JPH0374677B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic SF-2197B substance and a method for producing the same. More specifically, the present invention relates to a novel antibiotic SF-2197B substance and a method for producing the substance, which comprises culturing SF-2197B substance-producing bacteria belonging to the genus Microbispora and collecting the SF-2197B substance from the culture. . The present inventors searched for new and useful antibiotics that have antibacterial activity against various Gram-positive and -negative bacteria as well as anaerobic bacteria. As a result, the novel antibiotic SF-
The present invention was completed by discovering that substance 2197B was produced, isolating its effective substance pure from the culture, and determining its physicochemical and biological properties. An example of the SF-2197B substance-producing bacterium used in the present invention is the actinomycete SF-2197 strain isolated from Shiyang, 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
~0.6×0.6~1.2 μm. 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. Growth status on various media

[Table] Growth status of SF-2197 strain on various media:
As shown in this table. Culture was performed at 28°C, and observation was performed after 14 to 21 days of culture. Physiological properties (1) Growth temperature range: in yeast malt agar
It grows in a temperature range of 15-42°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 pigment: doubtful Utilization of carbon source (Pridham-Gottlieb agar medium) (1) Sugars used: D-glucose, D-fructose, D-xylose, glycerol, L
-arabinose, D. mannitol, raffinose, L-rhamnose, sucrose (2) Unutilized sugar: i-inositol Cell wall composition Method of Becker et al. [Appl.
Microbiol., 12 , 421 (1964)] and Lechevalier's method [J.Lab.Clin.Med., 71 ,
934 (1968)], the diaminopimelic acid in the whole cell hydrolyzate was of the DL type;
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). In addition, 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 seen in the case of other actinomycete strains, and can be mutated by artificial mutagenesis methods using, for example, ultraviolet rays, X-rays, radiation, chemicals, etc. Any mutant strain capable of producing the SF-2197B 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, edible oil, etc. can be used. Further, as the nitrogen source, soybean flour, wheat germ, cotton seed cake, tomato ketchup, cornstarch liquor, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, etc. can be used. Other calcium carbonate, sodium chloride, cobalt chloride, as needed.
In addition to adding inorganic salts such as phosphates and magnesium sulfate, organic and inorganic substances that aid the growth of bacteria and promote production of the SF-2197B substance can be added as appropriate. The most suitable culture method is a liquid culture method under aerobic conditions, especially a probe 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-2197B 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 for assaying SF-2197B substances. Gum agar medium (Nissui Pharmaceutical Co., Ltd.) is used as the assay medium. Bacteroides fragilis is used as the test bacterium. The SF-2197B substance exhibits a linear relationship between the logarithm of the concentration and the inhibition circle diameter at 0.25 μg/ml to 1 μg/ml, giving inhibition circle diameters of 16.4 mm to 23.3 mm, respectively (paper disk plate method). The SF-2197B 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), Cephadex LH-20, Toyopearl HW-
40 (manufactured by Toyo Soda Co., Ltd.), precipitation with hexane, solvent extraction with ethyl acetate, etc.
Column chromatography using silica gel is effective, but the following method is more efficient. That is, bacterial cells and other solid substances are filtered out from the culture solution using a filter aid such as diatomaceous earth, and then the active ingredients in the filtrate are adsorbed onto 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. The precipitate was chromatographed using an appropriate combination of Sephadex LH-20, column chromatography such as silica gel, high performance liquid chromatography, etc.
Pure SF-2197B substance can be obtained. The physical and chemical properties of SF-2197B substance are shown below. (1) Elemental analysis values: carbon 59.22%, hydrogen 9.31%, nitrogen 12.37%, oxygen 19.1% (difference) (2) Molecular weight: m/z 481 by mass spectrometry (SIMS)
Since a peak of (M ⁺ +1) is observed, the molecular weight is thought to be 480. (3) Melting point: 78℃ (4) Specific optical rotation: [α] ²⁵ _p = −20.2°
(Cl, methanol) (5) Ultraviolet absorption spectrum: Shows no characteristic absorption. (6) Infrared absorption spectrum: The spectrum measured in the potassium bromide tablet is as shown in Figure 1, and has the following absorption peaks. Absorption peak (νcm ^-1 ): 3300, 2950, 2920,
2850, 1720, 1660, 1620, 1540, 1440,
1420, 1360, 1250, 1160, 1060, 990, 920,
860 (7) Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. (8) Color reaction: Remieux, iodine reagent and ferric chloride reagent positive. Ninhydrin and sulfuric acid reagents negative. (9) Rf value of thin layer chromatography: Silica gel 60 F254 (manufactured by Merck & Co.) Chloroform-methanol (10:1) 0.51 Benzene-acetone (1:1) 0.42 (10) Distinction between neutral, acidic, and basic : Neutral (11) Color of substance: White (12) ¹ H-NMR spectrum: In deuterochloroform
Figure 2 shows the spectrum measured at 400MHz. (13) ¹³ C-NMR spectrum: in deuterated chloroform
The spectrum measured at 100MHz is shown in Figure 3, and has the following absorption peaks. Absorption peak (ppm): 11.65, 13.99, 16.10,
20.92, 22.43, 24.28, 26.20, 26.36, 28.53,
31.76, 32.71, 34.85, 36.69, 36.90, 47.36,
50.27, 63.05, 169.81, 172.20, 177.39,
208.13 Next, Table 1 shows the minimum inhibitory concentration (MIC) of SF-2197B substance against various bacteria, and Table 2 shows the minimum inhibitory concentration (MIC) against various anaerobic bacteria. As these tables show, SF−
Substance 2197B has antibacterial activity against various Gram-positive bacteria and anaerobic bacteria, and is used for the prevention and treatment of human and animal diseases caused by these bacteria and anaerobic bacteria.

【table】

[Table] Based on the properties described above, SF-2197A substance (patent application) is a known antibiotic similar to the compound of the present invention.
58-189591), but as it was clearly distinguished from the SF-2197A substance based on mass spectrometry values and NMR spectra, the SF-2197B substance 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 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 sp. SF-2197 strain (FERM
P-7213) was inoculated in 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 sterilized with this seed culture solution 30 (6.0% starch syrup, 0.3% soybean oil, 1.0% Sungrain (manufactured by Suntory), 1.5% cottonseed meal,
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 the culture solution was filtered using diatomaceous earth as an aid 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. Ethyl acetate (10%) was added to 10% of the concentrated solution to extract the effective substance. This extract was concentrated under reduced pressure to a volume of 100 ml, after which one portion of n-hexane was added and allowed to stand, causing the effective substance to precipitate. After removing the supernatant, the precipitate was dissolved in methanol and added to Cephadex LH-20.
(Manufactured by Pharmacia) When attached to a 700ml column and developed with methanol, fractions 24 to 36 were obtained in 20ml fractions.
The active substance was eluted. The active fractions were combined and concentrated to dryness under reduced pressure to form SF-2197B.
7.8 g of coarse powder of material was obtained. This crude powder was applied to a 250 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 to yield 1.9 g of a crude powder of SF-2197B material. When this 1.9 g was applied to a 500 ml column of Toyopearl HW-40 (manufactured by Toyo Soda Co., Ltd.) filled with methanol and developed with methanol, active fractions 24 to 30 were obtained in 20 ml fractions. The active fractions were combined and concentrated to dryness under reduced pressure to obtain 880 mg of a crude powder of SF-2197B substance. Example 3 Coarse powder of SF-2197B substance obtained in Example 2
Dissolve 300mg in 1ml of methanol and add YMC-
Using high performance liquid chromatography using a PACK S-343 (ODS) (manufactured by Yamamura Chemical Co., Ltd.) column (20φ x 250 mm), the active fraction was developed with 70% methanol at a flow rate of 4 ml/min, and the active fraction was concentrated to dryness. 80 mg of coarse powder was obtained. Furthermore, this crude powder was applied to an LH-20 200 ml column filled with acetonitrile, developed with acetonitrile, and the active fraction was concentrated to dryness under reduced pressure to obtain SF.
17 mg of white powder of -2197B substance was obtained. This substance has the above-mentioned physicochemical properties.

[Brief explanation of drawings]

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

Claims (1)

[Claims] 1. SF-2197B substance having the following physical and chemical properties. 1) Elemental analysis values: carbon 59.22%, hydrogen 9.31%,
12.37% nitrogen, 19.1% oxygen 2) Molecular weight: m/m by mass spectrometry (SIMS)
The molecular weight is thought to be 480 because a peak of (M ⁺ +1) is observed at z481. 3) Melting point: 78°C 4) Specific optical rotation: [α] ²⁵ _p = -20.2° 5) Ultraviolet absorption spectrum: No characteristic absorption is shown. 6) Infrared absorption spectrum: It has the following absorption peak (νcm ^-1 ). 3300, 2950, 2920, 2850, 1720, 1660,
1620, 1540, 1440, 1420, 1360, 1250,
1160, 1060, 990, 920, 860 7) Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. 8) Color reaction: Remieux, iodine reagent and ferric chloride reagent positive. 9) Rf value of thin layer chromatography: Silica gel 60F254 (manufactured by Merck) Chloroform-methanol (10:1) 0.51 Benzene-acetone (1:1) 0.42 10) Distinction between neutral, acidic, and basic: Neutral 11) Color of substance: White 12) ¹³ C-NMR spectrum: It has the following absorption peaks (ppm). 11.65, 13.99, 16.10, 20.92, 22.43, 24.28,
26.20, 26.36, 28.53, 31.76, 32.71, 34, 85,
36.69, 36.90, 47.36, 50.27, 63.05, 169.81,
172.20, 177.39, 208.13 2 SF-, an antibiotic belonging to the genus Microbispora
A method for producing a new antibiotic SF-2197B substance, which comprises culturing 2197B-producing bacteria in a medium and isolating an SF-2197B substance having the following physicochemical properties from the culture. 1) Elemental analysis values: carbon 59.22%, hydrogen 9.31%,
12.37% nitrogen, 19.1% oxygen 2) Molecular weight: m/m by mass spectrometry (SIMS)
The molecular weight is thought to be 480 because a peak of (M ⁺ +1) is observed at z481. 3) Melting point: 78°C 4) Specific optical rotation: [α] ²⁵ _p = -20.2° 5) Ultraviolet absorption spectrum: No characteristic absorption is shown. 6) Infrared absorption spectrum: It has the following absorption peak (νcm ^-1 ). 3300, 2950, 2920, 2850, 1720, 1660,
1620, 1540, 1440, 1420, 1360, 1250,
1160, 1060, 990, 920, 860 7) Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. 8) Color reaction: Remieux, iodine reagent and ferric chloride reagent positive. 9) Rf value of thin layer chromatography: Silica gel 60 F254 (manufactured by Merck) Chloroform-methanol (10:1) 0.51 Benzene-acetone (1:1) 0.42 10) Distinction between neutral, acidic, and basic: Medium 11) Color of substance: White 12) ¹³ C-NMR spectrum: It has the following absorption peaks (ppm). 11.65, 13.99, 16.10, 20.92, 22.43, 24.28,
26.20, 26.36, 28.53, 31.76, 32.71, 34.85,
36.69, 36.90, 47.36, 50.27, 63.05, 169.81,
172.20, 177.39, 208.13