JPS597437B2 - New antibiotic D-606A and its manufacturing method - Google Patents

Description

[Detailed description of the invention] The present invention relates to a new antibiotic and a method for producing the same.

More specifically, D- belonging to the genus Streptomyces
This invention relates to a new antibiotic D-606A, which is obtained by culturing 606A substance-producing bacteria in a medium and collecting the resulting culture, and a method for producing the same. The present inventors have discovered that a new antibiotic having antibacterial activity against various molds and yeasts is produced in a culture of a strain belonging to the genus Streptomyces, and that this can be collected from the culture. It was about to be completed.

To produce the antibiotic D-606A of the present invention, an antibiotic D-606A producing bacterium belonging to the genus Streptomyces is used; One example is the 606A strain.

The properties of the same bacterium are as follows. (I) Morphological properties Aerial mycelia are grown on starch agar, oatmeal agar, yeast
It adheres well to malt agar, tyrosine agar, etc. and forms abundant spores.

The branches are simple and no axle branches are seen. The tips of aerial hyphae exhibit a spiral or loosely wound spiral shape. Sporangia, sclerotia, and zoospores are not observed. The surface structure of the spores observed under an electron microscope is spiny. The shape of the spore is cylindrical or oval, and the size is 0.6 to 1.0 x 1.0.
It is 1.4 microns and usually contains more than 10 spores. (■) Growth status on various media The following properties were observed after culturing at 28°C for 14 days.

(Auto)Physiological properties (1) Growth temperature range: 26% in yeast/malt medium
Grows well in a temperature range of ~37°C.

Below 12℃ and above 45℃ It doesn't grow.

(2) Liquefaction of gelatin: Negative (20℃, 21 days culture) (
3) Starch hydrolysis: Positive (28°C, 14 days culture)
(4) Coagulation of skim milk: Negative (28℃, 14 days culture) Peptonization of skim milk: Negative (28℃, 14 days culture) (5) Production of melanin-like pigment: Positive (peptone/yeast/iron agar medium) (ISP-6) Tyrosine agar (ISP-
5) ([V) Utilization of carbon sources (Culture on Pridham-Gottlieb agar medium at 28°C for 14 days) (1) Utilize: L-arabinose, D-xylose, D-glucose, D-Flagdose, rhamnose, D-mannitol , raffinose, I-inositol (2) Slightly utilized: Sucrose and above To summarize the properties, strain D-606A belongs to the genus Streptomyces, the aerial mycelium tip is spiral, and the spore surface structure is barbed. has.

Aerial mycelia are gray and abundantly grown on starch agar, oatmeal agar, and tyrosine agar, and yellowish white and abundantly grown on yeast and malt agar. The underside is yellowish-white to blackish-brown, with no particular color tone. It produces melanin-like pigments as a soluble pigment, but no other characteristic pigments are produced. The above mycological properties are described in the ISP (International Streptomyces Project) (International Journal of Systematic Bacteriology).
Volumes 2 and 3, 1968, Volume 4, 1969, Volume 5, 19
72 years [InternationalOnalJournaIO
fSystematicBacteriOlOgy〕)
When we investigated related bacterial species of strain D-606A with reference to
esgannfllycicus) is the closest approximation.

That is, according to the description of ISP (International Journal of Systematic Bacteriology 1, Vol. 5, pp. 300, 302, 1972) and the comparison of the culture properties of the standard strain and this strain, it is found that the morphology is a helical thread. The culture characteristics are gray on starch agar, tyrosine agar, and oatmeal agar, yellow-white aerial mycelium on yeast malt agar, and formation of melaton-like pigment. In addition, the book D-60 is characterized by the fact that it does not produce any characteristic pigments, and that the back side has no special color tone ranging from light yellow to blackish brown.
There is good agreement between strain 6A and Streptomyces gammisix.

Regarding the assimilation of carbon sources, it is stated that sucrose and L-arabinose differ depending on the report in Streptomyces gammythicus, but D-60
The 6A strain uses sucrose to a small extent and L-arabizose to a large extent. For other carbon sources, the usability of the two is in good agreement. Also D-60 with nutritional agar
The two are different in that the 6A strain does not grow aerial hyphae, whereas Streptomyces gunmysix grows white aerial hyphae.

From the above, although there are some differences, the D-606A strain closely matches those of Streptomyces gammisix in terms of standard characteristics, so it is appropriate to identify it as Streptomyces gammisix. Therefore, the present inventors
6A strain was Streptomyces gammysix D-606A.
(StreptOmycesgirlnmycicus
D-606A). The present inventors D-606A
The strain was Streptomyces gammisix D-606A (S
treptOmycesgannnlycicusD-
606A). This strain has been deposited with the Institute of Fine Technology, and its application number is No. 5937. Streptomyces gammisix D-606A is a specific example of the bacteria used in the present invention, and mutations obtained by mutating this bacteria by artificial mutagenesis means using, for example, ultraviolet rays, X-rays, radiation, chemical agents, etc. The strain is of course a microorganism belonging to the genus Streptomyces,
Any product capable of producing antibiotic D-606A can be used in the present invention.

In the method of the present invention, the strain is cultured in a medium containing nutrients that can be used by common microorganisms.

As the nutrient source, any known nutrient source conventionally used for culturing Streptomyces bacteria can be used. For example, glucose, glycerol, starch, dextrin, molasses, etc. can be used as carbon sources. Also, as nitrogen sources, soybean flour, meat factory scratches, dry yeast, yeast factory scratches, corn staple, etc.
Water extractor, cottonseed meal, peptone, etc. can be used. In addition to adding other inorganic salts such as calcium carbonate, sodium chloride, and phosphates as necessary, D-60
Organic and inorganic substances that promote the production of 6A substances can be added as appropriate. The culture method is carried out in accordance with a general antibiotic production method, and a liquid culture method, particularly a deep aeration agitation culture method, is preferred.

Cultivation is carried out aerobically, and the pH suitable for cultivation is 5.5~
8.0, and the temperature suitable for culturing can be changed as appropriate within a range that allows microorganisms to grow and produce antibiotic D-606A, but a particularly preferred temperature is 25 to 30°C. The culture time varies depending on the culture conditions, but is approximately 50 to 120 hours. D-
The following method is used to assay the 606A substance.

A malt agar medium was used as the test medium, and Aspergillus nidorans was used as the test bacterium.
This is a paper disk plate method using nidulans). Antibiotic D-606A can be collected from the culture solution of the antibiotic D-606A-producing bacteria belonging to the genus Streptomyces obtained in this way using known means for separating and purifying antibiotics from microbial cultures. Selective combinations can be made.

In other words, since this antibiotic is an acidic and fat-soluble substance as confirmed by the physical and chemical properties described below, methods generally used to collect acidic and fat-soluble substances, such as solvent extraction and chromatography using various adsorbents, are used. B. The desired product can be obtained by combining gel F filtration using Cephadenx LH-20. The following collection method is recognized as the most effective means for separating and purifying antibiotic D-606A from the culture. That is, the culture solution is filtered to remove bacterial cells and other solid substances. The obtained culture solution is made acidic (PH4.O) with hydrochloric acid, and isopropyl ether, ethyl acetate, etc. are added to extract antibiotic D-606A. After concentration, it is applied to a column of silica gel (Kiesel Gel 60, Talc) and developed using a solvent system of benzene-lyacetone (5:1). Concentrate the active fraction to obtain a semi-purified product.
This was further subjected to silica gel column chromatography (n-
Hexaneliacetone (2:1)), Cephadex LH
-20 column chromatography (Pharmacia), high performance liquid chromatography using Nucleosil CN (Nagel) (n-hexane liisopropyl alcohol
96:4)) etc., a highly pure D-606A substance can be obtained. Below D-606
Shows the physical and chemical properties of substance A.

1. Appearance: Pale yellow oil 4. 5. 6. Infrared absorption spectrum: The infrared absorption spectrum measured by the liquid film method is shown in Figure 2.

Molecular weight: determined to be 606 from the mass spectrum (Figure 3).

Solubility: Examined at a concentration of 1-/d at room temperature.

Soluble: acetone, ethyl acetate, benzene, ethyl ether, chloroform, carbon tetrachloride, methyl alcohol, ethyl alcohol Slightly soluble (not completely soluble, but clear soil has antibacterial activity): n-hexane, petroleum ether, water 7. Negative color reaction: ferric chloride, Tollens reagent, aniline hydrogen phthalate 8. Distinguish between acidic, neutral and basic acidic substances9. Stability It is stable in the pH range of 5 to 8 when left in a culture solution for 24 hours at room temperature.

10. Performing silica gel thin layer chromatography using the Rf value increase method in silica gel thin layer chromatography (Talc Kiesel Gel 60F254), by spraying a 50% sulfuric acid aqueous solution and heating, brown spots were detected as brown spots, resulting in the following Rf values: Obtained.

Table 1 shows the anti-microbial activity of substance D-606A against various microorganisms.

The minimum inhibitory concentration was determined by the agar plate dilution method. As is clear from Table 1, substance D-606A has strong antibacterial activity against mold and yeast.

From the above physical and chemical properties and biological activity characteristics, D-60
Substance 6A has no equivalent to any known antibiotic, and therefore Substance D-606A is recognized as a new antibiotic. The present invention will be explained below with reference to Examples.

Example 1 After culturing Streptomyces gammysix strain D-606A (FEI Application Form Receipt No. 5937) on a yeast/malt agar slant medium for 10 days from 28)C7,
1 loop of platinum in 70ml of seed medium (2% glycerin, 1% cottonseed meal, 0.5% yeast scratches, 0.5% dried yeast, 0.5% sodium chloride, 0 disodium phosphate decahydrate) ．．
1%, magnesium sulfate heptahydrate 0.05%, sulfuric acid 1
Iron heptahydrate 0.01%, precipitated calcium carbonate 0.2%
, PH6. O, Adekanol LG-109 as antifoaming agent
(Asahi Denka Co., Ltd.)) was inoculated into a 500ml volumetric flask, and cultured in a reciprocating shaker soil for 28)C2 days.

420 ml of the obtained seed culture solution was inoculated into a 301 volume steam fermenter containing a main culture medium 171 having the same composition as the above seed culture medium, and the mixture was heated at 28°C with aeration (8.51/min) and stirring (200 r).
pm) conditions for 115 hours. The fermented liquid at the end of the culture was 16 m when measured using the paper disk plate method (malt agar medium pH 6.0) using Aspergillus nidorans as the test bacterium! It was found that the D-606A substance was produced which gave an inhibition circle of . After the culture was completed, the bacterial cells and solid matter were removed.

12.51 culture fluids were obtained.

When the obtained cultured solution is adjusted to pH 3.95 and extracted with 6e isopropyl ether, the active ingredients are transferred to the isopropyl ether layer. Collect the isopropyl ether layer,
Concentration under reduced pressure gave 1.69% of an oil. Dissolve 1.59% of the above oil in a mixture of benzene-lyacetone (5:1), and dissolve it in a silica gel column (Kiesel Gel 6, Talc Co., Ltd.).
0200rfL0 and benzeneliacetone (5:1
) and aliquoted in 14.5r111 portions. The active fractions (Fraction Falls 28 to 62) were collected and concentrated under reduced pressure to obtain a yellowish brown oil 190T19. Next, this oily substance was dissolved in n-hexane-lyacetone (1:1) and re-coated with a silica gel column (Kiesel Gel 6, manufactured by Talc Co., Ltd.).
0.60m0, n-hexanelyacetone (2:1
) and aliquoted into 10W11 portions. The active fraction was collected and directly concentrated under reduced pressure to obtain 28η of a pale yellow oil of substance D-606A. Furthermore, nearly pure substance D-606A was obtained by high-performance liquid chromatography using Nucleosil CN (Nagel) (n-hexane-lysopropyl alcohol (96:4) twice). The resulting chromatogram is shown in Figure 4.The conditions for high performance liquid chromatography are as follows.

O Packing material: Nucleosil CN (Nagel) o Column size: 4.6 ml φ x 30C7110 Developing solvent: n-
Hexane lyopropyl alcohol (96:4) O test
Ex: Ultraviolet absorption detector 263] 1rn Example 2 Culture solution 391, which was prepared in the same manner as in Example 1 and whose activity gave an inhibition circle of 19 mm, was adjusted to pH 3 and extracted with 292 ethyl acetate.

When the extract was concentrated under reduced pressure, a yellowish brown oil 9.1f1 was obtained.
I got it.

[Brief explanation of drawings]

FIG. 1 is an ultraviolet absorption spectrum of substance D-606A in ethyl ether. Figure 2 is an infrared absorption spectrum of D-606A substance (liquid film method). FIG. 3 is a mass spectrum (FD-mass spectrum) of the D-606A substance. Figure 4 shows νD-
6 is a chromatogram of high performance liquid talomatography using Nucleosil CN of substance 606A.

Claims (1)

[Claims] 1. Antibiotic D-606A having the following properties (1) Appearance: pale yellow oil (2) Elemental analysis value: C: 62.5-6
5.5%, H: 8.2-8.8%, N: 0% (3) Ultraviolet absorption spectrum: As shown in FIG. (4) Infrared absorption spectrum: As shown in Figure 2 (liquid film method). (5) Molecular weight: determined to be 606 from the mass spectrum (Figure 3). (6) Solubility: Examined at a concentration of 1 mg/ml at room temperature. Soluble: acetone, ethyl acetate, benzene, ethyl ether, chloroform, carbon tetrachloride, methyl alcohol, ethyl alcohol Slightly soluble (not completely soluble, but supernatant has antibacterial activity): n-hexane, petroleum ether, water (7 ) Negative color reaction: ferric chloride, Tollens reagent, aniline hydrogen phthalate (8) Distinction between acidic, neutral, and basic: acidic 2 Culture obtained by culturing D-606A substance-producing bacteria belonging to the genus Streptomyces A method for producing a new antibiotic D-606A, which comprises collecting D-606A substance from a substance. 3. The method for producing the new antibiotic D-606A according to claim 2, which uses Streptomyces gammisix D-606A strain as the D-606A substance-producing bacterium.