JPH01121293A - Novel antibiotic substance benanomicins a and b and production thereof - Google Patents

Abstract

NEW MATERIAL:Antibiotic substance benanomicins A and B. Appearance; reddish brown powdery acidic substance. Molecular formula: C39H41NO19. Melting point; >=220 deg.C. Solubility; extremely sparingly soluble in methanol, chloroform, ethyl acetate and acetone, soluble in dimethyl sulfoxide, dimethylformamide and alkalis and insoluble in water. USE:Useful as an antifungal agent having antifungal activity of inhibiting growth of various molds. PREPARATION:A microorganism (e.g. MH193-16F4), belonging to actinomyces and capable of producing antibiotic substance benanomicins A and B is cultivated under condition of, e.g. 27-37 deg.C for about 7 days, to afford the aimed antibiotic substance from the resultant culture.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention is directed to a novel anti-fungal and antibiotic drug Benanomycin (B
enanomicin) A and B or salts thereof,
and its manufacturing method.

(Prior art) KS-619-1 [
Matsuda et al.: rJ, Antibiotic
S J 40.

1104-1114 (1987)] G-2N and G-2A [Gerber et al.: 'Canad, J.
Chem,” 62.2818-2821 (1984
)'] etc. are known. However, the antibiotics benanomycin A and B have different physicochemical and biological properties from these substances and are clearly distinguished from each other.

(Problems to be Solved by the Invention) Various antibiotics produced by microorganisms have been known, but not many anti-fungal antibiotics with low toxicity have been found. There is a constant demand for new anti-fungal antibiotics in the medical field of disease.

An object of the present invention is to provide novel, low-toxicity antifungal antibiotics benanomycin A and B, and a method for producing the same.

Means for Solving Problem C) The first gist of the present invention is a novel antibiotic benanomycin A and its salt.

1. The details of the physical and chemical properties of benanomycin A are listed as follows.

(1) Color and shape: Reddish brown powder (2) Molecular formula: C311H41NO+9 (3)
Mass spectrum (FD-MS): m/z 827 (M
') (4) Melting point: >220℃ (5) Specific rotation: [α]n=unmeasurable (c O
, 05, DMSO) (6) Ultraviolet and visible absorption spectra λwax r+
+n (E1%) 1cm [in methanol] +206 (718), 230sh (
'600).

288 (482), 302sh (390).

400sh (120), 476 (197) [0, IN
8C1-in methanol] 207 (649), -233 (629), 298 (56
1), 395sh (140), 457 (223
) [0,1N NaOH-in methanol] 214 (12
70), 249 (637), 320 (289),
49B(287) (7) Infrared absorption spectrum (KBr cm-'n:
3350.2970.2890.1720.1620
゜16G0.1485.1440.1425.1390
゜1375, 1330.1295.1255.1235
゜1205, 11B0.1130.1070.1040
゜1000, ! 170. 9QO,870,830
゜800, 750 (8) 1H-NMR spectrum (400MHz, D
MSO-d, 40°C) δ (ppm): 1.14 (3H,
d), 1.35(3H,d,), 2.34(3)1
．． s), 3.09(1)1. dd), 3.13(
4N.

dd), 3.17 (Wataru Fl, dd), 3.32 (
II (,ddd).

3.56 (IH, dd), 3.62 (2) 1. m),
3.72 (IH, dd), 3.74 (II (, br)
, 3.92 (3H.

s), 4.43(IH,d), 4.43(IH,d
q).

4.53 (IH, d), 4.57 (IH, d), 4.
65: (IN, d), 4.90 (2H, br),
5.81(]Lbr), 6. (i5(IH,br)
, 6.86 (IH, d).

7.21 (IH, s), 7.24 (IH,' d)
, 8.05(1)1. s), 8.45 (IH, d
), , 12.47 (IH.

br), 12.77 (IH,s), 13.
69 (IH.

br) (9) “C-NMR spectrum (100MHz, DM
SO-da, 40°C) δ (ppm): 1B7.3
s, 184.9 s, 173.9 s.

1B6.9 s, 1[15,9,s, 164.7
s.

156.8 s, 151°Is, 147.7
s, 138.1s, 137.4s,
134.2 s, 131.3 s.

127.5 s, '125J s, 118.6
d, 115.5s, 115.4 d, 113.7
s, 110.0 s.

107.5 d, 106.8 d, 105.
2d, 104.4d, 83. Od, 81.7
d, 76, Od, 73. [id, 71.
! ] d, 70.3 d, 70.1 d,
70.1d, 69.4d, 65.6t,
56.3 q, 47.6 d, 19.1 q,
16,9 q, 16.3 q (72,9,'
The signal of 81.7.115.4.118.8 is broad. ) (10) Solubility: Slightly soluble in methanol, chloroform, ethyl acetate, and acetone, soluble in dimethyl sulfoxide, dimethyl formamide, and alkaline water, and insoluble in water.

(11) Distinction between basic, acidic, and neutral: Acidic substances The second aspect of the present invention lies in the novel antibiotic benanomycin B and its salts.

2. Details of the physical and chemical properties of benanomycin B hydrochloride are listed below.

(+) Color and shape: Reddish brown powder (2) Molecular formula 'C3sHI4zNxo, B・H
CII (3) mass spectrum (SI-MS): m/z
827 (MH”) (4) Melting point: >220°C (5) Specific rotation: [α) 22 = d60°
(c O, 05°H2O) (6) Ultraviolet and visible absorption spectrum λmax nm
(E] Responsibility.) [In methanol]: 205 (587), 233 (52
6), 29ft (426), 390sh (100)
, 458 [0,1N HCl-in methanol] 207 (514), 235 (530), 295 (4
42), 400sh (114), 457 (D, IN
NaDH-in methanol] 214 (121! l),
247 (518), 317 (238), 496 (2
15).

(7) Infrared absorption spectrum (KBrcm-1):
3350.2980.2900.1720.1610°1485, 1450. 430.1395.1380°1330, 1300.1260.1240.1210°1160, 1080.1045. 970. 955°900, 885. 840. 820(8) 1H-
NMR,2,'(at (400MHz, DMSO
-66,40℃) δ (ppm): 1.2Q
(31(,d), x, o(3H,d), 2.3
5 (3F1.s), 3.09 (I)I, dd
), 3.17 (1)1゜m), 3.19 (IH
,'m), 3.34 (IH, ddd).

3.44 (IH, br), 3.65 (1) 1. br
), 3.75 (IH, dd), 3.90 (IH, b
r q), 3.94 (3H, s), 3.97 (18
, dd), 4.44(II(.

dq), 4.57 (IH, d), 4.57 (1) 1
．． br d) 4.62 (IH, br d), 4.7
5(1)1. d).

fi, 90 (18, d), 7.27 (IH, b),
7.27(1)1. br s), 7.99 (3H,
br), 8.06(ILbr s), 8.45(1
)! , d), 8.45(l)l, br), 12
．． 79(1)1. s), 13.81 (IH.

br) 4.1-6.3 (5H, br) (9) ”C
-NMR spectrum 71. + (100'M) Iz, DM
SO-da, 40'e) δ (ppm): 187.4 s
, 184.9 s, 173.9 s, 1B6.9 S.

165.9s, 164.7s, 156.8
s, 151.0s, 148. Os, 13
7.8 s, 137.3 s.

134.2s, 131.2s, 127.5
s, 125.7s, 118.9d,
115.9 s, 115.5 d.

113.7 s, 110. Os, 107J d, 1
06, 8d, 104.4d, 104.1d, 8
1. Od, 77.4d, 75.1d, 73.3
d, 71.5 d, 89.8 d, II9.4
d, 67.0 d, 65.7 t, 56.3q
, 54.2 d, 47.6 d, 19.1 q,
1f1.9q, 16.3q (71,5,81,0,113,7,115,4,11
Signals of 8, 9, 125, and 7 are blows. ) (10) Solubility: Slightly soluble in chloroform, ethyl acetate, ethyl acetate, and acetone, and soluble in methanol, dimethyl sulfoxide, dimethyl formamide, and water.

(11) Distinction between basic, acidic, and neutral: Salts of the amphoteric substances benanomycin A and B include metal salts, particularly alkali metal salts such as sodium salts, alkaline earth metal salts such as calcium salts, Also included are acid addition salts with pharmaceutically acceptable inorganic acids such as hydrochloric acid or sulfuric acid, or pharmaceutically acceptable organic acids such as acetic acid and malic acid, and salts with amines as described below.

3. The antibacterial activities of benanomycin A and B are shown below.

The minimum inhibitory concentrations of benanomycin A and B according to the present invention against various bacteria and molds are shown in Tables 1 and 2, respectively. As shown in Table 2, benanomycin A and B exhibited antifungal activity against various molds.

4. Toxicity of benanomycin A and B Jcl of benanomycin A and B: ICR mouse (
In an acute toxicity test by intravenous administration to male males, body weight 19-20 g, 5 animals per group, benanomycin A was administered at a dose of 600 mg/K.
There were no deaths at a dose of 100 g, and benanomycin B
There were no deaths at the mg/Kg dose.

5. Infection treatment effect of benanomycin A Jcl: ICR mice (male, weight 10-20 g, 5 mice per group) were infected with Candida albicans
10' per mouse of CFII (0
, 2rnU dose intravenously, then benanomycin A
The therapeutic effects of benanomycin A on experimental candidiasis when administered subcutaneously and orally are shown in Table 3.

Table 3 The gist of the present invention in Table 3 is that bacteria producing the antibiotics benanomycin A and B belonging to actinomycetes are cultured, and the antibiotics benanomycin A and B, or either one thereof, are collected from the culture. A method for producing the antibiotic benanomycin A and/or benanomycin B.

An example of benanomycin A and B producing bacteria is the actinomycete Ml (strain 193-16P4 described below).

(a) Mycological properties of frozen bacterium MH1B3-16F4 This strain was isolated from the soil on the premises of the Institute at the Institute of Microbial Chemistry in March 1980.
The strain number of MH193-18F4 was assigned in [1lycete]. The mycological properties of this strain are as follows.

[1] The MH193-16P4 strain forms aerial hyphae from branched basal hyphae under a microscope. No division of basal hyphae is observed. Adhesion of aerial mycelium was at the same level as observed in l5P-medium 2 and rsp-3, and sporulation was normally observed in l5P-medium 2 and rsp-3.
It is observed from around the 18th day after culturing at 7°C.

Whorled branches, spiral formation, and sporangial formation are not observed in the aerial hyphae, and short sporophores are formed almost perpendicular to the aerial hyphae, and a chain of usually 3 to 7, but rarely 2 spores can be seen at the tip. It will be done.

Note that the spore chain may have a loop shape. Individual spores are cylindrical (0,8 x 1.0-1.2 μm) to spherical (0
, 8-1.2 μm), and the spore surface is smooth.

[2] Regarding the description of the growth state color in the seed medium, the standards shown in [ ] are for containers and
Corporation of America Color Harmony Manual
ion of America's Co1or Harm
onyMar+aal) was used.

(1) Sucrose/nitrate agar medium (cultured at 27°C; the seedling side is colorless, no aerial mycelia are attached, and no soluble pigments are observed. In the vitamin B group-enriched medium, growth is colorless to pink gray [5ec, Dusty Peach], aerial mycelia do not grow, and the soluble pigment is only slightly reddish.

(2) Glucose-asparagine agar medium (cultured at 27°C) The growth is colorless, no aerial mycelium is attached, and no soluble pigment is observed. In the vitamin B group-enriched medium, the growth is colorless to pale pink [4gc, Nude Tan], aerial mycelia do not grow, and soluble pigments are reddish.

(3) Glycerin-asparagine agar medium (ISP-medium 5.27°C culture) The growth is colorless, no aerial mycelium is attached, and no soluble pigment is observed. In the vitamin B group-enriched medium, the color was gray-red-purple [8℃g,
)lose Mauve] ～Nabu red purple [8℃e, R
On the growing oseWine, slightly thin white aerial mycelium grows, and it becomes reddish-purple [8pc, Cranberr]
y] to produce a soluble dye.

(4) Starch/Inorganic Salt Agar Medium (ISP-Medium 4.2
(cultivated at 7°C) The growth is colorless, no aerial mycelia are attached, and no soluble pigments are observed. In vitamin B group enriched media, the soluble pigments have a slight reddish tinge.

(5) Tyrosine agar medium (rsp-medium 7.27℃ culture) Growth is colorless to light yellowish brown [3ic, Lt Amber]
, no aerial mycelia were observed, and no soluble pigment was observed. In the vitamin B group-enriched medium, the growth is colorless to grayish-reddish-purple, no aerial mycelia are attached, and soluble pigments are slightly reddish.

(6) Nutrient agar medium (cultured at 27°C) Growth is pale yellow [2gc, Bamboo], no aerial mycelia are attached, and no soluble pigment is observed. Similar properties were observed in the case of the vitamin B group-enriched medium.

(7) Yeast/malt agar medium (IS'P-medium 2.2
7℃ culture) Light yellow [2ec, B15cuitl ~ dark red [7p
i, DK Wine-7%pe, DK Redl
~ On the growth of gray red [7 pg, Wine], 1 after culture.
From around the 4th day, gray-white aerial mycelia begin to grow. Nibuaka [61p
e, The soluble pigment of Tomato Redl is seen only around the growth at the early stage of culture, but gradually diffuses. Both the developmental pigment and the soluble pigment gradually change color with HCj2, but no change is observed with NaOH. Similar properties are observed in the vitamin B group-enriched medium.

(8) Oatmeal agar medium (ISP-medium 3.27℃
Culture) Light pink [4gc, Nude Tan] ~ Grayish red [6j2e.

Cedar]~Dark red [7pe, Cherry Wi
nel~Purple Gray [8'ig, Mauve Gray]
On the development of the cells, from around the 10th day after culturing, there was a dull grayish white color [3
cb, 5 and l aerial mycelia are attached. The soluble pigment has a reddish tinge.

Both the growth color and the soluble pigment gradually change color with HCI, but no change is observed with NaO1H. A vitamin B group-enriched medium also showed similar properties.

(9) Glycerin/nitrate agar medium (cultured at 27°C) Growth is colorless, no aerial mycelia are attached, and no soluble pigment is observed. Similar properties are observed in the vitamin B group-enriched medium.

(10) Starch agar medium (27℃ culture) Growth is colorless;
No aerial mycelia are attached, and no soluble pigments are observed. In the vitamin B group-enriched medium, the growth was colorless and aerial mycelia did not attach.
The soluble pigment gives it a slight reddish tinge.

(11) Malate lime agar medium (27°C culture) Growth is colorless, aerial mycelia do not adhere, and soluble pigment is slightly reddish. In the vitamin B group-enriched medium, the growth is colorless, no aerial mycelia are attached, and no soluble pigments are observed.

(12) Cellulose (synthetic solution with filter paper added, cultured at 27°C) Growth is not recognized.

(13) Gelatin puncture culture 15% simple gelatin medium (cultured at 20°C), glucose/
Growth was poor on both peptone and gelatin media (cultured at 27°C), with colorless growth, no aerial mycelium, and no soluble pigments.

(14) Skimmed milk (cultured at 37°C) Growth is extremely poor, colorless, aerial mycelia do not attach,
No soluble dyes are also observed.

[3] Physiological properties (1) Growth temperature range Starch yeast agar (1.0% soluble starch, 0.2% yeast extract, 3.0% string agar, pH 7.0
), 20°C, 24°C, 27°C, 30°C, 37°C,
As a result of testing at various temperatures of 50℃, growth occurred at all temperatures except 50℃, but the optimal growth temperature was 27℃ to 37℃.
It seems to be around ℃.

(2) Liquefaction of gelatin (15% simple gelatin medium, 20%
℃ culture; glucose peptone gelatin medium, 27℃
Culture) Both the simple gelatin medium and the glucose peptone gelatin medium were observed for 3 months after culture, but no liquefaction was observed.

(3) Hydrolysis of starch (starch/inorganic salt agar medium and starch agar medium, both cultured at 27°C) No ice-melting properties were observed in either the starch/inorganic salt agar medium or the starch agar medium.

(4) Coagulation and peptonization of skimmed milk (skimmed milk, 37℃
Culture) Growth was poor, and neither coagulation nor peptonization was observed during observation for 3 months after culture.

(5) Production of melanin-like pigments (tryptone, yeast,
Broth, l5P-medium 1; peptone yeast iron agar, l5P-medium 6; tyrosine agar, l5P-medium 7;
Both were cultured at 27°C) It was negative in all the media.

(6) Utilization of carbon sources (Pridham-Gotlieb agar medium, l5P-medium 9.27°C culture) glucose, L-
It grows using arabinose, D-xylose, D-fructose, sucrose, rhamnose, raffinose, and D-mannitol, but does not use inositol.

(7) Dissolution of malic acid lime (malic acid lime agar, 27℃
Culture) is negative.

(8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, ISP medium cultured at 8.27°C) is positive.

(9) Decomposition of cellulose (synthetic solution added with filter paper, cultured at 27°C) No growth.

To summarize the above characteristics, strain MH193-l6P4 forms a spore chain consisting of 3-7 (rarely 2) spores almost perpendicular to the main axis of aerial hyphae, and uses whorling, spiral formation, and spore formation. No caries formation is observed. In addition, no division of the basal hyphae is observed. The spore surface is smooth. l5P-medium 2,
l5P-2f of medium 3! In medium, gray-white aerial mycelia are formed on the gray-red to dark red growth. In addition, slant culture medium (yeast extract 0.2%, soluble starch 1.
0%, string agar 3.0%, p)I 7.0), a slight pink aerial mycelium may grow. In addition, 15P-medium 2 produces a dark red soluble pigment. On various other media, the growth is colorless, no aerial mycelia are attached, and almost no soluble pigments are observed. However, there is a culture medium that improves growth by adding B group vitamins to the culture medium, making the growth red. Both the growth color and soluble pigment) gradually changed color from red at ICj2, and Na0t! No change can be seen. Production of melanin-like pigment, proteolytic ability, and starch ice-melting ability were all negative, and nitrate reduction reaction was positive. In addition, growth may be good on a vitamin B group-enriched medium, and it is thought that the plant has a vitamin requirement.

By the way, the MH193-'16F4 strain contains meso-diaminopimelic acid as a bacterial component and glucose, ribose, and madulose as sugar components, as described by Lechevalier et al.

rInternational Journal of
Systematic Bacteriology” 2
0, p. 435, 1970) as the main constituents of the cell wall.

In addition, the type of phospholipid is PrV type (contains phosphatidyl ethanolamine and unknown glucosamine-containing phospholipid, but does not contain phosphatidyl glycerol),
The composition of menaquinone is MK-9 (H8) as the main component,
K-9 (Ha), MK 9 (! (4), MK -
! ] ()12), MK-9(l(to), [lN
A) GC content was 71.5%. Furthermore, as a result of gas chromatography analysis of bacterial cells, iso-branched fatty acids (i-18:0) and anteisi-branched fatty acids (a-17
: [1), 1o methyl fat rib H (10Me-17:0
) was found to be characteristically included.

Known actinomycetes with spore chains and cell wall type HI B
Those showing this are Actinoma-
dura), Microbispora
ra), Microtetras
pora).

Table 4 shows the characteristics of the MH193-16F4 strain and the three genera mentioned above. * for menaquinone indicates that it is included as a main component.

References 1) Identification experimental method for actinomycetes 1 edited by the Japan Actinomycetes Research Society.

1985 2) J, Po5chner et al.: r DNA-
DNA Reassociation and Chem
otaxonomic 5tudies on Act
inoma-dura, Mlcrobispora,
Microtetraspor6, Mlcropol
yspora and Nocardiopsis,
Systematic and Applied
Microbiology, vol. 16.

pp. 264-270, 1985.

3) A. Fischer et al.: rMolecul
ar-genetic and chemotaxono
mlc 5tudies on Actinomadu
raand Nocardlopsis, Jour
nai of GeneralMicrobiolog
y” Vol. 129, pp. 3433-3446, 1983 4) Thiemann et al.: rA New G
enus of the Acti-nomyceta
les : Microtetraspora g
en, nov,.

Journal of General Microb
iology J50, pages 295-303, 196
8 years.

5) Nonomura et al.: “Distribution of actinomycetes in soil” (
Report 11) Actinomadura Lecheva
ller et81, several new species of the genus, "Journal of Fermentation Engineering" 4
Volume 9.

pp. 904-912, 1971.

As shown in Table 4, there is no genus to which the MH193-16F4 strain easily falls. These genera have undergone very subtle changes and are described in the Vergey's Manual (B
ergey's Manual of Determi
We are looking forward to seeing how it will be featured on native Bacteriology clothing. However, many species of the genus Actinomadura have been described, and there is a wide range of properties, but among them, Actinomadura spadix (Actinomadura 5padix) is characterized by its morphology, bacterial cell fatty acid composition, and menaquinone composition. Considerable similarities can be found (references 1), 3), and 5) above). In the future, we plan to first conduct a comparative experiment between the MH193-16F4 strain and Actinomadura spasicus.

An application was made for depositing M) 1193-16F4 strain to the Institute of Microbial Technology, Agency of Industrial Science and Technology, and it was deposited as Microtechnology Research Institute No. 9529 on August 21, 1988.

(b) Cultivation method for strain MH193-16F4 The production of benanomycin A and B is performed using benanomycin A, which belongs to actinomycetes.
and B-producing bacteria are inoculated into a medium containing nutrients that can be used by ordinary microorganisms and grown under aerobic conditions. Mainly benanomycin A and B in the culture medium.
is produced and accumulated. Separate a target substance from a culture, especially a culture solution.

As the nutrient source in the medium used, any known nutrient source used as a nutrient source for actinomycetes can be used. For example, commercially available nitrogen sources such as soybean flour, nissan meat, peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, dried yeast, yeast extract, NZ amine, casein, sodium nitrate, ammonium sulfate, ammonium nitrate, etc. , and carbon sources such as glycerin, sucrose, starch, glucose, galactose, maltose, dextrin, lactose, molasses, soybean oil, fat, amino acids, and salt,
Inorganic salts such as phosphates, calcium carbonate, magnesium sulfate, cobalt chloride, manganese chloride, etc. can be used. In addition, trace amounts of metal salts, antifoaming agents, mineral oil, etc. may be added as necessary. These materials can be used as long as they are utilized by benanomycin-producing bacteria and are useful for the production of benanomycin A and B, and all known culture materials for actinomycetes can be used.

Liquid culture is preferred for mass production of benanomycin A and B, and the culture temperature can be applied within a range that allows the producing bacteria to grow and produce benanomycin A and B, usually 20-40°C,
Preferably it is 25-37°C.

Cultivation can be carried out by appropriately selecting the conditions described above depending on the properties of the benanomycin A and B producing bacteria.

(c) Purification of benanomycin A and B When collecting benanomycin A and B from the culture of benanomycin A and B obtained by the present invention, conventional separation methods utilizing their properties, such as solvent extraction, ion Extraction and purification can be carried out using an exchange resin method, an adsorption or distribution column chromatography method, a gel filtration method, a dialysis method, a precipitation method, etc. alone or in an appropriate combination. For example, benanomycin A
and B are extracted from the cultured bacterial cells with acetone-water or methanol-water. Furthermore, benanomycin A and B accumulated in the culture solution are adsorbed to a synthetic adsorbent such as Diaion H, P-20 (manufactured by Mitsubishi Kasei Corporation). Furthermore, if extraction is performed with an organic solvent that is immiscible with water, such as butanol, ethyl acetate, etc., benanomycin A and B substances will be extracted into the organic solvent layer.

To further purify benanomycin A and B, use silica gel (Wako Gel C-300, manufactured by Wako Pure Chemical Industries, Ltd., etc.)
, chromatography using an adsorbent such as alumina, Sephadex LH-20 (manufactured by Pharmacia), etc. may be performed.

Benanomycin A produced in culture in this way
and B can be isolated in free form, i.e. benanomycin A and B themselves.

Alternatively, a solution containing benanomycin A and B or a concentrate thereof may be mixed with a base, that is, an alkali metal compound such as sodium hydroxide or potassium hydroxide, or an alkaline earth metal compound such as calcium hydroxide or magnesium hydroxide.
Benanomycin A
and B are converted into their corresponding salt forms and separated.

Separately, benanomycin A produced in this way
The salts of and B can be converted into the free form, benanomycin A and B itself, by conventional methods. Furthermore, benanomycin A and B obtained in free form may be converted into the corresponding salts thereof using the above-mentioned bases in a conventional manner. Therefore, benanomycin A and B as well as their salts as described above are intended to be included within the scope of this invention.

Examples of the present invention are shown below. Since the properties of benanomycin A and B have been clarified by the present invention, various methods for producing benanomycin A and B can be devised based on their properties. . Therefore, the present invention is not limited to the examples,
Benanomycin A and B can be produced, concentrated,
Covers all extraction and purification methods.

Inukame■↓ Strain MH193-16F4 (Feikoken Bacteria No. 9529) cultured on an agar slant medium was mixed with 1.0% starch and 3% soybean flour.
0% (80 m9 in a 500 m + 51 volume Slope flask, pI 7.0 before sterilization) was inoculated and cultured with shaking (135 rpm) at 28° C. for 3 days to obtain a type 1 culture. This seed culture, each 3rl15I, was inoculated into the same medium as above and cultured with shaking under the same conditions for 3 days to obtain a second type culture. This seed culture at 2°C was inoculated into a 100°C culture tank containing medium 50J2 with the above composition that had been sterilized at 120°C for 15 minutes,
Culture was carried out at 28°C for 2 days with aeration and agitation at 20 rpm for 7 minutes at an aeration rate of 50. Cultures were obtained. 3 at 125℃ in advance
Glycerin 2.0% sterilized for 0 minutes, Nissanmeat 1
．． 5%, K2HPO40,0025%, KH2PO40
,1125%Cof:422 ・8820 0.00
05%, KM72 0.03%, Adekanol 0.05%, KM72 0.03%, Adekanol 0. O1
The third type culture 12JZ was inoculated into a 570°C culture tank containing 300J2 production medium consisting of 28°C for 7 days, with aeration rate of 150°C/min for the initial 24 hours of culture, and 3oox 7 minutes after 24 hours. Culture was carried out with aeration and stirring at 300 rpm. After culturing, diatomaceous earth is added as a filter aid and filtered.
Filtrate 25041 ('pH 6,0) was obtained.

The culture filtrate 25 obtained in Example 1 was adsorbed at 15°C on IP-20 (IIL as diamond ion), and water ion and 5
After washing with 0% methanol 4i, the active substance was eluted with 70% methanol 45 and then methanol 9i into the first fraction (531), second fraction (38J2) and third fraction (2
7Jlj) was obtained. The first fraction containing the active substance was concentrated under reduced pressure to 3° C. and the pH was adjusted to 3.5 using dilute hydrochloric acid, resulting in a red precipitate. This precipitate was collected by filtration and dried under reduced pressure to obtain 152 g of brown coarse powder containing mainly benanomycin A.

150 g of this crude powder was dissolved in 600 g of dimethylformamide and saturated with water vapor in a desiccator at room temperature for 3 days to form a crystalline precipitate. This precipitate was collected by filtration and dried under reduced pressure to obtain 29 g of benanomycin A dimethyl formaldehyde turbate. The second fraction was treated in the same manner as the first fraction to obtain 14 g of benanomycin A dimethylformamide turbate.

1 g of benanomycin A dimethylformamide turbate obtained from the first fraction was mixed with dimethyl sulfoxide (5 m
9. ) was added dropwise to 3 ounces of methanol with stirring, and the mixture was further stirred for 10 minutes to precipitate a reddish brown precipitate. This precipitate was collected by filtration and dried under reduced pressure to obtain 935 mg of pure benanomycin A reddish brown powder.

夾楌9113 The third fraction obtained in Example 2 was concentrated under reduced pressure.1.
When the pH was adjusted to 3.5 using dilute hydrochloric acid, a red precipitate was obtained. This precipitate was collected by filtration and dried under reduced pressure to obtain 99 g of brown coarse powder containing benanomycin B. 1 g of this crude powder was dissolved in 10 m9 of dimethylformamide under heating (40°C), applied to a column of LH-20 packed with dimethylformamide at 1°C, and developed with dimethylformamide.

Fraction No. 64-72 (1 fraction 6
ml>) was collected and concentrated to dryness under reduced pressure to obtain 657 mg of brown crude powder containing benanomycin B dimethylformamide turbate. 300 mg of coarse powder of
was dissolved in 100 ml of methanol, 1 ml of IN hydrochloric acid was added, and the mixture was concentrated to dryness under reduced pressure. The resulting brown coarse powder was dissolved in 3 liters of dimethyl sulfoxide and dropped into 200 liters of chloroform with stirring, and the mixture was further stirred for 20 minutes to precipitate a reddish brown precipitate. This precipitate was collected by filtration and dried under reduced pressure to obtain 258 mg of pure benanomycin B hydrochloride.

As explained in detail above, the actinomycete MH1 according to the present invention
Novel antibiotics benanomycin A and B were isolated from the culture solution of the 93-16F4 strain. The present antibiotic and its salts have antifungal activity that inhibits the growth of various molds.

[Brief explanation of the drawing]

Figure 1.2 Benanomycin A in methanol (20μ
The ultraviolet and visible absorption spectra at g/+++51) are shown. Figure 2: Shows the infrared absorption spectrum of benanomycin A in potassium bromide tablets. Figure 3: Shows a 400 MHz hydrogen nuclear magnetic resonance spectrum of benanomycin A in deuterated dimethyl sulfoxide solution. FIG. 4: Shows a 100 MHz carbon nuclear magnetic resonance spectrum of benanomycin A in deuterated dimethyl sulfoxide solution. Figure 5: Benanomycin B hydrochloride in methanol (2
The ultraviolet and visible absorption spectra at 0 μg/+nU) are shown. Figure 6: Infrared absorption spectrum of benanomycin B hydrochloride in potassium bromide tablets. FIG. 7: Shows a 400 MHz hydrogen nuclear magnetic resonance spectrum of benanomycin B hydrochloride in deuterated dimethyl sulfoxide solution. FIG. 8: Shows a 100 MHz carbon nuclear magnetic resonance spectrum of benanomycin B hydrochloride in deuterated dimethyl sulfoxide solution. ■ Dimensional Doto German σ7 0 Tsu's Fish Procedure? City official document (voluntary) December 10, 1986

Claims (1)

[Claims] 1. A reddish-brown powdery acidic substance having the following properties, with a molecular formula of C_3_9H_4_1NO_1.
_9, and the mass spectrum (FD-MS) is m/z8
27 (M^+), the melting point is higher than 220°C, the ultraviolet and visible absorption spectra (in methanol) are as shown in Figure 1 of the attached drawings, and the infrared absorption spectrum (
Potassium bromide tablets) are as shown in Figure 2 of the attached drawings, and the ^1H-NMR spectrum (400MHz, DMSO
-d_6, 40℃) is as shown in Figure 3 of the attached drawings, and has the characteristics of being extremely slightly soluble in methanol, chloroform, ethyl acetate, and acetone, soluble in dimethyl sulfoxide, dimethyl formamide, and alkaline water, and insoluble in water. The antibiotic benanomycin A, and its salts. 2. Benanomycin B hydrochloride has the following characteristics: it is a reddish-brown powdery amphoteric substance, the molecular formula is C_3_9H_4_1N_2O_1_8・HCl, and the mass spectrum (SI-MS) is m/z 827 (MH
＾+), the melting point is higher than 220℃, and the specific rotation is (
α) ^2^2_D=+360°(c0.05, H_2O
), and the ultraviolet and visible absorption spectra (in methanol) are as shown in Figure 5 of the attached drawings, and the infrared absorption spectra (potassium bromide tablets) are as shown in Figure 6 of the attached drawings. ,^1H-NMR spectrum (400
MHz, DMSO-d_6, 40°C) as shown in Figure 7 of the attached drawing.
As shown in the figure, the antibiotic benanomycin B and its salts have the property of being slightly soluble in chloroform, ethyl acetate, and acetone, and soluble in methanol, dimethyl sulfoxide, dimethyl formamide, and water. 3. Antibiotic benanomycin A, which is characterized by culturing antibiotic benanomycin A and B-producing bacteria belonging to Actinomycete, and collecting antibiotic benanomycin A and B, or either one thereof, from the culture. and/or a method for producing benanomycin B.