JPS59151896A - Novel antibiotic ss8201b and its preparation - Google Patents

Abstract

NEW MATERIAL:Antibiotic SS8201B of the formula. USE:It has high antibiotic activity against gram-positive bacteria and fungi. PREPARATION:A microorganism in Streptomyces, capable of producing an antibiotic SS8201B, e.g., Streptomyces sp. S8201 strain (FERM-6856) is aerobically cultured in an enriched medium at 25-32 deg.C, preferably about 30 deg.C for 3-10 days and the objective antibiotic SS8201B is collected from the resultant cell bodies and culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic B58201B and a method for producing the same.

The present inventors isolated a large number of microorganisms and conducted various studies on their products. As a result, a strain isolated from the soil at the foot of Mt. Myogi in Gunma Prefecture was found to have excellent antibacterial properties against clam-positive bacteria and fungi. A new powerful antibiotic 888
201B', and completed the present invention.

That is, the present invention provides a novel antibiotic ss8201B and a method for producing the same.

882 produced by the antibiotic 5s8201Bffi of the present invention
Is strain 01 the following mycological substance? have

α) Morphology The branches of aerial hyphae are simple branches, and no axle branching is observed. The tip of the aerial hyphae is spiral-shaped.

Substratum hyphae usually do not divide. No sporangia, flagellated spores, or sclerotia are observed. According to electron microscope observation,
The surface of the spore is smooth and oval to oval in shape. The thickness is 0.5~0.7X0.8~1.4μ
m, and usually 10 or more are chained together.

(21) Growth status on various media The growth status of strain 88201 on various media is as shown in the following table. Observations were made after culturing at 27°C for 14 days. ``Dictionary of Color Names'' was used to display the color names.

Blank space below (3) Physiological properties ■ Growth temperature range (Bennett agar medium, 14-day culture) 7
．． 13.16, 20, 23, 25.28, 32, 34,
The results of experiments at temperatures of 38°42 and 47°C were 20 to 3.
It grows at 2°C, but does not grow below 16°C or above 34°C. The optimum temperature for growth is 25-32°C.

■ Liquefaction of gelatin Positive ■ Hydrolysis of starch Positive ■ Coagulation of skim milk Positive Peptonization of skim milk Positive ■ Production of melanin-like pigments Positive ■ Reduction of nitrates Negative ■ Decomposition of cellulose Negative (4) Availability of carbon sources ( (Gridham-Godlieb agar medium, 27°C, 14 days culture) ■ L-arabinose + ■ D-xylose - ■ D-glucose + ■ D-fructose + ■ Sucrose - ■ Inositol - ■ L-rhamnose - ■ Raffinose - ■ D-Mannit + (Ta+ is used, - is not used.

(5) Cell wall composition The cell wall composition was analyzed for diaminopimeline and was found to be LL type.

From the above mycological properties, it is determined that this strain 88201 belongs to the genus Streptomyces.

Furthermore, the tip of the aerial hyphae of this strain is spiral-shaped and has 10 or more spores chained together, and the spore surface is smooth.The color of the aerial hyphae is gray color series, and the color of the underside is pale yellow to brownish olive. The soluble pigment is yellowish brown.
It has the following characteristics: it is olive, neither the color nor the soluble pigment on the back side is a pH-indicating color, and it produces melanin-like pigment.

Based on the above mycological properties and with reference to known literature, strain N-461, which is the same as a new subspecies of Diastatochromogenes
JP-A No. 55-310).

However, when comparing strain Et8201 and strain N-461, strain 88201 shows vigorous growth on sucrose e-nitrate agar medium, produces a dark brown to black soluble pigment, and utilizes sucrose. Therefore, the present inventors identified strain 58201 as a new strain,
Streptomyces sp. S 82011 Str
eptomycee 8p, 58201) and submitted it to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the 68th
No. 56 urrbRMF-6856).

Antibiotic 588201B of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. As the antibiotic SS8201B producing strain, not only the above-mentioned strains but also their artificial and natural mutant strains can be used.

The medium used for culturing may be any synthetic medium, semi-synthetic medium, or natural medium as long as it contains a nutrient source used by the bacteria.

Among the nutrient sources in the medium, examples of carbon sources include glucose, glycerol, arabinose, xylose, mannitol, rhamnose, fructose, starch, molasses,
Starch syrup, etc. or a mixture thereof may be used. Nitrogen sources include soybean flour, peptone, cottonseed meal, fishmeal, cornstap liquor, yeast extract, ammonium chloride, ammonium nitrate, or a mixture of these. can be used.

Calcium carbonate, sodium chloride, etc. as necessary.
Addition of inorganic salts such as phosphates, aiding the growth of bacteria, 5
Production of E18201B? For promotion, organic, inorganic and common antifoaming agents such as silicone oil or arabinose (trade name) can be added as appropriate.

As the culture method, methods commonly used for the production of antibiotics are employed, but liquid culture methods, particularly deep culture methods, are most suitable. Cultivation is performed under aerobic conditions, and the appropriate temperature for cultivation is 25 to 32°C, but it is generally preferable to culture at around 30°C. The amount of antibiotic 8S8201B accumulated reaches its maximum within 3 to 10 days after the start of culture, even in the case of collapse of shaking culture or deep culture.

Antibiotic B58201 from the culture thus obtained
Isolation of B is carried out by appropriately combining various methods in consideration of the physicochemical properties of this antibiotic, as shown in the Examples below. That is, since antibiotic 888201B normally exists in the bacterial cells and culture solution, the bacterial cells are isolated from the culture by centrifugation or filtration, and the bacterial cells and culture p solution are separated by conventional separation means 1, such as precipitation method. , solvent extraction method,
Antibiotic 5s8201Bk is separated and purified using ion exchange resin method, gel filtration method, adsorption or distribution column chromatography method, dialysis method, etc., or in combination as appropriate.

As an example of a preferable separation ff production method, the following method can be mentioned. First, add dilute hydrochloric acid to the culture solution.

The pH was adjusted to 4.0, and a precipitate containing bacterial cells was obtained by centrifugation. This precipitate is mixed with a suitable solvent, such as a dichloroform-methanol mixture, and extracted and filtered.

The entire residue was extracted again with a mixture of tetraform and methanol, the p solution was combined, and an appropriate amount of water was added to this extract to distribute the lower layer. Take. This lower layer was distilled off under reduced pressure and the residue was subjected to silica gel column chromatography. The active fraction was distilled off under reduced pressure, and the residue was subjected to Prepak 5001018 column chromatography. Concentrate to active fraction 1, add water to the concentrate, collect and dry the resulting precipitate kF, and recrystallize with a suitable solvent such as ethanol to obtain crystals of 888201B.

588201B obtained as described above has the following physicochemical and biological properties.

U) Physical and chemical properties Experimental value (%) 70.45 7.72 5.55 Theoretical value (%) 70,42 7,74 5.66
■ Molecular weight 494.63 ■ Melting point 254-257℃ (decomposed) ■ Specific rotation [α] = 278° (C-0,5,N,N'-dimethylformamide) ■ Ultraviolet absorption spectrum Figure 1: 320 nm (Crystalline) 12400) 325 nm (g 16900) 325 nm (g 16600) ∎ Infrared absorption spectrum t KBr method) Figure 2 ∎ 'H-NMI' t spectrum 190 MHz) Measured as TMSi reference substance in deuterated pyridine solution. Third
Figure ■ ``O-0-1t1 spectrum TMS in deuterated pyridine solution Measured as all reference substances.

δ (ppm) 196.8, 176.8, 173.4
,166.8°149.8,139.5,125.4,
124.0°102, 1, 62.3, 57.5,
53.4, 50.3.

49.3.47.8, 46.5.46.0, 41.
2.41.1.

39.4, 38.7, 36.9, 33.4.27.9,
26.2°23.1, 21.6, 17.5, 13
．． 3゜■ Mass spectrum (Chemical F 0 eV) M” m / z 494 0 Solubility in solvents Chloroform, ethanol, methanol, dichloroform-methanol mixture, dimethylformamide, dimethyl sulfoxide, soluble in pyridine, n-hexane , benzene, diethyl ether, water rtcrm■
Color reaction Represents reddish-brown color with ferric chloride reagent. It develops a reddish-purple color with the sulfuric acid reagent. Ninhydrin reagent and Dragendorff reagent do not show color.

■ Distinction between basic, acidic, and neutral Acidity 0 Color and properties of the substance Colorless acicular crystals 0 Thin layer chromatography carrier Nijiri Kakeru Prey) Fys+ (manufactured by Merck & Co., Ltd.) [Phase] Molecular formula % Formula % [Phase] Structure Formula Based on the above physical and chemical measurements, it was determined that the antibiotic 5B8201B of the present invention has a structure expressed by the following formula.

(2) Biological properties Antibacterial action Table 1 shows the minimum inhibitory concentration (MIC) of antibiotic B58201B against various microorganisms.

H゛, 4';': Mi1'□1 Test bacteria culture conditions: Inoculum size approximately 10@ce],
1s/mA! 0 For bacteria, Mueller-Hinton-Agar (D
ifco) and cultured at 37°C for 18 to 20 hours.

For yeast and mold, use glucose-Begton medium at 28℃
Cultured for 120 hours.

The properties described above were compared with those of known antibiotics similar to the compound of the present invention, but no corresponding substances were found.888201
B is judged to be a new antibiotic.

Next, the present invention will be explained with reference to examples.

Example S+38201B Producing Bacterium Streptomyces sp. 88201 (Feikoken Bacteria No. 6856) Total Soluble Starch 2
．． 0%, soytone 1.5%, glucose 0.2%, sodium chloride 0.2%, calcium carbonate 0.32% 1pH
7.0) and shaken at 30°C for 48 hours to prepare a seed culture. Liquid medium 1 consisting of the same medium composition
6j' was placed in a 30/volume fermenter, inoculated with 3001 nlp of the above seed culture solution, and the aeration amount was 16 I! The cells were cultured for 9 days under the following conditions: /min, stirring speed: 400 rpm, and culture temperature: 30°C. After culturing, add dilute hydrochloric acid to culture solution 151. Add pH
The precipitate containing bacterial cells was obtained by centrifugation.

Add chloroform-methanol to this precipitate in a ratio of 1:1.
) Add mixture 27=i, mix well, extract, filter, p
It was separated into a liquid and a filtration residue. The filtration residue was extracted again with 21 parts of a tetraroform-methanol (1:I) mixture, and the P solution was combined. Water 21 was added to this extract for distribution, and the lower layer was collected.

This lower layer was concentrated to dryness under reduced pressure, ether was added, stirred, and filtered to obtain brown coarse powder 203f. The crude powder thus obtained can be further purified as follows. That is, this coarse powder was dissolved in a chloroform-methanol-water (160:10:1) mixture, and the silica gel 1Wak was filled in advance with a chloroform-methanol-water (60:10:1) mixture.

ogLc-200) column l 55ZfX80crn
), acidified with the same solvent, collected active fractions, concentrated to dryness, and washed with a small amount of methanol to obtain 122 crude 5s82.
01B was obtained. This crude 5S8201B was mixed with dioxane-water-N, N'dimethy4-lformamide acetic acid +
50:29:20:1) Dissolve in the mixture and Prepa'k.
When the column is passed through a 5001018 column (manufactured by Waters Co.) and eluted with the same solvent, S+38201B, icargamycin (Japanese Patent Publication No. 46-28833) and N-461 (Japanese Patent Publication No. 55-310) are extracted. Prepak 5
Repeated purification with 001018 column, +3EI8
Seven active fractions of 201B were combined and concentrated. Next, add water to the concentrate, collect the resulting precipitate, and dry. As a result, a slightly yellow powder of 5s8201B500+xIF was obtained. Furthermore, this powder was recrystallized with ethanol to give 888'
Obtained colorless needle crystals 3801 of 201B.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antibiotic 5B8201B of the present invention, where (1) is 0. lNNaOH-1fl
tOH, ([1) is g, 1 h nc4-1Dto
u, ([) is l! This was measured by dissolving it in 1tOH. FIG. 2 is an infrared absorption spectrum (KBr method) of the antibiotic 5s8201B of the present invention. Figure 3 shows “H-IJ” of the antibiotic *8S8201a of the present invention.
MR spectrum (solvent double pyridine). Above is Figure 1 Wavelength + nm1 Procedural amendment (voluntary) April 1980 its '4';Yii' + Director-General Kazuo Wakasugi 1 Small angle table J 1988 Patent Application No. W'4489 2 Invention Name 3 Akane who does i mountain 11 = Ii (Relationship with 'l) Out JQ (j person i 1. Moe 2-12 Nihonbashihamacho, Chuo-ku, Tokyo Notification '4 Name
Nisnis Pharmaceutical Co., Ltd. Representative Nookata Yasumichi 4 Representative Attorney Name (7756) Patent Attorney 1 Toshi Takafu 11
・ ：Address Same as above
i-・-m=;6, [Claims] and "Detailed Description of the Invention" of the specification subject to amendment
Fence 7, Contents of amendment (1) In the specification, the "Claims" column is corrected as shown in the attached sheet. (2) In the specification, page 22, line 11 rN, h'-
Correct "dimethylformamide" to "rN,N-dimethylformamide." (3) Same, page 24, lines 9-10, “-methanol mixture, dimethylformamide” [-methanol mixture, N,N-dimethylformamide]
I am corrected. (4) Same, page 30, 15th to 31st negative line 1 rg-
Correct "N,N'dimethylformua Sd" to "r7)C-N,N-dimethylformuas)". 2. Claim 1: Novel antibiotic 1388 having the following physical and chemical properties
201B. ■ Elemental analysis OHN Experimental value (灼 70.45 7.72 5.55 Theoretical value (Kon 70.42 7.74 5.66 ■
Molecular weight 494.63 ■ Melting point 254-257℃ (decomposed) ■ Specific rotation ■ Ultraviolet absorption spectrum Figure 1 320nlε12400) 325 nm (ε16900) 325 nm (ε16600) ■ ■ Infrared absorption spectrum (KBr method) Figure 2
■■ ``H-NMR spectrum (9Q MH2) Deuterated pyridine solution A Measured using TMS as a reference substance. Figure 3 ■ ``O-NMR spectrum TM8 i in deuterated pyridine solution Measured as a reference substance. δ(pp!11) 196.8, 176.8, 173
．． 4,166.8,149.8°139.5,125.
4.124.0.102.1,62.3. 57.5.53.4, 50.3, 49.3, 47.8.
46.5. 46.0, 41.2, 41.1, 39.4, 3
8.7, 36.9. 33.4.27.9.26.2, 23.1, 21.
6.17.5. 13.3. Mass spectrum (gI70θV) M”m/z 494 Solubility in solvents Soluble in chloroform, ethanol, methanol, chloroform-methanol mixture, N,N-dimethylformamide, dimethyl sulfoxide, pyridino, n-hexane, benzene, diethyl ether, Slightly soluble in water. ■ Color reaction Represents a reddish-brown color with ferric chloride reagent. Represents reddish-purple color with vanillin sulfate reagent. Ninhydrin reagent,
No color develops in Dragenodorf's reagent. [F] Distinction between basic, acidic, and neutral Acidic ■ Color and properties of the substance Thin layer chromatography carrier of colorless needle crystals Nisilica gel plate F□4 (Merck & Co., Ltd.) O Molecular formula % Formula % A novel antibiotic 5S8201E03 according to claim 1, a novel antibiotic 888201B-producing bacterium belonging to the genus Streptomyces is cultivated,
From the culture, the following physical and chemical properties were obtained: ■ Elemental analysis CHR experimental value (Samurai 70.45 7.72 5.55 theoretical value) 70°42 7.74 5.66 ■ Molecular weight 494.63 ■ Melting point 254-257 °C (decomposition) ■ Specific optical rotation Figure 1 320 nm (12400) 325 nm (ε16900) 325 nm (ε16600) ■ Infrared absorption spectrum (L KBr method) Figure 2 ■ 'H-NMR spectrum (90 MHz) based on TMS in deuterated pyridine solution Measured as a substance. Figure 3 ■ ” C-N MItX vector measured in deuterated pyridine solution as TM day total reference substance. δ (ppm) 196.8, 176.8, 173.4
,166.8°149.8,139.5,125.4,
124.0°102.1.62.3, 57.5, 5
3.4, 50.3. 49.3, 47.8.46.5.46.0, 41.2°41.1, 39.4, 38.7.36.9.33.4°27.9, 26.2.23. 1,21.6.17.5°13.3. ■ Mass spectrum (drunk 7Qev) M”ro/z 494 0 Solubility in solvents Chloroform, ethanol, methanol, chloroform-meth/-Al solution, N,N-dimethylformamide, dimethylsulfoxide, pyridine. Soluble in n-hexane , benzene, diethyl ether, and water. ■ Color reaction: It exhibits a reddish-brown color with ferric chloride reagent. It exhibits a reddish-purple color with niline sulfuric acid reagent. It does not develop color with ninhydrino reagent and Dragendorff reagent. ■ Distinction between basic, acidic and neutral Acidic 0) Color and properties of the substance Colorless acicular crystals O) Thin layer chromatography carrier Nisilica gel plate F t: A method for producing the novel antibiotic 5s8201B, which is characterized by collecting.

Claims (1)

[Claims] 1. Antibiotic B5 containing the following physical and chemical substances
8201B. ■ Elemental analysis CHN Actual value (%> 70.45 7,72 5
．． 55 theoretical value) 70.42 7,74 5
．． 66 ■ Molecular Moon Z 494.63 (3) Point z+ 254-257℃ (resolved) ■ Specific rotation ■ Ultraviolet absorption spectrum Figure 1 320n, m 1m 12400) 32Snml & 16900) 325nml t16600) ■ Infrared absorption spectrum τKBr method) Figure 2 ■ 'H-NMR spectrum + 90 MHz) Measurements were made using TMS in a deuterated pyridine solution as a reference substance. Figure 3 ■ "C-NMR spectrum TMS in heavy pyridine solution Measured as i reference material. δIppm) 196.8, 176.8, 173.4
, 166.8, 149.8° 139.5, 125.4.
124.0.102.1.62.3. 57.5, 53.4, 50.3, 49.3.47.8,
46.5. 46.0, 41.2.41.1, 39.4, 38
．． 7.36.9. 33.4.27.9, 26.2, 23.1, 21.6.
17.5°13.3. ■ Mass spectrum (Revised 7Qev) M m/z494 (d Solubility in solvents Soluble in chloroform, ethanol, methanol, chloroform-methanol mixture, dimethyl sulfoxide, and pyridine. Slightly soluble in n-hexane, benzene, diethyl ether, and water. 0 Color reaction Represents reddish-brown color with ferric chloride reagent. Represents reddish-purple color with vanillin sulfate reagent. Ninhydrin reagent,
No color develops in Dragendorff reagent. ■ Distinction between basic, acidic, and neutral Acidic 0 Color and properties of the substance Colorless acicular crystals [Phase] Thin layer chromatography carrier Nisilica gel plate F□4 (manufactured by Merck & Co., Ltd.) Below, O molecular formula % formula % 2, The novel antibiotic 888201B according to claim 1, whose structure is represented by the following formula. 3. Novel antibiotic 888 belonging to the genus Streptomyces
201 e Cultivated on the producing bacteria, and obtained the following physical and chemical properties from the culture: ■ Elemental analysis OHN Experimental value (C 70.45 7.72 5.55 Theoretical value ←) 70.42 7.74 5.66 ■ Molecular weight 494.63 ■Melting point 254-257℃ (decomposed) ■Specific rotation (UV absorption spectrum 1KBr method) 320nm (ε12400) 325nm [" 16900) 325nm l g16600) ■Infrared absorption spectrum 1KBr method) Figure 2■ JR- NMR spectrum/1190 MHz) TMa in deuterated pyridine solution was measured as a reference substance.
Figure ■ "C-NMR spectrum TMS in deuterated pyridine solution (measured as a z reference material. δlppm) 196.8, 176.8, 173.4
,166.8°149.8,139.5,125.4,
124.0°102.1.62.3.57.5.53.
4,50.3. 49.3, 47.8, 46.5, 46.0, 4
1.2. 41.1, 39.4, 38.7.36.9, 33.4°27.9, 26.2, 23.1, 21.6, 17.5°13.3. ■ Mass spectrum (Ef0ev) M"m/z 494 [Phase] Solubility in solvents Soluble in chloroform, ethanol, methanol, chloroform-methanol mixture, dimethylformamide, dimethyl sulfoxide, pyridine. n-hexane, benzene, diethyl Difficult to ether and water. 0 Color reaction: ferric chloride reagent produces a reddish-brown color. Vanillin sulfate reagent produces a reddish-purple color. Ninhydrin reagent,
No color develops in Dragendorff reagent. 0 Distinction between basic, acidic, and neutral Acidic 0 Color and properties of the substance Colorless needle-like crystals 0 Thin layer chromatography J-type Nijiri gel plate F 2M & (manufactured by Merck & Co.) 6 Antibiotics with the molecular formula % formula % 5s82018 k A method for producing the novel antibiotic BB8201B, which is characterized by its collection.