JPH0448793B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel antibiotic OMR-59 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 an actinomycete OMR-59, 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 structural formula of the antibiotic OMR-59 according to the present invention is:
It is as follows. The physical and chemical properties of the constituent material OMR-59 according to the present invention are as follows. (1) Elemental analysis: C62.53% H6.23% (2) Molecular weight: 614, molecular ion peak in high-resolution mass spectrum, m/z 614, 2362, elemental analysis and C-13 nuclear magnetic resonance spectrum (4th figure)
The molecular formula C ₃₂ H ₃₈ O ₁₂ can be found from (3) Melting point: 230℃ (4) Specific optical rotation: [α] ²³ _D 0゜ (C=1, acetonitrile) (5) Ultraviolet absorption spectrum: 230 (s) nm, 291 nm and 347 nm in 50% acetonitrile aqueous solution
The absorption maximum is shown at m, and the molecular extinction coefficients are respectively
4770, 5760 (Figure 1). (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) The color reaction is positive for bromophenol blue, 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 OMR-59. 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 ₅₀ is 100 mg/Kg or more. A practical example of a microorganism used to produce the antibiotic OMR-59 of the present invention is Streptomyces OMR-59 isolated from soil by the inventor.
Stocks can be given. This bacterium was submitted to the National Institute of Industrial Technology and Microbiology with accession number 7988.
It has been deposited as "No." Its mycological properties are as follows. () Morphological properties Vegetative hyphae develop well on various agar media, and aerial hyphae grow abundantly on starch/inorganic salt agar media and glycerol/asparagine agar media, but may not colonize on other media. Or it is a poor epiphyte. When observed under a microscope, the sporophyte appears linear and chains of 10 or more spores are observed. () Properties on various media EB shearing (EB
Shirling) and Day Gottlieb (D.
Table 2 shows the culture properties of this producing bacterium, which were investigated by the method of J.D. Gottlieb (International Journal of Systematic Bacteriology, Vol. 16, p. 313, 1966).The color tones are as standard colors. , Color Harmony Manual, 4th Edition (Container Corporation of Omerica Chicago, 1958)
2007), 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] () Physiological properties (1) Production of melanin pigment (a) Tyrosine agar positive (b) Peptone/yeast iron agar negative (c) Glucose/peptone/gelatin medium puncture (21-23°C) Negative (d) ) Tryptone yeast solution negative (2) Tyrosinase reaction positive (3) Hydrogen sulfide production negative (4) Nitrate reduction positive (5) Liquefaction of gelatin (21-23℃) (glucose peptone gelatin medium) Negative (6 ) Hydrolysis of starch Positive (7) Coagulation of skim milk (37℃) Negative (8) Poupetonization of skim milk (37℃) Positive (9) Growth temperature range 15-38℃ (10) Availability of carbon source ( Freedom Godlieb Agar) Used: D-glucose, L-arabinose, D-xylose Not used: D-fructose, rhamnose, sucrose, D-mannitol, raffinose, melibiose, i-inositol (11) Degradation of cellulose Negative ( ) Chemical composition of the cell Diaminopimelic acid in the cell wall is of the LL type, and the sugar composition of the whole bacterial cell includes mannose, ribose, and arabinose. The mycological properties of this bacterium can be summarized as follows. Diaminopimelic acid in the cell wall is of the LL type,
The sugar composition of the entire bacterial cell is arabinose.
Morphologically, it forms long linear spore chains. Vegetative hyphae are light ivory or brown in color, and aerial hyphae are white or gray in color. Melanin pigment is produced on tyrosine agar medium, and other soluble pigments include yellow pigments. From these results, this strain does not match the usual Streptomyces genus because it has arabinose in the sugar composition of the whole cell, but in terms of morphology and diaminopimelic acid type,
It can be said that it is an actinomycete that closely resembles the genus Streptomyces. 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, fruit meal or its digested product, soybean flour or its digested product, defatted soybean or its digested product, pupa hydrolyzate, and various amino acids such as glycine, glutamic acid, and alanine are used. It is possible. 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 substances to the medium that satisfy its nutritional requirements, but this kind of nutrients must be added especially when using a medium containing natural substances. may not be necessary. 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 OMR-59 is produced and accumulated inside and outside the bacterial cells. Antibiotic OMR-59 from the culture after completion of culture
, for example, by the following method. The culture is separated into a filtrate and a precipitate by centrifugation. The filtrate is extracted by adsorption onto activated carbon, porous synthetic polymer resin, ion exchange resin, etc., and elution. The precipitate is extracted with an organic solvent such as ethyl acetate, n-butanol, or aqueous acetone. By appropriately concentrating the extract to dryness, OMR-59
of 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, a powder of antibiotic OMR-59 can be obtained. In the present invention, the antibiotic OMR-59 was detected and quantified using silica gel thin film chromatography (Silica gel 60F ₂₅₄ manufactured by Merck, thickness 0.2 nm, developing solvent benzene/acetone = 3:1, antibiotic
OMR-59 Rf value 0.44) and Clostridium perfringens (Clostridium perfringens)
perfringens) ATCC3624. (Effects of the Invention) As mentioned above, the antibiotic OMR-59 has low toxicity and exhibits activity mainly against anaerobic bacteria. Therefore, this substance is expected to be used as a therapeutic or preventive agent against microbial infections in humans and animals. (Example) Next, an example of the antibiotic OMR-59 of the present invention will be shown, but this example is merely an example and is not intended to limit the present invention. Inoculate 100 ml of one platinum loop from a slant culture of Actinomycetes OMR-59 strain (Feikoken No. 7988) with culture values (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 400 ml of this seed culture to 20 ml of production medium (glucose 0.1
%, starch 2.4%, peptone, 0.3%, meat extract
0.3%, yeast extract 0.5%, calcium carbonate 0.4%,
Cobalt chloride hexahydrate 20mg/agar 0.1%) was inoculated into a 30-volume jar fermenter, and cultured at 27℃ for 2 days with aeration (aeration rate 10/min, agitation).
300rpm). The culture solution 20 is centrifuged using a shear press 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. This acetone solution was concentrated under source pressure to remove acetone, and then combined with culture supernatant 18 and diluted with hydrochloric acid.
Adjusted to PH4. Add this to 750ml of Daikaiion Hp.
After passing through a column packed with -20, it was eluted with 80% acetone and fractionated into individual fractions. Active fraction (No. 2~
6) 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 800 ml of ethyl acetate. The extract was concentrated to dryness under reduced pressure to obtain 1 g of tar-like substance. Dissolve 1 g of tarry substance in 3 ml of benzene and add silica gel (40 g,
Merck & Co., Art. 9385) was added to the top of the column packed. The column was heated with benzene/acetone (5:
1) and fractionated into 10 ml portions. Active fraction (No.
60-100) and concentrated under reduced pressure.
150 mg of oily substance was obtained. 150 mg of the oily substance was dissolved in 1.5 ml of acetonyltrile and divided into 15 batches for rapid liquid chromatography (column, YMC A-324 manufactured by Yamamura Kagaku Kenkyusho; developing solvent).
65% acetonitrile water, flow rate 3ml/min; detection
UV (210 nm; retention time 13 minutes) was performed. By concentrating this active fraction to dryness under reduced pressure, 50 mg of white powder was isolated.Furthermore, by crystallizing from chloroform, antibiotic OMR-59 was isolated as colorless needle crystals (40 mg).

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of antibiotic OMR-59 (measured in 50% acetonitrile aqueous solution).
Figure 2 is an infrared absorption spectrum (KBr method), Figure 3 is
The figure shows a proton nuclear magnetic resonance spectrum (measured in deuterated chloroform), and FIG. 4 shows a C-13 nuclear magnetic resonance spectrum (measured in deuterated chloroform).

Claims (1)

[Claims] 1. Antibiotic OMR-59 represented by the following structural formula. 2. A method of culturing actinomycetes capable of producing the antibiotic OMR-59 represented by the following structural formula aerobically in a medium, producing and accumulating the antibiotic OMR-59, and collecting it. Antibiotic OMR−
59 manufacturing methods.