JPS6050440B2 - New ribostamycin manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for producing ribostamycin using bacteria belonging to the genus Bacillus.

Ribostamycin is an aminoglycoside antibiotic.
tomycesrilx sidifecus) (TheJoumalo
fAntibiotics, Vol. 23, p. 155 (1970), Special Publication No. 1715-1970).

The present inventor has already discovered that the same antibiotic can be produced using bacteria of the genus Bacillus (Patent Publication No. 1983-
879). However, in the invention, xylostacin may be produced together with ribostamycin, so
The present invention has now been completed by further improving the invention and obtaining a bacterium belonging to the genus Bacillus that produces only ribostamycin. The bacteria used in the present invention is a strain obtained by mutating B15M-30 strain, which the present inventors isolated from soil, and further subjected to mutation treatment with nitrosoguanidine, which will be described later. It has certain characteristics.

These properties are almost identical to the parent strain B15M-306, and while the parent strain can produce xylostacin together with ribostamycin and has spore-forming ability, this mutant strain B15M-451 differs only in that it produces only ribostamycin and lacks sporulation ability. Mycological properties (a) Morphological properties (on broth agar slant) It is a bacillus, 0.6 to 0.8 x 2 to 5 microns in size, 1 to 2 speeds connected, and motile.

Gram stainability is indeterminate.

(b) Characteristics on various media (1) Broth agar plate culture Amorphous, diffusive, smooth and glossy, wavy and flat from all edges, translucent, no diffusive pigments, bacterial cells pale yellow.

(2) Growth on broth agar slant culture Normal, diffusive, glossy, translucent, flattened, oily, no diffusible pigment, bacterial cells pale yellow.

(3) No growth on the liquid culture surface of meat juice, uniform turbidity.

(4) Soybean agar slope Growth is normal, and the bacterial body is pale yellow.

(c) Physiological properties (1) Methyl red test positive (2) Fouges-Proskauer reaction negative (3) Indole formation negative (4) Hydrogen sulfide formation negative (5) Starch hydrolysis positive (weak) (6 ) Utilization of ammonium salts Positive (7) Production of pigment Water-insoluble yellow pigment (8) Production of catalase Positive (9) Deamination of phenylalanine Negative (degradation of tyrosine 11 Negative (11) Attitude towards oxygen Facultative anaerobic ( 12) 0-F test Oxidative: acid production positive, gas production negative Fermentative: acid production positive, gas production negative (13) Growth salt concentration range 3% positive, 5% positive, 7% negative (d ) Generation of acids and gases from sugars 15M-3 strain (spore-bearing) and Bl5M-4.51
Both strains are facultative anaerobic rods and are positive for catalase production, so they belong to the genus Bacillus.

The shape and position of the spores, the shape of the sporangium, the fact that it produces acid from glycose and does not produce gas or asotoin, etc.
) is thought to be a closely related species. Virgie's Manual
Obdeterminative bacteriology (Ber?
y′SMManualOfDeterminativeB
Compared to the properties of Bacillus circulans described in the 8th edition of Bacillus circulans, it differs in that it does not produce acid from arabinose and xylose, but it also hydrolyzes starch, does not produce indole, and deaminates phenylalanine. They agree in that they do not decompose tyrosine, and they are highly motile, with the exception of the fact that they produce a water-insoluble yellow pigment. We agree that

Therefore, the B15M-451 strain was identified as a strain belonging to Bacillus circulans. In addition, Bl5M-45
One strain is Bacillus circulans Bl5M-451 (B
aclllL] Circulans Bl5M-451
) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 432 (G). Bl5M-
The method for obtaining the mutant strain B15M-451 from Tripartite is as follows.

Nitrosoguanidine was added to the cell suspension of the B15M-3 strain to a final concentration of 500 ml, and the mixture was shaken at 3rC for 1 hour.

After centrifugation, the cells are cultured at 37°C for 3 hours, diluted, and spread on an agar medium to obtain colonies. From the obtained colonies, Proteus vulgaris, which is sensitive to both ribostamycin and xylostacin, was detected.
and Serratia marcetuscens (Serratia marcetuscens), which is sensitive to ribos7tamycin but resistant to xylostacin.
latiamarcescens) to select colonies that do not have the ability to produce xylostacin but have the ability to produce ribostamycin. In the present invention, the above-mentioned Bacillus
Circulans Bl5IS/1-451 strain and its natural or artificial mutants can of course be used.

Next, the method for producing ribostamycin according to the present invention will be described.

Bacillus circulans Bl5M-451 is cultured under aerobic conditions in a medium containing various nutrients.

The culture conditions and medium composition may be similar to those commonly used in the production of antibiotics. That is, the medium basically contains a carbon source, a nitrogen source, and an inorganic salt.
Vitamins, precursors, etc. may also be added. As a carbon source, for example, glucose, mannose, sucrose, starch, dextrin, glycerin, mannitol, organic acid, molasses, potato, etc. are used alone or in a mixture, and as a nitrogen source, for example, pebtone, soybean flour, corn flour, etc. are used.
Steep liquor, yeast extract, amino sugar, rice sugar,
Wheat skin, urea, ammonium sulfate, ammonium nitrate, etc. are used in appropriate combinations or alone. Inorganic salts include calcium carbonate, sodium chloride, potassium chloride,
Examples include magnesium sulfate and other phosphates, which are added to the medium as necessary. The medium is preferably a liquid medium, and in the case of mass production, deep aeration culture is preferable.

The pH of the medium is preferably about 5.5 to 8.5, and the culture temperature is preferably adjusted to about 20 to about 40°C. Furthermore, an antifoaming agent may be added as appropriate before or during culturing, if necessary. After completion of the culture, ribostamycin may be collected from the culture in accordance with a conventional method for separating and collecting fermentation products from the culture. For example, the desired ribostamycin can be obtained by appropriately combining extraction methods using various organic solvents, chromatography using various active adsorbents, and the like. In the present invention, the bacterium Bacillus circulans Bl5M-451 used produces only ribostamycin, and no concomitant substances such as xylostacin are observed, so the operation for separating the target antibiotic ribostamycin from the culture is simplified. and high purity ribostamycin can be obtained.

The obtained ribostamycin may be converted into an acid addition salt, for example, a hydrochloride, a sulfate, an oxalate, a succinate, etc., according to a conventional method. Next, in Examples, embodiments of the method of the present invention will be shown, but these Examples are not intended to limit the present invention in any way.

Example a Medium (for seed culture) Soiton (manufactured by Difco) 1
．． 5%, sodium chloride 0.3%, agar 1.5%, water (
PH not adjusted).

b medium (for seed culture): glycerin 1.0%, soyton (
Manufactured by Difco) 1.5%, magnesium sulfate 0.3%,
Water (no PH adjustment).

c medium (for fermentation): 2.0% cornstarch, 3% soybean flour
．． 0%, magnesium sulfate 0.3%, calcium carbonate 1
．． 0%, water (PH not adjusted).

Dispense medium a into test tubes and add Bacillus circulans B.
l5r! Inoculum culture of 4-451 (FERM NO. 432O) is carried out at 28°C for 2 batches.

A 2000 ml Ellenmeyer flask containing 800 ml of B medium was inoculated with 1 test tube of the seed culture and heated at 28°C.
Perform rotary shaking culture between two plates.

The obtained culture solution was inoculated into a 30e capacity strainer containing 20' of c medium, and polypropylene glycol (P-2000
) and culture with aeration at 28°C ± 0.5°C for 4 days (aeration rate 201/min, pressure 0.5k91d1).
Stirring 200-350r'Pm. ．． pH 7.5 (automatic adjustment)).

After adding 15y of oxalic acid to a portion 7' of the thus obtained culture solution (titer: 795mlcg/M9 as ribostamycin sulfate), diluted sulfuric acid was added to adjust the pH to 4. Adjust the temperature to 0.05 °C and filter. Adjust the solution to pH 7 with dilute ammonia water. 0 and then applied to a 400 ml column of Amberite IRC-50 (NH type). Elute with 1N ammonia water, apply to Bacillus subtilis, collect fractions showing antibacterial activity, and concentrate under reduced pressure.

This concentrated liquid was added to Amberlite CG-50 (NHZ type) 17
Subjected to 0mt column chromatography. Elute with 0.3N aqueous ammonia, collect active fractions, and concentrate under reduced pressure.

Claims (1)

[Claims] 1. A novel method for producing ribostamycin, which comprises culturing Bacillus circulans B-15M-451 in a medium and collecting ribostamycin from the resulting culture.