JPS5920351B2 - New antibiotic acreasin Dα - Google Patents

Description

[Detailed description of the invention] The present invention relates to a novel antibiotic acreacin Aγ.

The present inventors discovered that M4845, a filamentous fungus belonging to the genus Aspergillus isolated from mountain forest soil in Omoto, Ishigaki City, Okinawa Prefecture, contained Candida albicans in its culture solution and within its cells.
To produce a new antibiotic that inhibits the growth of yeasts such as P. bicans at extremely low concentrations, and also inhibits the growth of filamentous fungi such as dermatophytes and plant pathogenic fungi at extremely low concentrations. was discovered and named the antibiotic acreacin Aγ.

The characteristics of the above-mentioned filamentous fungus M4845 when cultured on various media based on macroscopic and microscopic observations are as follows.

A. Macroscopic Observation 1. Growth on agar medium is fast, reaching a diameter of 55 to 65 mm in 10 days at 26°C.

The surface of the colony is flat and thin, and is white at the initial stage of culture, but as conidia are formed and mature, it gradually changes to phon (F).
amn, hue4lg). Conidia are formed in large numbers. The area around the village is somewhat spider web-like (Arachnoid). The color of the back side is colorless to pale yellow (Pale Yellow/Hue/Ca).No diffusible dye or exudate is released. Odorless. Growth at 37°C is slow, 14-17 m10 in 10 days.Growth fast on wort agar medium, 60-70v1t in diameter in 10 days at 26°C.
reach.

The surface of the colony is flat and thin, and is white at the initial stage of culture, but as conidia are formed and mature, Beav
er, hue4ll). Conidia are formed in large numbers. The area around the village is smooth or almost smooth. The color on the back is Light Yellow (LightYellOw?Hu)
ell/2ca) o Diffusible dyes and exudates are not produced. Odorless. Growth at 37℃ is slow, growing 10 to 12 times in 10 days.
關〇3 Potato Glucose Agar Medium The growth conditions are almost the same as those for the wort agar medium, except for the clear spider web-like appearance around the colonies.

Note) The color display description was made in 1958 by Container Corporation of America.
rpOratiOnOfAmerica) Published by ``Color Harmony Manual (COLOrHarmOny
The display method in the manual was followed.

B. Microscopic observation The conidial head is initially spherical and later becomes cylindrical with several radial cracks.

Conidiophore length 400-1500μ, width 8-13μ,
Colorless to pale yellow, walls smooth and somewhat thick, 1.5 mm thick.
~20μ. The apical sac is light brown, spherical or subspherical, and 3 in diameter.
0-70μ, mostly 40-60μ. 6 to 8 stalks in a single row
×3 to 4μ are lined up closely. Conidia range in shape from spherical to oval. 35~5.0 x 30~35
μ, pale yellowish brown, walls rough.

C growth conditions Growth temperature is 13-40°C, growth PH is 2-9, optimal growth temperature is 29-31°C, optimal growth PH is 3-5.

As a result of the above detailed observation, this bacterium has the mycological characteristics of the Aspergillus genus (Journal of the Agricultural Chemistry Society, 27, 806-809 (19
53), TheGenusAspergllusl3
28-331 (1965)), the Aspergillus niga group (Asp
ergillus nigerGrOup), and this group is further divided into two groups depending on whether the stalks are single-rowed or double-rowed, and this fungus belongs to the single-rowed group.

Aspergillus japonicus (Aspergillus japonicus) is a fungus with a single row of stalks.
s) and Aspergillus aculeatus (Aspergl.
In Aspergillus japonicus, the conidia are spherical or subglobose, with a size of 3 to 3.5μ, and the apical capsule is 15 to 45μ, mostly 20 to 35μ, and Aspergillus aculeatus is known as Conidia are subglobose or oval, size 4.5-5
X3~35μ, top capsule 35~100μ, mostly 60~
80μ, this bacterium has very similar mycological characteristics to Aspergillus aculeatus, and is also a member of the American Type Culture Collection.
As a result of comparison with Aspergillus aculeatus ATCClO34 from ypeCultureCOlle-CtlOn), there was an extremely good match, indicating that this bacterium is Aspergillus aculeatus.
This bacterium was named Aculeatus M4845, and has been submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology under microbial accession number 4023 (FERM-P4023). The present invention has been completed based on the above findings, and includes an antibiotic acreasin Aγ having the following physical and chemical properties: elemental analysis Pauli reaction, phorin reaction, periodic acid-benzidine reaction, permanganic acid Positive for potassium decolorization reaction, Moritsch reaction, Biuret reaction, xanthoprotein reaction,
Positive for Tollens reaction, ninhydrin reaction, Benedikt reaction, Sakaguchi reaction, Ehrlich reaction, ferric chloride reaction, Dragendorff reaction, negative for 2,4-dinitrophenylhydrazine reaction, Soluble in solvents, Soluble in lower alcohols. , Slightly soluble in ethyl acetate, Slightly soluble in acetone, chloroform, n-hexane, petroleum ether, and water; Distinguish between basic, acidic, and neutral. The substance is fair-skinned and stable at 37°C for 20 hours, stable in acidic and neutral conditions, unstable in alkaline conditions, and ultraviolet absorption spectrum measured in methanol.
The ultraviolet absorption spectrum of γ/MO is λMax278n
m. ．． E{? Maximum absorption at =15.5, 1 coefficient and λMa
x226nm. ．． ElCTn=145, λMax283
nm,. EI (! has shoulder 1 at n=13).

Liquid chromatography's highest retention time Rt=1
23 minutes (column diameter 5 x 500 mm 1 carrier polystyrene resin, developing solvent 0.01 (f) containing triethylamine 85
0/) methanol aqueous solution, column temperature 55°C),
The production method is Aspergillus genus Asteroides.
) is quantified by bioassay using the cup method or paper disk method.

Next, acreasin Aγ is collected from this culture, and it is preferable to collect acreasin Aγ accumulated within the bacterial cells.

Next, an example of means for separating and purifying acreasin Aγ will be shown. That is, microbial cells containing a large amount of acreasin Aγ are collected from the culture obtained by culturing acreacin Aγ-producing microorganisms as described above using a drum filter, filter press, centrifuge, etc., and the obtained wet microbial cells are Extract by adding a hydrophilic solvent such as alcohol or acetone, distilling off the solvent of the obtained extract, diluting this with water, and then adding a solvent such as n-butanol or ethyl acetate to this diluted solution. Re-extract and wash the extract with water, then concentrate under reduced pressure to obtain an oil. This oil is subjected to adsorption or partition chromatography using activated alumina, silica gel, etc. to obtain its active fraction, which is then concentrated under reduced pressure and subjected to thin layer chromatography in various solvent systems in a single spot. You get a powder that gives. The physical and chemical properties of acreasin Aγ thus obtained will be described below.

2. Molecular weight Last method: 1021 Amino acid analysis: (860)n (30μ from the hydrolyzate of acreasin Aγ2.6W9)
(Detection of threonine in MOL) Gel filtration method: 800±
110 (Using Sephadex LH-20 (manufactured by Pharmacia Huain Chemical Co., Ltd.), developing with ethanol, simultaneously developing an antibiotic with a known molecular weight, and estimating from the elution position)3. Melting point: 172-175 C (no clear melting point is shown) 4. Specific luminous intensity 5. Color reaction n''-1r-1胛▲1-111r? -d Todoroki-Cp beak-
♂Positive for Todoroki-Todorobenbenzidine reaction, potassium permanganate decolorization reaction, Moritzhu reaction, Biuretz reaction,
Xanthoprotein reaction, Tollens reaction with positive coagulation, ninhydrin reaction, Benedikt reaction, Sakaguchi reaction, Ehrrich reaction, ferric chloride reaction, Dragendorff reaction, 2
・Shows negative in 4-dinitrophenylhydrazine reaction.

6. Solubility in solvents: Soluble in lower alcohols, slightly soluble in ethyl acetate, slightly soluble in acetone, chloroform, n-hexane, petroleum ether, and water.

7. Distinguishing between basic, acidic, and neutral. Titration is impossible because alkalinity causes decomposition, and it is poorly soluble, so it does not move by electrophoresis, so the distinction between them is not clear, but it is possible to determine the pH between 2 and 9 from the butanol solution. It is presumed to be a neutral substance as it does not migrate to the water layer.

3. color of substance white crystalline powder ). Stability Stable at 37°C for 20 hours in acidic and neutral conditions.

Alkaline and unstable. 10. Ultraviolet absorption spectrum of acreasin Aγ measured in methanol (concentration 32.
The ultraviolet absorption spectrum of 4γ/MO is as shown in FIG.
Maximum absorption at
= 1CfL- with shoulder 1 at 13
011. The infrared absorption spectrum of acreasin Aγ measured by the infrared absorption spectrum KBr method is as shown in Figure 2, with 3350, 2910,
2840, 1620, 1510, 1435 (1771-
It has an absorption band at each wavelength of 1.

12. Rf value silica gel thin layer chromatography (manufactured by Eastman Kodak, Eastman Chromatogram Sheet 6060)
): Chloroform-methanol (10:3) Rf value 0
．． 47, Ethyl acetate-methanol:water (20:4:1)
Rf value 0.28, ethyl acetate-n-butanol (3:
1) Rf value 0.37 at water saturation.

(However, detection is by bioautography using Candida albicans and iodine vapor.)13
．． Retention time by liquid chromatography (
Rt) Acleacin Aγ5η/ml methanol solution 8μ
1 in a liquid chromatogram (column diameter 5 x 500 mm.
Finger body polystyrene resin, developing solvent 0.0101) Containing ethylamine 8501) Methanol aqueous solution, column temperature 55 μC: Hitachi 634 model manufactured by Hitachi, Ltd.) was charged and its retention time (RetentiOnt) was charged.
ime) was 12.3 minutes.

14. The hydrolyzate acreacin Aγ was hydrolyzed with 6N hydrochloric acid at 110°C, extracted by adding ether, and the extract was charged to a gas chromatogram to qualitatively detect free fatty acids, and palmitic acid was detected.

15. Amino acid composition: Hydrolyzed with 6N hydrochloric acid at 110°C for 20 hours, and ninhydrin-positive components were analyzed using an automatic amino acid analyzer (JLC manufactured by JEOL Ltd.).
As shown in FIG. 3, several types of threonine and ninhydrin-positive components were detected.

Accreacin Aγ obtained in the present invention is recognized as a poorly water-soluble peptide-like substance due to its physical and chemical properties, and among known antibiotics, the maximum ultraviolet absorption in methanol is 27.
If you search for antibiotics similar to acreacin Aγ, which has a particle diameter of around 8 mμ, you will find myrolysine (MyrOrldlnl Special Publication No. 12276/1981), athleistatin (Athlestatin), etc.
statinl Special Publication No. 41-1268), Monirin (
MOnillnl Takeda Research Annual Report 14, 8-101195
5), Oryzamycin (4) Ryzamycinl Special Publication No. 38-2800) Saramycetin
cetln,. AntlmicrObialagent
sandchemOtherapyl96l, 436
444), Unamycin (Unamycln,.Th
eJournalOf, AntiblOtics, Se
rAl3,ll4-120, 1960), benziside (Engiclde, British Patent No. 764198), acreasin A (US Patent No. 3978210), and echinochiandeine B (EchinOcarldlnBl Swiss Patent No. 568386: A322O4). , Myrolidine is distinguished from the water-soluble basic substance, and athleistatin is presumed to be closely related based on its physical, chemical, and biological properties, but the specific light extinction of athleistatin is (a) too? -64° (c=0.5,
methanol), and its maximum ultraviolet absorption is 278
In addition to nm, it shows a clear maximum absorption at 225 nm, and its elemental analysis values show C: 55.25 and H: 7.5.
001), N: 9,4101), and further distinguished by the fact that monilin is a basic nucleic acid-based substance and has a high nitrogen content in its elemental analysis value.
Oryzamycin is an acidic oil, salamycetin does not give aspartic acid, cystine, glycine-threonine, etc. by hydrolysis, unamycin is an acidic polyene-based substance, and benziside is a nucleic acid-based substance similar to monillin. Therefore, acreasin Aγ and echinokyandein B are presumed to be substances with extremely similar structures in terms of their physical, chemical, and biological properties.
γ is the highest retention time in liquid chromatography, 12
．． It is distinguished by the fact that it has a three-minute value and clearly liberates palmitic acid during its hydrolysis.Furthermore, the lattice constant of the crystal of acreasin Aγ was determined by the X-ray diffraction image of Tetrahedron Letters.
OnLettets) No. 46, pp. 4147-4150 (1976), the lattice constant of the crystal of echinokyandein B matches the lattice constant, and the two are indistinguishable in terms of crystal diffraction. According to the report by Echinokyandein B, a rigid crystal lattice is formed based on its cyclic peptide moiety,
It is formed with fatty acid (linoleic acid, stearic acid) residues and crystal solvents incorporated into the gaps in a relatively irregular manner, and this fact and the aforementioned acreacin Aγ Based on the properties of acreasin Aγ, it is assumed that the peptide portion of acreasin Aγ is the same as that of echinokyandein B, and that the fatty acid residue in acreasin Aγ is a palmitic acid residue, which distinguishes it from echinokyandein B. be. Therefore, acreacin A of the present invention
γ is recognized as a novel antibiotic different from conventionally known substances, and is presumed to have the structure shown in the following formula.
(R=palmitic acid residue) Next, the biological properties of acreasin Aγ will be described.

In the measurement, bacteria were inoculated onto Sabouraud agar plates containing various concentrations of acreasin Aγ, and cultured at 26°C (14 days for dermatophytes and 7 days for phytopathogenic fungi). The fungal moss area was calculated from the diameter, and this was the concentration that inhibited the growth of 80% of the fungal moss area of the control group without addition of acreasin Aγ, which was conducted in parallel.

Acute toxicity test for mice Using an aqueous solution of acreasin Aγ prepared with sodium deoxycholate as a solubilizing agent, its LD,O was determined.

EXAMPLES Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto in any way.

Example 1 2% glucose, 1% polypeptone, 1% corn steep liquor, KH2PO4O. 2%, MgSO4O.
100 ml of medium (PH 6.5) containing 1%
Dispense into a 0ml Erlenmeyer flask, heat sterilize at 120°C for 15 minutes, and add Aspergillus aculeatus M48.
One platinum loop of spores from a slant culture of 45 strains was inoculated, and 2
Shaking culture was carried out at 6°C for 48 hours, and then cultured at 120°C.
Satsucrose 1.5% obtained by heat sterilization for 15 minutes,
Dextrin 1.4%, polypeptone 2%, corn steep liquor 0.45%, KH2PO4O. 2%,
MgSO4O. 1%, BC-51Y (antifoaming agent: manufactured by Nippon Oil & Fats Co., Ltd.) in a 301 volume jar containing a medium (PH 6.5) 151, and this was incubated at 26°C.
Aerated agitation culture was carried out for 90 hours at 250 rpm and 301 liters of aeration, yielding about 151 liters of culture.

The activity of acreasin Aγ is present in the culture fluid and cultured cells of this culture, and acreasin Aγ is obtained as described in Examples 2 and 3 below. Example 2 About 101 pieces of the culture obtained in Example 1 were filtered by suction to obtain about 1 kg of wet bacterial cells.

Add methanol 51 to the wet bacterial cells, immerse and extract for 2 hours while stirring, collect the methanol solution by filtration under reduced pressure, and further add methanol 51 to the bacterial cells and extract in the same manner.
Both extracts were combined and methanol was distilled off under reduced pressure. A concentrated solution was obtained, water 21 was added thereto, and n-butanol 21 was further added twice for extraction, the n-butanol layer was collected, and the n-butanol solution was further washed with 51 water and then evaporated under reduced pressure. Concentrate while adding a small amount of water to obtain a dark brown viscous concentrate, and add 500ml of n-hexane to this.
The resulting precipitate was collected and washed with ethyl acetate.
12.59 ml of crude material containing acreasin Aγ was obtained. Example 3 12.59 of the acreasin Aγ-containing crude material obtained in Example 2 above was dissolved in a small amount of methanol, and 259
The silica gel obtained by adding and adsorbing silica gel (manufactured by Talc) and then removing methanol under reduced pressure was added to a column packed with separately prepared 6009 silica gel (inner diameter 50
m77! ) and was developed using a developing solvent of ethyl acetate lyisoprobanol:water (10:1.5:0.7), eluted as 1 fraction of 45 ml, and the fraction. /% 43-57 were recovered.

After combining these, they were dried under reduced pressure, and then dissolved in methanol.
Hitachi 634 model manufactured by Hitachi, Ltd.: Column diameter 5x500, carrier polystyrene resin, developing solvent 85(I) methanol aqueous solution containing 0.01% triethylamine, column temperature 55
℃) and its retention time is 11.
A fraction fractionated between 4 and 13.4 minutes was obtained, this was repeated, the fractions were combined, and then dried under reduced pressure, dissolved in a small amount of methanol, and ethyl acetate-n - Add a mixed solvent of hexane (1:1) and collect a lot of precipitate, about 50T! Purified acreacin Aγ of Z9 was obtained.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of acreasin Aγ, FIG. 2 shows the infrared absorption spectrum of acreasin Aγ, and FIG. 3 shows the amino acid analysis of acreasin Aγ.

Claims (1)

[Claims] 1 Antibiotic acreasin Aγ having the following physical and chemical properties: Elemental analysis C: 54.70% H: 8.23% N: 10.51%
Molecular weight Last method: 1021 Amino acid analysis: (860)n (30μ from a hydrolyzate of 2.6mg of acreasin Aγ
Mol threonine was detected) Melting point 172-175℃ Specific light intensity [α] ^2^3_D = -474° (C = 0.18, methanol) Color reaction Bowery reaction, Folin reaction, periodic acid-benzidine reaction , positive for potassium permanganate decolorization reaction, Moritsch reaction, Biuretz reaction, xanthoprotein reaction, coagulation positive for Tollens reaction, ninhydrin reaction, Benedikt reaction, Sakaguchi reaction, Ehrrich reaction, ferric chloride reaction, Dragendorff reaction, Negative for 2,4-dinitrophenylhydrazine reaction, Soluble in solvents Soluble in lower alcohols, Slightly soluble in ethyl acetate, Slightly soluble in acetone, chloroform, n-hexane, petroleum ether, water, basic, acidic, medium It is presumed to be a neutral substance because it does not migrate from the n-butanol solution to the aqueous layer with a pH of 2 to 9. White color stability of the substance Stable in acidic and neutral conditions at 37°C for 20 hours, stable in alkaline conditions Unstable ultraviolet absorption spectra of acreasin Aγ (concentration 32.
The ultraviolet absorption spectrum of 4γ/ml) is λmax27
Maximum absorption at 8 nm, E^1^%_1_c_m = 15.5, and λmax 226 nm, E^1^%_1_c_m
=145, λmax238nm, E^1^%_1_c_
It has a shoulder at m=13. Liquid chromatography glue tension time Rt=1
23 minutes (column diameter 5 x 500 mm, carrier polyethylene resin, developing solvent 85% methanol aqueous solution containing 0.01% triethylamine, column temperature 55°C).