JPS63107993A - Antibiotic substance tm-611 - Google Patents

Abstract

NEW MATERIAL:Antibiotic substance TM-611 exhibiting the following physical and chemical properties as Na salt. Appearance, white acicular crystal; m.p., 203-205 deg.C; elemental analysis (%), C 62.97, H 8.69, Na 3.16; molecular weight, FABMS m/z 705 (M+H)<+>; molecular formula, C37H61O11Na; [alpha]D<27>=+29.2 deg. (c=0.5, CHCl3); solubility, insoluble in water and soluble in methanol, ethanol, acetone, ethyl acetate, benzene, chloroform and n-hexane; color reaction, negative to ninhydrin and FeCl3 reaction and positive to vanillin-sulfuric acid and iodine reaction; nature, weakly acidic in isolated state; etc. USE:An antibiotic substance exhibiting proliferation-inhibiting action against Gram-positive bacteria. Useful as pharmaceuticals, agricultural chemicals and feed additives. PREPARATION:Streptoverticillium olivoreticuli TM-611 (FERM P-8972) is cultured under aerobic condition.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to antibiotics.

(Prior Art) The antibiotic of the present invention is a new substance, and the existence of a substance having the same physicochemical properties has not been reported to date.

(Problems to be Solved by the Invention) An object of the present invention is to provide a novel antibiotic that exhibits a growth-inhibitory effect on Gram-positive bacteria.

(Means for Solving the Problems) The strain producing the antibiotic of the present invention is a strain newly isolated by the present inventors from the soil of Sendai City, Miyagi Prefecture. Oripore Chikuri (
5treptoverticil'lium ollv
orticuli) TM-611J and microorganism deposit number ``FEBM P-89
72) The antibiotic of the present invention obtained by culturing this strain was named TM-6M.

The mycological properties of this strain are shown below.

1) Morphotrophic hyphae are well developed and irregularly branched on synthetic and natural agar media. Also, no bulkheads are allowed. Spores are moderately formed on sucrose/nitrate agar, starch agar, and yeast/malt agar.

When observed under a microscope, the branching method of spore-forming mycelium is primary or secondary axle branching, and the spores are formed in a linear chain. Spores usually have a chain of 10 or more spores and have a smooth surface.

The shape of the spore is oval, and its size is Q, 57-α55.
XO, 58-0, 98p. sclerotia, spore paste,
No flagellar spores are observed.

2) Growth status on culture media Table 1 shows the results of macroscopic observation when cultured on various media at 30°C for 14 days.

Table 1 Growth status 3) Physiological properties (1) Growth temperature range Grows well on oatmeal agar medium in the range of 25-37°C. It does not grow at temperatures below 10°C and above 45°C.

(2) Biochemical properties a) Distinction between aerobic and anaerobic: Aerobic b) Liquefaction of gelatin: Positive C) Coagulation of skim milk: Negative d) Peptonization of skim milk: Positive e) Hydrolysis of starch: Positive f) Melanin-like pigment production: Positive (3) Use of carbon source (on Pridham-Godreep agar medium): D-
Glucose, D-fructose, inositol, galactose.

starch.

Slightly used: Seaucrose, L-rhamnose,
D-mannit.

Not used: L-arabinose, D-xylose, raffinose.

From the above properties, it is clear that this strain belongs to actinomycetes, and based on the above properties, we have published the book "Manual of Determinative Bacteria" by Virgil. As a result of comparison with many known bacterial species reported in ``The Actinomycetes'' by Waxman, 8th edition (1974) and ``The Actinomycetes'' by Waxman, Volume 2 (1961), this strain was found to be S. It showed the properties closest to those of the 11 voreticua.

From the above results, this strain is Streptparticillium
This strain was determined to be the same species as Streptocarticillium oliporeticuli TM-
It was named 61j.

The production of antibiotic TM-611 is roughly similar to the production of general fermentation products, and TM-611 is produced in a medium containing various nutritional substances.
-6'11 strain is cultured under aerobic conditions.

The medium is mainly a liquid medium, and the carbon source is glucose, molasses, starch, etc., either alone or in combination. As the nitrogen source, meat extract, oatmeal, yeast extract, soybean flour, polypeptone, etc. may be used alone or in combination.

In addition, organic substances and inorganic salts that aid the growth of this strain and promote the production of antibiotic TM-611 can be appropriately added. As the antifoaming agent, commonly used antifoaming agents such as octyl alcohol, n-butyl phosphate, and silicone can be used.

As for the culture method, aerobic culture such as shaking culture or aeration agitation culture is suitable, and the culture is carried out at pH 4-8, 25-35°C for 3-6 days, preferably p)46-7.28-30°C for 4 days. .

The antibiotic TM-611 produced by this culture can be isolated according to a general method for collecting fermentation products. Since the antibiotic TM-611 is mainly accumulated within bacterial cells, the following method is effective, for example. That is, after the completion of culture, bacterial cells isolated by centrifugation or filtration are extracted with antibiotic TM-611 using an organic solvent such as low R alcohol or acetone, and this extract is concentrated and then extracted with an organic solvent such as ethyl acetate, benzene, or chloroform. Transfer to a water-soluble organic solvent and concentrate to form a syrup.

Add this syrup again to benzene, ethyl acetate, acetone,
Column chromatography using silica gel (Wako Tsumugi, Wako Gel C-200) and Sephadex LH-20 (trade name,
The antibiotic TM-611 can be purified and isolated by filtration using gel v5 (manufactured by Pharmacia).

The antibiotic TM-611 isolated by the above purification method has the following physical and chemical properties.

Physical and chemical properties a) Appearance White needle-like crystals b) m, p, 203-205°C C) Elemental analysis C: 62.97% H: a69% Na: 3.16% d) Molecular weight FABMS: m/z 705 (M+
H) + e) Molecular formula C37H61011Naf) Specific optical rotation [α)'D' = + 2 q, 2° (c =
0.5. CHC4) g) Ultraviolet absorption spectrum Terminal absorption h) Infrared absorption spectrum The results of measurements in KBr tablets are shown in FIG.

i) la-NMR spectrum The results measured at 400 MHz in cvctB are shown in FIG.

j) +3c-N MR spectrum The results measured at 100 MHz in cDct3 are shown in FIG.

k) Solubility in solvents Insoluble in water, methanol, ethanol.

Acetone, ethyl acetate, benzene, Soluble in loloform and n-hexane.

t) Color reaction Ninhydrin, ferric chloride reaction: Negative Vanillin-sulfuric acid, iodine reaction: Positive m) Distinction between basic, acidic and neutral: The free form is weakly acidic.

No antibiotics having properties consistent with the above-mentioned physicochemical properties have been reported, and antibiotic TM-611 is considered to be a new antibiotic.

In addition, polyether antibiotics such as laidromycin, alpolixin, and X206 can be mentioned as substances relatively similar to the antibiotic TM-611; M.R.
The spectrum and molecular weight are clearly different from those of the antibiotic TM-611.

(Action) Antibiotic TM-611 inhibits the growth of Gram-positive bacteria.
Has one for production.

Hereinafter, the action of antibiotic TM-611 will be specifically explained with reference to test examples.

Test example: According to the Japanese Society of Chemotherapy method, antibiotic TM-6 is used for bacteria.
11, using a 1fc sensitive disc agar medium, and for molds and yeasts, using a similarly treated Sabouraud agar medium.
C (minimum inhibitory concentration) was measured.

The results are shown in Table 2.

Table 2 Antibacterial action (effects of the invention) The antibiotic TM-611 of the present invention is a novel polyether antibiotic that has the above-mentioned properties and has a growth-inhibiting action against Gram-positive bacteria, and is a pharmaceutical.

Useful as a pesticide and feed additive. In addition, it can be used as a free form, potassium salt, ammonium salt, calcium salt, etc., if necessary.

(Example) The present invention will be specifically described below with reference to Examples.

Example (1) 2% by weight glucose, 2% by weight oatmeal, C13% by weight meat extract, 0.3% by weight salt, α2
Strain TM-611 was inoculated into a pH 7 sterile liquid medium containing 5% by weight of calcium carbonate, 0.04% by weight of ferric sulfate, and 4% by weight of manganese chloride, and cultured with shaking at 30°C for 72 hours. It was used as a culture solution.

Next, using a jar fermenter with a capacity of 5 tons, the seed culture solution 30- was inoculated into 3 tons of a sterile medium having the same composition as the seed culture, and cultured with stirring at 30° C. for 96 hours. After the culture was completed, 6 tons of the two plants were separated into supernatant and bacterial cells using a centrifuge.

400 microbial cells obtained were extracted twice with 1 t of acetone, and the extracts were combined and concentrated to remove acetone. The resulting aqueous solution was extracted twice with an equal amount of benzene, and the benzene fractions were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a brown syrup &42.

Conosyrup was dissolved in 10-benzene and adsorbed onto a 300-gluon of silica gel [Wakogel C-200 (trade name, Wako Pure Yakuri)] prepared by Hensen. benzene 6
After washing with 00ml, elute with a mixed solvent of benzene-acetone (90:10), fractionate into 151 fractions using a fraction collector, collect fractions up to N15S-47, and concentrate to dryness to obtain 230μ of a coarse powder. Obtained.

(2) Silica gel [Lycium 6 Gel C-200 (trade name, Wako Pure Chemical Industries, Ltd.) prepared by dissolving the crude powder obtained in the previous section 1 in chloroform and then using a mixed solvent of n-hexane-acetone (90:10) 1001n1. It was adsorbed onto the column.

Elute with the same mixed solvent used when preparing the column, and use a fraction collector to fractionate 1 fraction into 7 and 52 fractions.
Classifications up to la22-104 were collected.

After concentrating this fraction to dryness and converting it into a sodium salt in the usual manner, it was further dissolved in acetone and Sephadex LH-20 (
Gel filtration was performed with acetone using acetone (trade name; manufactured by Pharmacia), and the obtained active fractions were collected and concentrated to dryness.

This was dissolved again in a small amount of acetone and crystallized to obtain 93 white needle-like crystals of antibiotic TM-611.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of antibiotic TM-611 measured with KBr tablets, Figure 2 shows the infrared absorption spectrum of antibiotic TM-611 measured in CDCta at 400M
1H-N M of antibiotic TM-611 measured in Hz
R spectrum, Figure 3 is C! 100 MH in DOts
13C of antibiotic TM-611 measured by z! -N
MR spectrum is shown.

Claims (1)

[Claims] Antibiotic TM-611 having the following physical and chemical properties as a sodium salt. a) Appearance White needle-like crystals b) m. p. 203-205℃ c) Elemental analysis value C: 62.97% H: 8.69%
Na: 3.16% d) Molecular weight FABMS: m/z 705 (M+H)^
+e) Molecular formula C_3_7H_6_1O_1_1Naf
) Specific optical rotation [α] ^2^7_D = +29.2° (c = 0
．． 5. CHCl_3) g) Ultraviolet absorption spectrum Terminal absorption h) Infrared absorption spectrum The results of measurements in KBr tablets are shown in FIG. i)^1H-NMR spectrum The results measured at 400 MHz in CDCl_3 are shown in FIG. j)^1^3C-NMR spectrum The results measured at 100 MHz in CDCl_3 are shown in Figure 3. k) Solubility in solvents Insoluble in water, soluble in methanol, ethanol, acetone, ethyl acetate, benzene, chloroform, n-hexane. l) Color reaction Ninhydrin, ferric chloride reaction: Negative Vanillin-sulfuric acid, iodine reaction: Positive m) Distinction between basic, acidic and neutral: The free form is weakly acidic.