JPS5920352B2 - Parent antibiotic acreasin Dr. - Google Patents

Description

[Detailed description of the invention] The present invention relates to a novel antibiotic substance acreasin Dγ.

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 and Candida albicans in its culture solution and its bacterial bodies.
icanc) ξ yeasts at extremely low concentrations, and also inhibits the growth of filamentous fungi such as dermatophytes and plant pathogenic fungi at extremely low concentrations. The characteristics of the above-mentioned filamentous fungus M4845, which was found to produce an antibiotic and was named the antibiotic acreasin Dγ, were cultured on various media based on macroscopic and microscopic observations as follows.

A. Macroscopic Observation 1. Growth on Czapek agar medium is rapid, reaching a diameter of 55-651 mm in 10 days at 26°C.

The surface of the colony is flat and thin, and is white at the beginning of culture, but as conidia are formed and mature, it gradually changes to phon (F).
anlnhue4lg). Conidia are formed in large numbers. The area around the village is somewhat spider web-like (Arachnoid), and the underside is colorless to pale yellow (PaleYelIOwlhue/Ca).

Does not release diffusible dyes or exudates. Odorless. Growth at 37°C is slow, 14-2 in 10 days Growth on wort agar medium is fast, reaching a diameter of 60-700 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, they become Beay.
erlhue4ll). Conidia are formed in large numbers. The area around the village is smooth or almost smooth. The color of the back side is Light Yellow (LightYellOw,.h
ue/%Ca). Does not release diffusible dyes or exudates.
Odorless. Growth at 37°C is slow, with 10 to 3 potatoes per 10 days, and the growth conditions are almost the same as on the wort agar medium, except for the clear spider web-like appearance around the clusters on the glucose agar medium.

Note: Colors are listed in 1958 by Container Corporation. of. America (ContainerCOrpOratiOnOfAx
Erica) Published [Color. Harmony one. Manual (
The designation method of COIOrHarmOnyManuaI 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.
~2.0μ. The apical sac is light brown, spherical or subglobose, straight, 30-75μ, often 40-60μ.
8x3~4μ pieces are lined up closely. Conidia range in shape from spherical to oval. 3.5~5.0X3.0
~3.5μ, 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 properties of the Aspergillus genus (Journal of the Agricultural Chemistry Society, ?, 806-809 (195
3), The Genus Eight Spergi-11uss3
28-331 (1965)), Aspergillus with conidia of dark brown lineage. Nigaichi, 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.

In addition, Aspergillus is a fungus with a single row of stalks. Aspergillusiaponicus (Aspergillusiaponicus)
s) and Aspergillus. Acleatus (Aspergi)
llusaculeatus), and Aspergillus. The conidia of Jiaponycus are spherical or subglobose, and the size is 3 to 3.5μ, and the apical capsule is 15 to 45μ, often 20 to 35μ.Aspergillus and aculeatus have conidia that are subglobe or elliptic. Size 4.5-5
X3-3.5μ, apex 35-100μ, mostly 60
~80μ, and the present bacterium is Aspergillus. The mycological properties are very similar to Aculeatus, and it is also an American type. Culture Collection (AmericanTy
Aspergillus from peCultureCOllectiOn). As a result of comparison with A. aculeatus ATCClO34, it was found that this bacterium matched extremely well with Aspergillus. Aculeatus M4845, and this bacterium has been submitted to the National Institute of Microbiology, Agency of Industrial Science and Technology with the microbial accession number “Feikoken Bacteria Collection No.4”.
No. 023 FERM-P, l64O23. ] has been applied for. The present invention has been completed based on the above findings, and is an antibiotic acreasin Dγ having the following physical and chemical properties: Pauli reaction, phorin reaction, periodic acid-benzidine reaction, potassium permanganate decolorization. Positive for the reaction, Moritzsch reaction, Biuretz reaction, xanthoprotein reaction, positive for Tollens reaction, ninhydrin reaction, Benedikt reaction, Sakaguchi reaction, Ehrlich reaction, ferric chloride reaction, Dragendorff reaction, 2.4-dinitroph Negative for enylhydrazine reaction, Soluble in solvents Soluble in lower salcols, Slightly soluble in ethyl acetate, Slightly soluble in acetone, chloroform, n-hexane, petroleum ether, water Distinguish between basic, acidic and neutral n-butanol It is presumed to be a neutral substance since it does not move from the solution to the aqueous layer with a pH of 2 to 9.

White color stability of the substance Stable at 37°C for 20 hours in acidic and neutral conditions, unstable in altaric conditions.

Ultraviolet absorption spectrum Accreasin Dγ (concentration 40γ) measured in methanol
/ml) of ultraviolet light? ? The yield spectrum is λMax278n
Maximum absorption at mlEl;=17.0, 1% and λMax
226nmlE=137, λMaxlCfL284nm
lEZ? = 14.2 with shoulder 1.

Liquid chromatography's highest retention time Rt=1
4.1 minutes (column diameter 5 x 500 m 1t1 carrier polystyrene resin, developing solvent 0.01% triethylamine containing 85
% methanol aqueous solution, column temperature 55 °C), and the manufacturing method is to culture bacteria that produce the antibiotic acreasin Dγ belonging to the genus Aspergillus in a medium, and collect the antibiotic acreasin Dγ from the culture to produce a new antibiotic. Accreacin Dγ is obtained.

The bacteria used in the present invention include the above-mentioned Aspergillus. The acleatus M4845 bacterium is an example of this, and it is sufficient to use not only this bacterium, but also any species that can produce or synthesize acreasin Dγ (hereinafter simply referred to as acreasin Dγ) having the above-mentioned physical and chemical properties. All possible bacteria can be used in the present invention, and the acreasin Dγ of the present invention may be obtained using chemical means.

To carry out the present invention, as an example, first, an acreasin Dγ-producing bacterium belonging to the genus Aspergillus is cultured using a conventional method for producing antibiotics.

The form of culture at this time may be liquid culture or solid culture.Industrially, a spore suspension of acreasin Dγ-producing bacteria or a culture solution cultured for 1 to 2 days is inoculated into a production medium, followed by deep aeration and stirring. It is advantageous to carry out cultivation. As a culture source for the medium, a wide variety of media commonly used for culturing microorganisms can be used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, starch, dextrin, molasses, and glycerin. The nitrogen source may be any available nitrogen compound, such as corn. Steep. The following substances are used: liquor, soy flour, cottonseed flour, wheat gluten, peptone, meat scratches, yeast scratches, yeast, casein decomposition products, ammonium salts, nitrates, etc. In addition, salts such as phosphate, magnesium, calcium, potassium, sodium, zinc, iron, and manganese are used as necessary. The culture temperature can be changed as appropriate within a range that allows the bacteria to grow and produce acreasin Dγ, but is preferably 25 to 28°C.

Cultivation time varies slightly depending on conditions, but is usually 70 to 90 days.
The culture may be terminated at an appropriate time by determining the time when acreasin Dγ reaches its maximum titer. In the thus obtained culture of acreasin Dγ-producing bacteria, acreasin Dγ is mostly accumulated within the bacterial cells, and a portion is also produced outside the bacterial cells.

Also, when collecting acreasin Dγ, Candida. albicans or trichophyton. Asteroides (TrichOphytOnaster-01des
) is quantified by bioassay using the cup method or paper disk method. Next, acreasin Dγ is collected from this culture, and it is preferable to collect acreasin Dγ accumulated within the bacterial cells. Next, an example of a means for separating and purifying acreasin Dγ will be shown. That is, microbial cells containing a large amount of acreasin Dγ are collected from the culture obtained by culturing acreasin Dγ-producing microorganisms as described above using a drum filter, filter press, centrifuge, etc., and the obtained wet microbial cells are Extraction is carried out by adding a hydrophilic solvent such as alcohol or acetone, the solvent of the obtained extract is distilled off, this is diluted with water, and a solvent such as n-butanol or ethyl acetate is further added 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 concentrated under reduced pressure and collected in a single spot by thin layer chromatography in various solvent systems. Ask for a powder to give. The physical and chemical properties of acreasin Dγ thus obtained will be described below.

2. Molecular weight Last method: 1265 Amino acid analysis: (555)n (4.8μ from the hydrolyzate of acreasin Dγ2.6~
Gel filtration method that detected threonine in MOL: 850±
100 (Using Cephadex LH-20 (manufactured by Pharmacia Fine Chemical Co., Ltd.), developing with ethanol, simultaneously developing an antibiotic with a known molecular weight, and estimating from the elution position) 3. Melting point: 159-162C (no clear melting point shown) 4. Specific light intensity D5. Color reaction Benzidine reaction, potassium permanganate decolorization reaction positive, Moritsch reaction, Biuret reaction, xanthoprotein reaction, Tollens reaction positive, ninhydrin reaction,
Shows negative results for Benedikt reaction, Sakaguchi reaction, Ehrlich reaction, ferric chloride reaction, Dragendorff reaction, and 2,4-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 It is impossible to titrate because alkalinity causes decomposition, and it is poorly soluble and does not move by electrophoresis, so the distinction between them is not clear, but water with a pH of 2 to 9 can be extracted from the butanol solution. It is presumed to be a neutral substance since it does not migrate to the layer.

8. color of matter white crystal powder 9. Stability Stable at 37°C for 120 hours in acidic and neutral conditions.

Alkaline and unstable. 10. Ultraviolet absorption spectrum Accreasin Dγ (concentration 40γ) measured in methanol
The ultraviolet absorption spectrum of /MO is as shown in Figure 1, and is λMax278nrnlE{? : Maximum absorption is at 17.0 and λMax226n has a shoulder at 14.2.

11. The infrared absorption spectrum of acreasin Dr measured by the infrared absorption spectrum KBr method is shown in FIG.

12. Rf value silica gel thin layer chromatography (manufactured by Eastman Kodak, Eastman Chromatography Sheet 6060)
: Chloroform-methanol (10:3) Rf value 0.
67, Rf value 0.35s with ethyl acetate-methanol:water (20:4:1) ethyl acetate-n-butanol (3:1)
) Rf value 0.420 at water saturation (however, detection is by bioautography using Candida albicus and iodine vapor).

)13. U tension time (Rt) by liquid chromatography 8 μl of acreasin Dγ5η/ml methanol solution
71Lm1 carrier polystyrene resin, developing solvent 0.01%
85% methanol aqueous solution containing triethylamine, column temperature 55 °C, Hitachi 643 model (manufactured by Hitachi, Ltd.) was charged and its retention time (RetentiO
ntime) was 14.1 minutes.

14. The hydrolyzate acreasin Dγ was hydrolyzed with 6N hydrochloric acid at 110 iC, extracted by adding ether, and the extract was charged to a gas chromatogram to qualitatively detect free fatty acids. As a result, palmitic acid was detected.

15. Amino acid composition 6N hydrochloric acid, 11 hours of Cl2O hydrolysis, 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 Dγ 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 atareacin Dγ, which has a value of around 8 mμ, you will find Myronodine (MyrOridinl Special Publication No. 12276/1976), Athalestatin (Athles
tatiO, Special Publication No. 41-1268), Monilin (M
Onilinl Takeda Research Annual Report 14, 8-10, 1955
), oryzamycin (0ryZarr1yCin1 Special Publication No. 38-2800), salamycetin (Saramycetin
cetinsAnti-MicrObia agent
sandchemOtherapyl96l, 436
~444), Unamycin, The
JournalOfArltibiOti-Cs, Se
rAl3,ll4-120, 1960), Pendicide (Echinocand British Patent No. 764198), Acreasin D (U.S. Patent No. 3978210);
iuB. Swiss Patent No. 568366: 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. -164, (C=0.5, methanol), and its ultraviolet cathode absorption shows a clear maximum absorption at 225 nm in addition to 278 nm, and its elemental analysis value shows C: 55.25%, H: 7.50%,
It is distinguished by its N:9.41%, monilin is a basic nucleic acid substance, and its elemental analysis also has a high nitrogen content, oryzamycin is an acidic oil, and salamycetin gives aspartic acid, cystine, glycine-threonine, etc. by hydrolysis; unamycin is an acidic polyene substance; benziside is a nucleic acid substance similar to moniline; It is presumed that B is a substance having a structure that is extremely similar in physical, chemical, and biological properties, but the acreasin Dγ of the present invention has the highest retention time value of 14.1 minutes in liquid chromatography. death,
The structure of echinokyandeine B, which was reported in Tetrahedron Letters No. 46, pp. 4147-4150 (1976), is dihydroxy homozygous. While the formation of a cyclic peptide containing tyrosine was observed, amino acid analysis of atalesin Dγ confirmed α-hydroxyhomotyrosine in its cyclic structure.
Acreasin Dα and echinochiandein B are distinguished. Therefore, atalesin Dγ of the present invention is recognized as a novel antibiotic different from conventionally known substances. Next, the biological properties of acreasin Dγ will be described. ▲▲
Vl number 1b insult v Akira Vvrv Bihiro [Union-V●J-▼▼v
The measurement was carried out using samples containing various concentrations of acreasin Dγ.
1'-Ai-=Choyama Hitomi→Nanaphimisaki~Tetel Hachinohe08l+] A huge colony grown by culturing for 14 days for saponophilous fungi and 7 days for plant pathogenic fungi) The fungal moss area was calculated from the diameter of the microbial moss, and this was the concentration that inhibited the growth of the fungal moss by 80% compared to the fungal moss area of the control group without addition of acreasin Dγ, which was conducted in parallel.

Acute toxicity test for mice Using an aqueous solution of acreasin Dγ prepared with sodium deoxycholate as a solubilizing agent, its LD5O was determined.

Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto in any way.
1 glycose 2%, polypeptone 1%, corn. Steep. 1% KH2PO4O. 2% MgSO4O. l
100ml of medium containing % (PH6.5) to 500ml
Dispense into a 1 volume Erlenmeyer flask, heat sterilize at 120mC for 15 minutes, and add Aspergillus. Acreatus M4845
Inoculate one platinum loop of spores from a slant culture of the strain at 26°C.
Shaking culture was carried out for 48 hours at 120°C, 15
Satucarose 1.5%, dextrin 1.4%, polypeptone 2%, corn. Steve. 0.45% KH2PO4O. 2%, MgS
O4O. 1%, BC-51Y (antifoaming agent: manufactured by NOF Corporation)
The culture was transplanted into a 301-volume jar containing a medium (PH 6.5) consisting of 151 ml of the following culture, and cultured with aeration at 26° C. for 90 hours at an aeration rate of 250 rp11/min to obtain a culture of about 151 ml. Ta.

There is acreasin Dγ activity in the culture broth and cultured cells of this culture, and acreasin Dγ 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.

Methanol 51 was added to the wet bacterial cells, immersion extraction was carried out for 2 hours under stirring, the methanol solution was recovered by vacuum filtration, and methanol 51 was further added to the bacterial cells and extracted in the same manner.
Both extracts were combined and methanol was distilled off under reduced pressure to obtain a concentrated solution of 22, to which water 21 was added, and n-butanol 21 was further added twice for extraction, and the n-butanol layer was collected. After washing this n-butanol solution with 51 water, it was concentrated under reduced pressure while adding a small amount of water to obtain a dark brown viscous concentrate, to which 500 ml of n-hexane was added,
The resulting precipitate was collected and washed with ethyl acetate.
12.59 ml of crude material containing acreasin Dγ was obtained. Beautiful Example 3 12.59 of the acreasin Dγ-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
11) and developed using a developing solvent of ethyl acetate lyisopropanol:water (10:1.5:0.7), melted as 1 fraction 45d, and collected fractions 32 to 42. These were combined and dried under reduced pressure to obtain a white powder of 900~, which was dissolved in a small amount of methanol, and silica gel No. 29 was added thereto and adsorbed, then dried under reduced pressure, and this was prepared separately. 45f! Column (inner diameter 25 m) packed with silica gel (manufactured by Tokyo Kasei Co., Ltd.)
0 and then chloroformrimethanol (10
Developed with the developing solvent of :15), fractionated into 59 fractions and collected fractions /1676 to 141,
These were combined and concentrated to dryness to obtain an acreasin Dγ-containing product of 3707V, which was then dissolved in methanol. , developing solvent 0.01 triethylamine containing 85
% methanol aqueous solution, column temperature 55°C) to obtain an active fraction fractionated with a retention time of 14.4 to 16.4 minutes, this was repeated, the fractions were combined, and then This was dried under reduced pressure, dissolved in a small amount of methanol, and ethyl acetate-n-hexane (1:
Add the mixed solvent of 1), filter out the precipitate,
Approximately 15η of purified acreasin Dγ was obtained.

[Brief explanation of drawings]

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

Claims (1)

[Claims] 1 Antibiotic acreasin Dγ having the following physical and chemical properties: Elemental analysis C: 57.55% H: 8.32% N: 8.94
% molecular weight Last method: 1265 Amino acid analysis: (555)n (4.8 from a hydrolyzate of 2.6 mg of acreasin Dγ
μMol of threonine was detected) Melting point: 159-162°C Specific light intensity [α] D^2^3 = -46.5° (C = 0.25, methanol) Color reaction Pauly reaction, Folin reaction, periodic acid - positive for benzidine reaction, potassium permanganate 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; basic, acidic, and neutral; neutral as it does not migrate from the n-butanol solution to the aqueous layer with a pH of 2 to 9 Estimated to be a substance. White color of the substance, stability, 37°C, 20 hours, stable in acidic and neutral conditions, unstable in alkaline conditions, ultraviolet absorption spectrum measured in methanol Acleacin Dγ (concentration 40γ
/ml), the ultraviolet absorption spectrum is λmax278n
Maximum absorption at m, E^1^%_1_c_m=17.0 and λmax 226 nm, E^1^%_1_c_m=13
7, λmax284nm, E^1^%_1_c_m=1
Liquid chromatography glue tension time Rt = 14.1 min (column diameter 5 x
500mm, carrier polystyrene resin, developing solvent 0.01
85% aqueous methanol solution containing % triethylamine, column temperature 55°C)