JPS6210229B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to antibiotics and methods for producing the same. More specifically, it consists of culturing the novel antibiotic BN-227 substance and BN-227 substance-producing bacteria belonging to the genus Pseudomonas in a medium, and isolating and collecting the BN-227 substance from the resulting culture. This invention relates to a method for producing the antibiotic BN-227 substance. The present inventors discovered that a substance showing activity against Gram-positive and Gram-negative bacteria was produced in a culture of a strain belonging to the genus Pseudomonas, and the active substance was isolated purely from the culture. As a result of examining its properties, it was confirmed that it was a new antibiotic, and this effective substance was named BN-227 substance, and the present invention was completed. An example of a strain of the genus Pseudomonas used in the present invention is Pseudomonas sp. BN-227, which was newly isolated by the present inventors from a decaying plant in Shizuoka Prefecture. It is kept at the Institute of Microbial Technology, Agency of Technology under the accession number 4357. The mycological properties of Pseudomonas sp. BN-227 strain are as described below. (a) Morphological properties Cells cultured on broth agar are 0.6-0.8 x 1.0-
It is a 2.0μ bacillus and moves with polar flagella. No spores are formed, Gram staining is negative, and no pleomorphism is shown. (b) Culture properties (1) Broth agar culture: The bacterial cells are cream-colored;
It is actively proliferating. Does not produce pigment. (2) Broth liquid culture: The medium becomes cloudy throughout. (3) Meat juice gelatin puncture culture: Liquefied. (4) Milk culture: It is liquefied while exhibiting alkalinity. (c) Physiological properties 1 Nitrate reduction: Positive 2 Denitrification reaction: Negative 3 MR test: Positive 4 VP test: Negative 5 Indole production: Negative 6 Hydrogen sulfide generation: Negative 7 Starch hydrolysis: Negative 8 Citric acid Usage of inorganic nitrogen source: Available in ammonium salt form 10 Pigment production: Not produced 11 Urease: Negative 12 Oxidase: Weakly positive 13 Catalase: Positive 14 Arginine degrading enzyme (Meller's medium) Negative 15 Growth temperature: Does not grow at 4°C. 10℃～37℃
It grows at 41°C, but not at 41°C. 16 Does not grow under anaerobic conditions. 17 OF test: O type 18 No auxotrophy 19 Egg yolk reaction: Positive 20 Accumulation of polyvator hydroxybutyrate in bacteria: Positive 21 Utilization of carbon sources Grows using the following carbon compounds as the sole carbon source. Glucose, trehalose, 2-keto-gluconic acid, inositol, valine, alanine, arginine, arabinose, sucrose, L
-Fucose, succarate, propionic acid, butyrate, sorbitol, adonitol, 2.
3-butylene glycol, tributamine, cellobiose, sebacic acid, betaine, ribose,
Threonine, D-arabinose, O-hydroxybenzoate, citraconic acid The following carbon compounds are not available. Geraniol, propylene glycol, ethanol, D(-)tartaric acid, mesotartaric acid, erythritol.
Bergey's Manual of Determinative
It was identified according to Becteriology 8th Edition (1974) and the following conclusions were obtained. (i) This strain was identified as belonging to the genus Pseudomonas because it is a Gram-negative bacillus that does not produce spores, has polar hairs, and is obligately aerobic. (ii) This strain was determined to belong to Section of the genus Pseudomonas because it has no auxotrophy, intracellular accumulation of polybeta hydroxybutyrate was observed, and it can use arginine and betaine as the only carbon sources. (iii) Section lists 6 bacterial species, BN
Strain −227 is similar to the description of Pseudomonas cepacia, but it is not completely identical. Therefore, this strain
It was named Pseudomonas sp.BN-227. Pseudomonas sp. strain BN-227 can be mutated by artificial mutagenesis methods using ultraviolet rays, X-rays, radio frequency, radiation, chemicals, etc., and even mutant strains obtained in this way can
All Pseudomonas strains capable of producing the BN-227 substance can be used in the present invention. In order to culture BN-227 substance-producing bacteria and produce and accumulate BN-227 substance, various media used for ordinary microbial fermentation are used. That is, glucose, glycerin, sucrose, dextrin, starch, starch syrup, etc. are used as carbon sources, and peptone, meat extract, powdered bouillon, cornstarch liquor, soybean meal, wheat germ, fish meal, ammonium sulfate, and salt are used as nitrogen sources. An expression such as 安 is used. Further, inorganic salts such as common salt, potassium chloride, and calcium carbonate may be used in combination, and an antifoaming agent may be added if necessary. As a culture method, methods using liquid media such as shaking culture method and deep aeration agitation culture method are suitable, the culture temperature is selected in the range of 20 to 35°C, and the culture time is suitable for 24 to 40 hours. It is. The following method is used to test the BN-227 substance. As the assay medium, 3% Penatuse Agar (Kyoei Pharmaceutical Co., Ltd.) is used. The test bacterium is Staphylococcus aureus 209P (Staphylococcus aureus 209P).
-coccus aureus209P). BN-227 substance (pure product) was tested using the above method.
At 125 mcg/ml to 2000 mcg/ml, the relationship between the logarithm of the concentration and the inhibition circle diameter shows a linear relationship, giving inhibition circle diameters of 10.0 mm to 20.3 mm, respectively. (Paper disk method) Extraction and purification of the BN-227 substance are carried out in consideration of the physical and chemical properties of this substance, which will be described later, and the method shown below is efficient. That is, the solid content is removed from the culture solution containing the active ingredient, and the active ingredient is absorbed by an adsorbent such as Diaion HP-20.
% acetone water, and after distilling off the acetone, the active ingredients are extracted with an organic solvent such as ethyl acetate or n-butanol, and concentrated to dryness to obtain a crude substance of BN-227. For further purification, appropriate combinations of countercurrent distribution method, precipitation method, gel permeation chromatography using Cephadex LH-20, etc., chromatography using silica gel, alumina, florisil, cellulose, etc. are used. White plate-like crystals of the substance are obtained. When the crystals of the BN-227 substance thus obtained were subjected to silica gel thin layer chromatography in various solvent systems, a single spot was observed in each system, indicating that this crystal was a pure product of the BN-227 substance. It shows that. The physical and chemical properties of this BN-227 substance are as shown below. (1) Elemental analysis values: Carbon 53.79%, Hydrogen 5.84%, Nitrogen 8.73%, Oxygen 31.64% (difference) (2) Molecular weight: 155 (by mass spectrometry) (3) Molecular formula: From elemental analysis values and molecular weight
The formula C ₇ H ₉ NO ₃ is obtained. (4) Chemical structural formula: (5) Melting point: 115°C (6) Ultraviolet absorption spectrum: The spectrum measured in methanol is as shown in Figure 1. (7) Infrared absorption spectrum: The spectrum measured as a potassium bromide tablet is shown in Figure 2. (8) Solubility in solvents: Well soluble in methanol and ethanol, slightly soluble in benzene, acetone, ethyl acetate, and chloroform, but insoluble in petroleum ether and water. (9) Specific rotation: [α] ²² _D = 0 (C = 1, methanol) (10) Color reaction: positive for ferric chloride and potassium permanganate, negative for ninhydrin, Sakaguchi, and Biuret reactions . (11) Color and shape of substance: white plate-like crystals. (12) Rf in silica gel thin layer chromatography
Values Chloroform-methanol (9:1) 0.40 Benzene-ethanol (4:1) 0.47 Benzene-acetone (3:2) 0.08 The minimum inhibitory concentration of this BN-227 substance against various microorganisms is as shown in Table 1. It is used as a medicine, agricultural and livestock drug, or an intermediate for conversion thereof. In addition, the acute toxicity of this substance is
As a result of an intraperitoneal administration test using mice, all mice survived at 100 mg/Kg.

【table】

[Table] As mentioned above, the present BN-227 substance has the molecular formula shown as C ₇ H ₉ NO ₃ , and there is no substance matching this formula among known antibiotics. Furthermore, among natural products and chemically synthesized substances, many substances having the above formula are known, but there are no substances that match the physical and chemical properties and biological properties of the BN-227 substance.
Therefore, this BN-227 substance was determined to be a new substance. Next, an example of the method for producing the BN-227 substance will be shown.
It goes without saying that the present invention is not limited thereto, and that many variations and modifications not shown here can be used. Example 1 2% glycerin in 10 500ml Sakaguchi flasks,
Wheat germ 1.5%, soybean meal 1.5%, _NH4Cl 0.3%,
A liquid medium containing 0.05% K ₂ HPO ₄ and 0.2% CaCO ₃ was dispensed into 100 ml portions, capped with cotton plugs, and autoclaved at 120°C for 10 minutes to prepare Pseudomonas sp. BN-227.
Inoculate one platinum loop of the slant of the strain (Feikoken Bibori No. 4357). Shaking culture was performed at 28° C. for 72 hours to obtain 800 ml of a culture solution containing 1000 mcg/ml. This culture solution is centrifuged to remove bacterial cells, and the supernatant is passed through a tower filled with 80 ml of Diaion HP-20 (Mitsubishi Kasei) to adsorb the active ingredients. After washing this tower with water, the active ingredient is eluted with 50% acetone water. The active fractions are collected, concentrated under reduced pressure, adjusted to pH 2.0 with 1N hydrochloric acid, and extracted twice with the same volume of ethyl acetate. The extracts were combined, washed with water, dehydrated by adding Glauber's salt, and concentrated to dryness under reduced pressure to obtain 210 mg of a brown oil of substance BN-227. Mix this oil with 2 methanol
A column (1.5 cm) of Cephadex LH-20 (Pharmacia), which was dissolved in
The active fraction was collected and concentrated under reduced pressure to dryness to obtain 40 mg of a pale yellow powder of BN-227 substance. Example 2 Glycerin 2%, wheat germ 1.5%, soybean meal 1.5
%, _NH4Cl 0.3%, _{K2HPO4 0.05} %, _CaCO3 0.2%
Culture medium 20 containing Pseudomonas sp.
Inoculated with strain 227 (Feikoken Bacteria No. 4357) and incubated at 28℃.
Cultured for 40 hours with aeration rate of 20/min and stirring rate of 200 rpm.
A culture solution 35 with a concentration of 2000mcg/ml was obtained. After draining the culture solution, add 3 portions of Diaion HP-20 to the solution and stir for 30 minutes to adsorb the active ingredients to the resin. After washing Diaion HP-20 with water, it was packed in a column and the active ingredient was eluted with 50% acetone water. Active fraction 10 was combined and concentrated to 4 under reduced pressure, adjusted to pH 2.0 with 1N hydrochloric acid, and n-butanol 4 The active ingredients were extracted twice in each case, washed with water, concentrated under reduced pressure, and dried to obtain 9 g of powdered substance BN-227. Example 3 Sephadex was prepared by dissolving 5 g of the composition obtained in Example 2 in methanol and equilibrating with methanol.
The mixture is loaded into a LH-20 column (3 cm x 90 cm) and developed with the same solvent, and the active fraction is concentrated under reduced pressure to form a syrup. Furthermore, chromatography was performed again using Cephadex LH-20, and the active fraction was concentrated under reduced pressure to dryness. Approximately 10 ml of acetone was added and dissolved by heating, and plate-like crystals of the BN-227 substance were precipitated after being allowed to cool. 1.2
I took g.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the BN-227 substance.
0.1N caustic soda-methanol (b) respectively
This was measured after dissolving it at a concentration of 10mcg/ml.
Figure 2 shows an infrared absorption spectrum of the BN-227 substance, which was measured by mixing this substance with potassium bromide and making it into a tablet.

Claims (1)

[Claims] 1. A new antibiotic BN-227 substance having the following properties. Carbon 53.79%, Hydrogen 5.84%, Nitrogen 8.73%, Oxygen
31.64% (difference), molecular weight is 155 by mass spectrometry.
It has a molecular formula of C ₇ H ₉ NO ₃ and a melting point of 115°C.
The ultraviolet absorption spectrum in methanol is shown in Figure 1, and the infrared absorption spectrum in potassium bromide tablets is shown in Figure 2, showing positive in the ferric chloride reaction.
Ninhydrin, a substance that shows negative results in the Sakaguchi reaction and has the following structural formula. 2. BN-227 substance-producing bacteria belonging to the genus Pseudomonas were cultured in a medium, and BN-227 was extracted from the obtained culture.
New antibiotic BN characterized by collecting substances
−227 Substance manufacturing method.