JPH0123117B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides antibiotics AT265 and/or AT-
Concerning a new manufacturing method for 265-B. The purpose is to use AT-265 and/or antibiotics that have antibacterial activity against Gram-positive and Gram-negative bacteria.
Another object of the present invention is to provide a new method for producing AT-265-B. For this purpose, the present inventors obtained a large number of microorganisms from the natural world and conducted research on their antibiotic productivity. As a result, among the AT-265-producing bacteria, a strain [Streptomyces liciliensis subsp. SP-265,
(streptomyces rishiriensis subspecies SP−
265), hereinafter referred to as SP-265 strain],
We have discovered that antibiotics can be produced in culture. As a result of subsequent analysis of its chemical and physical properties, this antibiotic has the following general formula ()
It has been revealed that this is an antibiotic with the structure shown below. (However, R represents -H or -SO ₂ NH ₂ ) When R in the general formula () is -SO ₂ NH ₂ , AT-
265, and when R in the general formula () is -H, AT
It is called -265-B. AT-265-B is called 2-chloroadenosine. AT-265 is 5′-sulfamoyl-2-
It is also called chloroadenosine and is known to be derived from 2-chloroadenosine by organic chemical synthesis. (GRGough Journal of Medi−
cinal Chemistry 1978 vol21(6) 520-525) However, there has been no knowledge that both substances are produced by actinomycetes of the genus Streptomyces, and the present inventors discovered them for the first time. Below, this antibiotic AT-265 and AT-265-
The manufacturing method of B will be explained in detail. (A) Identification of strain SP-265 The morphological characteristics and physiological properties of the SP-265 strain were observed and tested, and the results are as follows. Morphological Properties Branching method of spore-forming hyphae: Simple branching Morphology of spore-forming hyphae: Spiral Number of spores: 10 or more Spore surface structure and size: Smooth 0.5~
0.7 x 1.0 to 1.5 microns Presence or absence of flagellar spores and sporangia: Not observed Location of sporophyte attachment: On aerial hyphae Growth status on various media

【table】

[Table] Physiological properties The physiological properties of SP-265 strain are shown below. (1) Growth temperature range: 20-40℃, optimal 34-36℃ (2) Tyrosinase reaction: Positive (3) Gelatin liquefaction: Positive (4) Nitrate reducing ability: Negative (5) Starch hydrolyzability: Positive (6 ) Coagulation of skim milk (26℃): Negative (37℃): Positive (7) Peptonization of skim milk (26℃): Positive (37℃): Positive (8) Production of melanin-like pigment (tyrosine agar medium and Peptone yeast iron agar): Positive Assimilation of each carbon source (on Pridham-Gottlieb agar) D-glucose, D-xylose, L-arabinose, L-rhamnose, D-fructose, D-galactose, D- Raffinose, i-inositol, sucrose, D-
Cellobiose, soluble starch, dextrin, D-mannose, D-trehalose,
Lactose, inulin, maltose, D-
Assimilates melibiose, glycerol, etc. well. Assimilates salicin and sodium succinate, but has difficulty assimilating mannitol, cellulose, dulcitol, sodium acetate, etc. Regarding bacteria with the above mycological properties, the International Journal of Systematic Bacteriology (International Journal of Systematic Bacteriology)
Bacteriology) Vol. 18, pp. 69-189, 279-392
Pages; Vol. 19, pp. 391-512; Vol. 22, pp. 265-394;
and Bergey's Manual 8th Edition
Manual of Determinative Bacteriology,
Streptomyces rishiriensis is considered to be the closest relative. However, SP-265 strain
Streptomyces liciliensis subsp. SP-265 has the characteristics of producing AT-265 and AT-265-B substances.
(Streptomyces rishiriensis subspecies SP
−265). This strain has been registered with the Institute of Microbial Technology, Agency of Industrial Science and Technology as application number 5921 for entrustment of microorganism storage. The strain isolated by the inventors is AT-
265 substance and AT-265-B substance, but bacteria belonging to the genus Streptomyces produce AT-265 substance and AT-265-B substance.
-265 and AT-265-B substances are not known to be produced. (B) Cultivation of strain SP-265 In culturing this strain, the usual culture method for actinomycetes is generally used. The culture medium used is AT belonging to the genus Streptomyces.
-265 Any medium containing a nutrient source used by the substance-producing bacteria may be used. That is, a synthetic medium, a semi-synthetic medium, or a natural medium is used, and the composition of the medium is, for example, glucose, sucrose, starch, etc., and the nitrogen source is meat extract, peptone, soy flour, corn steep liquor, dried yeast, ammonium sulfate, etc. Organic or inorganic nitrogen sources are used. In addition, metal phosphates, carbonates, and chlorides are added. If there is significant foaming during culture, it is advisable to add an antifoaming agent such as silicone, vegetable oil, mineral oil, etc. The appropriate culture temperature is around 25°C to 30°C, and as the culture volume increases, seed culture is appropriately performed and inoculated. The culture time for main culture is about 60 to 100 hours. The culture conditions described above are applied by selecting the optimum conditions for each depending on the characteristics of the production strain used. The AT− thus accumulated in the culture
Since the 265 substance and the AT-265-B substance are mainly contained in the culture solution, after removing the bacterial cells by centrifugation or filtration, the effective substance is purified and isolated from the solution. (C) Purification and isolation of AT-265 and AT-265-B The separation and purification method used to isolate microbial metabolic products from the culture fluid is used to purify and isolate this substance from the culture fluid. be done. That is,
Adsorption/desorption method using ion exchange resin, silica gel column chromatography, Sephadex LH
−20 column chromatography, Diamond ion
Adsorption/desorption methods such as HP-20, extraction methods using solvents, etc. can be used in appropriate combination. An example is shown below. That is, the culture solution is extracted with ethyl acetate. This extraction operation is repeated several times, and the extracted fractions are collected and concentrated under reduced pressure. This concentrated solution is suspended in chloroform in advance, passed through a column of silica gel, and eluted with methanol. Among the eluted fractions, the fractions containing AT-265 and AT-265-B were collected, concentrated under reduced pressure, suspended in methanol, passed through a Sephadex LH-20 column packed, and eluted with methanol. AT-265-B was eluted and then AT-
265 is eluted. After collecting each fraction and concentrating under reduced pressure to distill off methanol, crystals are obtained with methanol or water. AT−265 and AT− thus obtained
The physical and chemical properties of 265-B are shown in Table 1.

[Table] Table 2 shows the Rf values of thin layer chromatography for AT-265 and AT-265-B.

【table】

[Table] In addition to these properties regarding AT-265 and AT-265-B, based on the results of structural analysis by ¹³ C-NMR, FD mass spectrum, in-beam mass spectrum, etc., AT-265 is 5'-sulfamoyl. It was identified as -2-chloroadenosine. Also AT-
265-B was identified as 2-chloroadenosine. Antibacterial spectrum of AT-265 and/or AT-265-B against various bacteria (measured by agar dilution method using heart infusion medium (37
℃, 20 hours)) are shown in Table 3.

[Table] As mentioned above, AT-265 has an antibacterial effect, and AT-265-B has a weak antibacterial effect. −265 (GR
Gough Journal of Medicinal
Chemistry 1978vol 21(6) 520-525), can increase antibacterial activity, and can be used as a medicine or veterinary drug. Next, examples of this invention will be shown. Example 1 A medium with a composition of polpeptone 1%, yeast extract 0.5%, and glucose 0.5% was dispensed into 10 500 ml shaker flasks, 100 ml each, and after sterilization in a conventional manner, Streptomyces liciliensis was added to these. - Inoculate subspecies strain SP-265 (microorganism commission application acceptance number 5921) and culture at 27°C for 2 days. Separately, 1% soluble starch, 0.5% cottonseed meal,
Medium 15 with the composition of yeast extract 0.5%, monopotassium phosphate 0.2%, dipotassium phosphate 1.0%, magnesium sulfate (heptahydrate) 0.01%, and cobalt chloride 1ppm.
After sterilizing it as usual, add 5% of the above culture to it.
inoculate. Then pass 15 sterile air per minute,
Cultivate at 27°C for 5 days while stirring at 300 revolutions per minute. After completion of the culture, 3% High Flow Super Cell (manufactured by Johns-Manville Sales Corp.) was added to the culture solution and filtered. The obtained liquid 15 is extracted three times with ethyl acetate. The extract is concentrated by distillation under reduced pressure. Next, this concentrated solution was passed through a column packed with silica gel (manufactured by Merck & Co., Ltd.) 2 soaked in chloroform.
First, elution is carried out in the following order: 500 ml of chloroform, 500 ml of chloroform:methanol (9:1), 4 ml of chloroform:methanol (9:2), and 1 ml of chloroform:methanol (7:3). The target product is eluted with chloroform:methanol (8:2). Concentrate the eluate and transfer to Cephadex LH-20.
Column (diameter 50 mm, length
100cm) and elute with methanol. AT-265-B substance was eluted from the eluate of about 2, and then AT-265-B was eluted from the eluate of about 2.5.
−265 substances are eluted. Each fraction was concentrated under reduced pressure and dried to obtain a white powder. When the white powder of AT-265 is dissolved in water and recrystallized, white needle-shaped crystals are obtained. Yield is 7mg
It was hot. In addition, when the white powder of AT-265-B is dissolved in methanol and recrystallized, 10 white needle crystals are obtained.
mg was obtained. Example 2 A seed culture grown as in Example 1 was mixed with 2% soluble starch, 2% cottonseed meal, 0.2% monopotassium phosphate, 1.0% dipotassium phosphate, 0.01% magnesium sulfate (heptahydrate), and cobalt chloride. Medium composition of 5ppm
After sterilizing 15 in the usual manner, inoculate 5% and inoculate every minute.
Culture at 27°C for 5 days while stirring at 300 rpm. After the culture is completed, the bacterial cells are centrifuged and the removed liquid is passed through a column of Diaion HP-20 (manufactured by Mitsubishi Kasei) 1 for adsorption. Elution is performed with one each of 10% methanol, 20% methanol, 30% methanol, and 40% methanol. The active fraction is eluted with 30% methanol. The active fraction was concentrated and passed through ether-soaked silica gel (Merk).
Elution is carried out by gradually increasing the methanol content in the ether:methanol ratio. The desired fraction is ether:
It is eluted at a methanol ratio of 9:1. Combine the desired fractions, concentrate, and cephadex.
Pass through LH-20 column. When elution is performed with methanol, the AT-265-B substance and then the AT-265 substance are eluted. When each fraction is concentrated under reduced pressure and crystallized, both substances are obtained as white needle-like crystals. The yield is
AT-265 was 10 mg and AT-265-B was 15 mg.
The properties of these substances are shown in Table 1.

[Brief explanation of drawings]

Figures 1 and 2 show the ultraviolet absorption spectra of AT-265 and AT-265-B, respectively.
Figures 3 and 4 show infrared absorption spectra of AT-265 and AT-265-B, respectively.

Claims (1)

[Scope of Claims] 1 Antibiotic AT-265 belonging to the genus Streptomyces and having a structural formula in which R is SO ₂ NH ₂ in the following general formula () and a structure in which R is H in the following general formula () Antibiotic AT with the formula
A microorganism capable of producing -265-B is cultured in a nutrient medium, the antibiotic AT-265 and/or AT-265-B is accumulated in the culture, and the accumulated antibiotic is removed from the culture. Antibiotic AT-265 and/or AT-
265-B manufacturing method.