JPH0519556B2 - - Google Patents

Description

[Detailed description of the invention]

Field of the Invention The present invention relates to a novel antibiotic SA4-3 having antibacterial properties.
and its manufacturing method. More specifically, the present invention provides novel antibiotics produced by microorganisms belonging to the genus Streptomyces.
The present invention relates to a method for producing the antibiotic, which comprises culturing SA4-3 and the antibiotic-producing bacteria belonging to the genus Streptomyces in a nutrient medium, and collecting the antibiotic from the resulting culture. Disclosure of the Invention The present invention relates to the formula (): The present invention provides a novel antibiotic SA4-3 shown in the following and a method for producing the same. Antibiotic SA4-3 of formula () is produced by a new microorganism belonging to the genus Streptomyces. The microorganism was isolated from soil collected by the present inventors in Takatsuki City, Osaka Prefecture, and was identified as Streptomyces actamyceteix (S.
Streptomyces actamyceticus strain MS4-3, which is the representative strain, is named as Streptomyces actamyceticus nov.
It has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry. Streptomyces actamyceteix MS4
The mycological properties of -3 (Feikoken Bibori No. 8575) are shown below. Morphological characteristics Forms well-developed basal hyphae on both natural and synthetic media, and good aerial hyphae are observed on many media. Spore-forming hyphae are simply branched and can be straight or wavy. Flagellated spores and sporangia are not seen, and 10 to 20 or more spores form chains. Spores are spherical or oval in shape, size 0.7
The size is ~0.9 μm×1.2 to 1.6 μm, and the surface is smooth. Sporophytes are formed on aerial hyphae. The cell wall contains L,L-2,6-diaminopimelic acid (DAP) and glycine, but meso-
Contains no DAP, arabinose and glasstose. No sclerotia formation is observed. Properties on various media (i) Seuucrose-nitrate agar (incubated at 27°C) The basal hyphae show good growth and take on a white color. In addition, the growth of white aerial mycelium is observed, but the growth is poor. From around the 5th day, production of a fluorescent yellow-green water-soluble pigment is observed. (ii) Glucose-asparagine agar (incubated at 27°C) The basal hyphae show good growth and exhibit a yellow-white color. On the other hand, the growth of aerial mycelium is poor. Production of light brown soluble pigment is observed from around the 6th day. (iii) Glycerin-asparagine agar (incubated at 27°C) Both basal hyphae and aerial hyphae show vigorous growth. The basal hyphae exhibit a yellowish-white color. Aerial mycelia are initially white, but then develop a slightly brownish color. Production of light brown soluble pigment is observed from around the 6th day. (iv) Starch-inorganic salt agar (incubated at 27°C) Both basal hyphae and aerial hyphae show good growth. The basal hyphae exhibit a white to yellowish-white color.
Aerial mycelia are initially white, but then take on a pale greenish-white color. No production of soluble pigment is observed. (v) Tyrosine agar (incubated at 27°C) Basal hyphae show good growth, but aerial hyphae grow slowly. The basal hyphae are white in color. Aerial hyphae are initially greenish-brown, but then take on a gray tone. No production of soluble pigment is observed. (vi) Nutrient agar (incubated at 27℃) The basal hyphae show vigorous growth and exhibit a yellowish-white color. In addition, the growth of white aerial mycelium is observed at the edge of the colony, but the growth is poor. No production of soluble pigment is observed. (vii) Yeast-malt agar (incubated at 27°C) Both basal and aerial hyphae show good growth. The basal hyphae exhibit a yellow-brown color. Aerial mycelia are initially white, but then take on a light brown tone. Production of light brown soluble pigment is observed from around the 8th day. (viii) Oatmeal agar (incubated at 27°C) The basal hyphae show vigorous growth and exhibit a yellow-brown color. In addition, gray-white aerial mycelia are observed to grow on the edge of the village, but their growth is poor. Production of light brown soluble pigment is observed from around the 8th day. (ix) Peptone-yeast-iron agar (incubated at 27°C) The basal hyphae show good growth and exhibit a yellow-brown color. On the other hand, slight growth of aerial mycelia was observed;
It has a white color. Production of soluble pigment is observed from around the 6th day, giving a light brown color. (x) Maltose-yeast agar (incubated at 27°C) Both basal hyphae and aerial hyphae show vigorous growth. The basal hyphae exhibit a yellow-brown color. Aerial hyphae are initially flesh-colored, but then take on a brown tone.
Production of a light brown soluble pigment is observed in the medium from around the 6th day. (xi) Skim milk medium (incubated at 27°C) Forms a yellowish brown ring. No pigment production is observed in the medium. No coagulation of milk is observed and the milk becomes peptonized. (xii) Glucose-peptone-gelatin medium (incubated at 27°C) The basal hyphae show good growth on the surface of the medium and exhibit a yellow-brown color. Aerial mycelia and pigment production are not observed. Strong gelatin liquefaction is observed. () Tuapets agar (incubated at 27°C) Both basal and aerial hyphae show slow but good growth. The basal hyphae have a white color, and the aerial hyphae have a milky white color. Production of a yellow-green soluble pigment is observed from around the 6th day. Physiological and biochemical properties The temperature range suitable for the growth of MS4-3 strain is 27-35
℃, but growth is also observed at 15℃ and 37℃. However, it does not grow at 5°C. Strongly liquefy gelatin. It does not coagulate skimmed milk, but it converts it into peptonization. Perform starch hydrolysis. Peptone-yeast-iron agar, trypsin-yeast-
No melanin-like pigment production is observed in either broth or tyrosine agar. Assimilates glucose, xylose, arabinose, rhamnose, salicin, galactose, fructose, and mannitol, but does not assimilate ruffinose, inositol, and sucrose on Pridham-Gotlieb agar base medium. Bergey's Manual of Determinative Bacteriology, 8th edition (Bergey's Manual of Determinative Bacteriology)
Determinative Bacteriology, 8th edition) and International Journal of Systematic Bacteriology.
Bacteriology) Vol. 16, pp. 313-340, Vol. 18, pp. 69-189
pp., vol. 18, pp. 279-392, vol. 19, pp. 391-512 and vol. 22.
Various known bacterial species of the genus Streptomyces, S. halstedii, S. nitrosporeus, and S. flavovirens, described on pages 265-294. Compared with the nature. The results are shown in Table 1.

【table】

[Table] As is clear from Table 1, the MS4-3 strain is similar to known bacterial species, but they do not match, and therefore, it was determined to be a new bacterial species belonging to the genus Streptomyces. Production of antibiotic SA4-3 The novel antibiotic SA4-3 of the present invention is MS4-3
The antibiotic can be obtained by culturing the strain in a nutrient medium and collecting the antibiotic from the resulting culture. Like other actinomycetes, strain MS4-3 can be exposed to ultraviolet light, e.g.
^Co60 , which can be mutated by artificial mutagenic means such as irradiation with radiation such as X-rays, and the use of various mutagenic agents, and such mutant strains and naturally obtained mutant strains are also susceptible to the antibiotic SA4-3. It can be used in the present invention as long as it has production capacity. moreover,
Even with strains other than MS4-3 strain and its mutant strains,
Any strain capable of producing antibiotic SA4-3 can be used in the present invention. The production of antibiotic SA4-3 using MS4-3 strain will be described below as a representative example. Culture of MS4-3 strain The medium used for culturing this strain may be liquid or solid as long as it contains a nutrient source that can be used by the strain. When culturing is carried out, it is preferable to use a liquid medium. Examples of carbon sources for the medium include glucose,
Lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, or fats and oils such as soybean oil, lard oil, and chicken oil can be used alone or in combination. Nitrogen sources include, for example, meat extract, yeast extract, dried yeast, soy flour, corn stew liquor, peptone, casein, cotton flour, blackstrap molasses, urea or ammonium such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc. Salts and the like can be used alone or in combination. In addition, sodium, potassium, calcium, magnesium, iron, manganese,
Salts containing zinc, cobalt, nickel, etc., salts of inorganic acids such as phosphoric acid and boric acid, and salts of organic acids such as acetic acid and propionic acid are appropriately used. Furthermore, amino acids, such as glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline, etc., peptides, such as dipeptides, tripeptides, etc., vitamins, such as
Vitamin B ₁ , vitamin B ₂ , nicotinic acid, vitamin B ₁₂ , vitamin C, etc., as well as nucleic acids, e.g.
Purines, pyrimidines and their derivatives can be added as appropriate. In addition, inorganic or organic acids, alkalis, buffers, etc. are added as appropriate to adjust the pH of the medium, and appropriate amounts of oils and fats, surfactants, etc. are added to defoam. As a culture method, a static culture method, a shaking culture method, an aeration agitation culture method, etc. are used, and in the case of mass culture, it is preferable to use a deep aeration agitation culture method. Culture conditions vary depending on the condition, composition, culture method, etc. of the medium. Generally, under aerobic conditions, the culture temperature is 15-37°C, preferably 25-30°C and the initial temperature is in the neutral range, preferably PH6.0-8.0.
Culture at PH. It is best to culture the MS4-3 strain until the concentration of the antibiotic SA4-3 reaches its maximum.The culture period varies depending on various conditions, but when using a shaking culture method using a liquid medium or an aerated agitation culture method, In this case, the concentration of the antibiotic in the culture solution reaches its maximum after about 2 to about 8 days of culture. When the production amount of the antibiotic reaches the maximum, the culture is stopped, and the antibiotic is isolated and purified from the culture solution. Antibiotic SA4 produced in culture medium by culturing
-3 titer over time was measured in an anaerobic box (manufactured by Forma) purged with hydrogen, carbon dioxide, and nitrogen (10:5:85).
An overnight culture of Bacteroides gingivalis 381 test bacteria was grown on Anerovic Brain Heart Infusion Agar (yeast extract 1
%. Peptone 2%, Brain Heart Infusion 2%, Vitamin K 0.00002%, Hemin
Anaerobic culture is performed on an agar plate prepared by adding 0.0005% cysteine hydrochloride, 0.05% cysteine hydrochloride, pH 7,2), and the paper disk assay method is used. Purification of antibiotic SA4-3 For the isolation and purification of antibiotic SA4-3 from the culture medium, methods commonly used for isolating and purifying microbial metabolic products from culture medium are employed. . After the cultivation is completed, the antibiotic SA4-3 present in the culture solution is separated from the bacterial cells and other solid components by filtration using a filter aid such as diatomaceous earth or centrifugation, and extracted from the filtrate or supernatant. refine.
Antibiotic SA4-3 can be collected by utilizing its physicochemical properties, for example, using an adsorbent. Examples of absorbents include activated carbon, Amberlite XAD-2, 4 and 7, and Diaion HP.
Porous adsorption resins such as -10, 20 and 50 are used. After the antibiotic SA4-3-containing solution is passed through an adsorbent as described above to adsorb and remove impurities, or after the antibiotic SA4-3 is adsorbed, a methanol aqueous solution, an ethanol aqueous solution, a n-butanol aqueous solution, an acetone aqueous solution, etc. Elute using Antibiotic SA4-3 can be prepared in the stable PH range of Antibiotic SA4-3 using water-immiscible organic solvents such as dichloromethane, chloroform, ethyl acetate, butyl acetate, n-butanol, isobutanol, and ether alone or in combination. It can also be purified from culture filtrate or aqueous solution. For further purification, partition column chromatography using cellulose such as Azevil or Sephadex LH-20, extraction solution or countercurrent distribution method using the difference in the distribution ratio of antibiotic SA4-3 and impurities to the solvent. etc. are valid. Antibiotic SA4-3 can be purified by repeatedly using the above purification means alone or in appropriate combinations. Antibiotic SA4-3, like general lipid-soluble antibiotics, may exist in bacterial cells depending on culture conditions, and in such cases, it can be cultured with hydrophilic organic solvents such as alcohols and acetone. After extraction, the solvent is removed to obtain an aqueous solution, and then extraction and purification can be carried out in the same manner as described above. Biological Activity Data The novel antibiotic SA4-3 of the present invention exhibits antibacterial activity against both gram-positive and gram-negative bacteria, and also against both aerobic and anaerobic bacteria. show. The minimum inhibitory concentration (MIC) against aerobic bacteria and facultative anaerobic bacteria was determined by the agar dilution method using a heart infusion agar medium. The results are shown in Table 2. Also, the minimum inhibitory concentration (MIC) for facultative anaerobes and obligate anaerobes
was measured in an anaerobic box purged with hydrogen, carbon dioxide, and nitrogen (10:5:85) by the agar dilution method using an anerobic brain heart infusion agar medium. The results are shown in Table 3.

【table】

【table】

【table】

【table】

[Table] Examples Next, the present invention will be explained in more detail with reference to Examples. Example 1 Glucose 2%, starch 2%, soy flour 2%,
Yeast extract 0.2%, salt 0.25%, calcium carbonate
Inoculating antibiotic SA4-3 producing strain MS4-3 into a liquid medium having a composition of 0.3% and adjusting the pH to 7.4,
Culture was carried out at 27° C. for 2 days with shaking at 200 revolutions/min to obtain 220 ml of seed mother culture. Separately, 110 ml of the above liquid medium was added to a 500 ml flask (total of 70 bottles), each of which was inoculated with 1.5 ml of the above seed culture solution, and cultured with shaking (200 revolutions/min) at 27°C for 6 days. After the cultivation is completed, Hyflo Super Cell (manufactured by Johnsmanville Sales) is added as a filter aid to the collected culture to filter out the bacterial cells. The filtrate 7 was treated with porous adsorption resin Amberlite XAD-4 (manufactured by Rohm and Haas).
After applying the column (7.2 cm x 30 cm) and washing it with 10 parts of distilled water, it was eluted with 1.5 parts of a 50% acetone aqueous solution to collect the active ingredients, which was evaporated to dryness under reduced pressure to obtain crude antibiotic SA4-3 in the form of a brown powder. 40.5g was obtained. Example 2 40.5 g of the crude antibiotic SA4-3 obtained in Example 1 was suspended in 1000 ml of methanol and filtered. The filtrate was then concentrated, and the resulting brown crude powder was applied to a silylated silica gel column (5.5 cm x 20 cm).
Elution was performed with 80% methanol 1, and the solvent was distilled off under reduced pressure to obtain 20.5 g of a residue. The residue was applied to a silica gel column (6.5 cm x 60 cm) and eluted with 200 ml of chloroform-methanol (9:1) mixed solvent to obtain 4.1 g of powder.
LH-20 (manufactured by Pharmacia) column (4cm x 120
cm) and eluted with methanol to obtain 1.9 g of pure white powder antibiotic SA4-3. The powder was recrystallized from a benzene-methanol mixed solvent to obtain 1.1 g of antibiotic SA4-3 in the form of white columnar crystals. The physicochemical properties of antibiotic SA4-3 are shown below. (1) Appearance: White columnar crystals (2) Melting point: 85-89℃ (3) Elemental analysis: As C ₁₃ H ₁₆ N ₂ O ₃ Theoretical value (%): C, 62.90; H, 6.45; N, 11.29 Actual measurement Value (%): C, 62.83; H, 6.47; N, 11.30 (4) Molecular weight: 248 (5) Molecular formula: C ₁₃ H ₁₆ N ₂ O ₃ (6) Specific rotation: [α] ²⁰ _D +153.0 ° (C=0.62, MeOH) (7) Ultraviolet absorption spectrum: Maximum absorption 205.3 nm (ε=37440) in methanol (see Figure 1 below) (8) Infrared absorption spectrum: Characteristics in potassium bromide Absorption 3410, 3340, 1620, 1580, 1485, 1455,
1420, 1070cm ^-1 (See Figure 2 below) (9) ¹ H-nuclear magnetic resonance absorption spectrum: δ
(CDCl ₃ , TMS standard) = 7.4-6.2, 4.6, 3.3,
2.9, 2.2-1.4ppm (see Figure 3 below) (10) Solubility: Soluble in water (acidic), methanol, ethanol, butanol, acetone, chloroform, ethyl acetate, benzene, slightly soluble in water (basic) , insoluble in hexane. (11) Color reaction: iodine, ninhydrin, potassium permanganate, Ehritz, 2,3,5-triphenyltetrazolium chloride (TTC),
Positive for 10% sulfuric acid. (12) Acid-base: Basic.

[Brief explanation of the drawing]

Figures 1, 2 and 3 are the ultraviolet absorption spectrum, infrared absorption spectrum and ¹ H-nuclear magnetic resonance spectrum of antibiotic SA4-3, respectively.

Claims (1)

[Claims] 1 Formula: A novel antibiotic shown in 2 Antibiotic SA4, which is characterized by culturing a strain belonging to the genus Streptomyces and having the ability to produce the novel antibiotic SA4-3 in a nutrient medium, and collecting the antibiotic from the culture.
-3 manufacturing method. 3 Antibiotic SA4-3 producing bacteria belonging to the genus Streptomyces. 4. The producing bacterium of item 3 above, which is Streptomyces actamyceticus MS4-3 Microtechnical Research Institute No. 8575.