JPS6254431B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic and a method for producing the same, and more specifically to the antibiotic SF-
2107B substance or SF-2107C substance, and bacteria that produce SF-2107B substance or SF-2107C substance belonging to the genus Dactylosporangium are cultured in a medium, and the antibiotic SF-2107B substance is extracted from the obtained culture. Alternatively, the present invention relates to a method for producing a novel antibiotic SF-2107B substance or SF-2107C substance, which is characterized by collecting the SF-2107C substance. The present inventors have discovered that a substance exhibiting antibacterial activity against Gram-positive and Gram-negative bacteria is produced in the culture of certain bacterial strains, and the active substance has been isolated purely from the culture. As a result of investigating its properties, it was confirmed that it was a new antibiotic different from known substances, and the effective substances were named SF-2107B substance and SF-2107C substance. New antibiotic SF-2107B substance or SF-
As the producing bacteria of substance 2107C, in its culture,
Sufficient amount of SF-2107B substance or SF to collect
Any strain may be used as long as it has the ability to produce the -2107C substance, but as an example of such a strain, the present inventors have developed a strain from Jorinji Temple in Komaoka-cho, Tsurumi-ku, Yokohama City, Kanagawa Prefecture. There is SF-2107 strain newly isolated from soil. The mycological properties of this strain are as follows. Morphology The basal hyphae are well branched and elongate in a wavy manner, with a diameter of about 0.5-0.6 microns. Subdivision of basal hyphae is usually not observed in either agar or liquid media. Aerial hyphae are rarely seen and appear to be virtually unformed. The SF-2107 strain forms a single sporangium or a tuft-like sporangium on the surface of the agar medium. Many sporangia are observed on starch agar, glycerol-asparagine agar, etc. The sporangia are finger-shaped and approximately 0.8-1.1 x 2.5-4.0 microns in size. Each sporangium contains 3-4 spores in a row within it. Scrape the surface of the agar medium containing the sporangia, suspend it in sterile water, leave it to stand for at least 30 minutes, and then examine it under a microscope to find that the spores are actively migratory. When such spores are observed under an electron microscope, they are oval or short cylindrical with a smooth surface and several flagella can be seen at one end. Growth status on various media The growth status of SF-2107 strain on various media is as shown in the following table. Regarding the description of colors, the standards shown in [ ] are those of Container Corporation of
America (Container Corporation of
The one described in the "Color Harmony Manual" manufactured by America) was used. Observations were made after culturing for 14 to 21 days at 28°C.

[Table] Physiological properties (1) Growth temperature range: Grows in a temperature range of 20-42℃ on yeast malt agar medium, and grows well at 28-37℃. (2) Liquefaction of gelatin: Negative (20℃, 21 days culture) (3) Starch hydrolysis: Negative (28℃, 14 days culture) (4) Nitrate reduction: Positive (28℃, 14 days culture) ( 5) Peptonization of skim milk: Negative (28℃, 37℃, 14℃)
Coagulation of skim milk: Negative (28℃, 37℃, 14-day culture) (6) Salt tolerance: Grows at 1.5%, but not at 3.0% or higher. (7) Production of melanin-like pigments: How to use negative carbon sources

[Table] Basic medium used: (Yeast extract (manufactured by Difco): 1g Calcium carbonate: 0.2g Agar (manufactured by Difco): 15g Distilled water: 1000ml Cell wall composition Method of Becker et al. [Appl.
Microbiol, 13:236 (1965)] showed that diaminopimelic acid in the cell wall composition was mainly in the hydroxy type. Based on the above properties, the SF-2107 strain belongs to the genus Dactylosporangium among actinomycetes. The present inventors identified SF-2107 strain as Dactyrosporangium sp. SF-2107.
(Dactylosporangium sp. SF-2107). This strain has been deposited with the National Institute of Fine Technology, and its accession number is No. 5351. The SF-2107 strain is susceptible to changes in its properties, as is the case with many other strains of actinomycetes, and can be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, radiation, and chemicals. However, any strain of the genus Dactyrosporangium that has the ability to produce the SF-2107 substance, regardless of the mutant strain, 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 used by common microorganisms. As the nutrient source, any known nutrient source conventionally used for culturing actinomycetes can be used. For example, glucose, glycerol, sucrose, starch as a carbon source,
Dextrin, starch syrup, molasses, soybean oil, etc. can be used. Further, as the nitrogen source, soybean oil, wheat germ, meat extract, peptone, yeast extract, dried yeast, cornstap liquor, cottonseed meal, fish meal, ammonium sulfate, sodium nitrate, urea, etc. can be used. In addition to adding inorganic salts such as calcium carbonate, sodium chloride, cobalt chloride, and phosphates as necessary, organic and inorganic substances that can support the growth of bacteria and promote the production of SF-2107 substances are added as appropriate. Can be added. Any culture method may be used as long as it is carried out under aerobic conditions, similar to the method for general antibiotic production, but the deep culture method is particularly suitable. The appropriate temperature for culturing is 25 to 37°C, but in most cases it is preferable to culture at around 28 to 32°C. Production of SF-2107 substance reaches its maximum accumulation in 3 to 10 days in both shaking culture and tank culture. For quantitative determination of SF-2107B substance or SF-2107C substance, Vibrio percolans (Vibrio percolans)
percolans) ATCC 8461 and thin layer chromatography using silica gel. Since the SF-2107B substance and C substance have the physical and chemical properties described below, they can be extracted and purified according to their properties, and the method shown below is efficient. That is, water-containing acetone, water-containing methanol, etc. are added to the solid part after removing the liquid part from the culture containing the active ingredient, stirring and extracting the active ingredient, distilling off the organic solvent, and then extracting with a solvent such as ethyl acetate. If the liquid part of the culture also contains active ingredients, it is extracted with a solvent such as ethyl acetate. Next, the solvent layer containing the active ingredient, such as ethyl acetate, is concentrated and dried, and chromatography is performed using a carrier such as silica gel, alumina, Cephadex LH-20 (manufactured by Pharmacia), Florisil, or a countercurrent distribution operation. A pure SF-2107B substance or SF-2107C substance can be obtained by using an appropriate combination of these substances. The science fiction thus obtained
Thin layer chromatography of the -2107B or SF-2107C substances in various solvent systems gave a single spot, indicating that they were each pure product. The physical and chemical properties of the antibiotic SF-2107B substance and SF-2107C substance obtained by the above method are as shown in the following table.

【table】

[Table] The activity of SF-2107B substance and SF-2107C substance against various microorganisms was measured using the paper disk method (using a paper disk with a diameter of 8 mm). The results are shown in the following table. , effective against negative bacteria (numbers indicate inhibition circle diameter in mm). Therefore, SF−
Substances 2107B and C are useful as pharmaceuticals, veterinary drugs, disinfectants, or materials for conversion thereto.

【table】

〔Example〕

Dactyrosporangium sp. SF-2107 strain (Feikoken Microbiology Accession No. 5351) was used as a seed fungus.
2.0% soluble starch, 1.0% glucose, 0.6% wheat germ, 0.2% soybean flour, 0.5% polypeptone, 0.3% yeast extract, 0.2% meat extract as a seed medium.
%, calcium carbonate 0.1% (PH7.0 before sterilization) was used. Five platinum loops of inoculum cultured on yeast malt slant agar medium at 28°C for 14 days were placed in a 100 ml Erlenmeyer flask.
It was inoculated into 20 ml of the above seed medium and cultured with shaking at 32° C. for 96 hours, which was used as the first seed culture. Next, this seed culture solution was inoculated into 80 ml of seed medium in 10 tubes of 8 ml each in a 500 ml Erlenmeyer flask.
The culture was cultured with shaking at ℃ for 72 hours, and this was used as a second type culture. A jar fermenter with a capacity of 30 was filled with 20 production media, and 800 ml of the second type culture described above was inoculated thereto. As a production medium, glucose 1.7
%, sucrose 1.5%, wheat germ 2.0%, yeast extract 0.2%, gluten meal 0.3%, and sodium chloride 0.25% (PH 7.0 before sterilization). Culture was carried out at 28°C for 164 hours with aeration and agitation. After the cultivation was completed, the liquid was removed by filtration, and 12 80% acetone water was added to the solid content and stirred to extract the active ingredient. The acetone of the extract was distilled off under reduced pressure to obtain an aqueous solution of 2, and the pH was adjusted to 9 with ethyl acetate.
Extracted twice with 1.5 increments. The extracts were combined and concentrated to dryness under reduced pressure to obtain 800 mg of oil. Dissolve this in 5 ml of methanol and
The mixture was applied to a column packed with 500 ml of 20 (Pharmacia) and developed with methanol, and the active fraction was separated and concentrated under reduced pressure to dryness to obtain 280 mg of powder. This powder was applied to a column packed with 100 ml of Wako Gel C-200 (Wako Pure Chemical Industries, Ltd.), and a mixed solvent of chloroform and methanol (mixing ratio of 25:
(gradually changed from 1 to 15:1),
A fraction with activity was obtained. Each fraction obtained was mixed with chloroform-methanol (5:1) () and acetone-benzene (5:1).
1) Subjected to silica gel thin layer chromatography in the solvent system (), the fractions showing Rf 0.34 in the system () and Rf 0.51 in the system () were combined and concentrated to dryness under reduced pressure. 52 mg of SF-2107B substance was obtained. In addition, the fractions showing Rf0.27 in the system () and Rf0.07 in the system () were combined and concentrated to dryness under reduced pressure to obtain 93 mg of SF-2107C substance.
I got it. The SF-2107B and C substances obtained were dissolved in a small amount of methanol, and
-20 was applied separately to a column packed with 50 ml, developed with methanol, and the active fractions were subjected to thin layer chromatography using the two systems described above. Fractions giving a single spot were combined, and under reduced pressure Concentrate to dryness with
38 mg of pure SF-2107B substance and 76 mg of pure SF-2107C substance were obtained.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of SF-2107B substance, which was measured using a 25 mcg/ml methanol solution. Figure 2 shows an infrared absorption spectrum of SF-2107B substance, measured as a potassium bromide tablet. Figure 3 is SF-2107C
The ultraviolet absorption spectrum of the substance, 25mcg/ml
This was measured using a methanol solution of
FIG. 4 is an infrared absorption spectrum of the SF-2107C substance, measured as a potassium bromide tablet.

Claims (1)

[Scope of Claims] 1. Novel antibiotic SF-2107B substance or SF-2107C substance (new antibiotic SF-2107B substance and SF-2107C) characterized by having the following properties:
The substances are referred to below as B and C, respectively). (1) Elemental analysis values B: 53.66% by weight of carbon, 6.75% by weight of hydrogen (contains no nitrogen, sulfur, phosphorus, or halogen) C: 57.50% by weight of carbon, 7.04% by weight of hydrogen (contains no nitrogen, sulfur, phosphorus, or halogen) (2) Molecular weight B: 900 to 1100 (by gel filtration method) C: 900 to 1100 (by gel filtration method) (3) Melting point B: 170° to 175°C (gradually melting) C: 155° to 168°C (gradual melting) (4) Specific optical rotation B: [α] ²³ _D -5° (c1, methanol) C: [α] ²³ _D +29.8° (c1, methanol) (5) Ultraviolet absorption spectrum B: Shown in Figure 1 (in methanol) C: Shown in Figure 3 (in methanol) (6) Infrared absorption spectrum B: Shown in Figure 2 (potassium bromide tablet method) C: Figure 4 (Potassium bromide tablet method) (7) Color reaction B: Iodine reaction, Remieux reaction, positive Ninhydrin reaction, ferric chloride reaction Negative C: Iodine reaction, Remieux reaction, positive Ninhydrin reaction, ferric chloride reaction Negative (8) Appearance B: Slight yellow powder C: Slight yellow powder (9) Distinction between neutral, acidic, and basic B: Behaves as a neutral or weakly acidic substance (by electrophoresis) C: Neutral or weakly acidic substance (by electrophoresis) (10) Silica gel thin layer chromatography B: Rf = 0.34 (chloroform: methanol =
5:1) = 0.51 (acetone:benzene =
5:1) C:Rf=0.27 (chloroform:methanol=
5:1) = 0.07 (acetone:benzene =
5:1) (11) Solubility B: Soluble in methanol, acetone, benzene, chloroform, n-hexane,
Slightly soluble in water C: Soluble in methanol, acetone, benzene, chloroform, n-hexane,
SF-, an antibiotic belonging to the genus Dactylosporangium that is sparingly soluble in water2
A new antibiotic SF-2107B having the following characteristics, characterized by culturing a 2107B substance or SF-2107C substance producing bacterium and collecting the antibiotic SF-2107B substance or SF-2107C substance from the obtained culture.
substance or SF-2107C substance (new antibiotic SF-
2107B substance and SF-2107C substance (hereinafter referred to as B and C, respectively). (1) Elemental analysis values B: 53.66% by weight of carbon, 6.75% by weight of hydrogen (contains no nitrogen, sulfur, phosphorus, or halogen) C: 57.50% by weight of carbon, 7.04% by weight of hydrogen (contains no nitrogen, sulfur, phosphorus, or halogen) (2) Molecular weight B: 900 to 1100 (by gel filtration method) C: 900 to 1100 (by gel filtration method) (3) Melting point B: 170° to 175°C (gradually melting) C: 155° to 168°C (gradual melting) (4) Specific optical rotation B: [α] ²³ _D -5° (c1, methanol) C: [α] ²³ _D +29.8° (c1, methanol) (5) Ultraviolet absorption spectrum B: Shown in Figure 1 (in methanol) C: Shown in Figure 3 (in methanol) (6) Infrared absorption spectrum B: Shown in Figure 2 (potassium bromide tablet method) C: Figure 4 (Potassium bromide tablet method) (7) Color reaction B: Iodine reaction, Remieux reaction, positive Ninhydrin reaction, ferric chloride reaction, negative C: Iodine reaction, Remieux reaction, positive Ninhydrin reaction, ferric chloride reaction, negative ( 8) Appearance B: Slightly yellow powder C: Slightly yellow powder (9) Distinction between neutral, acidic, and basic B: Behavior as a neutral or weakly acidic substance (by electrophoresis) C: Behavior as a neutral or weakly acidic substance (by electrophoresis) (10) Silica gel thin layer chromatography B: Rf = 0.34 (chloroform: methanol =
5:1) = 0.51 (acetone:benzene =
5:1) C:Rf=0.27 (chloroform:methanol=
5:1) = 0.07 (acetone:benzene =
5:1) (11) Solubility B: Soluble in methanol, acetone, benzene, chloroform, n-hexane,
Slightly soluble in water C: Soluble in methanol, acetone, benzene, chloroform, n-hexane,
poorly soluble in water