JPS6122955B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention uses the antibiotic gentamicin C ₁ a
(Gentamicin C ₁ a). _{Micromonospora} purpurea (Special Publication No. 44-21394
No. 3,091,572, Antimicrob Agents
and chemother.1963P1, 8, 14, 17 and
116), Micromonospora echinospora and its two varieties (same document above), Micromonospora sagamiensis and its two varieties (Japanese Unexamined Patent Publication No. 49-42888), and Micromonospora echinospora・Micromonospora purpurea var・
nigrescens: Hungarian Patent No. 168778, J.
Antibiot. 30 :945 (1977) was known. As mentioned above, all of the bacteria producing the antibiotic gentamicin C _1a belong to the genus Micromonospora, and their morphological characteristics are that they form one spore each on the basal hypha; The genus Spora belonged to the family Micromonosporaceae (Bergeys manual of determinative
bacteriology 8th edition (1974)). The present inventors discovered that Streptomyces strain G367, isolated from field soil in Fuji City, Shizuoka Prefecture, produces the antibiotic gentamicin C ₁ a.
Strain G367 belongs to the genus Dactylosporangium, and its morphological characteristics are that it attaches sporangia to basal hyphae and forms spores with flagella inside the sporangium. The genus Dactylosporandium belongs to the family Actinoplanaceae, and there is _a clear taxonomic difference in the family level from the conventional genus Micromonospora. It was discovered that this is a new producing bacterium. Moreover, the mycological properties of the above-mentioned actinomycete strain G367 are as follows. [] Morphological properties Calcium malate agar medium [Bact.Rev.
21 :1 (1957)], and the following findings were observed after culturing at 30°C for 3-7 days. 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 mycelia 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) Decomposition of cellulose: negative (7) Decomposition of casein: positive (8) Decomposition of tyrosine: negative (9) Liquefaction of gelatin: positive (10) Production of hydrogen sulfide: weak positive (11) Nitrate reduction: Positive (12) Growth pH: PH5.5-9.0 As mentioned above, the characteristics of this fungus G367 are that it grows finger-shaped sporangia on the basal hyphae, and The spores are arranged vertically in a single line, and the spores have tufted flagella. 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 strain G367 has the characteristic that the basal hyphae exhibit an orange-brown to brown color and produce a brown soluble pigment on an organic medium, which makes it similar to Dactylosporangium thailandense [Arch.
Microbiol. 58 :42-52 (1967)]. Therefore, this bacterium G367 was named Dactyrosporandium thailandense G367, and this bacterium has been applied to the Institute of Microbial Technology, Agency of Industrial Science and Technology as ``Feikoken Bacteria Entrustment No. 4840.'' The present invention was completed based on the above findings, and involves culturing antibiotic gentamicin C ₁ a-producing bacteria belonging to the genus Dactyrosporandium in a medium, and collecting the antibiotic gentamicin C ₁ a from the culture. This is a method for producing the antibiotic gentamicin C ₁ a, which is characterized by: First, the bacteria producing the antibiotic gentamicin C ₁ a (hereinafter simply referred to as gentamicin C ₁ a) belonging to the genus Dactyrosporandium used in carrying out the present invention are the above-mentioned Dactyrosporandium thailandense strain G367. is cited as an example, and the bacteria used in the present invention are not limited to these, but may be any gentamicin C ₁ a-producing bacteria belonging to the genus Dactyrosporandium.
Natural or mutant strains may also be used. Next, in order to produce the gentamicin C ₁ a of the present invention, for example, the above-mentioned gentamicin C ₁ a producing bacteria belonging to the genus Dactyrosporandium are grown in a medium containing medium components used for the cultivation of ordinary microorganisms. Obtained by culturing aerobically. 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 a range that allows the bacteria to grow and produce gentamicin C ₁ a, but is particularly preferably 25 to 35°C. 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 gentamicin C ₁ a reaches its maximum titer. In the liquid culture of the gentamicin C _1a -producing bacteria thus obtained, gentamicin
C ₁ a is mostly produced in the fluid part. Next, gentamicin C 1 _a is collected from the culture of this gentamicin C ₁ a producing bacterium, but it is easy to separate and purify it by taking advantage of the fact that gentamicin C ₁ a is a water-soluble basic amino sugar compound. It is. The activity of the produced gentamicin C _1a was confirmed and quantified using Bacillus subtilis PC1219 as a test bacterium by the usual agar plate method. An example of a means for separating and purifying gentamicin C ₁ a is as follows. In other words, gentamicin C ₁ a-producing bacteria are cultured as described above to obtain a culture solution by removing solid matter from the resulting culture, but since gentamicin C ₁ a is an amino sugar compound, the pH of the culture is It is preferable to obtain a culture solution by adjusting the acidity and neutralizing the solution, and then charging this culture solution to a column of a cation exchange resin such as Amberlite IRC-50 (NH ₄ ⁺ type). At the very least, the active substance can be adsorbed.
After elution with 2N ammonia water and further concentrating the eluate, the pH was adjusted and a cation exchange resin such as CM-Sephadex C-25 (NH ₄ ⁺
Charge the column of type) to adsorb it, 0
The active fraction was obtained by elution with aqueous ammonia with a concentration gradient of ~0.35N, which was concentrated under reduced pressure and lyophilized to obtain a purified white powder of gentamicin C _1a in the form of its free base. It will be done. Furthermore, the gentamicin C _1a obtained in this manner can easily be one that shows a single spot in thin layer chromatography. Next, the physicochemical properties of the gentamicin C ₁ a of the present invention thus obtained are as follows. Molecular weight 449 (from mass spectrum) Molecular formula C ₁₉ H ₃₉ N ₅ O ₇ Specific optical rotation [α] ²⁶ _D = +96.2 (C = 0.39, H ₂ O) Paper chromatography Chloroform: Methanol: 17% aqueous ammonia ( 2:1:1) Rf=0.22 Propanol:Pyridine:Acetic acid:Water=(6:
4:1:3) Upper layer liquid Rf=0.29 Color property White powder Based on the above properties, mass spectrum peaks, nuclear magnetic resonance spectrum, etc., the compound obtained by the present invention is gentamicin C ₁ a described in the above-mentioned literature. It was identified as the same substance. 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 thailandense 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 heat-sterilized medium 20 having the same composition as above, and cultured with aeration at 30° C. for 72 hours under conditions of 300 rpm and sterile air at 20 per minute. 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
Culture with aeration and agitation under sterile air conditions, and culture approx.
Got 190. Next, as in Example 2, gentamicin C ₁ a was isolated 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 ⁺
The column was charged to a column packed with Type) 10, washed with water, and eluted with 2N ammonia water 20 to obtain the entire eluate, which was concentrated under reduced pressure to 100 ml. Next, this concentrated solution was adjusted to pH 7.0 with a 6N sulfuric acid aqueous solution, and then added to CM-Sephadex C-25.
(Manufactured by Pharmacia Huain Chemical Co.)
A column (diameter 4 cm) packed with 500 ml (NH ₄ ⁺ form) was charged to adsorb the active substance. After washing the column with water, the column was eluted with aqueous ammonia 5 with a concentration gradient of 0 to 0.35N, and the eluate was
It was fractionated into 20 ml portions. For each fraction, chloroform: methanol: 28% ammonia water = 1:1:1
Thin layer chromatography was performed using the lower layer as a developing solvent, and the target product was confirmed by ninhydrin color development. As a result, fractions 245 to 235 contained only gentamicin C ₁ a. The fractions were then collected, combined, concentrated under reduced pressure, and then lyophilized to obtain 85 mg of gentamicin C ₁ a.

Claims (1)

[Scope of Claims] 1. Antibiotic gentamicin C, characterized in that gentamicin C ₁ a-producing bacteria belonging to the genus Dactyrosporandium are cultured in a medium, and the antibiotic gentamicin C ₁ a is collected from the culture. ₁ Manufacturing method of a. 2. The method for producing the antibiotic gentamicin C ₁ a according to claim 1, wherein the antibiotic gentamicin C ₁ a-producing bacterium belonging to the genus Dactyrosporandium is Dactyrosporandium thailandense G367.