JPH0417199B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel antibiotic SF-2370 substance and a method for producing the same. Background Art and Problems to be Solved by the Invention Various antibiotics have been known in the past, but there is always a desire to discover and produce new antibiotics with new effects. Means, Action, and Effect for Solving the Problems The present inventors searched for new and useful antibiotics that have antibacterial activity against various Gram-positive and Gram-negative bacteria, and found that Actinomadura
It was discovered that by culturing actinomycetes belonging to the genus in a nutrient medium, substances with antibacterial and antifungal activities were produced in the culture, and the effective substances,
The present invention was completed by isolating the SF-2370 substance and determining its physicochemical and biological properties. Therefore, the present invention provides the following formula: The present invention provides a novel antibiotic SF-2370 substance represented by: Furthermore, the present invention provides antibiotic SF-2370 belonging to the genus Actinomadura.
Cultivate substance-producing bacteria and extract antibiotics from the culture.
The present invention provides a method for producing an antibiotic SF-2370 substance, which comprises collecting the SF-2370 substance. AM-2282 substance (Journal of Antibiotics, Vol. 30, Vol.
275-285 (1977), Japanese Patent Publication No. 53-73501 and Japanese Patent Publication No. 57-53076), but elemental analysis values, molecular weight, infrared absorption spectrum and
Since the ¹ H nuclear magnetic resonance spectra etc. are different,
It is clearly distinguished from this substance. Therefore SF−2370
The substance was determined to be a new substance. An example of the new antibiotic SF-2370 substance-producing bacteria used in the present invention is SF-2370, which was newly isolated from soil in Shimizu City, Shizuoka Prefecture by the present inventors.
The mycological properties of this strain are as follows. Morphology The basal hyphae are well elongated and branched and do not divide under normal conditions. Aerial mycelia generally adhere poorly, but on oatmeal agar they adhere relatively well and spore formation is abundant. Spore formation on aerial mycelium is also observed on glycerol-asparagine agar, starch agar, etc. The branches of the aerial hyphae are simple branches, and no axillary branches are observed. The spore chain at the tip of the aerial hyphae can be straight, wavy, hook-shaped, or loop-shaped. When observed using an electron microscope, the spores are elliptical to short cylindrical, and have a size of 0.4 to 0.8 x 0.8 to 1.2 μm.
The surface is smooth. There are often 10 to 20 spore chains. Growth status on various media

[Table] Physiological properties (1) Growth temperature range: 15% on oatmeal agar
It grows in a temperature range of ~37°C and grows well at 25-32°C. (2) Gelatin liquefaction: Negative (20℃, 21 days culture) (3) Starch hydrolysis: Negative (28℃, 14 days culture) (4) Nitrate reduction: Negative (28℃, 14 days culture) ( 5) Peptonization of skim milk: Positive (35℃), Negative (28℃) Coagulation of skim milk: Positive (35℃), Negative (28℃) (6) Production of melanin-like pigment: Negative Carbon source availability Basal medium [yeast extract (manufactured by Difco) 0.5%,
Calcium carbonate 0.1%, agar (manufactured by Difco) 1.5%]
1% of each carbon source was added and observed. (1) Used: D-glucose, D-fructose, L-rhamnose (2) Not used: D-mannitol, i-inositol, raffinose, sucrose (3) Suspected use: D-xylose, L-arabinose Cell wall composition 2,6-diaminopimelic acid in the whole cell hydrolyzate was in the meso form, and a small amount of madeulose was observed as a sugar. Based on the above properties, it is reasonable to consider that strain SF-2370 belongs to the genus Actinomadeura among actinomycetes. Therefore, the present inventors converted the SF-2370 strain into Actinomadeura sp. SF-2370.
(Actinomadura sp. SF-2370). Strain SF-2370 has been donated to the Institute of Microbial Technology, Agency of Industrial Science and Technology since July 28, 1981, under the microbial acceptance number FERM P-7760. The SF-2370 strain is susceptible to changes in its properties, as seen in the case of other actinomycetes. For example, S.F.
-2370 strain or derived from this strain, whether it is a mutant strain (naturally occurring or induced), a transzygote, or a genetically modified strain, which produces the antibiotic SF-2370 substance, is included in the present invention. Can be used. In the method of the present invention, the above-mentioned bacteria are cultured in a medium containing nutrients that can be utilized by common microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing actinomycetes can be used. For example, glucose, sucrose, starch, glycerol, starch syrup, molasses, animal/vegetable oils, etc. can be used as carbon sources. Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, yeast extract, cornstarch liquor, ammonium sulfate, sodium nitrate, etc. can be used. In addition, as necessary, sodium,
It is useful to add soluble inorganic salts capable of producing potassium, calcium, magnesium, cobalt, chlorine, phosphate, sulfate and other ions. It also helps the growth of bacteria, and antibiotics
Suitable organic and inorganic substances can be added to facilitate the production of SF-2370 materials. For the culture, cultivation methods under aerobic conditions, especially deep culture, are most suitable. The appropriate temperature for culturing is
The temperature is 15-37°C, but in most cases it is cultured at around 26-30°C. Production of the antibiotic SF-2370 substance varies depending on the medium and culture conditions, but the accumulation usually reaches its maximum within 2 to 10 days in both shaking culture and tank culture. When collecting the SF-2370 substance obtained according to the present invention from a culture, its extraction and purification was carried out using Amberlite made)
Synthetic adsorbents such as, gelling agents such as Cephadex LH-20 (manufactured by Pharmacia Fine Chemicals, Sweden), precipitation with hexane, solvent extraction with ethyl acetate, column chromatography with silica gel, etc. are used. However, the following collection method is more efficient. That is, the bacterial cells and other solid substances are separated from the culture solution using a supernatant such as diatomaceous earth, and then the active ingredients in the bacterial cells are separated by 70%
Extract with % acetone water. After the extract is concentrated under reduced pressure to remove acetone, the concentrated solution is extracted with ethyl acetate. The extract is dehydrated over anhydrous sodium sulfate, concentrated under reduced pressure to remove ethyl acetate, and dried. This is further treated with an appropriate combination of silica gel column chromatography using chloroform-ethyl acetate (10:1) as a developing solvent, and column chromatography such as Sephadex LH-20 using methanol as a developing solvent.
High purity SF-2370 material can be obtained. The physical and chemical properties of SF-2370 substance are detailed below. (1) Appearance Pale yellow crystal (2) Elemental analysis values C69.50%, H4.74%, N8.88% (3) Molecular formula C ₂₇ H ₂₁ N ₃ O ₅ (4) Molecular weight 467 (mass spectrometry, FD -MS) (5) Melting point 256℃ (6) Specific optical rotation [α] ²⁵ _D +57゜ (c0.1, methanol) (7) Ultraviolet absorption spectrum 228nm (E1%) in methanol as shown in Figure 1. Absorption characteristic of 1cm725), 250nm (E1% 1cm717), 265nm shoulder, 280nm shoulder, 290nm (E1% 1cm1708), 320nm shoulder, 335nm (E1% 1cm432), 350nm (E1% 1cm325) and 367nm (E1% 1cm353) These absorptions do not change significantly even when acid or alkali is added. (8) Infrared absorption spectrum The infrared absorption spectrum measured in potassium bromide tablets is shown in Figure 2. (9) Nuclear magnetic resonance spectrum The 400MHz ¹ H nuclear magnetic resonance spectrum measured in deuterated chloroform is shown in Figure 3, and the 100MHz ¹³ C nuclear magnetic resonance spectrum is shown in Figure 4. (10) Silica gel thin layer chromatography (Kieselgel 60 F 254) Developing solvent Rf value Ethyl acetate 0.58 Chloroform-methanol (20:1) 0.63 Benzene-acetone (2:1) 0.45 (11) Color reaction Positive for Greig-Leaback reagent, ninhydrin and chloride Negative for ferric reagents. (12) Solubility Soluble in pyridine, dimethylformamide, dimethyl sulfoxide, methyl cellosolve and chloroform, sparingly soluble in methanol and ethyl acetate.
Insoluble in water and n-hexane. (13) Classification of basic, acidic, and neutral Neutral Table 1 shows the antibacterial and antifungal spectra of the SF-2370 substance as measured by the agar plate dilution method.

【table】

[Table] Example Next, an example of manufacturing the new antibiotic SF-2370 substance is shown. Example 1 Glucose 2.0%, wheat germ 1.0%, peptone 0.5
%, yeast extract 0.5%, and calcium carbonate 0.1% (PH 7.0) was dispensed into a 100 ml Erlenmeyer flask and sterilized at 120° C. for 15 minutes. This was inoculated with Actinomadeura sp. SF-2370 (Feikoken Bacteria No. 7760), and was heated at 28°C for 7 days at a rate of 220 m/min.
Culture was performed by rotation. 20 ml of this culture was placed in a 1 volume jar fermentor containing 600 ml of production medium (PH 7.0) consisting of 1.5% glucose, 1.0% wheat germ, 1.0% cornstarch liquor, 0.5% firma media, and 0.3% calcium carbonate. The cells were inoculated and cultured with aeration at 28° C. for 5 days (aeration volume: 600 ml/min, rotation speed: 500 revolutions/min). After the culture was completed, diatomaceous earth was used as an auxiliary agent to obtain cultured bacterial cells. 500 ml of 70% acetone water was added to the bacterial cells to extract the active ingredient, and the bacterial cells were separated. Then, the bacterial cell extract was concentrated under reduced pressure to remove acetone, and the resulting concentrated liquid was
250 ml of ethyl acetate was added to 250 ml and shaken to extract the active ingredient. Repeat this extraction operation twice,
500 ml of the obtained ethyl acetate extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily substance. N-hexane was added to this oil and the resulting precipitate was collected to obtain 286 mg of crude material. This crude substance was dissolved in a mixture of chloroform and ethyl acetate (10:1), and 60 ml of silica gel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.) was added.
column and developed with 600 ml of a chloroform-ethyl acetate (10:1) mixture. The developing solution was subjected to silica gel thin layer chromatography (Merck & Co., Kieselgel, 60 F 254, 5714; developing solvent: ethyl acetate) and irradiated with ultraviolet light (254 nm) to obtain Rf.
Sarcina lutea (Sarcina lutea) shows a value of 0.58.
Fractions showing antibacterial activity in a paper-disc bioassay using M. lutea as the test bacterium were collected and concentrated to dryness under reduced pressure to obtain 216 mg of pale yellow powder. This crude powder was dissolved in ethyl acetate and subjected to preparative silica gel thin layer chromatography (Merck Kieselgel 60 F 254, 5744, developing solvent: ethyl acetate), the active portion (Rf value 0.58) was scraped off, and acetic acid Extracted with ethyl. After concentrating the extract under reduced pressure, methanol was added and left overnight to obtain 79 mg of pale yellow crystals of SF-2370 substance. Example 2 Seed culture 1 of Actinomadeura sp. SF-2370 obtained in the same manner as described in Example 1 was inoculated into two 50-volume jar fermenters each containing 35 of the above production medium, and incubated at 28°C. Aerated agitation culture for 5 days (aeration rate 35/min, rotation speed 200/min)
rotation). After the cultivation was completed, 25% of 70% acetone water was added to the microbial cells obtained by straining the culture to extract the active ingredients twice, and the microbial cells were separated to obtain an extract of 50%. This extract was concentrated under reduced pressure to remove acetone, and 30 ml of ethyl acetate was added to the resulting concentrate and stirred for 15 minutes to extract the active ingredients. The ethyl acetate extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 33 g of pale yellow crude crystals. The crude crystals were recrystallized twice from a chloroform-methanol mixture to obtain 15 g of pale yellow crystals of SF-2370 substance.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antibiotic SF-2370 substance in methanol, and Figure 2 shows the ultraviolet absorption spectrum of the antibiotic SF-2370 substance in methanol.
Figure 3 shows the infrared absorption spectrum of SF-2370 substance in potassium bromide tablets, Figure 3 shows the 400MHz ¹ H nuclear magnetic resonance spectrum of SF-2370 substance measured in deuterated chloroform, and Figure 4 shows the SF-2370 substance measured in deuterium chloroform. This shows the 100MHz ¹³ C nuclear magnetic resonance spectrum of substance 2370 measured in deuterated chloroform.

Claims (1)

[Claims] 1. The following formula: A novel antibiotic SF-2370 substance represented by 2. A method for producing an antibiotic SF-2370 substance, which comprises culturing an antibiotic SF-2370 substance-producing bacterium belonging to the genus Actinomadura, and collecting the antibiotic SF-2370 substance from the culture.