JPH043195B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention is directed to the antibiotic 5-0-mycaminosylnarbonolide (5-0-
Mycaminosylnarbonolide). BACKGROUND OF THE INVENTION 5-0-Mycaminosylnarbonolide is a known 14-membered ring macrolide antibiotic with antibacterial activity against Gram-positive bacteria. Streptomyces platensis subsp. malvinus, a strain that has traditionally produced the 16-membered ring macrolide antibiotic platenomycin
Using MCRL-0388, add the aglycone Narbonolide during the culture, or use Streptomyces
Platensis Subs My Venus MCRL−
Streptomyces platensis subs. It has been reported that 5-0-mycaminosylnarbonolide can be obtained [J. Antibiot. 29 1203-1208 (1976)]. Problems to be Solved by the Invention The conventional method for producing the antibiotic 5-0-mycaminosylnarbonolide is based on salpage synthesis by adding nalbonolide as an aglycone. It was not known that microorganisms directly produce lunarbonolide. Means for Solving the Problems The present inventors have discovered that the actinomycete L18-110 strain isolated from field soil in Kotohira-cho, Nakatado-gun, Kagawa Prefecture is an antibiotic.
-0-Mycaminosylnarbonolide was found to be produced. The mycological properties of the above Streptomyces L18-110 strain are as follows:
It is as follows. [] Morphological characteristics Tyrosine agar medium (ISP medium 7) [InterJ.
System.Bacteriol. 16 313-340 (1966)], 30
The following findings were observed after culturing at 10°C for 10 to 20 days. The basal hyphae have a diameter of 0.3 to 0.5μ, elongate in a curved manner with branches, and the hyphae divide with the passage of culture time, but no spores adhere to them. Aerial hyphae produced from basal hyphae have a diameter of 0.4 to 0.7μ.
The hyphae simply branch and elongate in a curved manner, and the entire aerial hyphae are divided to form multiple linked spores.
Although they do not form spirals, many aerial hyphae have spores chained together in a twisted fashion, creating a zigzag pattern. Spores vary in shape and size, with the shape being oval, short cylindrical, or cylindrical, and the size being 0.4 to 0.7 ×
It is 0.8 to 1.5μ, and when observed with an electron microscope,
The surface of the spores is smooth and no flagellated spores or sporangia are formed. Glycerin-asparagine agar medium (ISP medium 5) [Inter.J.System.Bacteriol. 16 313
~340 (1966)], an almost similar morphology was observed. [] Stainability Gram staining is positive and acid-fast staining is negative. [] Bacterial composition Method of B. Becker et al. [Appl. Microbiol. 12 ,
421-423 (1964)], the meso form of diaminopimelic acid was detected, and the LL-isomer was not detected, and the method of Lechevaller [J.Lab.Clin.
Med., 71 , 934-944 (1968)].
Ribose and glycose were detected, but arabinose, xylose, galactose or madeulose were not detected, and the method of Mordarska et al. [J.
Gen. Microbiol. 71 , 77-86 (1972)], nocardomicolic acid was not detected. []Culture properties The findings observed after culturing on various media at 30°C for 21 days are shown in Table 1. The color display follows the color display of the Color Harmony Manual, 4th edition (1958). Furthermore, no soluble pigment was produced in the various media shown in Table 1.

【table】

[According to J. Bacteriol 73 15-27 (1957)]

(13) Generation of acids from sugars Adonitol, L-arabinose, erythritol, D-fructose, D-galactose, D-glucose, glycerin, i-inositol, lactose, D-mannitol, D-mannose, L-rhamnose, salicin, D-ribose, cellobiose and starch; positive maltose and trehalose; false positive dulcitol, α-melibiose, α-methyl-D-cricoside, ruffinose, D-soldotol, D-xylose, melezitose, sucrose, L-sorbose and cellulose: Negative [according to J. Bacteriol. 69 , 147-150 (1955)] As mentioned above, the characteristics of the L18-110 strain of this bacterium are that it produces aerial hyphae rather than true basal hyphae that are fragmented in morphology; In the case of aerial bacteria, the entire body is divided to form a spore chain, and the spore chain has a zigzag shape. The spores have many sizes and shapes and the surface is smooth. In terms of the bacterial body composition, diaminopimelic acid is meso-type. It does not contain characteristic sugars or nocardomicolic acid. Gram staining is positive and acid-fast staining is negative. Physiological properties are positive for catalase and lysozyme. It is sensitive and aerobic. Judging from these characteristics, strain L18-110 belongs to the genus Nocardiopsis [Inter, J. System. Bacteriol 26 , 487-493 (1976)].
It was identified as belonging to Nocardiopsis
SP・L18−110 (Nocardiopsis.SP.L18−
110) [National Institute of Microbial Technology, Agency of Industrial Science and Technology, (accession number) FERMP-8176]. The present invention was completed based on the above findings, and is an antibiotic that belongs to the genus Nocardiopsis.
0-Mycaminosylnarbonolide-producing bacteria are cultured in a medium, and the antibiotic 5-0-mycaminosylnarbonolide is collected from the culture. It is a manufacturing method. First, the Nocardiopsis sp. L18-110 strain mentioned above is the producing strain of 5-0-mycaminosylnarbonolide, an antibiotic belonging to the genus Nocardiopsis, used in carrying out the present invention. Although mentioned as an example, the bacteria used in the present invention are not limited thereto, and may be any bacteria that produces the antibiotic 5-0-mycaminosylnarbonolide belonging to the genus Nocardiopsis, and may be a natural or mutant strain. can also be used. Next, to illustrate in producing the antibiotic 5-0-mycaminosylnarbonolide of the present invention, the above-mentioned 5-0 belonging to the genus Nocardiopsis is used.
- Obtained by aerobically culturing mycaminosylnarbonolide-producing bacteria in a medium containing medium components commonly used for culturing actinomycetes. As the medium, a solid medium or a liquid medium can be used, and especially for mass production, aerated agitation culture using a liquid medium, especially an aqueous medium, is suitable. As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. The carbon source may be any available carbon compound, such as glucose, starch syrup, starch, glycerin, molasses, dextrin, fructose, and soybean oil. The nitrogen source may be any available nitrogen compound, such as soybean flour, wheat germ, corn, etc.
Used include Steve liquor, meat extract, peptone, yeast extract, and ammonium sulfate. In addition, salts such as phosphate, magnesium, potassium, calcium, sodium, cobalt, iron, and manganese are used as necessary. The culture temperature can be changed as appropriate within a range that allows the bacteria to grow and produce the antibiotic 5-0-mycaminosylnarbonolide, but is particularly preferably 20 to 35°C. The culture time varies depending on the conditions, but is usually about 100 to 150 hours.
The culture may be terminated at an appropriate time by determining the time when mycaminosylnarbonolide reaches its maximum titer. In order to collect the antibiotic 5-0-mycaminosylnarbonolide produced in the culture from the culture, it is advantageous to take advantage of the physical and chemical properties of this antibiotic, which will be described later. It is done. In other words, since this antibiotic mainly exists in the culture solution, the solution from which the bacterial cells have been removed is made alkaline and extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, butanol, chloroform, benzene, etc. A crude product can be obtained by dissolving the substance in water at an acidic pH, then redissolving it in a non-hydrophilic organic solvent at an alkaline pH, and concentrating the extract. For further purification, silica gel,
Activated alumina, activated carbon, synthetic adsorption resins such as Diaion HP-20, Amberlite XAD-2
It can be purified by column chromatography using a carrier such as Chloroform as an eluent:
Elute with a mixed solvent such as methanol, chloroform: methanol: ammonia water, ethyl acetate: methanol, aqueous acetone, aqueous alcohol, etc. to obtain the active fraction, which is concentrated under reduced pressure.
A purified white powder of the antibiotic 5-0-mycaminosylnarbonolide is obtained by freeze-drying. Also, the antibiotic 5-0 obtained in this way
- Mycaminosylnarbonolide can be easily made to show a single spot in thin layer chromatography. Next, the physicochemical properties of the antibiotic 5-0-mycaminosylnarbonolide of the present invention thus obtained are as follows. Color property White powder Melting point 85-88℃ Elemental analysis C: 64.12%, H: 9.00%, N: 2.65% Molecular weight 525 (from mass spectrum) Molecular formula C ₂₈ H ₄₇ NO ₈ Specific optical rotation [α] ²⁰ _D = +28. 7゜±4.2 (C=0.5, MeOH) Thin layer chromatography [Silica gel f (manufactured by Tokyo Kasei Co., Ltd.)] Chloroform:methanol (10:1) Rf=
0.36 Chloroform: Acetone: Concentrated ammonia (10:
20:0.1) Rf=0.21 Color reaction Positive: Mauritius sulfuric acid reaction Negative: Ninhydrin reaction Solubility Soluble in organic solvents such as alcohol, ethyl acetate, chloroform, and acetone, and slightly soluble in water. Ultraviolet absorption spectrum λ ^EtOH _nax (nm) (log.ε) 226
(4.09), 281 (2.14) Infrared absorption spectrum (in KBr tablet) 3470, 2975, 2945, 2895, 2800, 1740, 1710,
1695, 1630, 1460, 1380, 1330, 1270, 1195,
1170, 1080, 1060 cm ^-1 . Nuclear magnetic resonance spectrum (in deuterated chloroform,
100MNz); As shown in Figure 1, the above properties and each peak of the mass spectrum indicate that the compound obtained by the present invention is an antibiotic 5-0-mica expressed by the following formula [] described in the above-mentioned literature. It was identified as the same substance as minosylnarbonolide. EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto in any way. Example 1 (Culture. Separation) Glucose 2%, starch 2%, soybean flour 2%,
A medium containing 0.25% sodium chloride, 0.5% yeast extract, 0.35% calcium carbonate, 0.0005% copper sulfate pentahydrate, 0.0005% manganese chloride heptahydrate, and 0.005% zinc sulfate heptahydrate (pH 6. 5) 100ml to 500
The mixture was aliquoted into a ml Erlenmeyer flask and sterilized by heating at 120°C for 20 minutes. One platinum loop from a slant culture of Nocardiopsis sp. L/18-110 strain was inoculated into each of four of the medium, and cultured with shaking at 28°C for 72 hours. Then a 500 ml Erlenmeyer flask containing 100 ml of heat-sterilized medium (pH 6.5) containing 3% starch, 2% soy flour, 0.5% yeast extract, 0.3% calcium carbonate, and 0.001% cobalt chloride hexahydrate.
100 plants were inoculated with the above seed culture solution and cultured with shaking at 28°C for 72 hours. After culturing, centrifugation
Both the culture supernatant 9 and the bacterial cells were separated. The majority of the antibiotic 5-0-mycaminosylnarbonolide is then present in the supernatant, and therefore from the supernatant, 5-0-mycaminosylnarbonolide is extracted as in Example 2.
-0-Mycaminosylnarbonolide is purified. Example 2 (Purification) Ethyl acetate from Step 5 was added to culture supernatant 9 obtained in Example 1, the pH was adjusted to 9 with 3N ammonia water while stirring, and ethyl acetate extraction was performed. Add 3 parts of water to the layer, adjust the pH to 2 with a 12N aqueous sulfuric acid solution, stir, and then separate the aqueous layer. Add chloroform 2 to this aqueous layer and add 3N
-Adjust the pH to 9 with ammonia water, stir and extract,
This chloroform layer was separated, dehydrated by adding anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 420 mg of crude antibiotic 5-0-mycaminosylnarbonolide in the form of a pale yellow oil with a purity of about 50%. Next, 400 mg of this crude antibiotic 5-0-mycaminosylnarbonolide was dissolved in 2 ml of chloroform, and a silica gel column prepared in advance with a mixed solvent having the composition of chloroform:methanol:concentrated aqueous ammonia (300:30:6) was applied. (210ml), eluted with the same solvent, and fractionated into 15g portions. Fractions showing activity were confirmed by paper disc assay using Sarcinalutea. As a result, 201 from the 159th fraction
The fractions were collected, combined, concentrated under reduced pressure, and then dried under vacuum to obtain 144 mg of the antibiotic 5-0-mycaminosylnarbonolide in free base form. Effects of the Invention The present invention is that the antibiotic 5-0-mycaminosylnarbonolide is produced directly from microorganisms.

[Brief explanation of drawings]

FIG. 1 shows the nuclear magnetic resonance spectrum of the antibiotic 5-0-mycaminosylnarbonolide.

Claims (1)

[Claims] 1. Antibiotics belonging to the genus Nocardiopsis 5-
0-Mycaminosylnarbonolide-producing bacteria are cultured in a medium, and the antibiotic 5-0-mycaminosylnarbonolide is collected from the culture. Manufacturing method. 2 Antibiotics belonging to the genus Nocardiopsis 5-
2. The production method according to claim 1, wherein the 0-mycaminosylnarbonolide-producing bacterium is Nocardiopsis sp. L18-110.