JPH0460599B2 - - Google Patents

Description

[Detailed description of the invention]

FIELD OF THE INVENTION The present invention relates to a novel antibiotic SA16-10 and a method for producing the same. More specifically, the present invention relates to a novel antibiotic SA16-10 produced by a microorganism belonging to the genus Streptomyces and a culture produced by culturing SA16-10-producing bacteria belonging to the genus Streptomyces in a medium. The present invention relates to a method for producing the antibiotic, which comprises collecting the antibiotic from a product. Disclosure of the Invention The novel antibiotic SA16-10 of the present invention has the following properties. (a) Melting point 85-89°C; (b) Elemental composition C, 61.44%; H, 6.72%; (c) Molecular weight 312.5; (d) Molecular formula C ₁₆ H ₂₁ O ₄ C (e) Ultraviolet absorption in methanol The spectrum is as shown in the attached Figure 1, and the absorption maximum is
221.5nm (ε=9.86×10 ⁵ ); (f) The infrared absorption spectrum in potassium bromide is as shown in Figure 2, and the characteristic absorption is 3460,
2850, 1760, 1695, 1635, 1390, 1258 ^{cm -1} ; (g) 1H nuclear magnetic resonance spectrum (CDC ₃ , TMS
Standard) is as shown in Figure 3, δ=1.00,
1.08, 2.75, 3.26, 3.59, 3.92, 4.07, 4.48,
4.72, 5.25, 5.42, 6.85ppm; (h) Specific rotation [α] ²³ _D = -159° (C = 0.7); (i) Sulfuric acid-ferric chloride, potassium permanganate reaction positive; (j) Soluble in chloroform, methanol, ethyl acetate, diethyl ether and benzene, insoluble in hexane and water. The R _f value of SA16-10 in silica gel thin layer chromatography is as follows.

[Table] The novel antibiotic SA16-10 of the present invention is produced by a new microorganism belonging to the genus Streptomyces. The microorganism was isolated from soil collected in Yamakei, Shimoge District, Oita Prefecture by the present inventors,
The respective representative strains, Streptomyces bungoensis MS16-10G strain and Streptomyces oitaensis MS16-10W strain, have been approved by the Agency of Industrial Science and Technology of the Ministry of International Trade and Industry under Microbiological Research Institute No. 8432 and No. 8433. It has been deposited at the Institute. Streptomyces bungoensis MS16−
The mycological properties of Streptomyces ooitaensis MS16-10G (FEI No. 8432) and Streptomyces ooitaensis MS16-10W (FEI No. 8433) are shown below. Morphological characteristics 1 Morphological characteristics of strain MS16-10G It forms well-developed basal hyphae in both natural and synthetic media, and good aerial hyphae are observed in many media. The spore-forming hyphae are simply branched and form a strong spiral. Flagellated spores and sporangia are not seen, but more than 20 segmented spores are formed and linked together. Long chains of 50 or more may be observed. The spores are spherical or oval in shape, measuring 0.8-1.0 μm x 0.9-1.3 μm, and have a spiny surface. Sporophytes are formed on aerial hyphae. The cell wall contains L,L-2,6-diaminopimelic acid (DAP) and glycine, but not meso-DAP, arabinose, and galactose. No sclerotia formation is observed. 2 Morphological characteristics of MS16-10W strain Same as MS16-10G strain. Properties on various media 1 Properties of MS16-10G strain on various media () Sucrose/nitrate agar (incubated at 30°C): The basal hyphae are white and grow slightly. No aerial mycelium is observed and no soluble pigment is produced. () Glucose-asparagine agar (incubated at 30°C): Both basal and aerial hyphae show good growth. The basal hyphae are white to yellow-brown in color;
Aerial mycelia exhibit a gray-brown color. No production of soluble pigment is observed. () Glycerin-asparagine agar (incubated at 30℃): Both basal and aerial hyphae show vigorous growth. The basal hyphae exhibit a white to yellowish-white color,
Aerial hyphae are initially flesh-colored but later turn grayish-white. No production of soluble pigment is observed. () Starch/inorganic salt agar (incubated at 30℃): Both basal hyphae and aerial hyphae show good growth. The basal hyphae are white to yellow-brown in color;
Aerial hyphae exhibit a gray tone. No production of soluble pigment is observed. () Tyrosine agar (incubated at 30°C): Both basal and aerial hyphae show good growth. The basal hyphae are white to yellow in color, and the aerial hyphae are initially flesh-colored but later turn gray. From around 10 days after culture, slight production of light brown soluble pigment is observed. () Nutrient agar (incubated at 30°C): The basal hyphae show good growth and exhibit a yellowish-white color. Aerial hyphae formation was poor, and a few white aerial hyphae were observed from around 5 days after culture.
The production of soluble pigment is vigorous, giving it a brownish color. () Yeast/malt agar (incubated at 30°C): Both basal and aerial hyphae show good growth. The basal hyphae exhibit a yellowish-white to brown tone;
Aerial mycelia are initially pinkish white, but later turn gray. The production of soluble pigment is observed from around the second day after culturing, giving it a brownish color. () Oatmeal agar (incubated at 30°C): Both basal and aerial hyphae show good growth. The basal hyphae change from white to grayish white, and the aerial hyphae are initially grayish brown but later turn gray. No production of soluble pigment is observed. () Peptone/yeast/iron agar (incubated at 30°C): The basal hyphae are white to yellowish white and show good growth. No formation of aerial mycelia was observed and the surface of the colony was moist. The production of soluble pigment is observed, giving it a brownish color. () Maltose yeast agar (incubated at 30℃): The basal hyphae are yellowish-white to blackish-brown and show vigorous growth. Aerial mycelium shows good growth and is initially white, but later turns gray. Slight production of light brown soluble pigment is observed. () Skim milk medium (incubated at 30℃): Forms a yellow-brown skin ring. The medium turns dark brown and peptonizes without solidifying. () Glucose-peptone-gelatin medium (incubated at 20℃): Basal hyphae showed vigorous growth on the surface of the medium,
It has a brown color. No formation of aerial mycelia was observed. Production of dark brown soluble pigment is observed. Liquefy gelatin. () Tzapetucus agar (incubated at 30°C): The basal hyphae are thin at first, but show good growth from around the 5th day after culturing, and take on a white color. No formation of aerial mycelia was observed, and no production of soluble pigment was observed. 2. Characteristics of strain MS16-10W on various media () Sucrose/nitrate agar (incubated at 30°C): The basal hyphae are white and grow slightly. 15 after culture
The formation of gray aerial mycelia is faintly observed before and after the first day. No production of soluble pigment is observed. () Glucose-asparagine agar (incubated at 30°C): Both basal and aerial hyphae show good growth. The basal hyphae are white to greenish-brown in color;
Aerial mycelia are initially white but later turn grayish-white. No production of soluble pigment is observed. () Glycerin-asparagine agar (incubated at 30°C): Both basal and aerial hyphae show good growth. The basal hyphae are white to yellow-brown in color;
Aerial hyphae are initially yellowish white but later turn gray. No production of soluble pigment is observed. () Starch/inorganic salt agar (incubated at 30℃): Both basal hyphae and aerial hyphae show good growth. The basal hyphae are white to yellow-brown in color;
Aerial hyphae are initially flesh-colored and white, but later turn gray. No production of soluble pigment is observed. () Tyrosine agar (incubated at 30℃): Both basal hyphae and aerial hyphae show vigorous growth. The basal hyphae are white to yellow in color, and the aerial hyphae are initially yellowish-white but later turn gray.
From around 10 days after culturing, slight production of light brown soluble pigment is observed. () Nutrient agar (incubated at 30°C): The basal hyphae are white and show good growth, but the formation of aerial hyphae is poor. From around the 5th day after culturing, slight formation of white aerial mycelium is observed. The soluble pigment exhibits a slight brown hue on the first day after culturing, and becomes clear brown on the third day. () Yeast/malt agar (incubated at 30°C): Both basal and aerial hyphae show good growth. The basal hyphae exhibit a yellowish-white to brown tone;
Aerial mycelia are initially white but later turn grayish-white. Production of a brown soluble pigment is observed from around the 5th day after culturing. () Oatmeal agar (incubated at 30℃): Both basal hyphae and aerial hyphae show vigorous growth. The basal hyphae are white to grayish-white in color;
Aerial hyphae are initially flesh-colored but later turn gray.
No production of soluble pigment is observed. () Peptone/yeast/iron agar (incubated at 30°C): The basal hyphae exhibit a white to yellowish-white color, indicating good growth. No aerial mycelium was observed;
The surface of the settlement becomes wet. Production of a brown soluble pigment is observed from the first day after culturing. () Maltose yeast agar (incubated at 30°C): Both basal and aerial hyphae show good growth. The basal hyphae are white to yellow-brown in color, and the aerial hyphae are initially white but later become grayish-white in color. Slight production of light brown soluble pigment is observed. () Skim milk medium (incubated at 30℃): Forms a brown skin ring. The medium turns dark brown and peptonizes without solidifying. () Glucose-peptone-gelatin medium (incubated at 20℃): Basal hyphae showed vigorous growth on the surface of the medium,
It has a brown color. Aerial mycelium is not formed, and production of black-brown soluble pigment is observed. Liquefy gelatin. () Tzapetucus agar (incubated at 30°C): The basal hyphae are thin at first, but show good growth from around 5 days after being cultured, and take on a white color. No formation of aerial mycelia was observed, and no production of soluble pigment was observed. Physiological and biochemical properties 1 Physiological and biochemical properties of the MS16-10G strain The temperature range suitable for the growth of the MS16-10G strain is 27
~35°C, and growth also occurs at 15°C and 37°C, although growth is slow. It does not grow at 5°C and 40°C. Liquefy gelatin. It does not coagulate skimmed milk, but it converts it into peptonization. Does not hydrolyze starch. Produces brown to black melanin-like pigment in peptone yeast iron agar and tryptone yeast broth. Glucose,
Assimilates xylose, fructose, galactose and mannitol and does not assimilate arabinose, rhamnose, raffinose, inositol, salicin and sucrose. 2 Physiological and biochemical properties of the MS16-10W strain The temperature range suitable for the growth of the MS16-10W strain is 27
~35°C, and growth also occurs at 15°C and 37°C, although growth is slow. It does not grow at 5°C and 40°C. Liquefy gelatin. It does not coagulate skimmed milk, but it converts it into peptonization. Does not hydrolyze starch. Produces brown melanin-like pigment in peptone, yeast, and iron agar. Assimilates glucose, xylose, fructose, galactose, mannitol, and inositol, but does not assimilate arabinose, rhamnose, raffinose, salicin, and sucrose on Pridham-Gotlieb agar. The above properties of MS16-10G strain and MS16-10W strain are described in Bergey's Manual of Determinative Bacteriology, 8th edition.
Manual of Determinative Bacteriology, 8th
edition) and International Journal of Systematic Bacteriology.
Bacteriology) Vol. 16, pp. 313-340, Vol. 18, pp. 69-189
Pages, Vol. 18, pp. 279-392, Vol. 19, pp. 391-512, Vol. 22, pp. 265
The known bacterial species of the genus Streptomyces, S. afganiensis, S. chromofuscus, and S. chromofuscus, which are described on pages 394 to
The properties of S. echinatus and S. lucensis were compared. The results are shown in Table 1.

[Table] As shown in Table 1, MS16-10G strain and
Although strain MS16-10W is similar to known bacterial species, they do not match, and therefore, it was determined to be a new bacterial species belonging to the genus Streptomyces. Production of SA16-10 Thus, the novel antibiotic SA16-10 of the present invention is
It is obtained by culturing the MS16-10G strain or MS16-10W strain in a nutrient medium, accumulating SA16-10 in the culture, and collecting SA16-10 from the culture. Similar to other actinomycetes, MS16−10G strain or
The MS16-10W strain can be mutated by artificial means such as ultraviolet rays, ^Co60 , X-ray irradiation, and the use of various mutagenic agents, and such mutant strains also lose SA16-10 production ability. It can be used in the present invention as long as it has. Furthermore, MS16−10G strain,
Any strain other than the MS16-10W strain and its mutant strains that has the ability to produce SA16-10 can be used in the present invention. The production of SA16-10 using the MS16-10G strain or the MS16-10W strain will be described below as a representative example. Cultivation of MS16-10G strain or MS16-10W strain A normal actinomycete culture method is used for culturing this strain. Various carbon sources can be used as the carbon source for the culture medium, including starch, glycerin, glucose,
It is preferable to use maltose, dextrin, fructose, molasses, etc. alone or in combination.
In addition, hydrocarbons, organic acids, vegetable oils, etc. can also be used. As the nitrogen source, soybean flour, yeast extract, dried yeast, peptone, meat extract, cornstarch liquor, casamino acids, fish meal, ammonium chloride, ammonium sulfate, urea, sodium nitrate, and the like can be used alone or in combination. In addition, inorganic salts such as common salt, potassium phosphate, sodium phosphate, magnesium sulfate, calcium chloride, calcium carbonate, calcium hydroxide, cobalt chloride, zinc sulfate, iron chloride, and iron sulfate, as well as trace amounts of heavy metals, may be added as necessary. Can be added. Furthermore, organic and inorganic substances that aid the growth of the bacterial strain and promote the production of antibiotic SA16-10 can be added as appropriate. Moreover, when using the aerated culture method, it is preferable to further add an antifoaming agent such as fatty oil, silicone oil, paraffin oil, or the like. Although culturing on a solid medium is also possible, it is preferable to use a liquid culture method, particularly a deep culture method, similar to a general antibiotic production method. Cultivation is carried out under aerobic conditions at 15 to 37°C, preferably 25 to 30°C, and a pH range of 3 to 10, preferably in a neutral region. Antibiotic SA16-10 may be produced using either shaking culture or tank culture, and the active substance is accumulated in the culture medium during 2 to 7 days of culture. The culture is stopped when the production amount in the culture solution reaches the maximum, and the antibiotic of interest is isolated and purified from the culture solution. Antibiotics produced in the culture medium by culturing
Measurement of the titer change over time of SA16−10 was performed using hydrogen,
Bacteroides gingivalis in an anaerobic box (manufactured by Forma) purged with carbon dioxide and nitrogen (10:5:85)
381 An overnight culture of the test bacteria was added to Anerovic Brain Heart Infusion Agar medium: yeast extract 1%, peptone 2%, brain heart infusion 2%, vitamin K 0.00002%, hemin.
Anaerobic culture is performed on an agar plate prepared with 0.0005% cysteine hydrochloride, 0.05% cysteine hydrochloride, and pH 7.2, and the paper disk assay method is used. Purification of Antibiotic SA16-10 For the isolation and purification of SA16-10 from the culture solution, methods commonly used for isolating and purifying microbial metabolic products from the culture solution are employed. After the culture is completed, it is present in the culture solution by filtration or centrifugation using a supernatant such as diatomaceous earth.
Separate SA16−10 from bacterial cells and other solid components,
Extract and purify from the liquid or supernatant.
SA16-10 can be collected by utilizing its physicochemical properties, for example, using an adsorbent. Adsorbents include activated carbon, amberlite, etc.
Porous adsorption resins such as XAD-2, XAD-4, XAD-7, Diaion HP-10, HP-20, and HP-50 are used. After the SA16-10-containing solution is passed through the adsorbent as described above to remove impurities or adsorb SA16-10, it is eluted using an aqueous methanol solution, an aqueous n-butanol solution, an aqueous acetone solution, or the like. SA16-10 can be extracted from culture broth or aqueous solution in the stable PH range of SA16-10 by using water-immiscible organic solvents such as dichloromethane, chloroform, ethyl acetate, n-butanol or methyl isobutyl ketone alone or in combination. It can also be purified. For further purification, cellulose such as Avicel or Cephadex
Partition column chromatography using LH-20 or the like, extraction method using the difference in distribution ratio of SA16-10 and impurities to the solvent, or countercurrent distribution method are effective. By repeatedly using the above purification methods alone or in appropriate combinations,
SA16-10 can be purified. SA16-10 also exists in bacterial cells depending on the culture conditions like general lipid-soluble antibiotics, and in such cases, after extraction with hydrophilic organic solvents such as alcohols and acetone, After removing and preparing an aqueous solution, extraction and purification can be carried out in the same manner as described above. Biological Activity Data The novel antibiotic SA16-10 of the present invention exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria, and also against both aerobic and anaerobic bacteria. The antibacterial activity of SA16-10 is shown below. 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. Minimum inhibitory concentration (MIC) for facultative and obligate anaerobic bacteria on anerobic brain heart infusion agar in an anaerobic box substituted with hydrogen, carbon dioxide and nitrogen (10:5:85). It was measured by the agar dilution method using a culture medium. The results are shown in Table 3.

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[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
In a liquid medium with a composition of 0.3% and adjusted to PH7.4.
The MS16-10G strain is inoculated and cultured at 27°C for 2 days with shaking at 200 rpm. 10 220ml of this seed medium
Inoculate the fermentation medium (same composition as the seed medium) 7 in glass jar fermenters (10 units). 27℃
The fermentation medium was cultured for 3 days using an aeration agitation method (400 revolutions/min, aeration rate 4/min). After the cultivation is completed, HyFlow Super Cell (manufactured by John Zumanville Sales) is added to the culture as a supernatant to remove the bacterial cells. liquid 7.5 to 2.5
Porous adsorption resin Amberlite XAD-4
(manufactured by Rohm and Haas) Column (7 x 30
cm), washed with 10 g of distilled water, eluted with 10 g of 50% acetone solution to collect the active ingredient, and evaporated to dryness under reduced pressure to obtain 21 g of brown crude powder. Recovery rate from liquid is 70%. Example 2 84 g of the crude powder obtained in Example 1 was applied to a silylated silica gel column (7 x 50 cm) and eluted with 2000 ml of methanol/0.1M acetic acid (concentration gradient method: methanol from 0% to 80%). pale brown oil 6.4
get g. This was further applied to a silica gel column (5×
45cm) and chloroform/methanol 1000ml
(7:3) and then applied to a silylated silica gel column (2.8 x 30 cm) with 30% methanol/
Elute with 200 ml of 0.1M acetic acid, collect the active fractions and evaporate to dryness under reduced pressure. This was extracted with diethyl ether, evaporated to dryness again, and then Cephadex LH
-20 (manufactured by Pharmacia) column (1.8 x 120cm)
27.3 mg of pure white powder SA16-10 having the above-mentioned physicochemical properties was obtained by gel filtration using
get.

[Brief explanation of the drawing]

Figure 1 is the ultraviolet absorption spectrum of SA16-10, Figure 2 is the infrared absorption spectrum, and Figure 3 is the 1H nuclear magnetic resonance spectrum.

Claims (1)

[Claims] 1. A novel antibiotic having the following physicochemical properties:
SA16-10: (a) Melting point 85-89℃; (b) Elemental composition C, 61.44%, H, 6.72%; (c) Molecular weight 312.5; (d) Molecular formula C ₃ H ₂₁ O ₄ C (e) In methanol Maximum absorption in the ultraviolet absorption spectrum at 221.5 nm (ε=9.86×10 ⁵ ); (f) Infrared absorption spectrum characteristic absorption in potassium bromide: 3460, 2850, 1760, 1695, 1635, 1390,
1258cm ^-1 ; (g) 1H nuclear magnetic resonance spectrum (CDC ₃ , TMS
Standard) δ=1.00, 1.08, 2.75, 3.26, 3.59,
3.92, 4.07, 4.48, 4.72, 5.25, 5.42,
6.85ppm; (h) Specific rotation [α] ²³ _D = -159℃ (C = 0.7); (i) Sulfuric acid-ferric chloride, potassium permanganate reaction positive; (j) Chloroform, methanol, ethyl acetate , soluble in diethyl ether and benzene, insoluble in hexane and water. 2. A strain belonging to the genus Streptomyces and having the ability to produce the novel antibiotic SA16-10 was cultured in a nutrient medium, and the antibiotic SA16-10 was accumulated in the culture. Antibiotics characterized by collecting substances
Production method of SA16−10.