JPS6126914B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel amino sugar antibiotic G-367-1 produced by an amino sugar antibiotic G-367-1 producing bacterium belonging to the genus Dactyrosporandium and a method for producing the same. Novel amino sugar antibiotic G-367-1 of the present invention
The physical and chemical properties of (hereinafter referred to as G-367-1) are as follows. Melting point: 130-133°C [α] ²⁴ _D : +188.9° (C = 1.0 H ₂ O) Elemental analysis Actual value Theoretical value C = 50.14 C = 50.51 H = 7.60 H = 7.84 N = 14.42 N = 14.73 Molecular weight : 475 (from mass spectrum) Molecular formula: C ₂₀ H ₃₇ N ₅ O ₈ Ultraviolet absorption spectrum: 220 ~ in aqueous solution
It does not show the characteristic maximum absorption at 360 nm, but only shows terminal absorption. Infrared absorption spectrum: (KBr method) As shown in Figure 1, 3350, 2920, 1660, 1590,
It has absorption bands at wavelengths around 1380, 1140, 1100, 1050, 1000, and 950 ^-1 cm. Nuclear magnetic resonance spectrum (hydrogen nuclei): As shown in Figure 2 (in D ₂ O, 100 MHz, internal reference DSS) Nuclear magnetic resonance spectrum (carbon nuclei): in D ₂ O,
Measurements were made at 25MHz using dioxane as an internal reference.

[Table] Solubility: Soluble in water and methanol, insoluble in acetone, benzene, ethyl acetate, and chloroform. Color reaction: ninhydrin reaction, potassium permanganate decolorization reaction positive, Elson Morgan reaction,
Biuretz reaction was negative. Color properties: white powder. Distinction between acids and bases: Basic, thin layer chromatography (silica gel) Lower layer of water (1:1:1) Rf = 0.36 10% ammonium acetate: methanol (1:1) Rf = 0.13 From the above physical and chemical properties, G -367-1 is recognized as a novel amino sugar compound, and its planar structure is predicted to be as shown below. Further, the antibacterial spectrum (indicating the minimum inhibitory concentration) of G-367-1 of the present invention obtained by the agar dilution method is shown in the following table.

【table】

[Table] Furthermore, G-367-1, which has antibacterial properties of the present invention, particularly has excellent antibacterial properties against Gram-negative bacteria, can be used in combination with non-toxic acid addition salts such as pharmaceutically acceptable mineral salts and organic acids. salts with acids such as hydrochloric acid, hydroiodic acid, sulfuric acid, phosphoric acid, carbonic acid, acetic acid, fumaric acid, malic acid, citric acid, mandelic acid, succinic acid, ascorbic acid, aspartic acid, glutamic acid, etc. It can be used in the form of Also, the G-
367-1 or its non-toxic acid addition salt, for example 20
Applied as an injectable formulation in vials for ~40 mg or ampoules containing 20-40 mg. The G-367-1-producing actinomycete of the present invention is an actinomycete G-367 isolated from field soil in Fuji City, Shizuoka Prefecture, and is Dactylosporangium thailandense G367, which belongs to the genus Dactylosporangium.
(Dactylosporagiumthailandense G 367) was identified and named (Microorganism Accession Number Notification Microorganism Accession Number "FERM-P No. 4840 FERM-P No.
4840). The mycological properties are described below. [] 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, undivided, and 0.5 to 0.8 μ in diameter.
Aerial mycelium does not form. 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 to 1.5 x 4.0
~6.5μ, with 3-4 spores arranged vertically in a row. The spores are motile in water and are spherical, oval, or pear-shaped, and the size is 1.0-1.5 x 1.5-
It is 2.5μ in length and has a polar, tufted flagellum. [] Diaminopimelic acid composition Diaminopimelic acid determined by whole bacterial cell analysis is meso-type and meso-type with a lower Rm value (slow
moving diaminopimellic acid) was detected. [] Growth status on various media, etc. Cultured on various media at 30℃ for 14 days, and observed findings are as shown in the table below.On oatmeal agar medium, a few terminally growing aerial mycelia are formed. No formation of aerial mycelium was observed, and sporangia were good on calcium malate agar, and on soil agar [J.gen.Microbiol.50:295 (1968)].
It was moderate, and little or no formation occurred on other media. Note that the color display is based on the color classification according to the Color Harmony Manual, 4th edition, 1958 (Container Corporation of America).

【table】

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

【table】

[Table] 2 Growth temperature range: 20-40℃ 3 Skim 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 of 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 to brown color and produce a brown soluble pigment on an organic medium.
thailandense) [Arch.Microbio1.58:42-52
(1967)]. Therefore, this bacterium G367 was named Dactyrosporandium thailandense G367. Next, G-367-, the novel antibiotic of the present invention
For example, in producing 1, the G-367-1 producing bacteria belonging to the genus Dactylosporandium is aerobically cultured in a medium containing medium components used for the cultivation of ordinary microorganisms. obtained by. As a medium, a solid medium or a liquid medium is used, but especially for mass production, a liquid medium,
Particularly suitable are aqueous media. 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.
As a nitrogen source, any available nitrogen compound may be used, such as corn stew liquor, soybean 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 G-367-1, but is particularly preferably 25
~35℃. The culture time varies slightly depending on the conditions, but is usually about 1100 to 200 hours.
-1 should reach its maximum titer and then terminate the culture at an appropriate time. In the liquid culture of the G-367-1 producing bacteria thus obtained, G-367-1 is mostly produced in the liquid portion. Next, from the culture of this G-367-1 producing bacteria, G
-367-1 is collected, but G-367-1
It is easy to separate and purify the compound by taking advantage of the fact that it is a water-soluble basic amino sugar compound. Also produced G-367-1 is Bacillus subtilis
Using PCI219 as the test bacterium, the active category was confirmed and quantified using the usual agar plate method. An example of separation and purification means for G-367-1 is as follows. That is, the solid content is removed from the culture obtained by culturing G-367-1 producing bacteria as described above to obtain a culture solution.
is an amino sugar compound, it is preferable to first adjust the pH of the culture to acidic and neutralize it to obtain a culture solution.Then, this culture solution is treated with a cation exchange resin such as Amberlite.
The active substance was charged and adsorbed onto an IRC-50 (NH ₄ ⁺ type) column, from which the active substance was eluted with 2N ammonia water, the eluate was further concentrated, the pH was adjusted, and the active substance was added to a cation exchange resin. for example
Charge and adsorb onto a column of CM-Sephadex C-25 (NH ₄ ⁺ type), elute with aqueous ammonia with a concentration gradient of 0 to 0.35N,
The active fraction is obtained, concentrated under reduced pressure, and lyophilized to obtain a purified white powder of G-367-1 in the form of a free base. Furthermore, G-367-1 obtained in this manner can easily be found to show a single spot in thin layer chromatography. Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto in any way. 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 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, G-367-1 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 (Rohm & 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 6N sulfuric acid aqueous solution, and then added to CM-Sephadex G-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, the 185th fraction from the 175th fraction was G-
It contained only 367-1. The fractions were then collected, combined, concentrated under reduced pressure, and then lyophilized to obtain a white powder, which was further dried under reduced pressure at 40°C for 48 hours in the presence of phosphorus pentoxide to obtain G-
750 mg of purified white powder (free base) of 367-1 was obtained.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of G-367-1 of the present invention, and FIG. 2 shows the nuclear magnetic resonance spectrum (hydrogen nuclei) of G-367-1 of the present invention.

Claims (1)

[Scope of Claims] 1 Amino sugar antibiotic G-367-1 and its non-toxic acid addition salt having the following physical and chemical properties Melting point 130-133°C [α] ²⁴ _D +188.9° (C= 1.0 H ₂ O) Elemental analysis Actual measurement Theoretical value C = 50.14 C = 50.51 H = 7.60 H = 7.84 N = 14.42 N = 14.73 Molecular weight 475 (from mass spectrum) Molecular formula C ₂₀ H ₃₇ N ₅ O ₈ Ultraviolet absorption spectrum 220 It does not show the characteristic maximum absorption at ~360 nm, and only shows edge absorption. Infrared absorption spectrum (KBr method) 3350, 2920, 1660, 1590, 1380, 1140,
It has absorption bands at each wavelength around 1100, 1050, 1000, and 950 cm ^-1 . Solubility: Soluble in water and methanol, insoluble in acetone, benzene, ethyl acetate, and chloroform; Color reaction: Positive for ninhydrin reaction and potassium permanganate decolorization reaction; negative for Elson-Morgan reaction and Biuret reaction. Color property: White Acid-base distinction: Basic 2 The amino sugar antibiotic G-367-1 producing bacteria belonging to the genus Dactyrosporandium is cultured in a medium, and the antibiotic G-367-1 is collected from the culture. Characteristic method for producing antibiotic G-367-1.