JPS6123996B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing the antibiotic sisomicin. Micromonospora inyoensis (Special Publication No. 49-1559
Micromonospora grisea (U.S. Patent No. 4009328), Micromonospora grisea
48-52991)], Micromonospora jionensis (Japanese Patent Application Laid-open No. 1973-
55895, U.S. Patent No. 4001209)], Micromonospora purpurea var. nigrescens
(Hungarian Patent No. 168778, J.Antibiot.30:
945 (1977)] was known. As described above, all of the bacteria producing the antibiotic sisomicin belong to the genus Micromonospora, and their morphological characteristic is that they form one spore each on the basal hyphae. Belongs to the family Micromonosporaceae (Bergeys manual of determinative
bacteriology 8th edition (1974)). The present inventors discovered that Streptomyces G367, isolated from field soil in Fuji City, Shizuoka Prefecture, produces the antibiotic sisomicin.
belongs to the genus Dactylosporangium, and its morphological characteristics are that it attaches sporangia to the basal hyphae and forms spores with flagella inside the sporangium; The genus Sporandium belongs to the family Actinoplanaceae, and it is a new producer of the antibiotic sisomicin, which is taxonomically distinct from the conventional genus Micromonospora. I found it. The characteristics of the actinomycete G367 on various media based on macroscopic and microscopic observations were as follows. [] Morphological properties Calcium malate agar medium [Bact.
Rev. 21:1 (197)] After culturing at 30°C for 3-7 days, the following findings were observed. The basal hyphae are curved or bent, branched and elongated, do not divide, and have a diameter of 0.5-0.8μ, and do not form aerial hyphae. Spherical or ellipsoidal objects measuring 1.5 to 2.0 x 2.0 to 2.5 microns are observed on the basal hyphae, buried in the agar medium. It produces sporangial stalks that are shorter than the basal hyphae, and the sporangia are finger-shaped and are formed singly or in clusters on the surface of the agar medium. The size of the sporangium is 1.0~
It measures 1.5 x 4.0 to 6.5μ, and contains 3 to 4 spores arranged vertically in a row. The spores are motile in water, spherical, oval, or pear-shaped, and 1.0 to 1.5 x 1.5 in size.
~2.5μ, with polar, tufted flagella. [] Diaminopimelic acid composition Diaminopimelic acid determined by whole cell analysis detected meso type and slow moving diaminopimelic acid with a lower Rm value than meso type. [] Growth status on various media, etc. Cultured on various media at 30℃ for 14 days, and the observed findings are as shown in the table below. A few undeveloped aerial mycelia were formed on oatmeal agar media. No formation of aerial mycelium was observed, and sporangia were good on calcium malate agar medium, and soil agar medium [J.Gen Microbiol.50:295
(1968)] and to a moderate extent on other media, and little or almost none on other media. The color display is based on the Color Harmony Manual, 4th edition 1958.
This is according to the color classification by Container Corporation of America.

【table】

【table】 [] Physiological properties Physiological properties are as follows. 1 Assimilation of carbon sources

【table】

【table】

[Table] 2 Growth temperature range: 20-40℃ 3 Skimmed milk: Positive with peptonization and coagulation 4 Production of melanin-like pigment: Negative (on tyrosine and peptone yeast extract iron agar medium) 5 Hydrolysis of starch: Positive 6 Cellulose decomposition: Negative 7 Casein decomposition: Positive 8 Tyrosine decomposition: Negative 9 Gelatin liquefaction: Positive 10 Hydrogen sulfide generation: Weak positive 11 Nitrate reduction: Positive 12 Growth pH: PH5.5-9.0 As above The characteristics of this fungus G367 are that it grows finger-shaped sporangia on the basal hyphae, the spores are arranged vertically in a single row, and the spores have tufted flagella. It is in. In this way, spores that form sporangia and have flagella inside them belong to the family Actinoplanaceae, and the sporangium is finger-shaped and there are upright spores inside. Those in which spores are formed in a row belong to the genus Dactyrosporandium. Furthermore, this fungus G367 has the characteristic that the basal hyphae exhibit an orange-brown color and produce a brown soluble pigment on an organic medium.
thailandense) [Acrch.Microbiol.58:42-52
(1967)]. Therefore, this bacterium G367 was named Dactyrosporandium thailandense G367, and this bacterium was applied to the Institute of Microbial Technology, Agency of Industrial Science and Technology as "Feikoken Bacteria Entrustment No. 4840". It was hot. The present invention has been completed based on the above findings, and is characterized in that an antibiotic sisomicin-producing bacterium belonging to the genus Dactyrosporandium is cultured in a medium, and the antibiotic sisomicin is collected from the culture. This is a manufacturing method. Next, the antibiotic sisomicin of the present invention (hereinafter referred to as
For example, when producing sisomicin (called sisomicin), the above-mentioned sisomicin-producing bacteria belonging to the genus Dactylosporandium are aerobically cultured in a medium containing medium components used for culturing ordinary microorganisms. can get. As the medium, a solid medium or a liquid medium can be used, and a liquid medium, especially an aqueous medium, is suitable especially for mass production. As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, maltose, starch, dextrin, and molasses.
The nitrogen source may be any available nitrogen compound, such as corn steep liquor, soy flour,
Cottonseed flour, wheat gluten, peptone, meat extract, yeast extract, casein hydrolyzate, ammonium salts, nitrates, etc. are used. In addition, salts such as phosphate, magnesium, calcium, potassium, sodium, cobalt, iron, and manganese are used as necessary. The culture temperature can be changed as appropriate within the range that allows the bacteria to grow and produce shisomycin, but is particularly preferably 25 to 35
It is ℃. The culture time varies somewhat depending on the conditions, but is usually about 100 to 200 hours, and the culture may be terminated at an appropriate time by determining the time when sisomicin reaches its maximum titer. In the thus obtained liquid culture of the sisomicin-producing bacteria, most of the sisomicin is produced in the liquid portion. Next, sisomicin is collected from the culture of this sisomicin-producing bacterium, and it is convenient to separate and purify it by taking advantage of the fact that sisomicin is a water-soluble basic amino sugar compound. Also, the produced sisomicin is Bacillus subtilis PCI219
As the test bacteria, the active category was confirmed and quantified using the usual agar plate method. An example of a means for separating and purifying sisomicin is as follows. In other words, a culture solution is obtained by culturing sisomicin-producing bacteria as described above and removing the solid content from the resulting culture, but since sisomicin is an amino sugar compound, the pH of the culture is first adjusted to acidic. , it is preferable to neutralize and filter this to obtain a culture solution, and then apply this culture solution to a cation exchange resin such as Amberlite IRC-50.
The active substance is charged to a column of (NH4 ⁺ type) and adsorbed, and the active substance is eluted with 2N ammonia water.The eluate is further concentrated, the pH is adjusted, and a cation exchange resin such as CM-Sephadex is used. Charge and adsorb onto a C-25 (NH4 ⁺ type) column, elute with aqueous ammonia with a concentration gradient of 0 to 0.35N to obtain the active fraction, concentrate under reduced pressure, and freeze-dry. A purified white powder of Sisomicin is thereby obtained in the free base form. Furthermore, the sisomicin obtained in this manner can easily be one that shows a single spot in thin layer chromatography. The physicochemical properties of the thus obtained sisomicin of the present invention are as follows, and are consistent with sisomicin, a known compound produced by a known sisomicin-producing bacterium belonging to the genus Micromonospora. (1) Molecular weight 447 (from mass spectrum) (2) Elemental analysis C = 49.39%, H = 8.03%, N = 14.75% (3) Specific optical rotation [α] ²⁵ _D = +184° (C = 0.3, H ₂ O) (4) Ultraviolet absorption spectrum: There is no absorption maximum in the region of 210 to 400 mm. (5) Solubility Easily soluble in water, slightly soluble in methanol, slightly soluble in chloroform, insoluble in ethyl acetate, acetone, and benzene. (6) Color reaction Remieux reaction, ninhydrin reaction + Sakaguchi reaction, ferrous chloride reaction, Moritsch reaction (7) R value: Silica gel (manufactured by Merck & Co., Ltd., Kieselgel 60) Chloroform: Methanol: Concentrated ammonia water = 1:1: Lower layer of 1 R=0.44 (8) Acid-base distinction Basic substances (9) Antibacterial spectrum (MIC: μg/ml) Staphylococcus aureus ATCC6538P
≦0.2 Staphylococcus aureus MS27 ≦0.2 Staphylococcus epidermidis SP−
al−1 ≦0.2 Streptococcus pyogenes NY5 1.6 Sarcina lutea ATCC9341 1.6 Corynebacterium diphtheriae PW8 ＜0.2 E. coli NIHJ 0.8 Citrobacter freundei GN346 0.8 Klebziira pneumoniae ATCC10031 0.4 Salmonella en Territides geertneri 0.4 Shigella sonnei E33 0.8 Enterobacter aerogenes 0655 0.4 Pseudomonas aeruginosa ML4561 0.8 From these results, it was confirmed that the compound obtained in the present invention is the same substance as sisomicin described in the above-mentioned publication. Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto. Example 1 Dextrin 1%, glucose 1%, casein hydrolyzate 0.5%, yeast extract 0.5%, calcium carbonate
100 ml of a medium containing 0.1% (PH7.0) was dispensed into a 500 ml Erlenmeyer flask and sterilized by heating at 120°C for 20 minutes. One platinum loop from a slant culture of Dactyrosporandium thailandensen strain G367 was inoculated into 10 bottles of this culture medium, and cultured with shaking at 30°C for 120 hours. This was then transferred to a 30-volume jar fermenter containing 20 heat-sterilized medium of the same composition as above, and incubated at 30°C for 72 hours at 300 rpm and 20 min.
Culture was carried out under sterile air conditions with aeration and agitation. Next, 5% dextrin, 0.5% glucose, 3% defatted soy flour, 0.7% calcium carbonate, and cobalt chloride.
Heat-sterilized medium containing 1.3ppm (PH7.2)
Culture 10 of the above into a 250 volume tank containing 200
transplanted, 30℃, 120 hours, 250 rpm, 100 rpm
The culture is incubated with aeration under sterile air conditions of approx.
Got 190. Next, as in Example 2, Sisomicin was separated and purified from the culture. Example 2 The culture obtained in Example 1 was adjusted to pH 2 with a 12N sulfuric acid aqueous solution, stirred for 30 minutes, adjusted to pH 7.0 with concentrated ammonia water, and further added with perlite as a super-aid. (Product name) 4Kg was added and filtered, and then the resulting culture solution was mixed with Amberlite.
IRC-50 (manufactured by Rohm and Haas) (NH ₄
Charge a column packed with + type) 10, wash with water, and elute with 2N ammonia water 20.
The total eluate was obtained and concentrated under reduced pressure to 100 ml. Next, the pH of this concentrated solution was adjusted to 7.0 with 6N sulfuric acid aqueous solution, and this was added to CM-Sephadex C-25.
(Manufactured by Pharmacia Huain Chemical Co., Ltd.) (NH ₄
A column (diameter: 4 cm) packed with 500 ml of the active substance was charged to adsorb the active substance. After that, the column was washed with water and eluted with ammonia water 5 with a concentration gradient of 0 to 0.35N, and 20ml of the eluate was
fractionated. For each fraction, chloroform:
Thin layer chromatography was performed using the lower layer of methanol:28% aqueous ammonia = 1:1:1 as the developing solvent. The target product was confirmed by ninhydrin color development. As a result, fractions 228 from fraction 210 contained only sisomicin. The fractions were then collected, combined, concentrated under reduced pressure, and then lyophilized to obtain 2.1 g of Sisomicin.

Claims (1)

[Scope of Claims] 1. A method for producing the antibiotic sisomicin, which comprises culturing an antibiotic sisomicin-producing bacterium belonging to the genus Dactylosporandium in a medium, and collecting the antibiotic sisomicin from the culture. 2 The antibiotic sisomicin-producing bacterium belonging to the genus Dactyrosporandium is Dactyrosporandium.
Claim 1 which is Tyrandense G367
The method for producing the antibiotic sisomicin described in Section 1.