JPH0321158B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing the antibiotic neoviridoglysein, more specifically, the formula In the antibiotic neoviridoglyzeins represented by the formula, R represents a hydrogen atom or a hydroxyl group,
Streptomyces sp.G- which has the ability to produce neoviridoglysein in which R is a hydrogen atom
The present invention relates to a manufacturing method with enhanced selectivity for the production of the antibiotic neoviridoglysein, which is characterized by culturing 89 strains in a nutrient medium and collecting the antibiotic accumulated in the culture. Conventionally, the antibiotic neoviridoglyzeins (hereinafter referred to as "neoviridoglyzeins") have been developed by some of the present inventors as Streptomyces sp. P8648 (FERM-3562). A method has been provided for simultaneously producing neoviridoglysein, , and by culturing them in a nutrient medium, and separating and collecting each composition as necessary (for example, see Japanese Patent Publication No. 55-3339). ). According to this known method, neoviridoglysein in which R in the formula () is a hydroxyl group is obtained as a main component, and the amount of neoviridoglysein that has higher antibacterial activity accumulated is small, and the composition ratio of these is small. In order to change this, it was necessary to culture in the presence of a prolyl hydroxylase inhibitor, such as zinc ion (Zn). As a means of solving the above technical problem, the present inventors conducted research to find a strain deficient in prolyl hydroxylase activity of a neoviridoglysein producing bacterium. Through treatment, we succeeded in obtaining a mutant strain that lacks prolyl hydroxylase activity but does not cause changes in the enzyme system involved in the neoviridoglysein biosynthesis system, thereby completing the present invention. . Therefore, according to the present invention, neoviridoglysein can be selectively produced when trans-4-hydroxy-L-proline is absent or in trace amounts in the medium, and neoviridoglysein can be produced selectively. Glysein production is not observed or is only produced in extremely small amounts, allowing for efficient use of the medium. The microorganism used in the present invention is Streptomyces sp.G-89 obtained by mutating Streptomyces sp.P8648 (FERM-p3562). This Streptomyces sp.G-89 exhibits the following mycological characteristics. This strain extends short, colorless aerial hyphae from well-branched simple basal hyphae, and forms a loose spiral spore chain at the tip. The surface of the spore is smooth. No whorl or ascospore formation is observed. The culture characteristics of this strain in various media are as follows. (1) Sucrose/nitrate agar medium Growth: Slight growth Aerial hyphae: Thin white aerial hyphae formed here and there Base hyphae: Colorless to grayish white Soluble pigment: None (2) Glucose/asparagine agar medium Growth: Good Air Medium hyphae: Hardly formed, white if formed Baseline hyphae: Pale yellow-white to bright yellow (2db-2fb) Soluble pigment: None (3) Glycerin/asparagine agar medium Growth: Moderate Aerial hyphae: Most They do not form, but if they do, they are white. Base hyphae: pale yellow to gray yellow (2 db to 2 dc)
3ec) Soluble pigment: None (4) Yeast malt agar medium Growth: Good Aerial hyphae: White to slightly grayish white Base hyphae: Bright yellow (2fb), but turns brownish-gray (3li) in the later stages of culture becomes. Soluble pigment: absent or sometimes slightly brown. (5) Starch agar medium Growth: Moderate Aerial hyphae: Hardly formed, or white if formed Baseline hyphae: Pale yellow (2db), center of colony exhibits bright grayish-yellowish brown (3ge) Soluble pigment : None Starch decomposition: Slightly decomposed (6) Tyrosine agar medium Growth: Moderate Aerial hyphae: Hardly formed, or occasionally scattered white aerial hyphae are observed. Base hyphae: Gray-yellow (3ec) to light gray-yellowish brown (3ge) Soluble pigment: At the early stage of culture, it produces light purple to light reddish-brown pigment, but after about 10 days of culture, it becomes light brown.
It then becomes thinner (slightly produces melanin pigment) (7) Nutrient agar medium Growth: Good Aerial hyphae: Forms thin white aerial hyphae Base hyphae: Pale yellow (2db) Soluble pigment: None (8) Oatmeal agar medium Growth: Good Aerial hyphae: White to grayish-white Baseline hyphae: Gray-yellow (3ec) to light gray-reddish brown (4ge) Soluble pigment: None This strain grows with an optimum temperature range of 25 to 33℃, but It can grow at ℃ or 37℃ with poor growth. However, it cannot grow above 52℃. This strain liquefies gelatin on glucose, peptone, and gelatin media, and liquefies gelatin on inorganic salt and starch agar media.
Hydrolyzes starch, albeit weakly. This strain peptonizes skim milk. However, no coagulation was observed. This strain does not produce melanin-like pigments in peptone-yeast-iron-agar medium or tryptone-yeast liquid medium, but it sometimes produces melanin-like pigment in tyrosine agar medium. This strain was tested for D-xylose, D-
Glucose, D-fructose, L-rhamnose, and D-mannite are used, L-arabinose, i-inositol, and raffinose are not used, and only a small amount of sucrose is used. This strain belongs to the S (Spirales) section according to morphological classification, grows white to light grayish-white aerial hyphae on a yeast malt agar medium, and does not utilize L-arabinose. This bacterial strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on September 20, 1982, and has been deposited under FERM-BP-184. Next, a method for producing the antibiotic of the present invention using the Streptomyces sp.G-89 strain described above will be described. The above-mentioned strain is inoculated into a nutrient medium suitable for production, cultured aerobically at 18°C to 37°C for 2 to 14 days, and extracted and purified from the culture. As the medium components, known ones used for normal Streptomyces culture can be used.
For example, carbon sources include glycose, glycerin, starch, dextrin, oatmeal, molasses, fats and oils,
Fat, etc. can be used, and as a nitrogen source, soybean flour,
Organic substances such as cottonseed flour, peptone, dry yeast, corn stable liquor, yeast extract, casein, and casein hydrolyzate, and inorganic substances such as ammonium sulfate and ammonium nitrate can be used. In addition, as necessary, inorganic salts such as calcium carbonate, common salt, potassium chloride, phosphate, magnesium sulfate, and other trace metal salts are added, and the antibiotic neoviridoglysein is added to help the growth of this bacteria. Organic and inorganic substances such as amino acids and vitamins that promote production can be added as appropriate. Also,
In order to simultaneously produce neoviridoglysein using this strain, in addition to the above medium composition, trans-4-hydroxy-L
- It is preferable to add proline or a substance containing it, such as fish meal, meat extract, etc. As a culture method, a liquid culture method such as shaking culture or tank culture, which is generally used for the production of antibiotics, is used, but a deep culture method in a tank is most suitable for industrial production. This culture is preferably carried out under aerobic conditions, and the culture temperature is preferably 25 to 33°C. With this method, the production of the antibiotics reaches its maximum concentration in 2 to 10 days in both shaking culture and tank culture. Depending on the type of culture medium, the pH may become highly acidic or alkaline during culturing, but in this case it is desirable to maintain the pH during culturing between 6 and 9. It is desirable to adjust the pH at the beginning of culture to 6.5 to 8.5. Neoviridoglysein accumulated as a main component in the culture solution can be recovered and isolated as a pure product using known methods for isolating depsipeptides and various purification methods based on the physical properties of this substance. . Furthermore, if necessary, it can be recovered as a mixture of neoviridoglyzeins or a mixture of these and griseoviriderin. When preparing the present antibiotic as a feed additive or veterinary drug, it is economically advantageous to use it without isolation. The antibiotic neoviridoglyzeins present in the culture solution include ethyl acetate, butyl acetate, benzene, toluene, n-butanol, methylene chloride,
Extracted with poorly water-soluble organic solvents such as chloroform and methyl isobutyl ketone. Since almost no neoviridoglyseins exist in cultured bacteria, the cultured bacteria should be removed and washed in advance by filtration or centrifugation, and the antibiotic should be extracted from the culture solution including the washing liquid. It is desirable to remove fat-soluble impurities in the bacterial cells. In addition to extraction methods using organic solvents, activated carbon and amberite
Adsorption/desorption using XAD (manufactured by Rohm and Haas), adsorption by cation exchange resins such as Amberlite IR-120 (manufactured by Rohm and Haas), Dowex 50W-X ₂ (manufactured by Dow Chemical), etc. , elution, gel filtration using Cephadex LH-20 (manufactured by Pharmacia), adsorption chromatography using alumina, silica gel, etc., are used in appropriate combinations using column methods or thin layer methods, and in some cases, appropriate solvents are used. This antibiotic can be recovered using a countercurrent distribution method. The present invention will be described in more detail below with reference to Examples. Example 1 Soluble starch 2%, soybean flour 0.5%, potassium phosphate 0.1%, salt 0.1%, magnesium sulfate 0.05%
(adjusted to pH 6.5) was placed in a 250 ml Erlenmeyer flask, and after sterilization, Streptomyces
sp.G-89 was inoculated and cultured with rotary shaking at 28°C for 48 hours, which was used as a seed mother. Soluble starch 2%, L-asparagine 0.5%, dibasic potassium phosphate 0.1%, magnesium sulfate 0.001%
(pH 6.5) was placed in a 500 ml Erlenmeyer flask, sterilized and inoculated with 2 ml of seedlings, and heated at 28°C.
Rotary shaking culture (200 rpm x 7 cm) was performed for 144 hours. Extract 5 ml of the culture solution with 1 ml of benzene, spot the benzene layer on a silica gel plate (Silica gel plate F ₂₅₄ manufactured by Merck & Co.), perform thin layer chromatography using a mixed solvent of chloroform:methanol = 30:1, and after development use Shimadzu. Quantification was performed using Chromato Scanner CS-910 manufactured by Seisakusho. The production amount of neoviridoglyseins was as shown in Table 1 below. In addition, Streptomyces cultured in the same way
The production amount of neoviridoglyseins of sp.p8648 (FERM P-3562) is also listed in Table 1 below.

[Table] Example 2 Soluble starch 2%, glucose 0.2%, soybean flour 0.5%,
Tryptone (manufactured by Difco) 0.4%, yeast extract
500ml of medium containing 0.2% potassium phosphate, 0.1% potassium phosphate, 0.1% sodium chloride, 0.05% magnesium sulfate (pH 6.5)
After sterilizing the flask at 120°C for 15 minutes, Streptomyces sp. Corn starch 10%, soybean flour 5%, potassium phosphate 0.1%, salt 0.1%, magnesium sulfate
0.05%, manganese chloride 0.1%, zinc sulfate 0.01%
(pH 6.5) was placed in a 500 ml Erlenmeyer flask, and after sterilization, 2% of the above seed culture was inoculated and cultured with shaking at 28°C. After inoculation, the cells were cultured for 5 days, and neoviridoglyseins in the culture solution were separated and quantified over time using thin layer chromatography. Streptomyces sp.p8648
(FERM P-3562) was cultured in the same manner, and the production amounts of neoviridoglyseins of both are listed in Table 2 below.

[Table] As described above, the present invention is a manufacturing method that improves the selectivity of the production of the antibiotic neoviridoglysein &.

Claims (1)

[Claims] 1. Streptomyces sp.
No. 184) was cultured in a nutrient medium, and the formula accumulated in the culture A method for producing the antibiotic neoviridoglysein, which comprises collecting the antibiotic neoviridoglysein shown in