min). ,2
150 revolutions/minute from day one). After the cultivation was completed, the pH of the culture solution was adjusted to 2.0, and diatomaceous earth was used as an auxiliary agent to filter the culture solution to obtain culture solution 280. Example 2 After adjusting the culture solution 280 obtained in Example 1 to pH 7.0, Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) 15
The active ingredients were adsorbed using the batch method. After collecting the resin, it was washed with 30 ml of water in a tower, and then pre-washed with 60 ml of 50% methanol water. Further, elution was performed with 80% acetone water. The active fraction 30 was collected and concentrated under reduced pressure to distill off acetone. 10 parts of ethyl acetate was added to 10 parts of the concentrated solution to extract the effective substance. This extract was concentrated under reduced pressure, and when the volume reached 100ml, 1 part of n-hexane was added and left to stand.
The active substance precipitated. After removing the supernatant, the precipitate was dissolved in methanol and added to Cephadex LH-
20 (manufactured by Pharmacia) and developed with methanol, the fraction 15 ~
The effective substance was eluted at 30%. The active fractions were combined and concentrated to dryness under reduced pressure, resulting in SF-
6.38 g of coarse powder of 2197A material was obtained. This crude powder was applied to a 200 ml column of Wako Gel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.) filled with chloroform and developed with a chloroform-methanol (50:1) mixture. It eluted. The active fractions were combined and concentrated to dryness, yielding 835 mg of a crude powder of SF-2197A substance. Of this, 830 mg was filled with 70% methanol.
When applied to a 500 ml LH-20 column and developed with 70% methanol, active fractions 20 to 25 were obtained in 20 ml fractions. The active fractions were combined and concentrated to dryness under reduced pressure to form SF-2197A.
347 mg of coarse powder of material was obtained. Example 3 Coarse powder of SF-2197A substance obtained in Example 2
Wakogel C-300 filled with 340mg benzene
Add to a 50ml column and add benzene-acetone (5:1)
The active substance was eluted. The active fraction was concentrated to dryness to obtain 30 mg of crude powder. Dissolve 30 mg of this crude powder in 1 ml of methanol, and use high performance liquid chromography using a column (10 φ x 250 mm) of Microbondapak C ₁₈ (manufactured by Waters Associates) at a flow rate of 2 ml/min to 70%
Developed with methanol and concentrated the active fraction to dryness.
10 mg of white powder of SF-2197A substance was obtained. This substance has the above-mentioned physical and chemical properties.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of SF-2197A substance in potassium bromide tablets. Figure 2 is SF
^-1H of substance 2197A in deuterated chloroform solution-
This is an NMR spectrum. Figure 3 is a ¹³ C-NMR spectrum of SF-2197A substance in deuterated chloroform solution.

Claims (1)

[Claims] 1. SF-2197A substance having the following physical and chemical properties. 1 Elemental analysis values: Carbon 60.43%, Hydrogen 8.64%, Nitrogen 10.32%, Oxygen 19.12% 2 Molecular weight: m/m by mass spectrometry (FDMS)
Since a peak of (M ⁺ +1) is observed at z438, the molecular weight is thought to be 437. 3 Melting point: Melts gradually from around 61℃, but does not show a clear melting point. 4 Specific rotation: [α] ²⁵ _D = -132° (C1, methanol) 5 Ultraviolet absorption spectrum: Does not show characteristic absorption. 6 Infrared absorption spectrum: As shown in FIG. 7 Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. 8 Color reaction: Remieux and iodine reagent positive. Ninhydrin and sulfuric acid reagents negative. 9 Rf value of thin layer chromatography: Silica gel 60F-254 (manufactured by Merck & Co., Ltd.) Chloroform-methanol (5:1) 0.65 Benzene-acetone (1:1) 0.63 10 Distinction between neutrality, acidity, and basicity: Neutral 11 Color of substance: White 12 ¹ H-NMR spectrum: As shown in Figure 2. 13 ¹³ C-NMR spectrum: As shown in Figure 3. 2 Antibiotic SF belonging to the genus Microbispora
A method for producing a new antibiotic SF-2197A, which comprises culturing 2197A-producing bacteria in a medium and isolating an SF-2197A substance having the following physicochemical properties from the culture. 1 Elemental analysis values: Carbon 60.43%, Hydrogen 8.64%, Nitrogen 10.32%, Oxygen 19.12% 2 Molecular weight: m/m by mass spectrometry (FDMS)
Since a peak of (M ⁺ +1) is observed at z438, the molecular weight is thought to be 437. 3 Melting point: Melts gradually from around 61℃, but does not show a clear melting point. 4 Specific rotation: [α] ²⁵ _D = -132° (C1, methanol) 5 Ultraviolet absorption spectrum: Does not show characteristic absorption. 6 Infrared absorption spectrum: As shown in FIG. 7 Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. 8 Color reaction: Remieux and iodine reagent positive. Ninhydrin and sulfuric acid reagents negative. 9 Rf value of thin layer chromatography: Silica gel 60F-254 (manufactured by Merck & Co., Ltd.) Chloroform-methanol (5:1) 0.65 Benzene-acetone (1:1) 0.63 10 Distinction between neutrality, acidity, and basicity: Neutral 11 Color of substance: White 12 ¹ H-NMR spectrum: As shown in Figure 2. 13 ¹³ C-NMR spectrum: As shown in Figure 3.