JPH0473439B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a novel antibiotic MG802-AF1-A and a method for producing the same. [Conventional technology] Until now, antibiotics produced by microorganisms have been
Over 5,000 types have been reported, and they are widely used to treat infectious diseases. Among these, the production of 18 types of antibiotics has been reported from microorganisms belonging to the genus Chainia [Published by Yusaco Co., Ltd. in 1981,
Index of Actinomycetes
Antibiotics (Index of
Actinomycetes Antibiotics)]. [Problems to be Solved by the Invention] When using antibiotics, the appearance of bacteria resistant to the antibiotics is inevitable, and new antibiotics that are effective against resistant bacteria are constantly desired. [Means for Solving the Problems] To summarize the present invention, the first invention of the present invention is an invention related to a novel antibiotic MG802-AF1-A, which has the following characteristics: Shape: yellow-brown candy-like element Analysis (actual values): Carbon 61.44%, Hydrogen 6.41%,
Nitrogen 0.00%, Oxygen 16.31%, Chlorine 15.16% Molecular weight: 480.1487 (by mass spectrometry) Molecular formula: C ₂₅ H ₃₀ O ₅ Cl ₂ Specific optical rotation: [α] ²⁷ _D = +40.6° (c = 0.5, ethanol) Ultraviolet absorption spectrum (in methanol): absorption maximum 205nm (logε4.25), 251nm (logε4.14),
270nm (sh.logε4.04), 295nm (sh.logε3.87),
360nm (logε3.80) Infrared absorption spectrum (KBr tablet): 3400, 2920,
1640, 1610, 1250, 1070cm ^-1 Solubility: methanol, acetone, chloroform,
Soluble in ethyl acetate, ethyl ether and benzene. Both the Gibbs reaction and the anisaldehyde reaction, which have color reactions that are poorly soluble in water and hexane, are characterized by having a weakly acidic substance that distinguishes between positive acidity, basicity, and neutrality. The second invention of the present invention is a novel antibiotic.
This invention relates to a method for producing MG802-AF1-A, which involves culturing MG802-AF1-A producing bacteria related to the genus Cainia, and producing a new antibiotic from the culture.
It is characterized by separating and collecting MG802-AF1-A. The present inventors have discovered that MG802-, which belongs to the genus Cainia,
AF1-A producing strain was cultured and MG802-AF1-A
MG802−AF1− was accumulated from this culture.
A was collected. Since MG802-AF1-A exhibits antibacterial activity equivalent to Gram-positive bacteria and multidrug-resistant Gram-positive bacteria, it can be used as an antibiotic against Gram-positive bacteria that is also effective against resistant bacteria. An example of a producing bacterium of MG802-AF1-A belonging to the genus Cainia is Cainia rubra, a strain of Cainia rubra ( Chainia
rubra) MG802-AF1 (Feikoken Bibori No. 8022). The mycological properties of this strain are as follows. [Mycological characteristics of strain MG802-AF1] 1. Morphology Under a microscope, strain MG802-AF1 forms hook-shaped to spiral-shaped aerial hyphae from branched basal hyphae, and no whorled branches are observed. The mature spore chain is
A chain of 10 or more spores was observed, and the size of the spores was approximately 0.4 to 0.5 x 0.6 to 0.7 μm. The surface of the spore has short thorn-like protrusions. A sclerotium-like structure was also observed, and its diameter was approximately 5.
~10 μm. 2. Growth status in various media The standards shown in parentheses for describing colors are the Color Harmony Manual of Container Corporation of America.
harmony manual) was used. (1) Sucrose/nitrate agar medium (cultured at 27℃) On the growth of light yellow daisies [2gc, Bamboo] to light brown [3ic, Lt Amber], white to
It has brownish white (3ba, Pearl to 3cb, Sand) aerial mycelium, and the soluble pigment is yellowish brown. (2) Glucose-asparagine agar medium (27℃
Culture) Growth is colorless to pale yellow [2gc,
Bamboo] ~ Dull Daidai [4pe, Orange
Rust], does not grow aerial mycelium, and the dissolved live pigment is only reddish brown. (3) Glycerin-asparagine agar medium (ISP
-Medium 5, 27℃ culture) Light brown [3ic, Lt Amber] ~ Light brown [3le, Maple] ~ Tea ash [4lg, Lt Spice]
Brown], a thin white aerial mycelium grows on it, and the soluble pigment is only reddish-brown. (4) Starch/inorganic salt agar medium (ISP-Medium 4, cultured at 27℃) Light yellow tea [3lg, Adobe Brown] ~ Light brown [4ie, Cork Tan] ~ On the growth of tea ash,
It grows white to brownish-white [3dc, Natural] aerial mycelia, and the soluble pigment is only reddish-brown. (5) Tyrosine agar medium (ISP-Medium 7, cultured at 27°C) White to brownish aerial mycelium grows on the growth of light brown to grayish reddish [5ng, Brich Red], and the soluble pigment of reddish brown grows. produces. (6) Nutrient agar medium (cultured at 27℃) Yellow tea [3ng, Yellow Maple] ~ Tea [4ni,
From around 20 days after culturing, a slight white aerial mycelium grows on the growing [Chestnut Brown], and the soluble pigment is only brownish. (7) Yeast/malt agar medium (ISP-medium 2,
27℃ culture) Yellow tea to light brown [3le, Cinnamon to 3lg,
Adobe Brown], white aerial mycelium grows on it, and the soluble pigment is only reddish-brown. (8) Oatmil agar medium (ISP-Medium 3, 27℃
Culture) Light Daidai [3gc, Lt Tan] ~ Dull Daidai [3ic, Lt Amber ~ 4ic, Pastel
Orange] ~ Light red color [5ic, Lt
Persimmon], white to brownish white [5cb]
The soluble pigment is aerial brownish. (9) Glycerin/nitrate agar medium (cultured at 27℃) Growth is colorless to pale yellow [2gc, Bamboo]
~Light yellow brown [3le, Cinnamon], no aerial mycelia are attached, and the soluble pigment is only brownish. (10) Starch agar medium (cultured at 27℃) Light yellowish brown to dull yellowish brown [4ic, Pastel
On the growth of [Orange], thin white aerial mycelium grows, and the soluble pigment is only slightly brownish. (11) Malate lime agar medium (cultured at 27°C) White to brown aerial mycelium grows on the colorless to pale yellowish brown growth, and the soluble pigment is only slightly yellowish. (12) Cellulose (synthetic solution added with paper strips, cultured at 27°C) White to brownish aerial mycelia are grown on the pale yellow to pale yellow brown growth, and no dissolved live pigments are observed. (13) Gelatin puncture culture In both simple gelatin medium (cultured at 20°C) and glucose peptone gelatin medium (cultured at 27°C), growth is colorless, no aerial mycelium is attached, and no soluble pigment is observed. (14) Skimmed milk (cultured at 37°C) Growth is light yellow to light brown, aerial mycelia do not attach, and soluble pigments are only brownish. 3 Physiological properties (1) Growth temperature range Yeast starch agar medium [soluble starch (manufactured by Koso Kagaku Co., Ltd.) 1.0%, yeast extract (manufactured by Daigo Nutrient Chemical Co., Ltd.) 0.2%, string agar 3.0%,
PH7.0], 20℃, 24℃, 27℃, 30℃,
As a result of testing at each temperature of 37℃ and 50℃, it grew at either temperature except 50℃.
The optimum temperature is estimated to be around 27°C to 37°C. (2) Liquefaction of gelatin (15% simple gelatin, 20℃
Culture: glucose, peptone, gelatin, 27
℃ culture) In simple gelatin medium, liquefaction begins around 14 days after culture, and its effect is weak.
In the glucose-peptone-gelatin medium, no liquefaction was observed when observed 30 days after culture. (3) Hydrolysis of starch (starch/inorganic salt agar medium and starch agar medium, both 27
(°C culture) In the case of starch/inorganic salt agar medium, water decomposition is observed from around 5 days after cultivation, and in the case of starch agar medium from around 10 days after cultivation, and the effect is moderate. (4) Coagulation and peptonization of skimmed milk (skimmed milk,
(37℃ culture) Coagulation begins around 14 days after culture, and immediately after completion, peptonization begins. Peptonization is completed around 25 days after culture. The effects are both moderate to strong. (5) Production of melanin-like pigment (tryptone yeast prosthesis, ISP-medium 1: peptone yeast, iron agar, ISP-medium 6: tyrosine agar, ISP-medium 7, all cultured at 27°C) In any medium, No melanin-like pigment formation was observed. (6) Utilization of carbon sources (Pridham-Godelive agar, ISP-medium 9, 27°C culture) D-glucose, L-arapinose, D-
It grows using xylose, D-fructose, L-rhamnose, raffinose, and D-mannitol, but does not use inositol.
It is presumed that sucrose is probably used. (7) Dissolution of malic lime (malic acid coal agar,
(27℃ culture) From around 14 days after culturing, malate lime around the growth is dissolved, and its effect is moderate. (8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, ISP-medium 8, cultured at 27°C) Positive. To summarize the above properties, the MG802-AF1 strain is
No sporangia are observed, aerial hyphae are hook-shaped to spiral-shaped, and no paraphytes are observed. The surface of the spore has short thorn-like projections and sclerotium-like structures are observed. In various media, white to brownish white aerial mycelium grows on the growth of light yellow to light brown to brown ash, and the soluble pigment is
Yellow to reddish-brown in color. The production of melanin-like pigments was negative, and the hydrolyzability of starch was moderate. Regarding protein decomposition power, gelatin has weak liquefaction ability, and milk coagulation and peptonization ability is moderate to strong. The cell wall composition of MG802 to AF1 strains is that 2,6-diaminopimelic acid is LL.
- type, does not contain arabinose as a sugar component. From these properties, it is estimated that the MG802-AF1 strain is an actinomycete belonging to the genus Cainia that has a sclerotia-like structure. Furthermore, we searched for known bacterial species similar to strain MG802-AF1 from the genera Cainia and Streptomyces, and found that Chainia rubra [International Journal of Systematic Bacteriology]
Bacteriology, Vol. 22, p. 347 (1972)] was listed as a closely related species. Table 1 shows Cainia rubra and MG802 in the literature.
-Comparison of AF1 strains is shown.

[Manufacturing method of MG802-AF1-A]

A culture containing MG802-AF1-A can be obtained by inoculating a MG802-AF1-A producing strain belonging to the genus Cainia into a nutrient-containing medium and growing it aerobically. As the nutrient source, those that can be used as a nutrient source for actinomycetes are used. For example, commercially available nitrogen sources such as peptone, meat extract, corn stave liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, casein hydrolyzate, sodium nitrate, ammonium nitrate, ammonium nitrate, and Commercially available carbohydrates such as glycerin, sucrose, starch, glucose, galactose, mannose, and molasses, or carbon sources such as fat, and inorganic salts such as common salt, phosphate, calcium carbonate, and magnesium sulfate can be used. . In addition, trace amounts of metal salts, antifoaming agents such as animal, vegetable, and mineral oils may be added as necessary. These substances are used by production bacteria, and MG802−
Any known culture material for actinomycetes can be used as long as it is useful for producing AF1-A. Liquid culture is preferred for mass production of MG802-AF1-A, and the culture temperature is set at a temperature that allows the production bacteria to grow.
Applicable within the range of producing MG802-AF1-A,
The temperature is usually 20-40°C, preferably 27-37°C. MG802−AF1− was cultured using the conditions described above.
The method can be appropriately selected depending on the properties of the A-producing bacteria. MG802-AF1-A is present in both the culture solution and the bacterial cells. The culture solution can be extracted with a water-immiscible organic solvent such as butyl acetate, chloroform, or butanol at a pH of 10 or less. Rather than bacterial cells,
After extraction with an organic solvent such as methanol or acetone, the extract can be concentrated under reduced pressure and further extracted in the same manner as the culture solution. In addition to the above-mentioned extraction methods, known methods used to collect fat-soluble substances, such as adsorption chromatography, gel permeation chromatography, scraping from thin layer chromatography,
Pure samples can be obtained by appropriately combining or repeating high-performance liquid chromatography and the like. [Physical and chemical properties of MG802-AF1-A] The physical and chemical properties are as described above, but the details will be explained below. Molecular weight: 480.1487 (based on high-resolution mass spectrum) Isotope peaks are seen at 480, 482, and 484 in the FD (field desorption) mass spectrum, and the intensity ratio matches that of two chlorine atoms, so it is considered a halogen in elemental analysis. is estimated to be chlorine. Therefore, the molecular formula is estimated to be C ₂₅ H ₃₀ O ₅ Cl ₂ based on elemental analysis and molecular weight. Ultraviolet absorption spectrum: MG802-AF1 of the present invention
FIG. 1 shows the ultraviolet absorption spectrum of -A.
In the graph of FIG. 1, the horizontal axis shows wavelength (nm) and the vertical axis shows absorbance. The absorption maximum of each curve is as follows.

[Table] Infrared absorption spectrum: The infrared absorption spectrum of potassium bromide tablets is shown in Figure 2. In the graph in Figure 2, the horizontal axis is the wave number (cm
^-1 ), the vertical axis indicates transmittance (%). Nuclear magnetic resonance spectrum: Figure 3 shows the proton nuclear magnetic resonance spectrum in deuterated chloroform ^.
The nuclear magnetic resonance spectrum is shown in FIG. In FIGS. 3 and 4, the horizontal axis indicates ppm. Rf value: Silica gel thin layer manufactured by Merck (Art.5715)
For chromatography, Rf = 0.47 when developed with chloroform-methanol (40:1) and Rf = 0.28 when developed with toluene-ethyl acetate (10:1).
Each shows a single spot. The inhibitory concentration of MG802-AF1-A against various bacteria on a nutrient agar medium (according to the agar plate dilution method) is as shown in Table 2.

【table】

【table】

[Table] Medium temperature 37℃

【table】

【table】

【table】

〔Example〕

Next, the present invention will be explained by examples, but the present invention is not limited thereto. Example 1 Cainia rubra cultured on agar slant medium
From MG802-AF1 strain (Feikoken Bibori No. 8022),
Galactose 2.0%, dextrin 2.0%, soy peptone (Bactosoitone manufactured by Difco) 1.0%,
Corn Stave Liquor (Nippon Shokuhin Kako Co., Ltd.)
A liquid culture medium (PH 7.4) consisting of 0.5% ammonium sulfate, 0.2% calcium carbonate, and 0.03% silicone oil for antifoaming was inoculated into two Erlenmeyer flasks with Watsufuru fittings, each containing 110 ml of liquid culture medium (PH7.4) for one night. , 27
The cells were cultured with shaking at ℃ for 5 days. Using this as a seed culture solution, 3 ml of the same medium was inoculated into 70 Erlenmeyer flasks with 110 ml aliquots and cultured with shaking at 27°C for 3 days. The liquid and the bacterial cells are separated by filtration, and the liquid has a pH of 8.
The mixture was extracted with an equal amount of butyl acetate. The bacterial cells were extracted with methanol (2), the extract was concentrated under reduced pressure, water (2) was added, and the mixture was extracted with butyl acetate (2) at pH 8. This was combined with a liquid butyl acetate extract and concentrated under reduced pressure to obtain 1.5 g of a brown oily substance. This was dissolved in a small amount of toluene and filled with toluene.
Chromatography was performed using a 140 ml silica gel column. First, elute with 500 ml of toluene, then 1.5 g of toluene-ethyl acetate (50:1), and 15 g
Fractionate each. Micrococcus luteus
Fractions No. 52-92 showing activity were collected by the paper disc method using IFO3333 strain as the test bacterium, and concentrated under reduced pressure.
430 mg of brown oil was obtained. 430mg of this oily substance
was dissolved in a small amount of methanol, applied to a Cephadex LH-20 (manufactured by Pharmacia) column (300 ml, diameter 2.1 cm) that had been swollen with methanol in advance, eluted with methanol, and determined by thin-layer chromatography to an Rf value of 0.47. The active fraction showing liquid chloroform-methanol (40:1) was collected and concentrated under reduced pressure to obtain 280 mg of MG802-AF1-A crude material.
MG802-AF1-A
The material was scraped off, extracted with methanol, and concentrated under reduced pressure. It was subjected to Sephadex LH-20 column chromatography (200ml, diameter 2.1cm) again.
Elute with methanol, collect active fractions and concentrate under reduced pressure.
45 mg of pure MG802-AF1-A was obtained as a tan candy. This is silica gel thin layer chromatography [chloroform-methanol (40:1)].
A single spot was obtained using high performance liquid chromatography [Sensyu Pack SSC-
ODS-1251N, Sensyu Science Co., Ltd., 4.6φ×250mm]
gave a single peak at 14.4 minutes at a flow rate of 1.5 ml/min using 85% methanol as the mobile phase. [Effects of the Invention] As explained in detail above, the present invention provides a novel antibiotic effective against Gram-positive bacteria and a method for producing the same. The novel antibiotic according to the present invention has a remarkable effect in that it is effective against multidrug-resistant bacteria.

[Brief explanation of drawings]

Figure 1 is an ultraviolet absorption spectrum diagram of MG802-AF1-A of the present invention, Figure 2 is MG802-AF1-A
Figure 3 is the infrared absorption spectrum (KBr tablet) of MG802-AF1-A, proton nuclear magnetic resonance spectrum (CDCl ₃ solution) of MG802-AF1-A, Figure 4 is MG802-
FIG. 13 is a ¹³ C nuclear magnetic resonance spectrum (CDCl ₃ solution) of AF1-A.

Claims (1)

[Claims] 1. The following properties: Shape: Yellow-brown candy-like elemental analysis (actually measured values): 61.44% carbon, 6.41% hydrogen,
Nitrogen 0.00%, Oxygen 16.31%, Chlorine 15.16% Molecular weight: 480.1487 (by mass spectrometry) Molecular formula: C ₂₅ H ₃₀ O ₅ Cl ₂ Specific optical rotation: [α] ²⁷ _D = +40.6° (c = 0.5, ethanol) Ultraviolet absorption spectrum (in methanol): absorption maximum 205nm (logε4.25), 251nm (logε4.14),
270nm (sh.logε4.04), 295nm (sh.logε3.87),
360nm (logε3.80) Infrared absorption spectrum (KBr tablet): 3400, 2920,
1640, 1610, 1250, 1070cm ^-1 Solubility: methanol, acetone, chloroform,
Soluble in ethyl acetate, ethyl ether and benzene. Poorly soluble color reaction in water and hexane: Both Gibbs reaction and anisaldehyde reaction are positive Distinction between acidic, basic, and neutral: New antibiotic MG802- characterized by having a weakly acidic substance
AF1-A. 2. Cultivate MG802-AF1-A producing bacteria belonging to the genus Cainia, and create a new antibiotic from the culture.
A method for producing a novel antibiotic MG802-AF1-A, which comprises separating and collecting MG802-AF1-A.