JPS6129715B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic M5070 produced by M5070, a filamentous fungus belonging to the genus Westerdykella, and a method for producing the same. The physicochemical properties of the novel antibiotic M5070 of the present invention are as follows. (1) Molecular weight 309 (from mass spectrum) (2) Melting point 130℃ (decomposition) (3) Light intensity [α] ²⁴ _D = -82° (C = 0.5% methanol) (4) Elemental analysis C = 65.91% H =8.56% N4.31% (5) Molecular formula This substance consists of C, H, N, and O. C ₁₇ H ₂₇ NO ₄
It is indicated by. (6) Ultraviolet absorption spectrum The ultraviolet absorption spectrum measured in methanol is as shown in Figure 1 (solid line).
mμ (E ¹ % ₁ cm=715), λmax274mμ (E ¹ % ₁ cm
=
900), with a characteristic absorption maximum at λmax 285 mμ (E ¹ % ₁ cm = 718). Moreover, no change in the spectrum was observed under the conditions of 0.1HCl-MeOH and 0.1N NaOH-MeOH. (The spectrum under alkaline conditions is as shown by the broken line in Figure 1) (7) Infrared absorption spectrum The infrared absorption spectrum measured by the KBr method is as shown in Figure 2, 3350, 2900, 1740,
1650, 1430, 1370, 1180, 1100, 1020, 980cm
It has an absorption band at each wavelength around ^-1 . (8) Nuclear magnetic resonance spectrum As shown in Figure 3. (in CDCl ₃
100MHz, internal reference TMS). (9) Appearance Pale yellow to yellowish white (same for its chlorine salt) (10) Solubility in various solvents Soluble in methanol, ethanol, acetone, ethyl acetate, chloroform, benzene, diethyl ether, water, n- Insoluble in hexane, petroleum ether. (11) Color reaction Positive for potassium permanganate decolorization reaction and ninhydrin reaction, negative for ferric chloride reaction, Fehring reaction, and Moritzhu reaction. (12) Distinction between basic, neutral, and acidic Weak basic (13) Stability A decrease in antibacterial activity was observed in a solvent at 60°C for 15 minutes. Moreover, it is quickly deactivated at pH 9 or higher. (14) Thin layer chromatography [using Sports Film (silica gel) manufactured by Tokyo Kasei Co., Ltd.] Chloroform: methanol = 25:1 Rf = 0.52 Benzene: methanol = 20:1 Rf = 0.15 Benzene: ethyl acetate = 1:1 Rf =0.05 Furthermore, the antibacterial spectrum of the novel antibiotic M5070 of the present invention is as follows. For bacteria, use ordinary agar medium (Escherichia coli; peptone medium),
For fungi and yeast, use Sabouraud medium and indicate the diameter of the inhibition circle on a paper disk (use concentration 500γ/ml)

[The color display follows the Color Harmony Manual (Container Corporation of America 1958)]

From the above mycological properties, the filamentous fungus M5070 of the present invention
The strain is recognized as belonging to the Westerdychem genus because it has a closed, mouthless ascus, and the ascospores contain 32 spores, and the ascospores do not have holes or clefts for budding. In 1655, the genus Westerdycera was
The genus was created by Stolk, and to date there are 6 species and 1
Variants are recognized. By the way, the shape, size, and other characteristics of the asci, ascospores, and ascospores indicate that this fungus is a filamentous fungus.
The M5070 strain matches well with the description of Westerdykella dispersa, but does not match with other species, and the results of comparison with Westerdykella dispersa IFO8431 and NHL2530 also show good agreement. , this strain was identified as Westerdeichella dispersa and named Westerdeichella dispersa M5070. [References; Trans Brit.mycol.Soc., 38 , 419
~424 (1955), Can.J.Bot., 39 . 1633-1666
(1961), Ceska Mykologin, 18 . 82～84
(1964), The Genera of Fungi Sporulating in
Pure Culture 315pp (1974)] Next, in manufacturing M5070 of the present invention,
Westerdychella Dispersa M5070 above
Bacterial strains are one example, and all M5070-producing bacteria belonging to the genus Westerdeichella can be used, even if they are mutant strains. Obtained by culturing aerobically in a medium. As the medium, a solid medium or a liquid medium is used, and especially for mass production, a liquid medium, especially an aqueous medium, is used. 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, such as glucose, sucrose, maltose, starch, dextrin, and molasses.
The nitrogen source may be any available nitrogen compound, such as corn steep liquor, soybean flour, cottonseed flour, wheat gluten, polypeptone, meat extract,
Yeast extract, casein hydrolyzate, ammonium salts, nitrates, etc. are used. In addition, salts such as phosphates, nitrates, magnesium, calcium, potassium, sodium, cobalt, iron, and manganese are used as necessary. The culture temperature can be changed as appropriate within the range that allows the bacteria to grow and produce M5070, but is particularly preferably 25 to 30°C.
It is. The culture time varies somewhat depending on the conditions, but is usually about 2 to 5 days, and the culture may be terminated at an appropriate time by determining the time when M5070 reaches its maximum titer. In the culture of M5070-producing bacteria thus obtained, most of M5070 is produced outside the cells. Next, M5070 is collected from the culture of this M5070-producing bacterium, but it is convenient to separate and purify it by taking advantage of the fact that M5070 is a weakly basic fat-soluble substance. An example of separation and purification means for M5070 is as follows. First, M5070-producing bacteria are cultured as described above, and solid contents such as bacterial bodies are removed from the resulting culture to obtain a culture solution. After extraction, concentration, and dissolution in a soluble solvent, adsorption chromatography using silica gel or alumina was performed to obtain the active fraction, which was then dried and purified.
It is sufficient to obtain M5070, and it is also possible to dissolve this M5070 in a soluble solvent, add water to this, add an aqueous acid solution, perform water inversion, and dry the aqueous layer to obtain a salt of M5070 with an acid. . Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. Example 1 1.5% sucrose, 1.5% dextrin, 2% polypeptone, 2% corn steep liquor,
KH ₂ PO ₄ 0.2%, MgSO ₄・7H ₂ O 0.1%, FeSO ₄・
Dispense 100 ml of a medium consisting of 0.01% 7H ₂ O and 1% Cetoite (trade name: Hyflo Supercell) into a 500 ml Erlenmeyer flask, sterilize it under high pressure at 120°C for 15 minutes, and add Westerdeichella dispersa to this. M5070
The mycelium containing spores from the slant culture of the strain was scraped and inoculated, and cultured with shaking at 27°C for 3 days. This culture was then transferred to 500 ml of medium containing 100 ml of the same composition.
Inoculate 1 to 2 ml each into 60 Erlenmeyer flasks, and inoculate 27
The cells were cultured with shaking at ℃ for 4 days. After culturing, merge this
A culture of about 5.5 was obtained, which was further filtered through Celite to remove bacterial cells, to obtain a culture solution of about 5 (PH of this solution was 7.8, containing 300 γ/ml of M5070). Furthermore, 1/2 amount of chloroform was added to this culture solution, and the mixture was extracted with PH9. The chloroform layer was then collected, dehydrated using anhydrous sodium sulfate, concentrated under reduced pressure, dried, and further dissolved in a small amount of chloroform and adsorbed on a silica gel column (diameter 2 x 30 cm) filled with chloroform. . After adsorption, wash with a small amount of chloroform, develop with a developing solvent of chloroform:methanol = 75:1, fractionate into 12 ml fraction, collect the active fraction, fractions No. 37 to 45, concentrate, and dry. Approximately 836 mg of pale yellow to yellowish white purified M5070 was obtained (recovery rate 50%, and thin layer chromatography showed a single spot). Example 2 400 mg of M5070 was dissolved in 50 ml of benzene,
After adding the same amount of water to this, add 0.5N hydrochloric acid to this until the pH becomes 3, collect the aqueous layer, repeat the same operation twice, collect the layer, freeze-dry it, and make about 350mg of it. The hydrochloride of M5070 was obtained.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of M5070 of the present invention, FIG. 2 shows the infrared absorption spectrum of M5070 of the present invention, and FIG. 3 shows the nuclear magnetic resonance spectrum of M5070 of the present invention.

Claims (1)

[Claims] 1. A novel antibiotic M5070 and its non-toxic salt having the following physicochemical properties ●Molecular weight 309 ●Melting point 130℃ (decomposition) ●Degree of light application [α] ²⁴ _D = -82° (C=0.5% methanol) ●Elemental analysis C=65.91% H=8.56 N=4.31% ●Molecular formula C ₁₇ H ₂₇ NO ₄ ●Ultraviolet absorption spectrum As shown in Figure 1 ●Infrared absorption spectrum As shown in Figure 2 Appearance: Pale yellow to yellowish white Potassium decolorization reaction, ninhydrin reaction are positive, ferric chloride reaction, Fehling reaction, Moritzhu reaction are negative ●Distinction between basic, neutral, and acidic Weak basic 2 Antibiotics belonging to the Westerdichella genus
A novel method for producing antibiotic M5070, which is characterized by culturing M5070-producing bacteria in a medium and collecting antibiotic M5070 from the culture. 3 Antibiotics belonging to the genus Westerdycera
3. The manufacturing method according to claim 2, wherein the M5070-producing bacterium is Westerdichella dispersa M5070 bacterium.