JPS5948991B2 - Antibiotic SF-837M↓1 substance manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a new antibiotic SF-837M obtained by culturing a strain belonging to Streptomyces.

The present inventors discovered that the new antibiotic 5F-837A1, A2, A3, and A4 substances were produced in the culture solution of a species of Streptomyces that was named Streptomyces myelofaciens ( Special Public Service 1977-2
8834, Japanese Patent Publication No. 47-3158), by examining the above-mentioned strain in more detail, the above-mentioned 5F-837A,
, A2, A3, and A4, a useful substance SF-837M1 having different physicochemical properties from the culture solution was successfully collected.

SF-837M1 material is 4" of 5F-837A1 material
It is already known that 5F-837A, which is a deacyl form, can be obtained in high yield by treating the substance with bacterial enzymes of microorganisms, such as molds of the genus Mucor, yeasts of the genus Cryptococcus, etc. It is also known to be one of the important substances in research into chemically processing macrolide antibiotics to produce useful derivatives.

That is, in the acylation reaction often used as a macrolide antibiotic derivative, SF-837M, the substance is 5F-
837A has better properties as a synthetic raw material than as a substance.

In addition, the SF-837M1 substance has activity against Durham-positive bacteria (see Table 1), and this substance itself is useful as an antibiotic.

2. Conidia: spherical to oval to oval, surface structure is barbed (relatively thin and long spines), spore size is 0.5 to 0.7
X0.8-1.0 micron.

III Physiological properties Hydrogen sulfide production reaction Negative tyrosinase production reaction Negative nitrite production reaction Positive skim milk coagulation reaction Positive skim milk peptonization reaction Positive starch hydrolysis Positive gelatin solubility Positive (weak) Loeffler coagulation Serum solubility Negative Fibrin degradation Negative IV Carbon source availability 1, Nigelcose, galactose, fructose, maltose, lactose, dextrin, starch, glycerol, inositol, mannose, salicin, sodium acetate, sodium citrate, Sodium succinate 2, Nialbinose whose use is suspected, Rhamnose 3,
Not used: xylose, saccharose, raffinose, inulin, dulcitol, sorbitol, mannitol, cellulose ■ Growth temperature: 15-38°C From the above, the mycological characteristics of Streptomyces mitophilus nobu sp. strain can be summarized. The conidiophores are spiral-shaped, and the conidia are thorn-shaped, and the findings on synthetic media are cream-brown to reddish-brown growth with rose-colored, cotton-like gray aerial hyphae.

Although no significant soluble pigment is produced, it becomes light brown in some media.

On the other hand, in organic media, white to cream-colored aerial mycelia are generally produced with brown growth, and no production of soluble pigments is observed.
On potato pieces, gray-brown aerial mycelium grows abundantly, and the soluble pigment is also black.

As seen in other Streptomyces, this strain is susceptible to changes in its properties and can be mutated by artificial mutagenesis methods using, for example, ultraviolet rays, X-rays, radio frequencies, radiation, chemicals, etc. Even if it is a mutant strain, it is a strain belonging to Streptomyces mycarofaciens.
Any substance capable of producing the F-837 substance can be used in the method of the present invention.

We have invented a method in which a strain having the properties described above is cultured under aerobic conditions, and the SF-837M1 substance in the culture solution is directly accumulated and collected.

According to this, the SF-837Ml substance was directly accumulated in the culture solution without the conventional method of deacylating the 5F-837A1 substance once produced by treating it with esterase from other microorganisms. This is extremely advantageous in terms of yield improvement, process simplification, etc.

Furthermore, in order to directly accumulate the SF-837M1 substance in the culture solution using Streptomyces mylofaciens, a method that combines general bacterial mutation treatment and changes in culture conditions can also be used.

For example, by using nitrosoguanidine, nitrous acid, ultraviolet rays, etc. as a mutagen, a strain that does not produce the 5F-837 substance or a mutant strain with strong esterase activity is obtained, and then SF is applied to these bacteria.
If secondary screening is performed using the production of the -837M1 substance as an indicator, it is possible to obtain bacterial cells that accumulate the substance with efficiency <SF=837M.

Further, as culture conditions, factors such as culture temperature, temperature, culture time, medium composition, and stirring can be considered.

By combining these factors, the SF-837M1 substance can be directly produced in the culture medium.

In this case as well, the accumulation of SF-837M1 substance is promoted if the culture is performed under conditions that increase esterase activity.

The culture temperature is preferably slightly higher than the normal growth temperature of Streptomyces myrofaciens (15 to 38°C).

In addition, it is best to maintain the pH at the neutral to weakly alkaline side (pH 7.0 to 8.0), and the accumulation of SF-837M and substances is promoted by higher aeration and stirring conditions, extended culture time, etc.

The standard culture period is 9 to 12 days. The SF-837M1 substance accumulated in the culture solution was collected and identified according to a conventional method.

That is, the culture P solution was adjusted to pH 9.5, an equal amount of butyl acetate was added thereto, stirred and extracted, the solvent was distilled off by vacuum concentration, dissolved in chloroform, and transferred to hydrochloric acid water (pH 2.2). dissolve

After extraction with butyl acetate and vacuum concentration in Step 5, a crude powder is obtained, which can be purified by alumina or silica gel chromatography.

The silica gel carme was developed with various solvents, and SF-83
7M, substances can be fractionated.

For example, when the crude powder is dissolved in chloroform, charged to a chloroform-adjusted column, and developed with ethyl acetate:chloroform-3:2, the SF-837M substance is eluted after the 5F-837A1 substance is eluted.

To give an example of an alumina column, when developed with n-hexane:ethyl acetate (1:1), SF-83
7M, the substance is eluted slightly later than the 5F-837A1 substance.

Perform rechromatography as necessary to prepare SF-837.
M1 material can be isolated.

Mass spectrometry, nuclear magnetic resonance spectroscopy,
Melting point, specific rotation, infrared absorption spectrum, thin layer chromatography, etc. were measured, and it was confirmed that the physical and chemical properties completely matched those of pure SF-837M1 substance.

Example 1 Streptomyces myrofaciens 5F-837
The strain was 2.0% glucose, 1.0% peptone, 0.5% meat extract, 0.4% C3L, 0.2% sodium chloride,
Calcium carbonate 0.3%, soybean oil 0.2%, pH 7.0
liquid medium 181 was inoculated and aerated at 28°C for 120 hours (
1817 m1n) agitation (400 RPM) culture was performed.

Part 101 of P solution obtained by filtering the culture solution (culture unit 3
After adjusting the concentration to 9.5 with IN sodium hydroxide and extracting with 101% butyl acetate, the organic solvent layer was concentrated under reduced pressure to obtain 51.48 g of yellow powder.

This was dissolved in chloroform 2.61, and hydrochloric acid acidic water (p
H'2.2) 5.21 to extract the aqueous layer (pH2.2).
, 75) was readjusted to pH 9.5, 5.21% butyl acetate was added, and the mixture was extracted and transferred to the organic solvent layer.

The upper layer (butyl acetate layer) was collected, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain 4.27 g of pale yellowish white powder.

The purity was 54% and the yield was 66%. Example 2 A part (1.25 g) of the powder containing 4.27 g of SF-837M1 substance obtained in Example 1 was dissolved in chloroform, and a silica gel column (370 m
Apply to l).

Next, it was developed with a mixed solvent of chloroform:ethyl acetate=4.5:2, and 10 ml portions were collected.

Each fraction was assayed by silica gel thin layer chromatography (developing solvent: benzene-acetone 1:1), and fractions 4115 to 235 were collected and concentrated under reduced pressure.
mg white powder was obtained.

The purity was 91% and the yield was 36%.

Example 3 264 mg of the white powder obtained in Example 2 was dissolved in chloroform and placed in a silica gel tower (200 mg) filled with benzene.
ml).

Developed with a mixed solvent of benzene-ethyl acetate 1:1,
A 1 ml portion was collected.

Each fraction was assayed in the same manner as in Example-2, and fractions 116.75 to 165 were collected and concentrated under reduced pressure.
08 mg of white powder was obtained.

When this sample was compared with a standard substance, it was found that the purity was 100%.

Yield: 87%
value, melting point (126-130°C), specific rotation ([α] to 8 = -61,8°), infrared absorption spectrum, ultraviolet absorption spectrum maximum value 230 mμ, mass spectrometry spectrum in methanol (M + 757) nuclear magnetic resonance In spectrum, etc.
SF-837M completely matched the substance standard.

Claims (1)

[Claims] 1. A method for producing SF-837M, which comprises culturing a strain belonging to the genus Streptomyces under aerobic conditions, accumulating SF-837M in a culture solution, and collecting the SF-837M.