JPH0468319B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel antibiotic SK-1071 and a method for producing the same. (Structure of the Invention) In the process of searching for antibiotics produced by actinomycetes, the present inventors discovered that actinomycetes SK-1071, which was newly isolated from soil, produced a new antibiotic effective against anaerobic bacteria. I discovered that. After identifying the production strain and isolating the substance, the present invention was completed by investigating its physicochemical and biological properties. The physical and chemical properties of the constituent material SK-1071 according to the present invention are as follows. (1) Elemental analysis: C60.50% H6.07% (2) Molecular weight: 630, molecular ion peak in high-resolution mass spectrum, m/z 630, 2315, elemental analysis and C-13 nuclear magnetic resonance spectrum (second Figure 4)
The molecular formula C ₃₂ H ₃₃ O ₁₃ can be found from (3) Melting point: 130-233℃ (4) Specific rotation: [α] ¹⁸ _D -0.1゜ (C=1, acetonitrile) (5) Ultraviolet absorption spectrum: Maximum absorption at 277nm and 350nm in 50% acetonitrile aqueous solution The molecular extinction coefficients are 6170 and 4660 (first
figure). (6) Infrared absorption spectrum: As shown in Figure 2. (KBr method) (7) Proton nuclear magnetic resonance spectrum: As shown in Figure 3. (8) C-13 nuclear magnetic resonance spectrum: As shown in Figure 4. (9) Solubility in solvents: soluble in acetonitrile, acetone, and ethyl acetate, and insoluble in water and n-hexane. (10) Color reaction is positive for potassium permanganate and H ₂ SO ₄ and negative for Dragendorff and ninhydrin. (11) It is an acidic substance. Since there are no known antibiotics that have the above-mentioned physicochemical properties and the biological activities described below,
The novel antibiotic according to the present invention was named SK-1071. The biological properties of this substance are as follows. (1) Antibacterial properties The minimum inhibitory concentration (MIC) determined by the agar dilution method is
As shown in Table 1.

[Table] (2) Toxicity When this antibiotic was administered intraperitoneally to mice,
LD ₅₉ is 100 mg/Kg or more. A practical example of the microorganism used to produce the antibiotic SK-1071 of the present invention is the actinomycete strain SK-1071, which was isolated from soil by the present inventor. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the accession number ``Feikoken Bacteria No. 8107''. Its mycological properties are as follows. () Morphological characteristics The vegetative hyphae of strain SK-1071 develop well on various agar media and usually do not have septa. Aerial mycelium is abundant on various agar media and appears velvety or powdery. When observed under a microscope, aerial hyphae appear spiral-shaped and form long chains of 20 or more spores. The spores are cylindrical and have a hair-like surface. The size of the spores is 1.2 x 0.7 μm. No sclerotia, sporangia and zoospores are observed. () Culture properties on various media Methods of EBShirling and D. Gottlieb [International Journal of Systematic Bacteriology (Int.J.Syst,
Bacteriol.) 16 , 313 (1966)], the culture properties of this producing bacterium are shown in the following table. The color tone is the standard color, and the Color Harmony Manual (Color
Harmony Manual) 4th Edition [Container Corporation of America
Corporation of America) in 1958], and the code is written in parentheses along with the color chart name. The following are the results of observations on each culture medium at 27°C for 2 weeks unless otherwise specified.

【table】

【table】

[Table] Selected culture medium
() Physiological properties (1) Production of melanin pigment (a) Tyrosine agar negative (b) Peptone/yeast iron agar negative (c) Glucose/beptone/gelatin medium (puncture (21-23℃) negative (d) Tryptone・Yeast liquid negative (2) Tyrosinase reaction negative (3) Hydrogen sulfide production negative (4) Nitrate reduction positive (5) Liquefaction of gelatin (21-23℃) (Glucose/peptone/gelatin medium) Negative (6) Starch Hydrolysis of skim milk Positive (7) Coagulation of skim milk (37℃) Negative (8) Peptonization of skim milk (37℃) Positive (9) Growth temperature range 15-45℃ (10) Availability of carbon source (Pleadam Codolive agar medium) Used: D-glycose, L-arabinose, D-xylose, raffinose, melibiose, D-mannitol, D-fructose, L-rhamnose, i-inositol, sucrose (11) Degradation of cellulose Negative () cells The chemical composition of diaminopimelic acid is the LL type, and arabinose and galactose were found in the sugar analysis of the whole bacterial cell.The mycological properties of this bacterium can be summarized as follows.The cell wall composition is LL. - Contains diaminopimelic acid. Morphologically, it forms a spiral spore chain, and the surface of the spore is hair-like. As for its cultural properties, the vegetative hyphae exhibit a bumpy or light ivory color. , the aerial mycelium exhibits a brownish gray color. It does not produce soluble pigments. Based on these results, this fungus belongs to the genus Streptomyces, and T.G. Pridham (TG
Pridham) and H.D. Tresner (HD
Tresner) classification [Bergeys Manual
Bergey's Manual of Determinative Bacteriology
Bacteriology) 8th edition, pages 748-829, 1974]
It is considered to be a bacterial species belonging to the Gray series. Mutant strains of the above-mentioned strains can also be used in the method of the invention. As a medium, a carbon source suitable for culturing actinomycetes,
Synthetic or natural media containing moderate amounts of nitrogen sources, minerals, and optionally other nutrients can be used. The carbon source and nitrogen source used in the culture medium may be any type as long as they are available to the strain used.
That is, carbon sources include, for example, glucose, glycerol, fructose, maltose,
Various carbohydrates can be used, such as mannite, xylose, galactose, ribose, starch or its hydrolysates. Its concentration is usually
0.1 to 5% (calculated as glucose) is preferable. Also,
Various organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, various amino acids such as glycine, glutamic acid, and alanine, alcohols such as methanol and ethanol, and various non-aromatic hydrocarbons such as normal paraffin, Various vegetable or animal fats and oils can also be used. Nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, urea, peptone, NZ-amine, meat extract, yeast extract, dried yeast, corn steep liquor, Nitrogen-containing organic substances such as casein hydrolyzate, fat meal or its digested product, soybean flour or its digested product, defatted soybean or its digested product, pupa hydrolysate, and various amino acids such as glycine, glutamic acid, and alanine can be used. It is. As inorganic substances, various phosphates, magnesium sulfate, common salt, etc., and trace amounts of heavy metal salts are used. Furthermore, when using a mutant strain that exhibits auxotrophy, it is of course necessary to add a substance to the medium that satisfies its nutritional requirements; however, when using a medium containing natural substances, There are cases where no particular addition is required. Fermentation is carried out under aerobic conditions such as shaking culture or submerged culture with aeration and stirring. The culture temperature is usually 20-40°C. The culture period is usually 1 to 8 days, and the antibiotic SK-1071 is produced and accumulated inside and outside the bacterial cells. After culturing, antibiotic SK-1071 was added to the culture.
For example, collect it using the following method. The culture is separated into a liquid and a precipitate by centrifugation. It is extracted from the liquid by adsorbing it onto activated carbon, porous synthetic polymer resin, ion exchange resin, etc. and eluting it. The precipitate is extracted with an organic solvent such as ethyl acetate, n-butanol, or aqueous acetone.
By appropriately concentrating the extract to dryness, SK-
1071 crude material is obtained. The crude substance is further purified by appropriately combining known methods commonly used in the purification of fat-soluble substances, such as concentration methods such as silica gel column chromatography. By concentrating and drying the active fraction obtained by these purification methods, powder of antibiotic SK-1071 can be obtained. In the present invention, the antibiotic SK-1071 was detected and quantified using silica gel thin layer chromatography (Silica gel 60F ₂₅₄ manufactured by Merck & Co., Ltd., thickness 0.2 nm, developing solvent benzene/acetone = 3:1, antibiotic
SK-1071 Rf value 0.40) and Clostridium perfringens (Clostridium perfringens)
A biological assay using ATCC 3624 was used. (Effect of the invention) As mentioned above, antibiotic SK-1071 has low toxicity;
Mainly active against anaerobic bacteria. Therefore,
This substance is expected to be used as a therapeutic or preventive agent for microbial infections in humans and animals. (Example) Next, an example of the antibiotic SK-1071 of the present invention will be shown, but this example is merely an example and does not limit the present invention. A platinum loop from a slant culture of Actinomycetes strain SK-1071 (Feikoken No. 8107) was added to 100 ml of seed medium (glucose 0.1%, starch 2.4%, peptone 0.3%, meat extract 0.3%, yeast extract 0.5%, Calcium carbonate 0.4
%) into a 500 ml Sakaguchi flask,
A seed culture was obtained by culturing with shaking at 27°C for 2 days. Add 400ml of this seed culture to 40ml of production medium (glucose 0.1%,
Starch 2.4%, peptone 0.3%, meat extract 0.3%,
Yeast extract 0.5%, calcium carbonate 0.4%, ferrous sulfate heptahydrate 1mg/, manganese chloride tetrahydrate 1mg/
, zinc sulfate heptahydrate 1 mg/, copper sulfate pentahydrate 1
mg/, cobalt chloride dihydrate 1 mg/30
The vaccine was divided into two separate units and 27
Culture with aeration at ℃ for 3 days (aeration rate 10/mm, stirring
250rpm). The culture solution 40 is centrifuged using a shear press type centrifuge to separate the bacterial cells and the culture supernatant. 2.5 g of 90% acetone solution was added to the bacterial cells and stirred to perform re-extraction. After concentrating this acetone solution under reduced pressure to remove acetone, it was combined with culture supernatant 18, and the pH was adjusted with hydrochloric acid.
Adjusted to 4. Add this to 650ml of Daikaiion Hp−
After passing through a column packed with 20% acetone, it was eluted with 70% acetone water and fractionated into individual fractions. Active fraction (No. 1~
3) was collected and concentrated under reduced pressure to remove acetone, the pH was adjusted to 3 with hydrochloric acid, and the mixture was extracted twice with ethyl acetate. The extract was concentrated to dryness under reduced pressure to obtain 840 mg of a tar-like substance. 840 mg of tar-like substance was dissolved in methanol, mixed with Celite, and then concentrated to dryness. This powder is 3
Silica gel (40 g, manufactured by Merck, pre-suspended in benzene)
Art.9385) was added to the top of the column packed. The column was eluted with benzene/acetone (4:1);
It was fractionated into 10 ml portions. By collecting the active fractions (No. 23-39) and concentrating under reduced pressure, 150 mg of oily substance was obtained.
I got it. 150 mg of the oily substance was dissolved in 1.5 ml acetonitrile, and divided into 15 times, high performance liquid chromatography (column, YMC A-324 manufactured by Yamamura Kagaku Kenkyusho; developing solvent 65% acetonitrile water, flow rate 3 ml/mi, detection UV 210 nm; retention time 9.8 minutes). By concentrating this active fraction to dryness under reduced pressure,
39 mg of white powder was isolated.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of antibiotic SK-1071 (measured in 50% acetonitrile aqueous solution).
Figure 2 is an infrared absorption spectrum (KBr method), Figure 3 is
The figure shows the proton nuclear magnetic resonance spectrum (measured in deuterated chloroform), and FIG. 4 shows the C-13 nuclear magnetic resonance spectrum (measured in deuterated acetone).

Claims (1)

[Claims] 1. Having the following physical and chemical properties and having a molecular formula of C ₃₂ H ₃₈ O ₁₃
Antibiotic SK-1071 represented by. Specific optical rotation: [α] ¹⁸ _D −0.1° (C=1, acetonitrile) Ultraviolet absorption spectrum: λ ^CH ₃ ^CN _nax nm (ε) 277
It has absorption bands at (6170) and 350 (4660). Infrared absorption spectrum: 3400, 1760, 1740,
It has absorption bands at 1710, 1640, and 1600 cm ^-1 . C-13 nuclear magnetic resonance spectrum: chemical shift in heavy acetone ppm 172.3 (s), 157.5
(d), 144.0 (s), 139.5 (s), 135.3 (s),
131.6(d), 125.7(d), 123.4(d), 98.4(d)
，
88.9(d), 79.5(d), 76.8(s), 74.3(d), 73
.6
(t), 69.7(d), 67.4(d), 66.3(d), 63.3
(t), 61.0 (q), 59.6 (q), 46.5 (d), 39.6
(d), 38.3 (t), 38.0 (d), 34.5 (t), 33.5
(t), 30.3 (d), 19.8 (t), 15.9 (q) s: singlet, d: doublet, t: triplet, q: qultet 2 Has the following physical and chemical properties and has a molecular formula
A microorganism belonging to the genus Streptomyces that has the ability to produce the antibiotic SK-1071 represented by C ₃₂ H ₃₈ O ₁₃ is cultured aerobically in a medium, and the antibiotic SK-1071 is grown in a medium.
A method for producing antibiotic SK-1071, which comprises producing and accumulating 1071 and collecting it. Specific optical rotation: [α] ¹⁸ _D −0.1° (C=1, acetonitrile) Ultraviolet absorption spectrum: λ ^CH ₃ ^CN _nax nm (ε) 277
It has absorption bands at (6170) and 350 (4660). Infrared absorption spectrum: 3400, 1760, 1740,
It has absorption bands at 1710, 1640, and 1600 cm ^-1 . C-13 nuclear magnetic resonance spectrum: chemical shift in heavy acetone ppm 172.3 (s), 157.5
(d), 144.0 (s), 139.5 (s), 135.3 (s),
131.6(d), 125.7(d), 123.4(d), 98.4(d)
，
88.9(d), 79.5(d), 76.8(s), 74.3(d), 73
.6
(t), 69.7(d), 67.4(d), 66.3(d), 65.3
(t), 61.0 (q), 59.6 (q), 46.5 (d), 39.6
(d), 38.3 (t), 38.0 (d), 34.5 (t), 33.5
(t), 30.3 (d), 19.8 (t), 15.9 (q) s: singlet, d: doublet, t: triplet, e: qultet