JPH03219887A - Production of stereptovaricin - Google Patents

Abstract

PURPOSE:To obtain the subject substance with a high productivity by culturing a specified producing-bacillus in the presence of a nonionic adsorbent. CONSTITUTION:To a medium containing a nitrogen source such as beef extract, hydrocarbon source, etc., such as sugar, potassium fumarate, etc., is added in an amount of 0.1-10wt.% based on fumaric acid) and further 0.1-20wt.% nonionic adsorbent such as a styrene-divinylbenzene-based synthetic resin adsorbent (e.g. HP-20) having 50-1000mum particle size, 50-100m<2>/g specific surface area and 0.2-1.5ml/g pore volume is added thereto to obtain a culture medium. Into the resultant culture medium, Streptomyces spectabilis (ATCC 27465) is inoculated and aerobically cultured at pH5.5-7.5, at 23-37 deg.C for 4-8 days to obtain a cultured material. The obtained cultured material is filtered through a net, etc., and the separated nonionic adsorbent is subjected to washing and elution using acetone, etc. The resultant eluate is purified by silica gel column chromatography, etc., thus producing the objective streptovaricin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing streptovaricin with high production efficiency.

[Conventional technology]

Streptovaricin is mainly composed of five types, A, B, C, D and E, and initially attracted attention for its usefulness as an anti-tuberculous antibiotic. Recently, a derivative obtained by chemically modifying it has been developed as an anti-tuberculous antibiotic. It has been found to be useful as a retroviral agent, anticancer agent, etc., and has attracted attention (for example,
(See Publication No. 4-110000). Since such useful derivatives are derived from streptovaricin C, streptovaricin C has attracted particular attention.

Conventionally, the method for producing streptovaricin is as follows:
A method of fermentation production using deep culture of Streptomyces spectabilis is known. However, the above-mentioned conventional production method has low production efficiency and only a very small amount of streptovaricin can be obtained. Therefore, it has the disadvantage that it is difficult to put it into practical use industrially. In the conventional method described above, it has been found that the produced streptovaricin is rapidly degraded in the culture medium, and because the produced streptovaricin is highly fat-soluble, it accumulates on the hyphal surface, causing production suppression. It is thought that this is because of this.

[Failure to solve the problem]

Therefore, an object of the present invention is to improve the conventional method and provide a production method that can produce streptovaricin with high production efficiency.

That is, the present invention solves the problems of the above-mentioned conventional methods, and provides a method for producing streptovaricin, which comprises a step of culturing streptovaricin-producing bacteria belonging to the genus Streptomyces in the presence of a nonionic adsorbent. The present invention provides a method for manufacturing.

Microorganisms Streptovaricin-producing bacteria belonging to the genus Streptomyces used in the method of the present invention include, for example, Streptomyces spectabilis (available from ATCC under deposit number ATCC 27465).

Non-ionic adsorbent According to the method of the present invention, streptovaricin produced by culturing the bacteria described above is quickly transferred to the non-ionic adsorbent and stored. Since it is difficult to accumulate, it rarely inhibits bacterial activity. As a result, streptovaricin production is maintained efficiently.

Such nonionic adsorbents have a large specific surface area,
Various synthetic resins, such as styrene, divinylbenzene,
Porous fine particles made of a polymer of one or more compounds such as acrylic esters can be used. Specific examples include adsorbents made of styrene-divinylbenzene synthetic resins, such as IP-10, 8P-20, H
P-30, HP, 40. HP-50 (product name, manufactured by Mitsubishi Chemical Industries, Ltd.), Amberlite XAD-2, XAD-4
(trade name, D-A7', manufactured by Ha7s), adsorbents made of acrylic acid ester resins, such as Amberlite XAD-7 (trade name, manufactured by Rohm and Haas)
etc. can be mentioned. Among such nonionic adsorbents, particularly preferable ones have a particle size of 50 to 1.00.
It has a specific surface area of 0 μm/1, 50 to 1.000 m'/g, and a pore volume of 0.2 to 1.5 m/g. Examples of the commercially available products include HP-20°XAD-4, but are not particularly limited thereto.

In carrying out the method of the invention, the nonionic adsorbent is
Although it may be added after the start of fermentation, it is usually preferable to add it to the fermentation medium before inoculating the bacteria into the fermentation medium. The appropriate amount to add to the medium is about 0.1 to 20%, more preferably 0.5 to 20%.
10% is preferred.

Preferred Embodiments When carrying out the method of the present invention, it is preferable that at least one kind selected from fumaric acid and its water-soluble salts is further added to the medium containing the nonionic adsorbent and allowed to coexist. . By adding fumaric acid or its salt to the medium, the production of streptovaricin, especially streptovaricin C, which is highly useful, is promoted and the production amount is further increased.

Examples of water-soluble salts that can be used include potassium fumarate, sodium fumarate, potassium sodium fumarate, potassium fumarate, and sodium fumarate. Fumaric acid and the fumarate salts exemplified above can be used alone or in combination of two or more.

The amount of fumaric acid or its water-soluble salt added to the medium is 0.1 to 10%, and further 0.5 to 10% of the medium as fumaric acid.
About 5% is preferable. This fumaric acid or its salt may also be added to the medium before fermentation or after the start of fermentation, but it is usually better to add it before the start of fermentation.

Other Culture Conditions There are no particular limitations on the culture medium and other conditions used in the method of the present invention, and conditions commonly used when producing antibiotics and the like by culturing microorganisms can be used. That is, it is common to perform deep culture under aerobic conditions using an aqueous medium containing a nitrogen source, an assimilable carbon source, and an inorganic salt.

Both inorganic and organic nitrogen sources can be used, such as beef extract, peptone, vegetable proteins such as soybean flour, casein, malt extract, fish meal, cotton flour, keiso (defatted fine soybean powder), peanut flour, Examples include organic nitrogen sources such as brewer's yeast, corn gluten flour, and corn steep liquor; and inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, and potassium nitrate.

Examples of assimilable carbon sources include glucose, dextrin, molasses, starch, maltose, galactose, mannitol, sucrose, lactose, and soybean oil.

Examples of nutrient inorganic salts include salts that produce ions such as IJ, calcium, phosphate, and sulfate, specifically, calcium carbonate, potassium phosphate, magnesium sulfate, potassium chloride, and IJ chloride. These include zinc sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, and ammonium molybdate.

In culture, the appropriate pt (pt) of the medium is about 5.5 to 7.5, the temperature is preferably 23 to 37°C, especially 25 to 30°C, and the maximum yield can be achieved by culturing for about 4 to 8 days. is obtained.

Collection of streptovaricin According to the method of the present invention, streptovaricin produced by fermentation is obtained in the form of being adsorbed to a nonionic adsorbent. It is necessary to separate syn from non-ionic adsorbents.

In order to separate the nonionic adsorbent from the medium, for example, there is a method of filtering it using a net, etc., a method of utilizing the difference in specific gravity between the nonionic adsorbent and the medium (specifically, for example, Examples include a method of adding the fermentation medium after fermentation to a saline solution, a method of using a centrifuge, etc.).

To separate streptovaricin from the nonionic adsorbent, the nonionic adsorbent recovered from the culture medium is washed using a suitable organic solvent or a mixed solvent of organic solvent and water as an eluent. The streptovaricin adsorbed on the adsorbent may be eluted. Examples of organic solvents used include methanol, ethanol, acetone,
Acetonitrile, ethyl acetate, dichloroethane 1. Examples include chloroform or a mixed solvent of two or more thereof; a mixed solvent of one or more of these organic solvents and water. In order to effectively elute streptovaricin, it is preferable to first wash the adsorbent on which streptovaricin has been adsorbed with an aqueous solution with a low organic solvent concentration, and then wash it with an aqueous solution with a high organic solvent concentration. .

Among the streptovaricins obtained by fermentation, streptovaricin C is the most useful, so it is desirable to be able to selectively separate streptovaricin C when separating it from the adsorbent. For this purpose, an acetonitrile-water mixed solvent is preferred.

The obtained streptovaricin can be further purified, for example, by repeated recrystallization, silica gel column chromatography, etc.

〔Example〕

Next, the present invention will be specifically explained using examples.

Example I NZ amine A 1.25g, glucose 0.63g.

Enzymatic soybean extract 0.63g, potassium phosphate 0.1
Streptomyces spectabilis ATCC in a 500 ml shake flask containing a seed medium prepared by mixing 6 g of dipotassium phosphate, 0.16 g of dipotassium phosphate, and 100% distilled water.
A culture of 27465 strains was inoculated. This flask was loaded into a rotary shaker and cultured at 27° C. and 20 rpm for 72 hours to obtain a seed culture.

Next, 1 g of defatted soybean powder, Corn Stieble force -181
2g cornstarch, 0.25g brewer's yeast.

The above seed culture 2- was inoculated into a 500 ml shaking flask containing a preculture medium prepared by mixing 0.3 g of potassium chloride, 0.4 g of calcium carbonate, and 100 mf of distilled water. Next, attach this flask to a rotary shaker,
Culture was carried out for 48 hours under the conditions of 27° C. and 20 rpm to obtain a preculture.

Next, 500 ml of the preculture obtained in this way was prepared in advance.
Prepared in a shaking flask were 48 g of soybean flour, 4 g of glucose, 0.25 g of brewer's yeast, and 0.0 g of sodium chloride.
3g, calcium carbonate 0.05g, magnesium sulfate 0.25g, potassium hydrogen phosphate 0.25g, polystyrene adsorbent (product name: Diaion HP20) 3g
and 100 d of distilled water were inoculated into a fermentation medium. This flask was attached to a rotary shaker and heated to 28°C for 6 hours.
Cultured for 1 day. Thereafter, the culture solution was filtered using a mesh to separate the adsorbent. This adsorbent was washed with a mixed solvent of ethyl alcohol and ethyl acetate (1:1) to elute the adsorbed substances. When the washed solvent was subjected to high pressure liquid chromatography, it was confirmed that it contained 0.4 mg of streptovaricin C.

Comparative Example 1 Culture was carried out in the same manner as in Example 1, except that a medium having the same composition but not containing a polystyrene adsorbent was used as the fermentation medium, and streptovaricin C was separated from the medium and the bacterial cells. 0.05 mg of streptovaricin C was obtained.

Example 2 As a fermentation medium, additionally 1.2 g of sodium fumarate
A medium with the same composition as that used in Example 1 except that it contains
That is, 4 g of soybean flour, 4 g of glucose, 0.0 g of brewer's yeast.
25g 1 sodium chloride 0.3g, calcium carbonate 0.
05g, magnesium sulfate 0.25g.

Example 1 except that a medium prepared by mixing 0.25 g of potassium hydrogen phosphate, 1.2 g of sodium fumarate, 3 g of polystyrene adsorbent (trade name: Diaion HP20) and 100 g of distilled water was used. Culture was carried out in the same manner, and streptovaricin C was separated from the adsorbent separated from the medium using the same solvent as in Example 1. It was confirmed that the solvent after washing contained 3.6 mg of streptovaricin C.

Comparative Example 2 Culture was carried out in the same manner as in Example 2, except that a medium with the same composition except that it did not contain a polystyrene adsorbent was used as the fermentation medium, and streptovaricin C was separated from the medium and the bacterial cells. . Streptovaricin C is 0.08
mg was obtained.

Example 3 In Example 2, the proportion of streptovaricin C in the substance adsorbed on the polystyrene adsorbent separated from the fermentation medium after culturing was determined by high performance liquid chromatography (analyzed at 254 nm) as follows: It was 10% by weight. After washing this adsorbent with a 15% acetonitrile aqueous solution, the adsorbed material was eluted with a 50% acetonitrile aqueous solution. After the elution operation, the acetonitrile aqueous solution was evaporated to dryness, and the resulting solid crude product contained 50% streptovaricin C.

〔Effect of the invention〕

Conventionally, only a very small amount of streptovaricin could be obtained by culturing Streptomyces microorganisms, but according to the method of the present invention, streptovaricin can be obtained with an efficiency of more than 10 times, and in a particularly preferred embodiment, more than 20 times. It is a manufacturing method that can produce baricin and is extremely practical from an industrial perspective.

Claims (2)

[Claims] (1) A method for producing streptovaricin, which comprises the step of culturing streptovaricin-producing bacteria belonging to the genus Streptomyces in the presence of a nonionic adsorbent. (2) The method for producing streptovaricin according to claim (1), wherein the culturing is carried out in a medium further supplemented with at least one member selected from the group consisting of fumaric acid and a water-soluble salt thereof. .