JPH0656860A - Antibiotic substance oh-4156 and its production - Google Patents

Abstract

PURPOSE:To provide an antibiotic substance having antibacterial activity and proliferation suppressing activity and to provide a process for the production of the substance. CONSTITUTION:This antibiotic substance OH-4156 can be produced by culturing a microbial strain belonging to the genus Streptomyces and capable of producing OH-4156 in a medium and separating the substance from the cultured product.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel antibiotic OH-
4156 manufacturing method. More specifically, the present invention is an antibiotic produced by culturing an antibiotic-producing bacterium belonging to the genus Streptomyces, showing antibacterial activity against Gram-positive bacteria and having growth inhibitory activity against B16 mouse melanoma cells. It relates to the substance OH-4156 and a method for its production.

[0002]

BACKGROUND OF THE INVENTION Although many antibiotics having antiproliferative activity have been developed so far, most of them are not yet complete therapeutic agents.

[0003]

Therefore, there is a strong demand for an antibiotic which exhibits antibacterial activity against such Gram-positive bacteria and has growth inhibitory activity against B16 melanoma cells. In such a situation, the present invention has been researched and developed in view of its being extremely important as a solution to human medical or social problems.

[0004]

In order to solve the above problems, the present inventors isolated strain OH-4156 from various soils for the purpose of searching for new antibiotics, and produced metabolites thereof. As a result of continuing research on products,
In a culture of strain OH-4156 isolated from soil of Edogawa, Tokyo, B showed antibacterial activity against Gram-positive bacteria, and B
16 It was found that a substance having an inhibitory action on the growth of 16 mouse melanoma cells was produced. Next, the effective substance was separated from the culture and purified. Since no substance having physicochemical properties has been known so far, this substance was designated as antibiotic OH-4156. The present invention has been completed based on such findings.

According to the present invention, the substance OH-4156 and the substance OH-4156-producing bacterium belonging to the genus Streptomyces are cultured in a medium to accumulate the substance OH-4156 in the culture, and the substance OH-4156 is obtained from the culture. Substance to collect
It is a manufacturing method of H-4156.

As a strain used for producing the substance OH-4156 of the present invention, for example, OH-465 belonging to the genus Streptomyces isolated by the present inventors.
The two strains are examples of the most effectively used strains of the present invention, and the bacteriological properties of the strains are as follows.

(I) Morphological Properties Vegetative hyphae are well developed on various agar media and fragmentation is observed. Aerial mycelium is starch, inorganic salt agar, glycerol
It grows abundantly on asparagine agar and has a white to gray color tone. When observed under a microscope, the aerial hyphae are linear and 20 or more spore chains are observed. The spore size is 1.4 × 0.7 μm and is columnar. The surface of spores is smooth. No sclerotia, sporangia and zoospores are found.

(II) Properties on various media EB shirring [E. B. Shirlin
g] and the method of D. Gottlieb (International Journal of Sististic Bacteriology, Vol. 16, 313).
Table 1, Table 2 and Table 3 show the culture properties of the presently produced strains, which were investigated by pp. 1966). The color tone was determined as a standard color using the Color Harmony Manual Fourth Edition (Container Corporation of America Chicago, 1958), and the code is also shown in parentheses together with the color chart name. The following are the observation results in each medium at 27 ° C. for 2 weeks, unless otherwise specified.

[0009]

[Table 1]

[0010]

[Table 2]

[0011]

[Table 3]

(III) Physiological properties (1) Formation of melanin pigment (a) Tyrosine agar positive (b) Peptone yeast iron agar positive (c) Glucose / peptone / gelatin medium positive (21-23 ° C) (d) Tryptone / Yeast solution negative (2) Tyrosinase reaction positive (3) Hydrogen sulfide production positive (4) Nitrate reduction negative (5) Gelatin liquefaction (21-23 ° C) positive (Glucose-peptone-gelatin medium) (6) Hydrolysis of starch Positive (7) Coagulation of skim milk (37 ° C) Positive (8) Peptone conversion of skim milk (37 ° C) Negative (9) Growth temperature range 10-37 ° C Optimal growth temperature 26 ° C (10) Carbon Source availability (Pleedam-Gotrieve agar) Use; glucose, arabinose, xylose Some use; fructose, sucrose Not; raffinose, melibiose, mannitol, rhamnose, inositol (11) Decomposition negative cellulose

(IV) Cell Wall Composition The cell wall diaminopimelic acid is of the LL type. The following is a summary of the mycological properties of this bacterium. Diaminopimelic acid in the cell wall is LL type. The aerial hyphae are linear and form long spore chains. The surface of spores is smooth. As various properties of the culture, the vegetative mycelium has a brown color tone, and the aerial mycelium has a gray or white color tone. Soluble pigments are produced on glucose / nitrate agar and glucose / peptone agar.

From these results, this strain is a strain belonging to the genus Streptomyces, and is classified into Pridham and Tresner (Virgin's Manual of Determinant Bacteriology, 8th Edition, 748 to 829).
Page, 1974) and is considered to be a bacterial species belonging to the gray series or white. In addition, this strain is
Streptomyces SP OH-4156 ( Str
eptomyces sp. OH-4156 has been deposited with the Institute of Microbial Technology, Institute of Industrial Science and Technology. (FE
RM P-12947).

Although the OH-4156-producing strain of the substance has been described above, the general characteristics of actinomycetes are such that the mycological properties are extremely variable and are not constant, and the UV irradiation or UV irradiation which is performed naturally or normally It is a well-known fact that mutation is carried out by means of artificial mutagenesis using X-ray irradiation or a mutagenesis agent such as N-methyl-N-nitrosoguanidine, ethyl methanesulfonate and the like. All strains, including strains, belonging to the genus Streptomyces and having the ability to produce the substance OH-4156 can be used in the present invention.

In the present invention, first, the OH-4156-producing bacterium belonging to the genus Streptomyces is cultured in a medium. In culturing this bacterium, ordinary culturing of actinomycetes is generally used. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and an inorganic salt, if necessary, is used. As the assimilable carbon source, glucose, sugar concentrate, starch, dextrin, cellulose, corn steep liquor, glycerin, organic acid or the like is used alone or in combination. As a digestible nitrogen source, peptone, meat extract, yeast extract, dry yeast, soybean powder, corn staple liquor, cottonseed meal, casein, soybean protein hydrolyzate, amino acids, organic nitrogen sources such as urea, nitrates, Inorganic nitrogen compounds such as ammonium salts are used alone or in combination. In addition, if necessary, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt and phosphate are added.
In addition, if necessary, the medium may contain growth or substance O of this bacterium.
Micronutrients, growth promoting substances, and precursors that promote the production of H-4156 may be added appropriately.

The culture is usually carried out under aerobic conditions such as shaking or aeration-agitation culture. From the industrial point of view, deep aeration stirring culture is preferred. The pH of the culture is preferably around neutral. The culturing temperature may be 20 to 37 ° C, but is usually 24 to 30 ° C (preferably around 27 ° C). The culture time is usually 3 to 6 in the case of liquid culture.
When the culture is continued for a day, the antibiotic is produced and accumulated. The culture may be terminated when the accumulated amount of the substance OH-4652 in the culture reaches the maximum. The culture conditions such as the medium composition, the liquidity of the medium, the culture temperature, the stirring speed, and the aeration amount should be appropriately adjusted and selected so that preferable results can be obtained depending on the type of the strain used and external conditions. Needless to say. In liquid culture, when foaming occurs, a defoaming agent such as silicone oil, vegetable oil, or surfactant can be used as appropriate.

Since the substance OH-4156 accumulated in the culture thus obtained is contained in the culture filtrate and the cultured cells, the substance OH-4156 is obtained from the culture filtrate.
If necessary, a filter aid such as Celite, High Flow Supercell, etc. is added and filtered, or centrifugation is performed to separate the culture filtrate and the cells, and the culture filtrate and the organic solvent extract of the cells are concentrated. OH-4 from a mixture of things
It is advantageous to collect 156. Further, the culture filtrate and the separated product can be directly extracted with a non-hydrophilic organic solvent without separation. Further, when the content of the substance OH-4156 is extremely high in either the culture filtrate or the bacterial cells, it may be extracted from the larger one. Substance from cultured cells OH-4156
In order to separate and purify OH-4156, the substance OH-4156 is usually obtained by extracting a mixture of the culture filtrate and a concentrated organic solvent extract of bacterial cells with a non-hydrophilic solvent such as ethyl acetate or chloroform. It is redissolved in an organic solvent.

The organic solvent layer thus obtained may optionally contain various dehydrating agents such as anhydrous sodium sulfate,
After bead gel or the like is added for dehydration, the organic solvent is distilled off under reduced pressure. In this concentration operation, the substance OH-41
Although 56 is a stable substance, it is preferable that the heating temperature is usually 50 ° C. or lower. The substance OH-4156 can be precipitated by adding an organic solvent such as hexane or petroleum ether to the residue. The obtained precipitate is washed several times with hexane or the like, and then suction filtered or centrifuged to remove the substance O.
The crude product of H-4156 can be collected. In order to further purify the above crude product, many means utilizing the difference in the solubility between the substance OH-4156 and the contaminant, the difference in the distribution of immiscible two-liquid correlation, and the difference in the adsorption force for various adsorption carriers. In particular, chromatography is an effective method for purifying the substance OH-4156.

Chromatography effective for purification of the substance OH-4156 includes adsorption chromatography using silica gel, alumina, activated carbon cellulose, hydroxyapatite HP-20 and other adsorption resins, silanized silica gel, octadecyl silanized silica gel and the like. Reversed-phase partition chromatography used, Sephadex LH-20,
Examples include ion exchange chromatography, which uses gel filtration chromatography, which is based on molecular sieves that use Toyopearl, etc.

The substance OH-4156 can be separated and purified by using these means such as chromatography, electrophoresis, countercurrent distribution, ultrafiltration and the like individually or in combination or repeatedly in any order. . For example, the above composition is dissolved in a small amount of chloroform and adsorbed on silica gel, column chromatography is performed using a chloroform-methanol mixed solution, and the active fraction is concentrated under reduced pressure and dissolved in a small amount of chloroform, which is then chloroform. -The substance OH-4 was obtained by performing gel filtration chromatography based on molecular sieves with a methanol-based mixed solution.
156 can be separated and purified.

Next, the physicochemical and biological properties of this substance OH-4156 will be described. Physicochemical properties (1) Molecular weight; 2096 (according to fast atom bombardment (FAB) mass spectrum) (2) Melting point; 188-193 ° C (3) Specific optical rotation; [α] _D ²⁵ = -70.3 ° (c = 1.
(0, methanol) (4) Ultraviolet absorption spectrum (measured in methanol);
As shown in FIG. 1, 203, 225 (shoulders), 26
It has a maximum absorption at 5 (shoulder) nm. (5) Infrared absorption spectrum (measured by KBr disk method); as shown in FIG. 2, 3450, 3300,
3000, 1660, 1530, 1340, 1260 cm
^-Has a characteristic absorption band near ^-1

(6) Solubility in solvent; sparingly soluble in water, acetone, ethyl acetate, ethyl ether and hexane, soluble in methanol, ethanol, chloroform and benzene (7) Color reaction: positive for sulfuric acid and iodine (8 ) 1H nuclear magnetic resonance spectrum (CDCl ₃ in 30
0MH _z); as (9 shown in FIG. 3) @ 13 C nuclear magnetic resonance spectrum (in CDCl _3, 7
As shown in FIG. 4; 5MH _z)

Biological properties (10) Antibacterial spectrum (Agar dilution method) The minimum inhibitory concentration (MIC) for each test bacterium by the agar dilution method is shown in Table 4 below.

[0025]

[Table 4]

From the above results, the substance OH-4156 exhibits antibacterial activity against Micrococcus rutheus. (11) Cell-killing activity of substance OH-4156 Substance OH-4156 is B16 melanoma (malignant melanoma)
When the cells were allowed to act for 3 days, the IC was 5.4 μg / ml.
₅₀ (50% inhibitory concentration) is shown.

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereby.

Example: Glucose 0.1 was added to a 500 ml Erlenmeyer flask.
%, Starch 2.4%, peptone 0.3%, meat extract 0.3
%, Yeast extract 0.5%, calcium carbonate 0.4%, 100 ml of a liquid medium (pH 7.0) was charged and steam sterilized, and then 1% of starch, NZ amine 0.3%, yeast extract of 0. 1%, meat extract 0.1%, calcium carbonate 0.3
%, Streptomyces sp. OH-4165 cultured at 27 ° C. for 6 days on an agar slant medium containing 2% agar.
One platinum loop was inoculated from the slant culture of (FERM P-12947), and the seed mother was obtained by shaking culture at 27 ° C. for 72 hours on a reciprocal shaker having an amplitude of 17 cm and reciprocating 120 times per minute.

Next, 30 l jar jar amenta was added with 5% maltose, 1.5% dry yeast, 2.5% Ebios and K.
Br 1.0%, KH ₂ PO ₄ 0.05%, MgSO
₄ After sterilizing by charging into 20 l of medium containing 0.05%, 200 ml of the seed obtained by the above method was aseptically transplanted, and 96
About 20 liters of culture was obtained by culturing with aeration and stirring for an hour. The cells were collected from this culture by centrifugation, methanol was added to the cells, and the mixture was stirred well, and then the methanol extract was separated by filtration. The ethyl acetate layer was concentrated under reduced pressure, and the oily residue obtained was adsorbed on a silica gel (Merck) column (50 mm x 250 cm) pre-filled with chloroform and eluted with a chloroform-methanol mixed solvent for chromatography. I went. The obtained active fraction was concentrated under reduced pressure to obtain 142 mg of a crude product containing the substance OH-4156.

The crude product containing the substance OH-4156 was then loaded onto a column (15 cm) packed with Sephadex LH-20.
Column chromatography using 70 cm) was performed. The crude product dissolved in a small amount of methanol was conducted, and the product was developed and eluted with methanol to obtain 93 mg of the crude product containing the substance OH-4156. This is dissolved in a small amount of methanol and subjected to high performance liquid chromatography (Shiseido Capsule Pack C18SG120A, 20 × 250 mm) to give 80
42 mg of a purified product of the substance OH-4156 was obtained by fractionating the active fraction using a% methanol solution as a mobile phase.

[Brief description of drawings]

1 is an ultraviolet absorption spectrum of substance OH-4156. FIG.

FIG. 2 is an infrared absorption spectrum of the substance OH-4156.

FIG. 3 is a 1H nuclear magnetic resonance spectrum of the substance OH-4156.

FIG. 4 is a 13 C nuclear magnetic resonance spectrum of the substance OH-4156.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 465)

Claims (3)

[Claims] 1. A substance OH- having the following physicochemical properties:
4156. (1) Molecular weight: 2096 (according to fast atom bombardment (FAB) mass spectrum) (2) Melting point: 188 to 193 ° C. (3) Specific optical rotation; [α] _D ²⁵ = -70.3 ° (c = 1.
(0, methanol) (4) Ultraviolet absorption spectrum (in ethanol); 20
3, 225 (shoulder), 265 (shoulder) has maximum absorption at nm (5) Infrared absorption spectrum (KBr): 3450, 3
300, 3000, 1660, 1530, 1340, 1
It has a characteristic absorption band near 260 cm ^-1. (6) Solubility in solvent: sparingly soluble in water, acetone, ethyl acetate, ethyl ether, hexane, soluble in methanol, ethanol, chloroform, benzene (7) Color Reaction: Positive for sulfuric acid and iodine reaction 2. A substance OH belonging to the genus Streptomyces
The -4156-producing bacterium is cultivated in a medium, and the substance O is added to the culture.
H-4156 was allowed to accumulate and the material OH-4 from the culture.
OH-4156, characterized by collecting 156
Manufacturing method. 3. A substance OH belonging to the genus Streptomyces
-4156 producing Streptomyces sp. OH
The method according to claim 2, which is -4156 (FERM P-12947).