JPH0153675B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic, Saccharocin, and a method for producing the same. The physicochemical properties of the novel amino sugar antibiotic satukalosin (hereinafter referred to as satukalosin) of the present invention are as follows. Melting point: 188℃ - 190℃ (decomposed) [α] ²⁴ _D +163.5゜ Elemental analysis (for C ₂₁ H ₄₀ N ₄ O ₁₂・2H ₂ O) Actual value Theoretical value C: 43.35 C: 43.74 H: 7.46 H: 7.69 N: 9.74 N: 9.72 Molecular weight: 540 (from field desorption mass spectrum) Molecular formula: C ₂₁ H ₄₀ N ₄ O ₁₂ 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, 2900, 1585, 1460, 1380,
It has absorption bands at wavelengths around 1340, 1140, 1090, and ^{990 cm -1} . 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): 25M in D ₂ O
Measurements were made at Hz with dioxane as an internal reference. No. ppm No. ppm 1 101.4 12 67.9 2 96.5 13 67.4 (dioxane) 3 95.1 14 66.4 4 87.6 15 62.3 5 78.1 16 61.3 6 76.7 17 51.1 7 73.5 18 50.2 8 73 .4 19 49.8 9 71.4 20 36.3 10 71.0 21 33.0 11 70.2 22 32.7 Solubility: Soluble in water, slightly soluble in lower alcohols such as methanol, insoluble in acetone, benzene, ethyl acetate Color reaction Positive for ninhydrin reaction, potassium permanganate decolorization reaction, negative for Elson Morgan reaction and Sakaguchi reaction . Color property: White powder Acid-base distinction: Basic Thin layer chromatography (silica gel, manufactured by Merck & Co., Ltd., Art. 5735) Chloroform-methanol-28% aqueous ammonia (2:3:2) Rf=0.32 Chloroform-methanol- 14% ammonia water (1:2:1) Rf=0.26 Based on the above physicochemical properties, satukalosin is recognized as a new amino sugar compound, and its planar structure is predicted to be the following structure. In addition, the antibacterial spectrum (indicating the minimum inhibitory concentration) of satukalosin determined by the agar dilution method is as shown in the table below. [Table] [Table] [Table] Furthermore, satukalosin, which has antibacterial properties of the present invention, especially excellent antibacterial properties against Gram-negative bacteria, can be used with non-toxic acid addition salts such as normally pharmaceutically acceptable mineral acids and organic acids. For example, hydrochloric acid, hydroiodic acid, sulfuric acid, phosphoric acid, carbonic acid, acetic acid, fumaric acid,
It can be used in the form of salts with acids such as malic acid, citric acid, mandelic acid, succinic acid, ascorbic acid, aspartic acid, and glutamic acid. In addition, the satukalosin or its non-toxic acid addition salt of the present invention can be applied as an injection preparation, for example in the form of a 20 to 100 mg vial or an ampoule containing 20 to 100 mg.
It may be formulated as a suppository formulation containing 20 to 100 mg per g. Furthermore, satukalosin or its non-toxic acid addition salt of the present invention can be used as an antibacterial therapeutic or prophylactic preparation for livestock, poultry, fish and shellfish, or as a feed composition. In these applications, satukalosin is highly soluble in water, but preferably in the form of an acid addition salt, and is added to the animal's drinking water as a solution at an effective concentration for treatment or prophylaxis, growth promotion, or in feed. May be added to. Furthermore, it is also effective to administer it as an injectable preparation together with an injectable diluent such as water or physiological saline. Furthermore, due to its formulation design, satukalosin
It may also be used as a prodrug to exert its effect as satukalosin in vivo. The Saccharopolyspora-producing bacterium of the present invention is an actinomycete strain Ac3440 isolated from field soil in Ato Town, Takebe District, Yamaguchi Prefecture, and was identified as belonging to the genus Saccharopolyspora.
Satsukalopolispora sp. Ac3440
(Saccharopolyspora sp. Ac3440) [National Institute of Microbiology, Agency of Industrial Science and Technology, contract number: FERMP-6238]. The mycological properties of this bacterium are described below. Morphological properties Starch/inorganic salt agar medium (IPS medium 4)
[Inter.J.System.Bacteriol. 16 :313-340
(1966)], cultured at 37°C for 10 to 14 days, and the following findings were observed. The basal hyphae are curved or linear, elongate with branching, and are divided by hyphal parts or at the late stage of culture, with a diameter of 0.4 to 0.6 μ, and no saplings are attached. Aerial hyphae produced from basal hyphae are curved or straight with simple branches, and have a diameter of 0.5 to 0.7μ.
The tip of the tip may be a loop or a loose spiral wound two or three times, or it may be curved or straight. Aerial hyphae are segmented to form numerous bead-like chains (usually 10 or more), and the spores are often separated by empty hyphae. Spores are oval or short cylindrical, size 0.5
It measures ~0.7 x 0.7 to 1.3μ, and its surface is covered with a shell with many tufts of straight or curved long hair-like material. It does not form sporangia, sclerotia, or flagellated spores in basal hyphae or aerial hyphae. Stainability: Gram staining is positive, acid-fast staining is negative. Bacterial cell composition (1) Method of B. Becker et al. [Appl. Microbiol 12 :
421-423 (1964)], diaminopimelic acid (DAP) was meso-type (meso-type).
type) was detected, and Lechevalier's method [J.
Lab.Clin.Med. 71 :934-944 (1968)], arabinose and galactose were detected. (2) The method of H. Mordarska et al. [J. Gen. Microbiol.
71:77-86 (1972)], lipid LCN-A was not detected, and D.
E. Minnikin et al. [J. Gen. Microbiol. 88 :200−
204 (1975)], no cardomycolic acid or mycolic acid was detected. Culture properties Table 1 shows the findings observed after culturing on various media at 37°C for 14 days. Color display is Color
harmony manual 4th edition 1958 (Container
Corporation of America) color classification. [Table] [Table] Culture was observed at 37°C for 14 days, except for physiological properties [1]. ] (1) Growth temperature range: 22~53℃ (optimum temperature: 30~
45℃) [Tested in ISP medium 2] (2) Liquefaction of gelatin: Positive (3) Hydrolysis of starch: Positive (4) Skimmed milk: Peptonization: Positive, coagulation: Negative (5) Production of melanin-like pigment: Negative on ISP medium 7 and 6 (6) Oxygen requirement: aerobic (7) Hydrogen sulfide production: positive [tested on ISP medium 6 with filter paper containing lead acetate] (8) Nitrate reduction: negative [J .Bacteriol, 73 :
15-27 (1957). ] (9) Resistance to lysozyme: non-resistant [J.Gen.Microbiol, 45 :355−
364 (1961). ] (10) Resistance to sodium chloride: 0-10%
It grows at 11% or higher, but does not grow at 11% or higher. [Basal medium: ISP medium 2] (11) Resistance to antibiotics [J.Antibiot. 32 :
180-186 (1979). ] [Table] [Table] [Table] [Ecology of soil, edited by TRGGray et al.
bacteriap.293−321, Liverpool University
Press, Liverpool, 1967, J.Gen.Microbiol,
69:33–80 (1971) and J.Gen.Microbiol. 88 :
75-85 (1975). +: Positive, -: Positive〓 [Table] [Table] Bad, -〓Negative〓
[Table] [Table] I followed. +〓Positive, -〓Negative〓
From the above, the characteristic properties of this fungus Ac3440 are (1) Morphology, in which loosely wound spiral, linear, or curved spore chains are formed on aerial hyphae that are generated from fissile basal hyphae. However, the spores are covered with a shell with long hair-like substances, and (2) meso-diaminopimelic acid, arabinose, and galactose were detected in bacterial cell analysis, and lipids
LCN-A, nocardomicolic acid, and mycolic acid were not detected; (3) Gram staining was positive, acid-fast staining was negative, and (4) it was aerobic. Based on these characteristic properties, we searched various documents and found that the genus Saccharopolyspora (Saccharopolyspora Lacey & Goodfellow)
[J.Gen.Microbiol. 88 :75-85 (1975)], the present bacterium Ac3440 was identified as belonging to the genus Satucharopolyspora, and was identified as Satucharopolyspora sp. Ac3440.
(Saccharopolyspora sp. Ac3440). Next, in carrying out the present invention and obtaining satukalosin, the above-mentioned Satucalopolyspora sp.
Collection from a culture using Ac3440 is simple and good. In addition, as for the bacteria to be used, the above-mentioned bacteria are one example, and in addition to this bacteria, all bacteria belonging to the genus Satucalopolyspora that produce satucalosin can be used in the present invention, such as those belonging to the genus Satucalopolyspora Examples include satukalosin-producing bacteria or mutant strains thereof. Next, in order to produce satukalosin, which is the novel antibiotic of the present invention, the above-mentioned satukalosin-producing bacteria belonging to the genus Satucalopolyspora are aerobically grown in a medium containing medium components used for the cultivation of ordinary microorganisms. Obtained by culturing. 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, for example,
Glucose, sucrose, dextrin, starch, molasses, etc. are used. As a nitrogen source, any available nitrogen compound may be used, for example,
Corn steep liquor, soybean flour, cottonseed flour, peptone, meat extract, yeast extract, ammonium salts, nitrates, etc. are used. In addition, if necessary, phosphate, carbonate, sulfate, hydrochloride, calcium,
Salts such as potassium, sodium, copper, iron, zinc, manganese, and cobalt are used. The culture temperature can be changed as appropriate within the range where the bacteria can grow and produce satukalosin, but it is particularly preferable to
The temperature is 25-35℃. The culture time varies somewhat depending on the conditions, but is usually about 72 to 140 hours, and the culture may be terminated at an appropriate time by determining the time when satukalosin reaches its maximum titer. In the liquid culture of the satukalosin-producing bacteria thus obtained, most of the satukalosin is
It is produced in the liquid part. Satucalosin is then collected from this culture, and it is convenient to separate and purify it by taking advantage of the fact that satukalosin is a water-soluble basic amino sugar compound. In addition, the produced satukalosin was tested using Bacillus and subtilis PCI219 as test bacteria.
The active category can be confirmed and quantified using the usual agar plate method. An example of a means for separating and purifying satukalosin is as follows. That is, the satukalosin-producing bacteria are cultured as described above, and the solid content is removed from the resulting culture to obtain a culture solution. However, since satukalosin is an amino sugar compound, the pH of the culture is first adjusted. It is preferable to obtain a culture solution by making the solution acidic and neutralizing it, and then adsorbing this solution on a column of cation exchange resin such as Amberlite IRC-50 (NH ⁺ ₄ type), From this, the active substance was eluted with 1N ammonia water, and the eluate was further concentrated.
PH is adsorbed onto a prepared cation exchange resin column, such as CM Sephadex C-25 (NH ⁺ ₄ type), eluted with 0.03N ammonium water, and the eluted active fraction is concentrated under reduced pressure and frozen. Upon drying, a purified white powder of satukalosin is obtained in the form of the free base. In addition, the satukalosin thus obtained can easily 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. Example 1 100 ml of a medium (PH7.0) containing 1% dextrin, 1% glucose, 0.5% casein hydrolyzate, 0.5% yeast extract, and 0.1% calcium carbonate was dispensed into a 500 ml Erlenmeyer flask and incubated at 120°C for 20 Heat sterilized for minutes. A loopful of Saccharopolyspora sp. Ac3440 strain was inoculated into this medium,
This was cultured with shaking at 30°C for 72 hours, and this was used as a seed culture. Separately, glucose 0.2%, glycerin 4%, peptone 5%, starch 0.2%, defatted soybean flour 0.5%,
Dry yeast 0.5%, salt 0.5%, calcium carbonate 0.2
% (PH7.0) was dispensed into 500 ml flasks, and after heat sterilization, 3% of the above-mentioned seed culture was transplanted into 100 bottles of this medium. Culture at 30℃
After 96 hours of shaking culture, they were combined to obtain approximately 10 cultures. Example 2 The culture obtained in Example 1 was adjusted to pH 2.0 with 12N sulfuric acid aqueous solution, stirred for 10 minutes, adjusted to pH 7.0 with concentrated aqueous ammonia, and concentrated by centrifugation. Clear liquid 9 was obtained. The obtained supernatant liquid was transferred to Amberlite IRC-50 (NH ⁺ ₄ type) (Roam & Co., Ltd.).
The column was charged to a column packed with 1 (manufactured by Haas), washed with water, and eluted with 1N ammonia water 2 to obtain the entire eluate, which was concentrated under reduced pressure to 30 ml. Next, this concentrated solution was adjusted to pH 7.0 with 6N sulfuric acid, and then added to CM Sephadex C-25 (manufactured by Pharmacia Fine Chemicals) (NH ⁺ ₄ type).
It was charged to a column (diameter 3 cm) packed with 300 ml and adsorbed. Thereafter, it was eluted with 0.03N ammonia water, and the eluate was fractionated into 20ml portions. Each fraction was subjected to thin layer chromatography using chloroform-methanol-concentrated aqueous ammonia (2:3:2) as a developing solvent, and the target product was confirmed by ninhydrin coloring. As a result, the 143rd fraction contained only satukalosin compared to the 124th fraction. 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 a purified white powder of satukalosin (free). base)
Obtained 110 mg.

[Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum of satukalosin, and Figure 2 shows the nuclear magnetic resonance spectrum (hydrogen nuclei) of satukalosin.

Claims (1)

[Scope of Claims] 1. An amino sugar antibiotic satukalosin or its non-toxic acid addition salt having the following physicochemical properties. Melting point: 188℃ - 190℃ (decomposition) [α] ²⁴ _D : +163.5゜ (C = 1.0, H ₂ O) Elemental analysis (for C ₂₁ H ₄₀ N ₄ O ₁₂・2H ₂ O) Actual value C: 43.35, H: 7.46, N: 9.74 Theoretical value C: 43.74, H: 7.69, N: 9.72 Molecular weight: 540 (from mass spectrum) Molecular formula: C ₂₁ H ₄₀ N ₄ O ₁₂ Ultraviolet absorption spectrum: Terminal absorption red External absorption spectrum: 3350, 2900, 1585,
Has absorption bands at wavelengths around 1460, 1380, 1340, 1140, 1090, and 990 ^{cm -1} (KBr method) Nuclear magnetic resonance spectrum (carbon nuclei) No. ppm No. ppm 1 101.4 12 67.9 2 96.5 13 67.4 (Dioxane ) 3 95.1 14 66.4 4 87.6 15 62.3 5 78.1 16 61.3 6 76.7 17 51.1 7 73.5 18 50.2 8 73.4 19 49.8 9 71.4 20 36.3 10 71.0 21 33.0 11 70.2 22 32.7 Solubility: Soluble in water Acetone, Benzene, Ethyl acetate Insoluble color reaction: ninhydrin reaction, potassium permanganate decolorization reaction positive, Elson Morgan reaction,
The Sakaguchi reaction is negative Color property: White powder Acid-base distinction: Basic 2 A bacterium that produces the amino sugar antibiotic satukalosin belonging to the genus Satucalopolyspora is cultured in a medium, and satukalosin is collected from the culture. Production method of Satsukalosin. 3. The production method according to claim 2, wherein the amino sugar antibiotic satucalosin-producing bacterium belonging to the genus Satucalopolyspora is Satucalopolyspora SP AC3440FERMP-6238.