JPS5813156B2 - Shinko Seibutsutsu SF-1854 Shinkou Seizouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a new antibiotic substance SF-1854 obtained by culturing a strain of the genus Micromonospora.

The present inventors have discovered that a novel antibiotic that exhibits antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria was produced in a culture of a strain belonging to the genus Micromonospora, and was collected from the culture. After realizing that it is possible, he completed the present invention.

This new antibiotic was named SF-1854 substance.

The Micromonospora strain used in the method of the present invention is Micromonospora sp. SF-1854, which was newly isolated from soil in Fukushima Prefecture by the present inventors.
Stocks are used.

This strain has been deposited with the Microbial Technology Research Institute (Feikoken Bibori No. 3141).

Micromonospora sp. SF-1854 strain (Mi
cromonospora sp. The mycological properties of SF-1854) are as follows.

■． Form strain SF-1854 does not form aerial mycelium on various commonly used agar media.

When observing the surface of agar such as starch agar under a microscope, one spore can be seen attached to each part of the long, elongated basal mycelia.

When observed with an electron microscope, the spores are approximately spherical and have a diameter of approximately 1.
．． The diameter of the spore is 2 microns, and the surface of the spore has many protrusions with slightly rounded tips, giving it a conpeitou-like appearance at first glance.

■． Growth status on various media ■. Physiological properties (1) Optimal growth temperature: 26-37°C (2) Optimal growth pH: 6.8-7.8 (3) Liquefaction of gelatin: Positive (4) Hydrolysis of starch: Positive (5) Effect on skim milk: No peptonization or coagulation (6) Production of melanin-like pigment: Negative ■. Carbon source availability (medium: yeast extract 0.5%, calcium carbonate 0.1%, agar 1.5%) D-glucose
++ Rhamnose + D-fructose ++ Sucrose + D-xylose ++ Raffinose
-L-arabisose + cellobiose -D-mannitol- glycerol -I-inositol- α-melibiose-D-galactose++ starch -Strain SF-1854 does not form aerial hyphae on an agar medium, but only spores on the basal hyphae. It is a mesophilic bacterium that produces meso-diaminopimelic acid (Meso-DAP) through whole cell analysis.
It is a strain belonging to the genus Micromonospora.

Based on the above, the SF-1854 strain was isolated from Micromonospora sp.
．． SF-1854).

The SF-1854 strain is susceptible to changes in its properties, as seen in the case of other actinomycetes strains, and can be mutated by artificial mutagenesis methods using, for example, ultraviolet rays, X-rays, radio frequencies, radiation, chemicals, etc. Even if such mutant strains exist, all Micromonospora bacteria capable of producing the SF'-1854 substance can be used in the method of the present invention.

In the method of the present invention, strain SF-1854 is cultured in a medium containing nutrients that can be used by common microorganisms.

As the carbon source, glucose, starch, sucrose, starch syrup, molasses, soybean oil, etc. can be used.

Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, dry yeast, cornstarch liquor, sodium nitrate, ammonium sulfate, etc. can be used.

In addition, in addition to adding inorganic salts such as calcium carbonate, common salt, potassium chloride, and phosphates as necessary, organic and inorganic substances that support the growth of bacteria and promote the production of SF-1854 substances may be added as appropriate. I can do it.

As for the culture method, the liquid culture method, especially the deep culture method, is most suitable, as is the case with general antibiotic production methods.

Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 26°C.
~37°C, and the pH is preferably neutral to weakly alkaline.

When culture is carried out in liquid culture for usually 3 to 10 days, the antibiotic is produced and accumulated in the culture solution.

The following method is used for assaying the SF-1854 substance.

Mycin assay agar is used as the assay medium.

The test bacterium is Bacillus subtilis.
s subtilis) ATCC6633.

SF-1854 substance is 400% in assay using this substance.
At ~50 meg/ml, the relationship between the logarithm of the concentration and the inhibition circle diameter shows a linear relationship, and is 18.0 to 13.9, respectively.
Gives an inhibition circle of mm (paper disk plate method).

The SF-1854 substance has physical and chemical properties as described below, so it can be extracted and purified using an adsorbent, ion exchange resin, etc. according to its properties, or extracted into a derivative soluble in organic solvents and then extracted using a solvent extraction method. It can be extracted and purified, but the method shown below is efficient.

First, a filter aid is added to the culture filtrate, and solid matter containing bacterial cells is filtered out.

Next, the active ingredients of the culture solution are adsorbed onto a cation exchange resin,
Elute with an aqueous acid or alkali solution, and if necessary, neutralize and concentrate under reduced pressure or freeze-dry to obtain a coarse powder.

This crude powder is further subjected to appropriate combinations of column chromatography using a cation exchange resin, solvent extraction method using derivatives, etc. to further improve purity, thereby isolating a pure substance of SF-1854. be able to.

The physical and chemical properties of this substance (sulfate) are as follows.

This substance is a white amorphous powder.

It is well soluble in water and insoluble or poorly soluble in organic solvents such as benzene, chloroform, ethyl acetate, petroleum ether, and butanol.

It is stable in acidic and neutral conditions, but slightly unstable in alkaline conditions.

The melting point turns slightly brown at 210°C and decomposes above 220°C.

The Rf value of silica gel thin layer chromatography is chloroform-methanol-17% ammonia water (2:1:1
The Rf value for cellulose thin layer chromatography is 0.19 for the system of chloroform-methanol-17% aqueous ammonia (upper layer) and 0.31 for the system of 10% ammonia acetate-methanol (2:1). 1:1 upper layer) system with 0.
It is 09.

In addition, the Rf value of paper chromatography using the ascending method is chloroform-methanol-17% ammonia water (2
:1:1 lower layer) system, each giving a single spot.

The specific optical rotation measured at a 1% concentration in an aqueous solution (temperature: 25°C) was +67.0°.

The ultraviolet absorption spectrum measured in an aqueous solution of this substance did not show a characteristic absorption maximum between 220 and 350 mμ,
It only shows terminal absorption.

The infrared absorption curve measured in potassium bromide tablets is also shown in FIG.

The color reaction of this substance is positive with ninhydrin, Lemieux, and Gray-Cool-Layback reagents, but negative with Sakaguchi and silver nitrate reagents.

The elemental analysis values of this substance (sulfate) are C 31.04%, H
6.58%, N10.49%.

In addition, the molecular weight of this substance (free base) according to the vapor pressure method is approximately 5.
It is 00.

Further, the SF-1854 substance according to the present invention has a structural formula shown in formula (1).

The molecular formula and molecular weight calculated from this structural formula are C18H35
It is N5O7 (433).

The in vitro antibacterial properties of SF-1854 substance are shown in Table 1.

Next, we will compare this substance with known antibiotics.

Water-soluble basic antibiotics produced by Micromonospora include dientamimine group antibiotics [M. J. Wei
Nstein et al.: AntimicrobialAgen
ts and Chemctherapy (1963)
, and D. J. Cooper et al., J. Infect D
is 119, 342, (1969), J. A. Wa
iter et al., An-timicrob Ag. Chem
oth2, 464, (1972)], Antibiot
ic NO. 460 (Special Publication No. 46-16153)
, Sisomicin (M. J. Weinstein et al. J. An
tibiotics 23,551,555,559(
1970), XK-62-2 (patent application 1970-42696)
), hortimycin A and B (Special Publication No. 50-2978
Publication No. 9) etc.

First, in direct comparison with Horteimycin A and B, the Rf value of paper chromatography using the ascending method (lower layer of chloroform-methanol-17% ammonia 2:1:1) was 0 for Horteimycin A and B, respectively. .25,0.
86, and that of the SF-1854 substance was 0.30.

Therefore, it is clearly distinguished from Horteimycin A and B.

Next, when directly compared with each component of dientamicin, the Rf values in the above paper chromatography are 0.00,
SF-18 at 0.04, 0.10, 0.19, 0.42
It is clearly distinguished from the 54 substances.

Next, regarding the differences between XK-62-2 and Sisomicin, SF-
1854 substances include sisomicin, around 3.5 ppm,
A signal of a methoxy group that does not exist in XK-62-2 was observed, and it was clearly distinguished from XK-62-2 and sisomicin.

Furthermore, water-soluble basic antibiotics produced by actinomycetes other than Micromonospora include lipostamycin, lipidomycin, streptomycin, kanamycin, paromomycin, nebramycin, neomycin, and kasugamycin, but the SF-1854 substance is mentioned above. The Rf value in the paper chromatography system is completely different from these, and further, it is clearly distinguished from any of these antibiotics based on its physicochemical and biological properties.

Based on the above, it was determined that substance SF-1854 is a new antibiotic.

Examples are shown below, but in the present invention, examples are shown below.
Of course, many other variations or modifications may be used.

Example 1 Using Micromonospora sp. SF-1854 strain (Feikoken Bibori No. 3141) as a seed culture, glucose 0.5%, starch 0.5%, soybean flour 3.0 (pH
7.0) was used.

Two to three platinum loops of inoculum are inoculated into 10 ml of the above medium in a 50 ml large test tube, and cultured with shaking at 28° C. for 3 days.

This seed culture is inoculated at 2% into 100 ml of the same medium in a Sakaguchi flask, and cultured with shaking at 28°C for 3 days (5 Sakaguchi flasks are used).

This was further inoculated into 20 liters of the same medium placed in a 30 liter jar fermenter, cultured with aeration and stirring at 28° C. for 48 hours, and the culture was used as a seed mother.

Five seeds were added to 200 liters of a liquid medium consisting of 3.0% starch, 3.5% soybean flour, 1.5% wheat germ, 0.25% salt, and 0.2% soybean oil (pH 7.0). % inoculation amount, and cultured with aeration at 28° C. for 80 hours (using a 300 liter tank).

After adjusting the culture solution (pH 8.6) to pH 4 with hydrochloric acid, Hyflo Super Cell (filter aid) is added to filter out the bacterial cells.

Adjust the filtrate to pH 7.5 with caustic soda, and filter out the precipitate again. Passing the filtrate thus obtained (150 liters) through a column (15 liters) of cation exchange resin Amberlite IRC-50 (sodium type) is effective. The components are adsorbed to the resin.

After washing with water, elute with 0.5N hydrochloric acid water.

Discard the first 45 liters and add the next 30 liters to the anion exchange resin I.
After neutralizing with R-45 (OH type) and adjusting the pH to 9, it is passed through an activated carbon column (1 liter).

The active ingredients are adsorbed on activated carbon, so after washing with water, 0.1
Elute with a mixture of N-hydrochloric acid and acetone (1:1).

After neutralizing the active fraction with Amberlite IR-45 (OH type), it is concentrated to 1.5 l under reduced pressure.

Subsequently, the concentrated solution was treated with cation exchange resin Amberlite CG-
Pass through a column packed with 500 ml of 50 (ammonia type).

After washing the resin with water, it is eluted with 0.1N aqueous ammonia.

The active fractions were combined, concentrated under reduced pressure to 100 ml, adjusted to pH 6.5 with sulfuric acid, and then freeze-dried.

920 mg (purity, about 25%) of crude powder of SF-1854 substance (sulfate) was obtained.

Example 2 300 mg of the coarse powder obtained in Example 1 was dissolved in 20 ml of water, and cation exchange resin Amberlite CG50 (N
When passed through a 20 ml column (H4+ type), the active ingredient is adsorbed to the resin.

After washing with 200 mg of water and developing with 0.075N ammonia water, active fractions are obtained in fractions 40 to 52 (5 g fraction).

This is collected and concentrated to approximately 4 ml to remove ammonia.

When an ethanol solution containing 10% salicylaldehyde is added to this, a yellow salicylidene derivative of SF-1854 substance is precipitated.

After concentrating this to dryness, equal amounts of chloroform and water are added and the mixture is shaken well in a separating funnel to extract the salicylidene derivative into the chloroform layer.

After further washing with water and dehydration with sodium sulfate, the chloroform fraction was concentrated to dryness to obtain 99 mg of a salicylidene derivative.

When equal amounts of chloroform and sulfuric acid water (pH 2.0) are added to this and shaken well in a separating funnel, the SF-1854 substance is reverse extracted as sulfate.

This was neutralized with Amberlite IR-45 (OH type) and then concentrated to dryness to obtain 31 mg of pure white SF-1854 substance (sulfate).

[Brief explanation of the drawing]

FIG. 1 shows SF-185 obtained by the method of the present invention.
Four substances, sulfate, were measured in potassium bromide.

Claims (1)

[Claims] 1. SF-1854, a new antibiotic characterized by culturing SF-1854 substance-producing bacteria belonging to the genus Micromonospora, accumulating SF-1854 substance, and collecting the same.
Method of manufacturing a substance.