JPS5818384A - Antibiotic substance and its preparation - Google Patents

Abstract

NEW MATERIAL:The antibiotic substance KA-6643-G of formula, its salt or its ester. USE:Antibiotic substance. PROCESS:A bacterial strain belonging to Streptomyces genus and capable of producing antibiotic substance KA-6643-G (e.g. streptomyces KC-6643-M-162-175 strain; FERM-R No.5890), is cultured under aerobic conditions at 3-35 deg.C, preferably 27-33 deg.C, especially by submerged technique, and the antibiotic substance KA-6643-G of formula can be separated from the cultivation liquid. The compound is stable in the living body and has the folowing physical properties: appearance, white powder; molecular weight (demetermined by mass spectrometry of methyl ester), measured value 342.1224, calculated value 342.1246 (as C15H22N2O5S); molecular formula, C14H19N2O5O.Na; solubility, soluble in water and methanol, insoluble in acetone, ethyl acetate, and chloroform; etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic KA-6643-G or a salt or ester thereof, and a method for producing the same.

The present inventors first cultured Streptomyces KC-6643 strain isolated from soil, and found that the culture solution showed excellent antibacterial activity and β-
Antibiotic KA-6643 with lactamase inhibitory activity
-A substance, KA-6643-B substance, KA-66
The KA-6643-F substance and the KA-6643-F substance were isolated and collected, and a mutant strain thereof, KC-6643M-16, was isolated and collected.
Antibiotic KA-6643-X from the culture solution of strain 2-175
Succeeded in separating and collecting it, and filed a patent application fC.
.

While investigating the above culture solution, the present inventors discovered that Streptomyces KC-6643M-162-
The above antibiotic KA-6643- was found in the metabolites of strain 175.
Discovered the existence of a new moth antibiotic different from X.
We succeeded in isolating and obtaining this, and completed the present invention.

KC-6643 used in the production of antibiotics of the present invention
The strain M-162-175 has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under accession number 5890, and its mycological properties are described in JP-A-55-133980 and %1-31983. It is detailed in the book. In the present invention, not only the strain concerned, but also natural mutant strains and artificial mutant strains thereof can be used.

As is clear from the physicochemical properties and biological properties described below, the new antibiotic of the present invention is the antibiotic KA-6643-
X and different from known antibiotics, antibiotic KA-
It was named 6643-G (hereinafter sometimes referred to as KA-6643-G substance).

A normal method for culturing actinomycetes is used to culture this strain. Yuzu can be used as a carbon source for the culture medium, but starch, glycerin, maltose, dextrin,
It is preferable to use fructose, molasses, etc. alone or in combination, and it is also possible to use hydrogen hydroxides, organic acids, vegetable oils, etc. Nitrogen sources include soybean flour, yeast extract, dried yeast, veptin, cornstarch extract, cornstarch liquor, Casamino version,
Tastyler's Solyupur, ammonium chloride, ammonium chloride, urea, sodium nitrate, etc. can be used alone or in combination. Other prizes, as required, include inorganic salts such as potassium phosphate, sodium phosphate, magnesium sulfate, calcium chloride, calcium carbonate, water, northern lucium, cobalt chloride, zinc sulfate, iron chloride, iron sulfate, and trace amounts of metals. can be added to the sister. Furthermore, organic and inorganic '4'a substances, such as sulfur-containing amino acids such as methionine, cysteine, and cystine, which help the growth of KA-6643-G and promote the production of the KA-6643-G substance, can be added as appropriate. When using the strained and aerated culture method, it is preferable to add an antifoaming agent such as fatty oil, silicone oil, paraffin, etc. to the heating layer.

Regarding the culture method, although culture on a solid medium is also effective, it is preferable to use a liquid culture method, especially a deep culture method, as in the general antibiotic production method. Cultivation is carried out under aerobic conditions at 3-35°C, preferably at 27-33°C.

1 (A-6643 substance production is performed using shaking culture, tank j@
Any of the nutrients may be used, and the hydrating substance can be completely accumulated in the culture solution by culturing at 1'iJ for 2 to 6 days. When the production level in the culture solution reaches a critical level, the culture is stopped, and the antibiotic of interest is isolated from the culture solution.

As a method for isolating the KA-6643-G substance from the culture solution obtained in this way, various known methods that can be used in such cases can be adopted. Centrifuge all of the culture medium without vomiting.Z:
The cells are removed by IAi treatment, and the culture 21-I solution is subjected to any combination of the following treatments. That is, the culture P solution is passed through a basic ion exchange resin to adsorb the KA-6643 substance, and then the KA-6643 substance is leached out with a salt bath or an aqueous methanol bath of salt. Examples of side fat include DOWEX 1x2 (manufactured by Dow Chemical Company), DIAION PA-318, 316,
306, 308, 312 (manufactured by Mitsubishi Chemical Corporation), Ryonnoku-
Strongly basic anion exchange resins such as Lite IRA-400, 401, 410 (manufactured by Rohm and Haas); Medium basic anion exchange resins such as Amberlite IRA-68; DEAE-Sephadex, LIEAE-
A weakly basic anionic father exchange resin such as cellulose is used. Next, the above culture solution F was adsorbed onto an adsorbent such as activated carbon, and then K was added with aqueous methanol, aqueous acetone, etc.
The A-6643 substance can then be eluted by passing the active fraction through a column of strongly basic ion exchanger resin, such as Dowex 1×2.
After the three substances are adsorbed, they are eluted in stages by gradually changing the salt content in the phosphorous buffer.

In addition, the above active category is intermediate polarity -! or non-polar hydrophobic cross-linked polymers, such as Amberlite XAD-2,
The KA-6643 substance can also be separated and collected by passing it through a column packed with Diaion HP-20, DEAE-Sephadex A-25 (manufactured by Almasia), etc.The KA-6643vI substance thus obtained is , and also Biogel I)-2 (manufactured by Bio-Rad), Sephadex G
Gel Δ1 using a gel such as -10 (manufactured by Pharmacia)
Bonda Pack C1a (manufactured by Waterus)
KA-6643-X substance and KA-6 by subjecting to reverse phase chromatography using a column packed with
643-G substance.

This KA-6643-G substance can be converted into alkali metal salts, alkaline earth 6-metal salts, primary, secondary, or tertiary amine salts, or quaternary ammonium salts or esters by a suitable method as required. can do.

The KA-6643-G substance of the present invention has the following physicochemical and biological properties, and is stable in vivo.

Physical and chemical properties of I KA-6643-G substance sodium salt (1) Appearance: White powder (2) Molecule f: Mass spectrum value of methyl ester Actual value: 342.1224 Calculated value = 342.1246 (Cx5H++zNzOs
(3) Molecular formula: C14HtgN105S-
C14Ht ultraviolet absorption spectrum: The ultraviolet absorption spectrum of the aqueous solution is as shown in FIG.

(5) Infrared absorption spectrum: The infrared absorption spectrum of the pellets in beautified potassium is as shown in Figure 2.

Main peak (6J 1H-NMR spectrum) The IH-NMR spectrum measured at 1001eIz in heavy water is shown in Figure 3.

Main peak δD20 ppHla 1.78 (3H, s, C-Ckls) 1.83
(3) i, s-'C-Ckb)2.25(3
H, s, COO mountain) -11- Cellulose plate (manufactured by Merck & Co.) Solvent system used
Rf value (8) High performance liquid chromatography: Column: Radialback A (Waters Inc. ff) OJ3X10c1n solvent system Flow rate Retention time (9) Solubility Soluble in water and methanol; insoluble in acetone, ethyl acetate, and chloroform.

CIQ Color reaction positive: Ehrlich reaction negative: Ninhydrin reaction l KA-6643-G substance) Biological properties of IJum salt (1) Antibacterial activity of this compound Staphylococcus aureus 209
The minimum inhibition line for PJC-1 was 6mcP/-.

(2) Enhancement of antibacterial activity against β-lactam resistant bacteria Aminobenzylpenicillin (ABPC) 500μF/
β-
Escherichia coli ML-1410REC-1m'e, a lactamase (TEMu) producing bacterium, was inoculated, and 10ml of this
The mixture was poured into a petri dish with a diameter of 9crIl and stirred, and this was used as a test medium. KA-6643- of each concentration was added to this agar medium.
Material G) Pulp disks with a diameter of 8 mm each coated with 30 μt of IJum salt aqueous solution were placed, and after culturing at 37° C. for 18 hours, the size of the inhibition circle on the side of the disk was measured. The results are shown in the table below.

Table (3) Stability Decomposition rate of dihydrobebutigase extracted and purified from pig kidneys for KA-6643-A substance 1
, 6643-G substance was 2.5×10 −3 .

Based on the above properties, the KA-6643-G substance is
i is estimated to have the formula (1).

(1) Next, I will explain it with an example.

Example Corn starch 3.6%, soybean flour 2.5%, cottonseed 1.
5%, magnesium sulfate heptahydrate 0.05%, L-
cysteine 0.02%, potassium phosphate 0.28%,
Liquid medium 30 with the composition of sodium diphosphate 12 hydrate 0.18%, cobalt chloride 6 hydrate 0.000515-1 KM-700, 1%
d into a 500 m/volume Mayer and sterilized. In this medium, Streptomyces sp.

KC-6643M-162-175 is inoculated and cultured with shaking at 30°C for 3 days to obtain the first type of bacteria.

Two 20 tV Jar Fermentors were charged with 10 tons of the medium having the above composition, and the first type bacteria were transplanted into each.
t/min, internal pressure 0.5 K9/cm”)
Culture for 1 day.

After the culture is completed, the culture solution is centrifuged at 5.00 Orpm to obtain 13 tons of centrifuged supernatant.

A column (6X
After washing with water, the supernatant was passed through a 0.05 MIJ acid buffer (pH 8.45 cm) containing 20% sodium chloride.
, 6) and collect the active fraction. This active category 4t
0.05 phosphate buffer containing 20% sodium chloride (pH
It was adsorbed onto a column (4.5 x 50 cm) packed with Diaion HP-20 which had been washed with 0.8.
After washing with 1 t of 05M phosphoric acid buffer pHs, o), it is eluted with dilute ammonia water, pH 8.0, and the active section is stained. The active fraction (1 t) was diluted with deionized water to a conductivity of 3 mΩ, purified with 1 t of methylene chloride containing 1% benzylmethylcetylammonium chloride, and pre-buffered to pH 8.0. Passed through 2 x columns (3.5 x 40 m) and the salt concentration was 0 and 0.3.
0.05 M IJ acid buffer (pH 8,
0) Bathe by concentration gradient method for each 1t. Collect the active fraction 400-, add 0.05M phosphate buffer (pH 8,0) 400- containing 20% sodium chloride, and add 400-
A column (2X
45 cm). Then, 0.05 MIJ acid buffer containing 10% sodium chloride (pH 8,0) 15
After washing with 0 ml, elute with dilute ammonia water of pH 8.0,
The active fraction is collected and pulverized to about 21 nt under reduced pressure at below 30°C. The condensate was adsorbed on a column (2.2 x 90 cm) packed with Sephadex G-10, and the pH was adjusted to 8.
, 0 dilute ammonia water. The active fractions are collected and, after dashing, lyophilized to yield 50.4 mg of a pale yellow solid.

The above-mentioned facepiece was bath-dissolved in 0.2 M IJ acid buffer 2- of pi (7,8), and then placed on a column (O, SX 120 cm) of Bondabac C18/Porasil B (manufactured by Waters).
buffered gx at a flow rate of 6 td/min, KA at 5 t.
After bathing out the -6643-X substance, add 400 ml of deionized water.
The fraction eluted with C deionized water is dyed, dried under reduced pressure, and then lyophilized to yield 2.5 ml of KA-6643-G'ltJ salt.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the sodium salt of the substance KA-6643-G, Figure 2 shows the infrared absorption spectrum of the same substance 19-, and Figure 3 shows the "H-N" spectrum of the same substance.
This is an MR spectrum. Above 1 person Kowa Co., Ltd. 120-5 Director % La 2 Figure 6 5 4 3
Z L OWW Continued from page 1 7z Inventor: Hisakatsu Ito, 461 Imafuku, Kawagoe City, 72) Inventor: Toshito Mori, 4-16-3 Fujimi-cho, Higashimurayama City, 753-

Claims (1)

[Scope of Claims] 1. Antibiotic KA-6643-G, a salt thereof, or an ester thereof, represented by the following formula (1) or (1). 2. Antibiotic I belonging to the genus Streptomyces KA-6
643-G producing bacteria are cultured, and antibiotic K is extracted from the culture solution.
A method for producing antibiotic KA-6643-G, which comprises collecting A-6643-G;