JPH032519B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing antibiotic AC-4437. The present inventors isolated various Actinobacteria in a pure form for the purpose of searching for amino sugar antibiotics, and continued research on the metabolites produced by them. As a result, Actinobacteria AC- We found that a substance with antibacterial activity against Gram-positive and Gram-negative bacteria was produced in the culture of strain 4437. Next, the effective substance was isolated and purified from the culture, and its physical and chemical properties were investigated, and as a result, it was found to have various properties as described below. Antibiotics AC-
I decided to name it 4437. The present invention was completed based on such knowledge. The antibiotic AC-4437 obtained by the present invention is
formula However, a substance with the same chemical structural formula as this is 4-O-(5-desoxy-3
-C-hydroxymethyl-α-L-lyxofuranosyl)streptidine is known [Chem.
Ber., 110 , 1908-1915 (1977)]. Therefore, the present invention provides an antibiotic AC- belonging to the genus Streptomyces.
4437-producing bacteria are cultured in a medium and antibiotics are added to the culture.
AC-4437 was allowed to accumulate and antibiotics were removed from the culture.
Antibiotics characterized by collecting AC-4437
This is a method for producing AC-4437 or an acid addition salt thereof or a hydrate thereof. The bacteria producing the present antibiotic AC-4437 belong to the genus Streptomyces, and for example, the AC-4437 strain belonging to the genus Streptomyces isolated by the present inventors is an example of a strain most effectively used in the present invention.
The mycological properties of this strain are as follows. a Morphological characteristics AC-4437 strain was grown on starch/inorganic salt agar medium (ISP medium 4) [Inter.J.System.Bacteriol., 16 ,
313-340 (1966)] at 27°C for 10-14 days, and the following findings were observed. In addition, similar morphology was observed on various media shown in the growth state on each medium in the next section b. The basal hyphae are curved and elongate with branching.
The diameter is 0.4 to 0.5μ, and there is no hyphal division or spore settlement. Aerial hyphae formed from basal hyphae extend on a curved line with simple branching, and have a diameter of 0.4 to 0.6μ.
and forms numerous linked spores. The spore chains are connected in a curved manner and do not form a spiral. The shape of the spore is short cylindrical, and its size is
It is 0.4-0.6 x 0.7-0.9μ, and its surface is smooth. Does not form flagellated spores or sporangia. b Growth status on each medium Cultured on each medium at 27°C for 14 days, and observed findings are shown in Table 1. Color display is Color
Harmony Manual 4th edition 1958, Container
Color classification according to Corporation of America.

【table】

[Table] c The following physiological properties Optimal growth conditions: PH6-7, 25-30℃, aerobic Growth temperature range: 13-37℃ Liquefaction of gelatin: positive Hydrolysis of starch: positive Production of melanin-like pigments ; Negative on tyrosine agar and peptone/yeast extract/iron agar medium Peptonization of skim milk; positive Coagulation of skim milk; negative d Availability of each of the following carbon sources (+: positive, -: negative) L-arabinose; - D-xylose; + D-glucose; + D-fructose; + inositol; - L-rhamnose; - raffinose; - D-mannitol; + sucrose; - e Bacterial body composition Becker et al.'s method [Appl. Microbiol., 12 ,
421-423 (1964)], LL-
type of giamino pimelic acid was detected. Based on the above mycological properties, this strain AC-4437 forms aerial hyphae with more chains of spores than true basal hyphae, has LL-type diaminopimelic acid, and forms flagellated spores and sporangia. Judging from the characteristic properties such as the absence of a bacterial strain, it is clear that the strain belongs to the genus Streptomyces. Therefore, this strain was designated as Streptomyces sp. AC-4437. This strain has been given a certificate of entrustment to the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the accession number is Microbiological Research Institute.
It has been deposited as No. 7240 (FERM P-7240). As mentioned above, we have explained the antibiotic AC-4437-producing bacteria, but as a general property of actinomycetes, their mycological properties are extremely variable and not constant, and they are exposed to ultraviolet irradiation or radiation that is naturally or normally carried out. or mutagenic agents, such as N-methyl-N-
It is a well-known fact that mutations occur through artificial mutation means using nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc., and such artificial mutant strains as well as natural mutant strains belong to the genus Streptomyces. Any strain capable of producing the antibiotic AC-4437 can be used in the present invention. In the present invention, first, antibiotic AC-4437-producing bacteria belonging to the genus Streptomyces are cultured in a suitable medium. In culturing this bacterium, the actinomycete culture method is generally used. As the culture medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, an inorganic salt is used. Assimilable carbon sources include glucose, fructose, glycerin, molasses, starch, dextrin, corn steep liquor,
Organic acids and the like can be used alone or in combination. Digestible nitrogen sources include Huama mediaa,
Organic nitrogen sources such as peptone, meat extract, yeast extract, dried yeast, soybean flour, corn steep liquor, cottonseed meal, casein, soy protein decomposition products, amino acids, urea, and inorganic nitrogen compounds such as nitrates and ammonium salts. Used alone or in combination. As inorganic salts, sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, and other heavy metal salts may be added as appropriate. Furthermore, micronutrients, growth-promoting substances, etc. may be added as appropriate. Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation culture. Industrially, deep aeration agitation culture is preferred. It is preferable to culture the medium at a slightly acidic or neutral pH. The culture temperature is usually kept at 22-32°C, particularly preferably around 27-30°C. In the case of liquid culture, the culture time is usually 2
When cultured for ~5 days, the antibiotic is produced and accumulated. Preferably, the culture may be terminated when the amount of antibiotic accumulated in the culture reaches the maximum. These culture compositions, medium liquid properties, culture temperature, stirring speed,
It goes without saying that culture conditions such as the amount of aeration are appropriately adjusted and selected depending on the type of bacterial strain used, external conditions, etc. so as to obtain preferable results. When foaming occurs in liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants are used as appropriate. Since the antibiotic accumulated in the culture obtained in this way is mainly contained in the culture medium, the culture may be cultured with the addition of a super-adjuvant such as Celite, Perlite, High Flow Supercell, etc. Alternatively, it is advantageous to separate the culture fluid and the bacterial cells by centrifugation and collect the present antibiotic from the culture fluid. In order to separate and purify the present antibiotic AC-4437 from the culture solution, various known methods known in the technical field of separating and purifying amino sugar antibiotics can be used. It can be separated from the culture medium by methods such as chromatography using cation exchange resins or other solid adsorbents. Preferably, the culture solution is adjusted to pH 7, and Amberlite IRC-50,
Chromatography using a weakly acidic cation exchange resin such as CG-50, most preferably in the ammonium form, or chromatography using an adsorbent or polymer adsorbent such as activated carbon or silica gel, CM-cellulose, CM Cephadex, etc. This is a chromatographic method using an ion exchanger. To elute the adsorbed antibiotic from the carrier, if it is adsorbed on a weakly acidic cation exchange resin, it is eluted with a dilute acid aqueous solution, and when it is adsorbed on activated carbon, it is eluted with a hydrophilic organic solvent, For example, if the sample is eluted with a dilute aqueous acid solution containing acetone or the like and adsorbed onto silica gel, it may be eluted with a dilute aqueous acid solution containing a hydrophilic organic solvent such as propanol. Furthermore, if purification is required, separation and purification can be achieved by repeating the above chromatography or by performing it in an appropriate combination. Since the antibiotic AC-4437 thus obtained is a basic substance, an acid addition salt can be formed by a known method. Such salts include pharmaceutically acceptable non-toxic salts. For example, acid addition salts with inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; acid addition salts with organic acids such as acetic acid, propionic acid, malic acid, succinic acid, tartaric acid, citric acid, L-glutamic acid, and L-aspartic acid. It is. Next, the physicochemical and biological properties of this antibiotic AC-4437 will be described. Physical and chemical properties Elemental analysis: C ₁₄ H ₂₈ N ₆ O ₈・3/2H ₂ O C% H% N% Actual value 38.75 7.32 19.54 Theoretical value 38.62 7.13 19.31 Molecular weight: 409 (MH ⁺ ) (First Atom Bombard Melting point: 168-172℃ Specific rotation: [α] ²³ _D -32.4゜ (C=1, water) Ultraviolet absorption spectrum: Only terminal absorption Infrared absorption spectrum (KBr method): Figure 1 On the street, 3360, 1670, 1400, 1110,
1010 ^-1/ cm Solubility in solvents; soluble in water, slightly soluble in lower alcohols such as methanol, acetone,
Insoluble in benzene, ethyl acetate Color reaction; positive for ninhydrin reaction, potassium permanganate decolorization reaction, Sakaguchi reaction, negative for Elson-Morgan reaction, ferric chloride reaction, basic, acidic, neutral distinction; basic Substance Color of substance; white Thin layer chromatogram; Support: silica gel (manufactured by Merck & Co., Ltd.,
Art5735) Developing solvent; A: Propanol-pyridine-acetic acid-water (15:10:3:12) B: Butanol-acetic acid-water (2:1:1) C: Propanol-0.5N acetic acid (2:1)

[Table] ^13C -NMR (25MHz, in _D2O , internal standard:
Dioxane 67.6ppm); 59.8 (C-1), 71.0
(C-2), 58.9 (C-3), 79.0 (C-4),
74.0 (C-5), 72.1 (C-6), 108.8 (C-
1'), 80.4 (C-2'), 80.2 (C-3'), 78.6 (C
−
4'), 13.7 (C-5'), 63.0 (C-6'), 158.8 (1
-guanidyl), 158.8 (3-guanidyl) Chemical structural formula: As described above, and corresponds to a substance obtained by eliminating N-methyl-glucosamine from dihydrostreptomycin. The present antibiotic AC-4437 is a basic substance in aqueous solution, and the infrared absorption spectrum shows that it contains sugar, so it is clear that it is an amino sugar antibiotic. Moreover, since the Sakaguchi reaction is positive, it has a guanidyl group,
It is also clear from the ¹³ C-NMR spectrum that it is a stoptomycin antibiotic. Biological properties Antibacterial spectrum: Minimum inhibitory concentration (MIC) of antibiotic AC-4437 by agar dilution method (μg/ml)
is as shown in Table 2. Test bacteria MIC Staph.aureus ATCC6538P 12.5 Staph.aureus MS27 ＞100 Staph.aureus0119 〃 Staph.epidermidis sp−al−1 6.3 Stapt.pyogenes NY5 12.5 Bacillus subtilis ATCC6633 1.6 E.coli NIHJ−JC2 6.3 E.coli W3630 3.1 E . coli W3630 RGN14 100 Cit.freundii GN346 ＞100 Kleb.pneumoniae ATCC10031 6.3 Salm.enteritidis Gaertner 6.3 Shigella sonnei E33 12.5 Proteus morganii0239 50 Proteus rettgeri ACR ＞100 Enterobac.aerogenes0655 6.3 Enterob ac.cloacae GN336 6.3 Serratia marcsscens 6.3 Ps.aeruginosa IAM1095 ＞100 Next, production examples of the present invention will be specifically described with reference to Examples. Example: Dispense 100 ml of liquid medium (PH7.0) containing 1% glucose, 1% dextrin, 0.5% casein decomposition product, 0.5% yeast extract, and 0.1% calcium carbonate into a 500 ml Erlenmeyer flask and incubate at 120°C for 20 minutes. After sterilization, 5 bottles of each medium were inoculated with Streptomyces sp.
One platinum loop of cultured AC-4437 (FERMP-7240) was inoculated onto a slanted agar medium, and cultured with shaking on a rotary shaker incubator at 30°C for 72 hours to obtain seeds. Soluble starch 4 in 30 jar fermentor
%, soy flour 2%, corn steep liquor 1
%, dipotassium phosphate 0.05%, magnesium nitrate
0.05%, potassium chloride 0.05%, cobalt chloride
Prepare liquid medium (PH7.0) containing 0.0013%,
After heat sterilization, transplant about 0.5 of the seed mother above,
96 hours at 30℃, stirring speed 200r.pm, aeration rate 15/
Culture 19 was obtained by culturing with aeration under conditions of 10 minutes. This culture was filtered, and the resulting culture solution was charged to a column of Amberlite IRC-50 (manufactured by Rohm and Haas) (NH ₄ type) 1, washed with water, and eluted with 0.5N hydrochloric acid 5. . After adjusting the pH of all eluates to 8.0 with concentrated ammonia water,
ml column was charged. , after washing with water
Elution was performed with a mixture of 0.01N hydrochloric acid and acetone (1:1). The eluate was fractionated into 500 ml portions, and each fraction was tracked by disk bioassay on an agar plate using Bacillus subtilis ATCC6633 as the test bacterium.
I collected the 2nd to 10th fractions. After neutralizing this with Amberlite IRA-410 (manufactured by Rohm and Haas) (OH type), it was concentrated under reduced pressure.
The concentrate was freeze-dried to obtain 2 g of crude powder of antibiotic AC-4437. This powder was dissolved in as little water as possible, charged to a 200 ml column of silica gel, and eluted with propanol-0.5N acetic acid (4:1). The eluate was fractionated into 5.0 g portions, and each fraction was separated using silica gel (manufactured by Merck & Co., Ltd.) using butanol-acetic acid-water (2:1:1) as the developing solvent.
Art5735) Antibiotic AC-4437 was tracked by thin layer chromatography and determined by ninhydrin color development.
It was confirmed. Collect the 21st to 28th fractions,
After neutralizing this with concentrated aqueous ammonia, it was concentrated under reduced pressure. Charge the concentrate to a 150ml column of activated carbon.
After washing with water, elution was performed with 0.01N hydrochloric acid-acetone (1:1). The eluate was fractionated into 150 ml portions, and each fraction was tracked using the same bioassay method described above.
~Collected the 8th fraction. After neutralizing this with Amberlite IRA-410 (OH type),
It was concentrated under reduced pressure. The concentrated solution was passed through a 100 ml column of Dowex 1×2 (manufactured by Dow Chemical Company) (OH type), and the passed solution was freeze-dried and treated with the antibiotic AC-
150 mg of purified powder of 4437 was obtained.

[Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum of antibiotic AC-4437.

Claims (1)

[Claims] 1 Antibiotic AC- belonging to the genus Streptomyces
4437-producing bacteria are cultured in a medium and antibiotics are added to the culture.
AC-4437 was allowed to accumulate and antibiotics were removed from the culture.
Antibiotics characterized by collecting AC-4437
A method for producing AC-4437 or an acid addition salt thereof or a hydrate thereof.