JPS5918991B2 - Antibiotic AM-2947 and its manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The object of the present invention is to provide a novel antibiotic AM-2947 and a method for producing the same.

The present inventor researched antibiotics produced by actinomycetes, discovered that a certain type of actinomycetes produces new antibiotics, investigated the mycological properties of this strain, and isolated this antibiotic. The present invention was completed by investigating its physicochemical and biological properties. The isolated and purified antibiotic provided by the present invention is an orange powder and has the following physical and chemical properties. 1. Elemental analysis C69.88%; H5.09%: No%) 2. Molecular weight 580 (M+1)0=581 was observed in the FD mass spectrum.

Elemental analysis values, proton nuclear magnetic resonance spectrum (Figure 3)
The molecular formula C34H2809 was determined from the C-13 nuclear magnetic resonance spectrum (Figure 4). 3. Melting point 1
76-178℃ 4, Specific rotation [α]24=+5020 (C=1, chloroform) 5, Ultraviolet absorption spectrum '5 228, 268 and 422 nm in methanol
The absorption maximum is shown in , and the molecular extinction coefficient is 49400, respectively.
:42500 and 13600 (Figure 1)6. The infrared absorption spectrum is shown in Figure 2 (KBr method) 7. Solubility in solvents: Soluble in lower alcohols such as methanol and ethanol, and organic solvents such as acetone, ethyl acetate, chloroform, and benzene, but not in n-hexane, petroleum ether, and water.

8. It is positive for the color reaction ferric chloride reaction, and negative for the ninhydrin reaction, Lydon-Smith reaction, and Ehrlich reaction.

9. It is a slightly acidic substance.

10. The proton nuclear magnetic resonance spectrum and the Cl3 nuclear magnetic resonance spectrum are shown in FIGS. 3 and 4, respectively.

Based on the above physicochemical properties, this antibiotic is considered to be an anthracycline type antibiotic. However, since there is no known antibiotic with the above physicochemical properties, the new antibiotic according to the present invention is called AM-2947. I named it.

Furthermore, as a result of structural analysis of AM-2947, it was found that it has the following structure. Antibiotic AM-2947
Table 1 shows the results of determining the minimum inhibitory concentration on a nutrient agar medium (PH7nθ) using the conventional agar dilution method.

As can be seen from the above table, this antibiotic has relatively strong antibacterial activity against Gram-positive bacteria, and also has activity against penicillin-resistant bacteria and erythromycin/tetracycline-resistant bacteria. Therefore, this antibiotic is used as an antibacterial agent, medicine,
It is expected that it can be used for purposes such as veterinary medicine. LD investigated by intraperitoneally administering this antibiotic to mice
, O were 200-400η/Kg.

According to the method of the present invention, antibiotic AM-2947 can be obtained by aerobically cultivating a microorganism that belongs to the genus Streptomyces and has the ability to produce antibiotic AM-2947 in a medium, and then injecting the present antibiotic into the medium or bacterial cells. A practical example of a possible microorganism is Stresses sp. AM-2947, which the present inventor isolated and screened from the soil of Shodoshima. The mycological properties of the strain Atsuhi are as follows.

? Physical properties? The nutrient mycelia are well developed on synthetic agar and natural agar5, branching irregularly, but without transverse septa.

Aerial mycelium grows on starch, inorganic K medium, yeast/malt agar medium, etc., and is velvety or cotton-like. /coase nitrate agar medium, peptone agar medium, iron agar medium, nutrient agar medium, glucose 7゜ton agar medium, etc., which do not grow aerial mycelia or even if they do, there are few. Its color is brownish-gray to brownish-white like a medium. When observed under a microscope, the main axes of aerial hyphae are irregularly branched.

The sporophyte is linear and usually contains chains of spores.

The spore n has no wrinkles and is almost smooth. r is almost elliptical, its size is 0.4-0.8 x 0.8-
1.3μ is normal. No sclerotia, sporangia I, or zoospores were observed. }Properties in seed medium (Table 2) I. B. Shearing method [Int. J. 5t.
BacterlOl. , VOl. l6, 3131966
)], and a commonly used culture medium and testing method were also used.

As a standard color table, Gala Harmony Manual, No. 4
The color tones were determined using a printing plate (Conte Corporation of America, Chicago, 1958) and are shown in the diagram. The results shown are observations made on each medium after two weeks of culture at 27° C. unless otherwise specified.

．． Physiological properties (1) Formation of melanin pigment 0) Tyrosine agar positive (to) Peptone/yeast iron agar positive ←→ Glucose/peptone/gelatin medium, puncture (2
1-23℃) Positive test Triftone yeast solution Positive (2) Tyrosinase reaction Positive (3) Nitrate reduction Negative (4) Gelatin liquefaction (glucose-peptone-gelatin medium) Negative (5) Starch hydrolysis Positive (6) ) Coagulation of skim milk (37℃) Negative (7) Peptonization of skim milk (37℃) Positive (8) Decomposition of cellulose Negative (9) Growth temperature range 20-38℃ QO) Utilization of carbon sources (Bridham Gotrib agar medium) D-glucose, L-arabinose, sucrose, D-flagdose, D-xylose, L-rhamnose, i-inositol, raffinose, and D-mannite are often used.

．． It contains diaminopimelic acid and glycine of cell wall composition LL type, but arabinose and galactose are not observed.

The mycological properties of this strain are summarized as follows.

The cell wall composition contains LL-diaminopimelic acid and glycine.

In addition, morphologically, it forms a linear sporophyte, and the surface of the spore is smooth. As for various cultural properties, the vegetative hyphae are pale yellow to light yellowish brown and do not change with changes in pH, and the aerial hyphae are brownish-gray to brownish-white in color. Production of melanin pigment is positive, and other soluble pigments produce yellowish-brown-white pigments. These results indicate that this bacterial strain belongs to the genus Streptomyces, and is classified according to Pridham and Tresna's classification (Burgess Manual of Determinative Bacteriology, 8th edition, 1974).
Streptomyces pseudovenezuelae (StreptOmyc
It was determined that it belongs to the genus espseud Ovenezuelae).

This strain is Streptomyces sp. AM2947 (
StreptOmycessp. AM-2947) has been deposited with the National Institute of Industrial Science and Technology (National Institute of Industrial Science and Technology).
No. 360). This strain has also been deposited in the United States as NRRLll269. In addition to the above-mentioned Streptomyces sp. AM-2947 and its mutant strains, any microorganism that belongs to Streptomyces and has the ability to produce the present antibiotic can be used in the method of the present invention. In the method of the present invention, the medium is
Any synthetic medium or medium using natural products containing carbon sources, nitrogen sources, inorganic substances, and other nutrients as necessary that can be used for the cultivation of microorganisms belonging to the genus Streptomyces can be used. can.

Various medium compositions and culture methods of this type are known per se. The concentration of carbon source is 0.1-5.07/
dl (glucose equivalent) is preferred. Fermentation is carried out under known aerobic conditions. The culture temperature is also known and is usually 2.
The temperature should be in the range of 0-38°C. The culture period is usually 2-8 days, which results in a significant amount of antibiotic A in the culture medium and bacterial cells.
M-2947 is generated and stored. After completion of the culture, the antibiotic is collected from the culture solution, the bacterial cells, or a mixture thereof.

Various methods are known for collecting lipid-soluble substances such as the present antibiotic from cultures, and an appropriate method can be selected and applied. For example, after separating the culture into bacterial cells and sap, the bacterial cells are treated with an organic solvent such as acetone or ethyl acetate, and the sap is treated with an organic solvent such as ethyl acetate, butyl acetate, or benzene. The antibiotic can then be recovered by various techniques commonly used for the purification of fat-soluble substances. For example, after concentrating the extract, the antibiotic can be isolated by silica gel column chromatography or the like. Next, examples of the present invention will be shown, but the present invention is not limited thereto.

Example 1 One platinum loop was taken from a slant culture of Streptomyces sp. 0t/Dll soybean flour 2.07/d
1 and sodium chloride 0.3f7/dl, PH, 7. O
The cells were inoculated into sterilized and sterilized cells, and cultured for 2 days at 27°C.

Fermentation medium 201 (prepared in the same manner as the seed medium) in which this culture was placed in Jafermenta 1 (301 volumes)
The cells were inoculated at 1.0% (V/V) and cultured with aeration and agitation in the same manner as the seed culture. Add 10 parts of ethyl acetate to 20 parts of the culture solution.
f! After stirring well, the ethyl acetate layer was separated by centrifugation, concentrated under reduced pressure to approximately 20 ml, and then filled with silica gel (Silica Gel G, manufactured by Talc) 4007 pretreated with 2% sulfuric acid. The column was eluted by chromatography using a chloroform-methanol (200:1) solvent system. Eluted fraction (
Bacillus subtilis PC has antibacterial activity of approximately 15 m0 each.
The inhibition circle was determined by measuring the circle of inhibition by the paper disc method using an agar plate containing I2l9. The active fractions (No. 54 to No. 118) were combined (950 ml) and concentrated to dryness under reduced pressure to obtain a yellow-orange powder of 2.37 (purity 96%,
melting point 174-177°C). 200 watts of this powder was further purified using thin layer chromatography. That is, after developing using a silica gel plate manufactured by Talc (2 mm thick) and a mixed solvent of benzene monoethyl acetate (1:3), the active fraction was scraped off, extracted with ethyl acetate, and the extract was concentrated under reduced pressure. The mixture was dried to obtain an orange powder 113η (purity of 99% or more). The physical and chemical properties of this product were as follows. F581 was observed.

Elemental analysis values, proton nuclear magnetic resonance spectrum (Figure 3)
The molecular formula determined from the C-13 nuclear magnetic resonance spectrum (Figure 4) was C34H28O9. Example
2 Culture solution obtained in the same manner as in Example 1 (19.5f!)
Culture supernatant after removing bacterial cells by centrifugation (1
6f! ) was added with ethyl acetate (101), stirred thoroughly, and extracted.

The extract was concentrated to dryness under reduced pressure and treated in the same manner as in Example 1 to obtain a yellow-orange powder of 2.97% (purity 97%, melting point 175-177°C). This powder was further purified in the same manner as in Example 1 to obtain an orange powder (yield: 1.8V), and the physical and chemical properties were similar to those in Example 1. Example
3 Culture solution obtained in the same manner as in Example 1 (19.5f!
) to obtain 1.31<g (wet weight) of bacterial cells by centrifugation, and then the bacterial cells were suspended in 51 ethyl acetate, stirred well, and extracted.

The extract was concentrated to dryness and treated in the same manner as in Example 1 to obtain a yellow-orange powder 2.2r (purity 95%, melting point 17
4-176°C). This powder was further purified in the same manner as in Example 1 to obtain an orange powder (yield: 1.4 tons), which had the same physical and chemical properties as those in Example 1.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum (methanol) of the antibiotic AM-2947, and Figure 2 shows the infrared absorption spectrum (methanol).
3 shows the proton nuclear magnetic resonance spectrum (deuterochloroform), and FIG. 4 shows the C-13 nuclear magnetic resonance spectrum (deuterochloroform).

Claims (1)

[Claims] 1. Antibiotic having the following physical and chemical properties 1. Elemental analysis: C 69.88%; H 5.09%; N0% 2. Molecular weight 580 3. Melting point 176-178°C 4. Specific rotation [α] ]^2^4_D=+502°(C=1
, chloroform)5, ultraviolet absorption spectrum, as shown in Figure 16, infrared absorption spectrum, as shown in Figure 27,
Solubility in solvents Soluble in lower alcohols such as methanol and ethanol, organic solvents such as acetone, ethyl acetate, chloroform, and benzene, but not soluble in n-hexane, petroleum ether, and water 8. Color reaction Ferric chloride reaction positive, ninhydrin reaction, lydon
Smith and Ehrlich reactions are negative. 9. Slightly acidic. 10.The color is orange. 2 Antibiotic AM-2947 belonging to the genus Streptomyces
An antibiotic AM-2947 is produced by aerobically cultivating a microorganism capable of producing AM-2947 in a medium, accumulating the antibiotic AM-2947 in the medium or in the bacterial cells, and collecting the antibiotic AM-2947.
2947 manufacturing method. 3 The microorganism is Streptomyces sp. AM-29
47. The manufacturing method according to claim 2, which is No. 47.