JPH0337559B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel macbecin analogs and methods for producing the same. In order to search for new antibiotics, the present inventors collected a large number of microorganisms from soil, etc., and searched for the antibiotics they produced. We learned that the microorganism belongs to the genus Actinosynnema and that by culturing the microorganism in an appropriate medium, antibiotics that exhibit activity against Gram-positive bacteria, fungi, and protozoa can be accumulated in the medium, and we purified and isolated this microorganism. Based on its properties, the above antibiotic was recognized as a novel macbecin analogue, and based on these findings, further research was conducted, and as a result, the present invention was completed. The present invention provides (1) antibiotic C-33196E-4 or antibiotic C-33196E-4-R, and (2) antibiotic C-33196E-4 and/or antibiotic C belonging to the genus Actinosynnema.
-33196E-4-R (hereinafter sometimes referred to as "the antibiotic concerned") is cultivated in a medium, and the compound is collected from the culture. -33196E-4 "and/or" A method for producing antibiotic C-33196E-4-R. The microorganism used in the method of the present invention may be any microorganism as long as it belongs to the genus Actinosynnema and has the ability to produce the antibiotic. As a specific example, for example, actino synnema
sp.No.C-33196 strain (hereinafter sometimes abbreviated as "C-33196 strain"). The taxonomic properties of strain C-33196 were determined by the Shearing and Gottlieb methods [International Journal of Systematic Bacteriology].
Systematic Bacteriology) Volume 16, pp. 313-340
Page, 1966)], and the results of observation over 21 days at 28°C are as follows. 1 Morphological characteristics The basal hyphae are colorless to pale yellow or orange-yellow, elongate and branch well both on agar and liquid media. Most of the diameters of basal hyphae are 0.5~
It is 1.2μm and divides into rod-shaped, rod-shaped, or branched hyphae in the late stage of culture. This fungus grows well on various classification media, and aerial hyphae grow on the basal hyphae, but many bundle-like bodies (50-180 μm x 400-
1500 μm) and are often observed as growing on them. Most of the aerial hyphae are curved or straight, and in rare cases, they are loosely spiral. When a mature culture of this bacterium is examined under a microscope, there are few cases where spores are thought to be chained, and so-called conidia or spores are rarely formed. When the bacterial suspensions collected from the culture surfaces were examined under a microscope, they were found to have an oblong shape (0.5~
1.2μm×4.8~6.8μm) and oval (0.8~1.2μm
Many dividing cells or segmented spore-like cells (×1.5 to 4 μm) were observed, and their surfaces were smooth when observed using an electron microscope. In addition, the growth of aerial mycelia is generally poor, and although they grow relatively well until 3 to 7 days of culture on many media, they may no longer be observed after 10 days or more of culture. When mature aerial hyphae are immersed in a liquid medium, motile cells are released after 15 to 30 minutes. When these motile cells are observed using an electron microscope, long pericytal flagella are observed around the cells. When this fungus is cultured in a liquid medium, it sometimes produces pleomorphic fractured hyphae in the later stages. Some of the torn hyphae are motile. 2 Bacterial composition This strain was grown in a modified medium of ISP No. 1 at 28℃, 66-90℃.
After culturing with shaking for hours, the cells that had grown sufficiently and reached the stationary phase were collected and washed. The above bacterial cells were prepared using the method of B. Betzker et al. [Applied Microbiology, Vol. 12, p. 421, 1964]
2009] and M.B. Lucivarier's method [Journal of Laboratory and Clinical Medicine]
Clinical Medicine, Vol. 71, p. 934, 1968], the composition of diaminopimelic acid and sugar in bacterial cells was confirmed to be meso, and the latter was found to have a spot equivalent to galactose. Its existence was recognized. Also, the method of B. Betzker et al. [Applied Microbiology, Vol. 17, p. 236, 1965]
Prepare cell walls according to diaminopimelic acid,
The sugar and amino acid content was as follows. That is, the meso form of diaminopimelic acid was detected, and the presence of a large amount of galactose was observed, but arabinose was not detected. Regarding amino acids, glutamic acid and alanine were clearly detected, but only trace amounts of lysine and glycine were detected. That is, it is a strain belonging to the cell wall type. 3 Properties on classification media This strain grows relatively well on various media, and its basal hyphae are colorless or pale yellow at the initial stage of culture, and then turn pale yellowish brown or yellowish brown. It also does not produce soluble pigments in most sorting media. Aerial mycelia are powdery and generally of moderate growth;
Shows white to yellow or pale yellowish brown color. These aerial hyphae disappear when cultured for a long period of time (about 2 weeks or more) on many media, and the surface of the basal hyphae becomes glossy. The properties of this strain on various classification media are shown in Table 1. Table 1 Characteristics of C-33196 strain on classification medium Growth on closed nitrate agar medium (G): Moderate, thin growth, pale yellow (2ca) ^* Aerial mycelium (AM): poor; White Soluble Pigment (SP): None Glycerol Nitrate Agar G: Moderate, pale yellow (2ca) ^* AM: Poor, white SP: None Glucose Asparagine Agar G: Moderate, yellow (3ga) ^* , fascicle formation AM: poor, pale yellow (3ea) ^* SP: none Glycerol-asparagine agar medium G: moderate, pale yellow (2ca) ^* , fascicle formation AM: poor, white to pale yellow (2ca) ) ^* SP: None Nutrient agar G: Rich, yellow (31a) ^* AM: None SP: None Calcium malate agar G: Poor, light yellow (2ca) ^* AM: Poor, white SP: None Yeast Extract/malt extract agar medium G: rich, yellow (31a) ^* AM: poor, white SP: none Oatmeal agar medium G: medium, light wheat color (2ea) ^* , fascicle formation AM: poor, white SP : None Inorganic salt/starch agar medium G: Medium, pale yellow to yellow (2ca or
3ea) ^* AM: Poor, light wheat color (2ea) ^* SP: None Peptone/yeast extract/iron agar G: Medium, yellow (31a) ^* AM: None SP: None Tyrosine agar G: Medium , yellow (31a) ^* , bundle formation AM: poor, white SP: none *: Color Harmony Manual, 4th edition (Container Corporation of
(Published in the United States in 1958) Color name symbol 4 Physiological properties The physiological properties of this strain are shown in Table 2. That is, the growth temperature range is 12°C to 35°C, and the temperature range for good growth of aerial mycelia on agar medium (ISP No. 2) is 16°C to 32°C. Table 2 Physiological properties of strain C-33196 Growth temperature range: 12°C to 35°C Aerial mycelial growth temperature: 16°C to 32°C Gelatin liquefaction: Positive starch hydrolysis: Positive nitrate reducing ability: Positive Milk peptonization: Positive milk/coagulation: Negative casein resolution: Positive melanin-like pigment formation: Negative tyrosine resolution: Positive xanthine resolution: Negative hypoxanthine resolution: Negative lysozyme resistance: Positive salt tolerance: 2% 5 Availability of various carbon sources Asparagine 0.05%, phosphorus Potassium acid 0.05
%, magnesium sulfate 0.02%, ferrous sulfate 0.001
The usability of various carbon sources was investigated using a basal medium containing 0.1% ammonium sulfate, 0.1% ammonium sulfate, and 2% agar, and the results are shown in Table 3. Table 3 Carbon source utilization of strain C-33196 Carbon source Growth D-xylose L-arabinose ± D-glucose D-galactose D-fructose L-rhamnose D-mannose Seuculose Maltose + lactose Trehalose Raffinose Melibiose i-inositol ± D - Sorbitol ± D-mannitol Glycerol + Soluble starch Control ± Note: Grows abundantly + Grows moderately ± Grows slightly 6 Other properties Bacterial cells were grown using the method shown in “2) Bacterial cell composition” above. These were collected using the method of J. Marmer et al. [Journal of Molecular Biology, vol. 3, p. 208,
DNA was prepared according to [1961], and the DNA G-C
The (guanine-cytosine) content was about 72 mol%. Gram staining of the vegetative hyphae of this strain was positive. The properties of the C-33196 strain described above are described in S.A. Waksman, The Actinomycetes, Volume 2, published by The Williams & Wilkins Company.
1961, R.E. Puthuanan &
Bergey's Manual of Determinative Bacteriology, edited by N. E. Gibbons.
Bacteriology) 8th edition, 1974 and other literature. As mentioned above, 1) this strain produces pleomorphic fractured hyphae in the late stage of vegetative growth, some of which are motile. 2) Peritrichous motile cells are generated from mature aerial hyphae. 3) Aerial hyphae form bundles on some agar media. 4) Belongs to cell wall type. From other properties, it is clear that this bacterium belongs to the genus Actinosynnema. Therefore, this bacterium was named Actinosynnema sp. No. C-33196. The genus Actinosynnema is a genus of the order Actinobacteria, and its mycological properties were published in the International Journal of Systematic Bacteriology by Hasegawa et al.
of Systematic Bacteriology) Volume 28, 304~
Reported on page 310 (1978), and further published in volume 30 of the same book,
Also published on page 245 (1980). This strain C-33196 was entrusted to the Fermentation Research Institute on August 11, 1981, and was designated as IFO14127.
August 1981: Institute of Microbial Technology, Agency of Industrial Science and Technology
FERM P-6138 (FERM BP-
166), respectively. In general, the properties of Actinosynnema bacteria are easily variable, and they can mutate naturally or by mutagens. For example, there are many mutant strains obtained by irradiation with radiation such as X-rays, gamma rays, and ultraviolet rays, isolation of monospores, cultivation on media containing various drugs, and other methods, or mutations obtained naturally. Even if it is a strain, all those that have the mycological properties listed above or those that are not substantially different from each other when compared with the mycological properties shown below, and that also have the property of producing the antibiotic concerned. It can be used in the method of the present invention. For example, by subjecting the antibiotic-producing strain to various mutation treatments, there are strains that produce light yellow to light yellowish brown or brown soluble pigments, those whose basal hyphae are colorless, those whose basal hyphae are colorless, those whose color is reddish brown or orange-red, and those whose color is yellowish-green. A strain that produces basal hyphae or soluble pigment, a strain that produces abundant white aerial hyphae, and a mutant strain in which the hyphae are easily fragmented can be obtained. The medium used for culturing the antibiotic-producing bacteria may be liquid or solid as long as it contains a nutrient source that can be used by the bacteria, but it is more appropriate to use a liquid medium when large quantities are to be treated. A carbon source that can be assimilated by the antibiotic-producing bacteria, a nitrogen source that can be digested, inorganic substances, and micronutrients are appropriately blended into the culture medium. Examples of carbon sources include glucose, lactose,
Sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, fats and oils (e.g., soybean oil, lard oil, chicken oil, etc.), n-paraffin, etc. Nitrogen sources include, for example, meat extract, yeast extract, dried Yeast, soybean flour, corn stew liquor, peptone, cottonseed flour, molasses, urea, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.), and others are used. Further, salts containing sodium, potassium, calcium, magnesium, etc., metal salts such as iron, manganese, zinc, cobalt, nickel, etc., salts of phosphoric acid, boric acid, etc., and salts of organic acids such as acetic acid, propionic acid, etc. are used as appropriate. In addition, amino acids (e.g., glutamic acid, aspartic acid, alanine, lysine, methionine, proline, etc.), peptides (e.g.,
dipeptides, tripeptides, etc.), vitamins (e.g., B ₁ , B ₂ , nicotinic acid, B ₁₂ , C, etc.), nucleic acids (e.g., purines, pyrimidines, their derivatives, etc.)
etc. may be included. Of course, inorganic or organic acids, alkalis, buffers, etc. may be added for the purpose of adjusting the pH of the medium, or appropriate amounts of oils and fats, surfactants, etc. may be added for the purpose of defoaming. The culturing method may be static culture, shaking culture, or aerated agitation culture. It goes without saying that for large-scale treatment, it is desirable to use so-called deep aeration agitation culture. It goes without saying that culture conditions vary depending on the state and composition of the medium, the type of strain, the culture method, etc., but they are usually about
It is best to select a temperature of 20°C to 32°C and an initial pH of around neutrality. In particular, the temperature during the middle stage of culture is approximately 25°C.
Conditions of ~28°C and initial pH of approximately 6.5 to 7.5 are desirable. The culture period also varies depending on the conditions described above, but it is preferable to culture until the desired antibiotic concentration is maximized. This usually takes approximately 48 hours for shaking culture or aerated agitation culture using a liquid medium.
~96 hours. To collect the antibiotic produced in the culture, since the antibiotic is neutral fat-soluble, separation and purification methods normally used to collect such microbial metabolites can be used as appropriate. be done. For example, means utilizing the difference in solubility with impurities, adsorption chromatography using various adsorbents such as activated carbon, nonionic macroporous resin, silica gel, alumina, etc. are used alone or in combination. The antibiotic is generally contained in a larger amount in the culture solution, but it can also be extracted from the bacterial cells if necessary. For extraction from bacterial cells, organic solvents that are miscible with water, such as lower alcohols such as methanol and ethanol, ketones such as acetone and methyl ethyl ketone, or a mixture of these and water are used. Extraction can also be carried out using water-immiscible organic solvents, for example esters such as ethyl acetate. Furthermore, the whole culture can be extracted by adding a water-miscible organic solvent such as methanol or acetone. To collect the antibiotic from the culture solution, use an organic solvent that is immiscible with water, such as fatty acid esters such as ethyl acetate and butyl acetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform, methyl isobutyl ketone, etc. Extraction with ketones is used. Wash the extract with water,
After concentration, n-hexane or the like can be added to obtain a crude material. At that time, if one component is contained in a large amount as a main component in the antibiotic, it may be isolated as crystals simply by extraction and concentration using this organic solvent. In addition, when collecting the antibiotic from the culture solution,
Adsorption with a nonionic macroporous resin such as Diaion HP-10 (manufactured by Mitsubishi Kasei), elution with hydrous alcohol, hydrous ketones, etc. can also be used. When the crude material obtained from the culture fluid through extraction, concentration, etc. as described above consists of a mixture of the antibiotics, various types of adsorption chromatography are used to separate each component. As the adsorbent, commonly used carriers such as silica gel, alumina, adsorbent resins, etc. can be used. When using silica gel as an adsorbent, it is generally a combination of a polar organic solvent and a non-polar organic solvent,
For example, development with a mixed solvent such as ethyl acetate and n-hexane or methanol and chloroform is used to separate the components. For example, each component is separated by first developing with a non-polar solvent and then eluting with a gradually increasing proportion of a polar solvent. If the crude powder (mixture) consists of multiple components and contains many impurities, it can be separated into single components by changing the combination of these organic solvents and repeating chromatography. The antibiotic consists of a benzoquinone type and a hydroquinone type, which are mutually convertible, so when purifying it, if it is converted to a benzoquinone type or a hydroquinone type by oxidation or reduction, the number of components will be halved. It's convenient. As the oxidation method, the method normally used for oxidizing hydroquinone is used. For example, oxidizing agents include ferric chloride, ferric sulfate,
Examples include potassium ferricyanide, silver oxide, etc.
Further, as the reaction solvent, any solvent can be used as long as it does not interfere with the reaction. for example,
Examples include esters such as ethyl acetate, ketones such as acetone, water, etc., or a mixed solvent thereof may be used. Furthermore, two-phase systems with water and water-immiscible organic solvents are advantageously used. Although the reaction temperature is not particularly limited, it is usually carried out at about 0 to 40°C, generally at room temperature, and the reaction is usually easily completed within about 30 seconds to 24 hours, depending on the reaction time and temperature. As the reduction method, a method normally used for reducing benzoquinone is used. For example, reducing agents used in the reaction include sodium hydrosulfite, sodium acid sulfite, sodium borohydride, etc., and as a reaction solvent, any solvent can be used as long as it does not inhibit the above reaction. It is. For example, esters such as ethyl acetate, methanol,
Examples include alcohols such as ethanol, water, etc., or a mixed solvent thereof may be used. Furthermore, two-phase systems with water and water-immiscible organic solvents can be advantageously used. The reaction temperature is usually about 0 to 40°C, generally room temperature, and the reaction is easily completed in about 30 seconds to 24 hours, depending on the reaction time and temperature. In silica gel chromatography, purification is generally performed using benzoquinone type, but in some cases it may be convenient to use hydroquinone type. Each component is separated using oxidation and reduction. Each of the obtained components is crystallized from a solvent such as ethyl acetate, methanol, methylene chloride, or methanol-water. Antibiotic C-33196E-4 and antibiotic C-33196E obtained in Examples/and 4 below
Table 4 shows the physicochemical properties of -4-R (containing one molecule of ethyl acetate as a crystal solvent).

[Table] The antibiotic has antibacterial, antifungal, and antiprotozoal effects. In addition, since it exhibits a cytocidal effect on tumor cells, antitumor effects are also expected. Furthermore, it can also be used as a raw material for the synthesis of useful derivatives. The antibiotic exhibits an antibacterial effect against Staphylococcus aureus and Bacillus subtilis with a MIC of 50 to 100 mcg/ml, which is approximately equivalent to that of macbecin, and can be used as an antibacterial agent. Shin (Japanese Unexamined Patent Publication No. 58-74651) has an acute toxicity of LD ₅₀ 100 to 200 mg/Kg (mouse, intraperitoneal administration).
The acute toxicity of this antibiotic is considered to be low because it is weaker than that of macbecin and macbecin.The antibiotic is used, for example, as an aqueous solution containing 10 to 100 μg/ml of ethanol to disinfect bird cages,
It can be used as a disinfectant to disinfect laboratory equipment, livestock pens, and animal pens. The present invention will be explained in more detail with reference to Examples below. Percentages (%) refer to weight/volume percentages unless otherwise specified. Example 1 Actinosynnema sp.No.C-33196 (IFO14127,
FERM P-6138) in a 200 ml Erlenmeyer flask with 2% glucose, 3% soluble starch, 1% raw soybean flour, 1% cornstarch liquor, 0.5% polypeptone, 0.3% NaCl, _0.5 % CaCO3, (PH7 0) was inoculated into 40 ml of seed medium and cultured on a rotary shaker at 28°C for 48 hours. Use 10ml of the obtained culture solution as a seed medium.
Transfer to a 2-volume Sakaguchi flask containing 500 ml and incubate at 28°C.
Cultured on a reciprocating shaker for 48 hours. 1 of this culture solution
was inoculated into a 200-volume stainless steel tank containing 100% seed medium and incubated at 28°C, aeration 100/min, and agitation.
A seed culture solution was obtained by culturing at 200 revolutions/min for 48 hours.
60% of the resulting seed culture was mixed with 5% glycerol, 2% cornstarch liquor, 2% dry yeast,
Transplanted into a 2000 volume stainless steel tank containing a main medium 1200 with the composition of MgCl ₂ 0.5%, KH ₂ PO ₄ 2%, CaCO ₃ 0.1%, heated at 28℃, aeration 1200/min.
Culture was carried out for 114 hours under conditions of stirring at 150 rpm and internal pressure of 1 Kg/cm ² . Hyflo Supercell (manufactured by Johns Manville Sales Corp.) 68.1 was added to the obtained culture solution 1100.
Kg was mixed and passed. The culture solution and the water washing solution were combined, 1200 ml was adjusted to pH 6.5, and extracted with 600 ml of ethyl acetate. After washing the extract with 300 g of water, it was concentrated under reduced pressure, and when it reached about 40 g, 20 g of a 2% aqueous ferric chloride solution was added and stirred for 1 hour. After the oxidation was completed, the ethyl acetate layer was washed twice with 20 g of water, and then concentrated under reduced pressure. Add n-hexane 1 to 2 times to approximately 200 ml of the oily concentrated residue, wash it, and remove the residue (approximately 100 ml).
500 ml of ethyl acetate was added to the mixture, and the mixture was allowed to stand in a cool place. After filtering out the precipitated crystals, the mother liquor was concentrated, and the residue was subjected to column chromatography using 450 g of silica gel (manufactured by Merck & Co., Ltd.). Column with n-hexane (500ml)
After washing with n-hexane-ethyl acetate (4:1
→ 2:1 → 1:1 → 1:2 → 1:4 → 1:8), the antibiotic C-
33196E-4 was eluted. Since the component thus obtained was not yet a single component, it was purified again by silica gel chromatography. The mixture mainly consisting of antibiotic C-33196E-4 is
Adsorb onto a column of silica gel (150 g), develop in the order of hexane, chloroform, and chloroform-methanol (50:1 → 10:1), collect and concentrate the desired fraction, dissolve in ethyl acetate, and add 2% hydrosulfite. Reduced with sodium water. The reduced product was again subjected to column chromatography on silica gel (130 g), and then treated with hexane, chloroform,
By developing in the order of chloroform-methanol (25:1→5:1) and collecting and concentrating a single fraction, crystals (430 mg) of antibiotic C-33196E-4-R were obtained. Example 2 Culture solution 1 obtained by culturing in the same manner as Example 1
After adding 1 part of methanol to the solution and filtering it, the methanol of this liquid was distilled off, and the pH was adjusted to 6 to 7 using ethyl acetate.
It was extracted twice with 500 ml, washed with water, and then concentrated. When this concentrated solution was examined by TLC, antibiotic C-33196E-4 was detected.
and the presence of antibiotic C-33196E-4-R was confirmed. Example 3 Antibiotic C-33196E-4 produced in Example 1
100 mg of R was dissolved in 100 ml of ethyl acetate, and 50 ml of a 2% aqueous ferric chloride solution was added and stirred. After 1 hour, remove the ethyl acetate layer, wash with water, and concentrate to obtain antibiotic C.
-33196E-4 88 mg was obtained.

Claims (1)

[Claims] 1. Antibiotic C-33196E-4 having the following properties:
Or C-33196E-4-R: a Antibiotic C-33196E-4: Melting point: 209-210℃ Shape: Yellow crystal Light intensity: [α] ²⁵ _D +53゜±5゜ (c=0.5,
CHCl ₃ ) Elemental analysis value (%) C 63.14±0.5 H 7.57±0.5 N 5.26±0.5 Mass spectrum (M ⁺ ): m/z532 Ultraviolet absorption spectrum: λ ^MeOH _nax 276nm±2 (E1% 1cm272±20) 390nm ±2 (E1% 1cm33.5±5) Infrared absorption spectrum, main peak (cm
^-1 ): 1735, 1700, 1670, 1650, 1625, 1605,
1500, 1380, 1305, 1290, 1210, 1050 Solubility: Slightly soluble; petroleum ether, hexane, water soluble; chloroform, methylene chloride, toluene, diethyl ether easily soluble; acetone, ethyl acetate, methanol,
Dimethyl sulfoxide Color reaction: Positive; Decolorizes potassium permanganate test solution Negative: Ninhydrin, Ehrlich reaction, Burton reaction Acidic, neutral, basic: Neutral substance b Antibiotic C-33196E-4-R (Ethyl acetate) (Physical properties for those containing one molecule of as a crystal solvent): Melting point: 224-225°C Shape: Colorless crystal Light intensity: [α] ²⁵ _D +32° ± 5° (c = 0.5,
MeOH) Elemental analysis value (%): C 60.96±0.5 H 8.25±0.5 N 4.59±0.5 Mass spectrum (M ⁺ ): m/z534 Ultraviolet absorption spectrum: λ ^MeOH _nax 307nm±2 (E1% 1cm75±8) Red External absorption spectrum, main peak (cm
^-1 ): 1720, 1670, 1630, 1600, 1535, 1465,
1380, 1325, 1045 Solubility: Slightly soluble; petroleum ether, hexane, diethyl ether, chloroform, ethyl acetate soluble; methanol easily soluble; dimethyl sulfoxide) Color reaction: Positive; Burton reaction Negative; ninhydrin, Ehrlichi reaction Acidic, medium Characteristic and basicity: Neutral substance 2 Antibiotic C- belonging to the genus Actinosynnema
33196E-4 and/or antibiotic C-33196E
Antibiotic C-33196E-4 and/or Antibiotic C-33196E-4-R, which is characterized by culturing a microorganism capable of producing -4-R in a medium and collecting the compound from the culture. Manufacturing method.