JPH0331194B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to substance P-59B1, a novel 14-membered ring macrolide antibiotic, and a method for producing the same. More specifically, the present invention relates to a new antibiotic P-59B1 substance-producing bacteria belonging to the genus Micromonospora, which is collected from a culture obtained by culturing the P-59B1 substance-producing bacteria in a medium.
-59B1 substance and its manufacturing method. The present inventors have discovered that a culture of a certain strain belonging to the genus Micromonospora produces a substance that exhibits a control activity against wheat rot.
As a result of pure isolation of the active substance from the cultured material and examination of its properties, it was confirmed that it was a substance with a new structure different from known substances, and this active substance was named P-59B1 substance. The invention has been completed. The new antibiotic substance P-59B1 according to the present invention has an excellent control effect on wheat rot, particularly by inhibiting the elongation of germ tubes of spores of the wheat rot fungus. The physical and chemical properties of the P-59B1 substance of the present invention are as follows. 1 Appearance: Yellow oily neutral substance 2 Molecular formula: C ₂₁ H ₃₂ O ₆ (Actual value 380.2196 by high-resolution mass spectrometry, calculated value 380.2200) 3 Ultraviolet absorption spectrum: In methanol
It shows maximum absorption at 215nm (ε6800) and 240nm (ε1400). 4 Ultraviolet absorption spectrum: Shown in the attached drawing. 5 Rf value for thin layer chromatography: Developing solvent hexane-ethyl acetate (1:1) 0.66 6 Solubility: Soluble in lower alcohols, ethyl acetate, acetone, chloroform, and benzene, slightly soluble in water. 7 Color reaction: Sulfuric acid reaction, iodine reaction: Positive Ninhydrin reaction, Remieux reaction: Negative 8 Stability: Unstable under acidic and alkaline conditions. The chemical structure of the P-59B1 substance was determined to be as follows based on the above-mentioned physicochemical properties, hydrogen nuclear magnetic resonance spectrum, etc. When examining the antibacterial activity of the P-59B1 substance of the present invention,
The germ tube elongation of wheat rot fungus diaspores was inhibited at a concentration of 1 μg/ml, and a solution of 4 μg/ml was applied on wheat leaves to control the onset of black rust. The present invention also provides P. belonging to the genus Micromonospora.
−59B1 substance-producing bacteria are cultivated, and P from the culture is
The summary is a method for producing the new antibiotic P-59B1 substance, which is characterized by collecting the -59B1 substance. The P-59B1 substance-producing bacteria used in the method of the present invention may be any species as long as it has the ability to produce a sufficient amount of P-59B1 substance to be collected in its culture. An example of such a bacterial strain is strain 980-MC1 (P-59), which was newly isolated by the present inventors from a soil sample collected from a bamboo forest in Makisono Town, Kagoshima Prefecture. The mycological properties of the 980-MC1 (P-59) strain are as follows. The basal hyphae of this strain elongate while branching radially, and are usually septate-free or undivided. Spores are formed directly on basal hyphae or singly at the tips of short stalks with uniaxial branches, spherical to pear-shaped, 0.6 to
0.8 x 0.7 to 1.1 μm, the surface is almost smooth and slightly uneven, and when it matures, it easily separates from the hyphae and spreads in the medium or on the surface of the medium. This strain may form hyphal bundles of Coremia or similar hyphae to the air, but true aerial hyphae are not observed. Sporangia, motile spores, and sclerotia are not observed. As shown in Table 1 below, the culture characteristics of this strain generally form yellow-orange colonies at the early stage of growth, and after 2 to 3 days, dark olive-gray spores begin to settle, and the amount of colonization increases. The color of the colony changes from brown to dark brown-gray or black depending on the maturity level. The color and soluble pigment of the basal hyphae are pH-indicating, with pale yellow in acidic conditions and pale orange to pale pink in basic conditions. The physiological and biochemical properties of this strain are shown in Table 2. This bacterium is mesophilic, does not produce melanoid pigments, and has positive amylase and protease activities and nitrate reduction ability. Regarding the glucosidase activities of this bacterium, α-galactosidase and β-xylosidase are positive, and α-mannosidase is negative. The availability of carbon sources is positive for α-melibiose and raffinose, and negative for L-rhamnose and mannitol. Diaminopimelic acid (A ₂ pm) contained in this bacterium is mainly of the meso type, with a small amount of the EL type, but does not contain the 3-hydroxy type. When searching for the above properties using the search table in Mr. Burgee's Bacteria Identification Handbook, we found that this strain belongs to the genus Micromonospora and is closely related to three species: M. charsii, M. halohuiteica, and M. narasino. be. M. halofiteica differs from this strain in that the size of its spores is 1.2 μm or more and that A ₂ pm (diaminopimelic acid) contains the 3-hydroxy type and does not contain the L type. M. narasino differs from this strain in that it is negative for nitrate cyclogen ability and negative for β-xylosidase activity. Although M. charsii differs from this strain in that it grows poorly on Tuapetsk's agar medium and grows well on potato sections, it is judged that the differences are not enough to make it a different species. Therefore, this bacterial strain belongs to M. chalcii, Micromonospora chalcea,
Let's call it 980−MC1 strain. This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as Micromonospora chalcea 980-MC1 strain in April 1982.
It has been deposited since April 21st, and the deposit number is:
It is FERM P-7046.

【table】

【table】

[Table] This strain, 980-MCI (P-59) strain, is susceptible to changes in its properties, as seen in the case of other actinomycetes, such as artificial mutation using ultraviolet rays, X-rays, radiation, drugs, etc. It is possible to mutate by means, and even such a mutant strain has P-
All micromonospora bacteria capable of producing the 59B1 substance can be used in the method of the present invention. In the method of the invention, strain 980-MC1 (P-59) is typically cultured in a medium containing nutrients available to the microorganism. For example, glucose, sucrose, dextrin, starch, starch syrup, molasses, vegetable oil, animal oil, etc. can be used as the carbon source. In addition, soybean flour, wheat germ, peptone,
Meat extract, yeast extract, corn staple liquor, sodium nitrate, ammonium sulfate, etc. may be used. In addition to adding inorganic salts such as calcium carbonate, potassium chloride, and phosphates as necessary,
Organic and inorganic substances that aid the growth of bacteria and promote the production of P59B1 substances can be appropriately added. Any culture method may be used as long as it is carried out under aerobic conditions, as is the case with general antibiotic production methods, but deep culture is most suitable. The temperature suitable for culturing is 20-35°C, but in most cases it is preferable to culture around 26-32°C. Production of P59B1 substance reaches its maximum accumulation in 2 to 7 days for both shaking culture and tank culture. In assaying the P-59B1 substance of the present invention, a method is used in which inhibition of germ tube elongation is observed on an agar medium of wheat black rot fungus (Paccinia graminis) as a test bacterium. That is, Puccinia graminis f.sp. tritici was placed on an agar medium containing the P-59B1 substance.
The purpose is to place pedicel spores of Race 21 strain on the tube and observe the degree of inhibition of germ tube elongation. According to this method,
Even at a concentration of 1 μg/ml, the P-59B1 substance can inhibit the elongation of the germ tube of Paccinia graminis. The P-51B1 substance obtained by the present invention is a neutral fat-soluble substance and has the above-mentioned physical and chemical properties, so when collecting the P-59B1 substance from the culture, It can be extracted and purified using its properties. That is, the P-59B1 substance accumulated in the culture solution is adsorbed to a synthetic adsorbent such as Diaion HP-20. In addition, if extracted with an organic solvent that does not mix with water, such as ethyl acetate, P-
The 59B1 substance is extracted into the organic solvent layer. In addition, it can be extracted from the cultured bacterial cells with acetone-water or methanol-water. To further purify the P-59B1 substance, use adsorbents such as silica gel and alumina or Cephadex LH.
-20 (Pharmacia) or high performance liquid chromatography using Radial Pack 8C18 (Waters) or the like. By using the methods described above or by appropriately combining these methods, a highly purified P-59B1 substance can be obtained as an oily substance. Although the present invention will be specifically explained with reference to the following examples, it is not limited thereto, and it goes without saying that many variations and modification means not exemplified here can be adopted. Example 1 Production of P59B1 substance Spores of Micromonospora charsii strain 980MC1 (FEI accession number FERMP-7046) were placed in a liquid medium 1 containing 1% starch and 3% soybean flour (PH7).
(500ml Erlenmeyer flasks, 10 flasks used) at 28°C.
The seeds were cultured with shaking for 43 hours. 25% starch, 1.5% soybean flour, 0.2% dry yeast,
The seed mother was inoculated into liquid medium 35 having a composition of 0.4% calcium carbonate (PH 7.4), and cultured with aeration and stirring at 28° C. for 94 hours (50 jars). The cultures of two jar fermenters were centrifuged using a shear press centrifuge. The obtained supernatant 50% was mixed with synthetic adsorbent resin Diaion HP-20.
(5), washed with 10 portions of 50% methanol water, and eluted with 10 portions of methanol. The obtained eluate was concentrated under reduced pressure in 2 parts, and then 1 part in ethyl acetate was added.
It was transferred to Add 100ml of sodium bicarbonate solution to the ethyl acetate layer.
After sequentially washing with 0.1 mL of 0.1N hydrochloric acid, the mixture was concentrated to dryness.
The obtained crude extract was placed on a Sephadex LH-20 column (diameter: 3 cm, length: 50 cm) filled with methanol, and chromatography was carried out by developing with methanol. The eluate is fractionated into 6 g portions, fractions Nos. 25-29 having activity against Paccinia graminis are collected and concentrated under reduced pressure. This yielded 91 mg of an oil with a purity of 20-30%. Radial Pack for 80mg of this oily substance
High performance liquid chromatography was performed using an 8C18 (Waters) column and 70% methanol water as the developing solvent. When eluted at a rate of 2 ml per minute, the P59B1 substance is eluted around 12 minutes after injection. The active fraction was concentrated to dryness to obtain 18 mg of the P59B1 substance of the present invention.

[Brief explanation of the drawing]

The figure is an infrared absorption spectrum diagram (in chloroform) of the P-59B1 substance of the present invention.

Claims (1)

[Claims] 1. The following structure: A novel 14-membered ring macrolide antibiotic P-
59B1 substance. 2. A new antibiotic P- characterized by culturing P-59B1 substance-producing bacteria belonging to the genus Micromonospora and collecting the P-59B1 substance from the culture.
59B1 Manufacturing method of substance.