JPH02312596A - Production of antibiotic 4-thiouridine - Google Patents

Abstract

PURPOSE:To obtain the subject compound having antibacterial and anticancer action by culturing a specific microorganism belonging to the Genus Streptomyces in a culture medium. CONSTITUTION:A microorganism [e.g. Streptomyces espi-NK-110134 (FEHM- P-10635)] belonging to the Genus Streptomyces and having antibiotic 4- thiouridine-producing ability is cultured in a culture medium to provide the antibiotic 4-thiouridine, which is then collected.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides an antibiotic 4-
This invention relates to a method for producing thiouridine.

[Conventional technology]

The antibiotic 4-thiouridine stimulates t-RN in E. coli
Minor constituents of A (J, Biol, chem, 24
0, 3975 (1965)), and until now it has been known as microbial conversion (Japanese Patent Publication No. 52-1176 or synthetic method CJ, 8m).

chem, Soc, 8L 178 (1959);
There is a manufacturing method disclosed in Japanese Patent Application Laid-Open No. 4EI-14678).

[Problem to be solved by the invention]

Since the antibiotic 4-thiouridine has antibacterial and anticancer effects and is expected to be used as a pharmaceutical product, it is desired to develop a new method for producing this substance for better manufacturing development.

[Means to solve the problem]

The present invention involves culturing a microorganism belonging to the genus Streptomyces and having the ability to produce the antibiotic 4-thiouridine in a medium, producing and accumulating the antibiotic 4-thiouridine, and collecting the antibiotic 4-thiouridine from the resulting culture solution. The present invention relates to a method for producing the antibiotic 4-thiouridine, with special feature a.

The bacterium that produces the antibiotic 4-thiouridine belonging to the genus Streptomyces used in the present invention is Streptomyces sp.
P, NK-110134, Microtechnical Research Institute No. 10638).

The mycological properties of the NK-110134 strain are shown below.

1. Morphology Strain NK-110134 forms hook-shaped or spiral-shaped aerial hyphae from basal hyphae separated under a microscope, and no whorls or sporangia are observed.

A mature spore chain is a chain of 10 or more spores, the size of the spores is about 0.5 to 0.6 Xo, 9 to 1.2 microns, and the surface of the spores is smooth or rough.

2. Regarding the description of live bone state colors in various media, the color standards of the Japan Color Research Institute were used.

(1) Sucrose/nitrate agar medium (27°C culture) White to brownish aerial mycelia are grown on the growing light yellow brown, and the soluble pigment is only slightly brownish.

(2) Glucose-asparagine agar medium (cultured at 27°C) White to brownish-white to gray-black aerial mycelium grows on the pale yellow to brown growth, and the soluble pigment is slightly yellowish. It is.

(3) Starch/inorganic salt agar medium (rsp-medium 4.2
(7°C culture) On the pale yellow growth, white to grayish white to light brownish gray aerial mycelium grows, and the soluble pigment is only slightly yellowish.

(4) Tyrosine agar medium (15P-medium 7.27'C culture) White to brown-gray to gray-black aerial mycelia are grown on the growing yellow tea, and no soluble pigment is observed.

(5) Nutrient agar medium (27°C culture) White aerial mycelium grows on the pale yellow growth, and no soluble pigment is observed.

[6] Yeast/malt agar medium (l5P-medium 2.27
(°C culture) On the growing yellow tea, white to grayish white to light brownish gray aerial mycelium grows, and the color is only slightly brown.

(7) Oatmeal agar medium (ISP-medium 3.27°
C culture) On the colorless growth, watery black (h
ygroscopic), and no soluble pigments are observed.

(8) Starch agar medium (27°C culture) Aerial mycelium of white to light brown ash to brown ash was grown on the growing light yellow tea, and no soluble pigment was observed.

(9) Malate lime agar medium (cultured at 27°C) White to grayish-white aerial mycelia are grown on the pale yellow growth, and no soluble pigment is observed.

(10) Gelatin puncture culture In simple gelatin medium (20°C culture), white aerial mycelium grows on the growing yellow tea, and the soluble pigment is yellowish.

Glucose-peptone-gelatin medium (24°C culture)
In this case, white aerial mycelium grows on the colorless growth, and no soluble pigment is observed.

(11) Skimmed milk (cultured at 37°C) Colorless to pale yellow, no aerial mycelium attached and no soluble pigments observed.

3. Physiological properties (1) Growth temperature range Yeast starch agar medium [Soluble starch 1.0%
, yeast extract (Otama) 0°2%, powdered agar (Eiken) 2.
0%, pH 7,0) using 5.10.24.27.32
．． Test results at each temperature of 37.45°C, 5.45°C
It grew at all temperatures except for , but the optimum temperature seems to be around 24-32°C.

(2) Liquefaction of gelatin [15% simple gelatin (20°C
culture), glucose-peptone-gelatin medium (24°
C culture)] No liquefaction was observed in each culture medium for 21 days.

(3) Hydrolysis of starch (starch/inorganic/salt agar medium and starch agar medium, both cultured at 27°C) Ice-melting properties were observed from around 7 days after culture in both media, and the effect was moderate. degree.

(4) Coagulation and peptonization of skim milk (37°C culture) 2
Although cultured for 1 day, no coagulation or peptonization was observed.

(5) Production of melanin-like pigments (tryptone, yeast,
Broth (l5P-Medium 1), Peptone Yeast Iron Agar (ISP=Medium 6), Chiron Agar (ISP-
Medium 7), both cultured at 27°C] Not observed in any medium.

(6) Utilization of carbon source (Bridham-Gottlieb agar medium Hsp-medium 9), 27°C culture] glucose,
D-fructose, D-mannitol, raffinose,
It uses inositol, D-galactose, and sucrose, but does not use rhamnose. Furthermore, L-arabinose and D-xylose are probably not used.

(7) Dissolution of malate lime (malate lime medium 27°C
Culture) Solubility is observed.

(8) Nitrate reduction reaction [peptone water containing 0.1% potassium nitrate (15P-medium 8] 27°C culture) is negative.

To summarize the above characteristics, strain NK-110134 belongs to the genus Streptomyces, and 2,6-diaminopimelic acid contained in the cell wall is LL.
-It is a type. Also, no sporangia are observed, the aerial mycelium has a hook-like or spiral shape, and no whorl technique is observed. The surface of the spore is smooth or rough. In various media, white to light brownish gray aerial mycelia grow on pale yellow to yellowish brown growth, and the soluble pigment is slightly yellowish. Melanin-like pigment, proteolytic ability, and nitrate reduction are negative, and starch ice-melting ability and malate lime solubility are positive. Additionally, this strain is characterized by its aerial hyphae being watery black (hygroscopic).

Based on these properties, we searched for known strains that are closely related to the NK-110134 strain from the description of ISP strains in Bursey's Manual of Determinative Hateriology, 8th edition, by Schirling and Gottlieb. dandan” and “” 10,000, Streptomyces rehanishi 8A
uAm-c and 65 Libani, and this strain is thought to be closely related to these.

To produce the antibiotic 4-thiouridine according to the present invention, a microorganism belonging to the genus Streptomyces and having the ability to produce the antibiotic 4-thiouridine is cultured in a medium, and the antibiotic 4-thiouridine is produced and accumulated in the culture. , and then collect this. The culture method is basically similar to that of actinomycetes, but deep culture using liquid culture is usually advantageous. The culture medium used is strain N.
Any medium may be used as long as it contains a nutrient source used by K-110134. As a nutrient source, known ones that have been conventionally used for culturing actinomycetes can be used. For example, as a carbon source, glucose, galactose, mannitol, dextrin, starch, starch syrup (starch malt saccharide), soybean oil, etc. can be used alone or as a carbon source. Can be used in combination. Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, peptone,
Meat extract, yeast extract, dry yeast, corn/steve/
Liquor, soybean oil cake, oatmeal, casamino acid, batato soyton, soluble vegetable protein, etc. can be used alone or in combination.

Other salt, magnesium sulfate, copper sulfate, as needed.
Inorganic salts such as zinc sulfate, manganese chloride, calcium carbonate, and phosphates can be added, as well as organic substances such as nucleic acids, amino acids, vitamins, and inorganic substances that promote the growth of wood grains and the production of the antibiotic 4-thiouridine. can be added appropriately.

It is desirable to culture at a culture temperature of 25°C to 30°C and at a neutral or slightly acidic pH. Usually 3~ for liquid culture
After culturing for 6 days, the antibiotic 4-thiouridine is accumulated in the culture solution. When the production amount in the culture solution reaches the maximum, the culture is stopped, the bacterial cells are filtered off, and the target product is purified and isolated from the obtained culture solution.

For the purification and isolation of this substance from the culture filtrate, an appropriate combination of methods such as adsorption/desorption using an adsorption resin or activated carbon, column chromatography using Sephadex R, or silica gel can be used. Examples of the present invention are shown below, but these are merely illustrative and do not limit the present invention in any way, and various modifications are possible.

Example: 2% soluble starch, 0.5% glucose, 0.5% peptone, 0.5% yeast extract, 0.5% soybean flour, 0 dibasic potassium phosphate in a 500 d Erlenmeyer flask for rotary shaker.
．． 0.05% magnesium sulfate, 0.05% magnesium sulfate, and 0.2% calcium carbonate (pf 17.2) was dispensed and sterilized in an autoclave at 120°C for minutes. This was combined with the NK-110134 strain (Feikoken Bacterium No. 106).
One platinum loopful of No. 38) was inoculated and shaken at 28° C. at 1192 revolutions/min for 2 days. Apart from this, there is also a 500i for rotary shakers! 4% glycerin, 0.5% polypeptone, 0.3% yeast extract, 0.5% meat extract in a Erlenmeyer flask.
, sodium chloride 0.3%, magnesium sulfate 0.05
Dispense 100 ml of % medium (pH 7.0) and
2 mfl of the above culture solution was transferred to a flask that had been autoclaved for 20 minutes at Cl2O, and cultured with shaking at 28°C and 192 revolutions/min for 5 days. Filter the culture solution and use filtrate 1
Obtained 0β.

Diaion Il! -20■ (To Mitsubishi Chemical Industries, To,)
2. Adsorb to 100 mfl and add 50% acetone water. It eluted when ON. The active fraction was collected and concentrated under reduced pressure to obtain 8.3 g of a dark brown solid. After treatment with 150 mR of methanol, 6.6 g of a dark brown oil was also obtained. This was subjected to silica gel column chromatography (400 g of silica gel, developing and dissolving agent chlorophorus/methanol = 4). The active fraction was collected and concentrated under reduced pressure to obtain 350 mg of a yellow oil. 5eph
It was adsorbed onto a dex LH-20 900ml column and eluted with methanol. The active fractions were collected and concentrated under reduced pressure to obtain 100 mg of a yellow oil, which was collected as a yellow powder from ether. This substance had an M'260 in FD-MS, and was confirmed to completely match the standard 4-thiouridine (manufactured by Aldrich) in Uv, II-HMR, and l''C-NHR.

〔effect〕

According to the present invention, 4-thiouridine can be obtained by direct fermentation.

Claims (1)

[Claims] A microorganism that belongs to the genus Streptomyces and has the ability to produce antibiotic 4-thiouridine is cultured in a medium to produce antibiotic 4-thiouridine.
A method for producing the antibiotic 4-thiouridine, which comprises producing thiouridine and collecting the antibiotic 4-thiouridine from the resulting culture solution.