JPS6143036B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic AF-8 and a method for producing the same.

The antibiotic AF-8 substance of the present invention is a novel antibiotic having the following physical and chemical properties.

(1) Elemental analysis values: C58.52%, H8.64%, N9.04% (2) Melting point: 145-147℃ (3) Specific optical rotation: [α] ²⁵ _D = +12.0° (C = 1. Chloroform) (4) Ultraviolet absorption spectrum: Shown in Figure 1 (methanol solvent).

(5) Infrared absorption spectrum: As shown in Figure 2 (KBr method).

(6) Solubility in solvents: Soluble in benzene, ethyl acetate, chloroform, and butanol, poorly soluble in methanol, ethanol, and ether,
Insoluble in n-hexane, acetone, and water.

(7) Color reaction: Positive for Dragendorff and Lydon-Smith reactions, negative for ninhydrin and para-anisidine reactions.

(8) Distinction between acidic, neutral, and basic: Acidic (9) Color of substance: White (10) Proton nuclear magnetic resonance spectrum: As shown in Figure 3 (measured in deuterated chloroform).

(11) C-13 nuclear magnetic resonance spectrum: As shown in Figure 4 (measured in deuterated chloroform).

(12) Rf value: Silica gel thin layer chromatography (Merck Kieselgel G, thickness 300μ)
The Rf value when carried out in the usual manner is as follows.

Rf value (a) n-butanol: acetone: water (4:
5:1) 0.40 (b) Chloroform: methanol: acetic acid (15:1:1) 0.50 (c) n-butanol: acetic acid: water (3:1:
1) 0.72 (13) Acid hydrolysis: When hydrolyzed with 6N hydrochloric acid for 16 hours, it is a substance containing leucine, valine, glutamic acid, and aspartic acid in a molar ratio of 4:1:1:1.

The biological properties of the antibiotic AF-8 are as follows.

(1) Antibacterial properties Minimum inhibitory concentration (MIC) by agar dilution method
are shown in Table 1.

Table 1 Antibacterial spectrum of antibiotic AF-8 Test bacteria name Minimum inhibitory concentration (mcg/ml) Candida albicans ＞100 Satucharomyces cerevisi ＞100 Axanthomonas oryzae ＞100 Erwinia aroide ＞100 Glomerella sigrator ＞ 100 Altaneria mari ＞100 Fusarium oxysporum ＞100 Cladosporium vernice ＞100 Cochliobolus miyabaena ＞50 Spiricularia oryzae ＞50 Botrytis cinerea ＞50 Alterneria quictiana ＞100 Sclerotinia cinerea ＞50 Pericularia sasaki 50 supergillus・Niger 100 Trichophagiton inter 25 Digithalebacillus subtilis >100 PCI219 Staphylococcus ole >100 Us FDA 209P Mycobacterium smegma >100 Tess ATCC607 Escherichia coli >100 NIHJ AF-8 substance 1000mcg/ml As shown in Table 2, the antibacterial effect against various test bacteria when using the paper disk method with a solution of 100% is shown to have a remarkable effect on Pyricularia oryzae and Alterneria quictiana.

Table 2 Test Bacterium Inhibition Circle Diameter (m/m) Staphylocotcus aureus - FDA209P Bacillus subtilis PCI219 - Mycobacterium smegmates - Sartina lutea PCI1001 10.8 Escherichia coli NIHJ - Xanthomonas oryzae - Pseudomonas aeruginosa - P-3 Candida albicans - Satucharomyces cervicia - Pyricularia oryzae 33.5 Aspergillus niger - Alterneria quictiana 25.7 (2) Toxicity The acute toxicity of the AF-8 substance was investigated by intraperitoneal (ip) administration to mice. No abnormalities were observed after administration of 100mg/Kg.

As mentioned above, since this antibiotic shows antibacterial activity against fungi, this antibiotic AF-8
is a substance that can be used as an antifungal medicine, agricultural chemical, veterinary medicine, etc.

Based on the above physical and chemical properties, this antibiotic AF-8
is presumed to be a compound having a peptide skeleton. Tetrahedron A is a substance that has a peptide skeleton and is active against fungi.
29 , 3455-3459, 1973), mycosubtilin (Eur.
J.Biochem. 63 , 391-398, 1976), bachiromycin L (Proc.Soc.Exp.Bio.&Med. 63 , 539-541,
1948). However, when comparing the properties of this antibiotic with iturin A, mycosubtilin, and batiromycin L, it is found that this substance does not contain tyrosine in its amino acid composition;
Ityurin A, mycosubtilin, and batiromycin L are clearly different in that they contain tyrosine. Therefore, since no substance has been found in the physicochemical properties of this antibiotic that match those of known antibiotics, this antibiotic is determined to be a new antibiotic.

The antibiotic AF-8 substance of the present invention is antibiotic AF-8.
-8 is produced by culturing a microorganism belonging to the genus Bacillus in a medium, and collecting the antibiotic from the culture.

As an example of the microorganism belonging to the genus Bacillus that produces the antibiotic AF-8 substance used in the present invention, Bacillus subtilis AF, which was newly isolated by the present inventors from an airborne bacterium in Tottori Prefecture,
-8 (Bacillus subtilis AF-8) strain. The mycological properties of this strain are as follows.

A Morphology Shape and size: Bacillus 0.8 - 1.0 x 2.0 - 2.5μ Pleomorphism: Series or double Motility and hairs: Yes, Pericrichous hairs Presence or absence of spores: Yes Spore shape: Oval Spores Site of formation: Center Gram staining: Positive Acid-fastness: None B Growth condition: Cultured on broth agar plate. Good growth, amorphous or diffused. The surface becomes wrinkled with the passage of culture days, and the contents are butter-like, opaque, and milky white.

Juicy agar slant culture: Good growth, tree-like growth, wrinkled surface, buttery, opaque, milky white content.

Meat juice liquid culture Growth is good, a bacterial film grows on the surface, and as the number of days of culture passes, it becomes slightly cloudy and the bacterial cells take on a milky white color.

Cornish section: Good growth, diffused growth and formation of grayish-white colonies.

Litmus milk quickly converts to peptone and slightly decolorizes with litmus.

gravy gelatin puncture It grows on the surface and liquefies in layers.

C Physiological properties Nitrate reduction: Yes Denitrification reaction: No MR test: Negative VP test: Positive Indole formation: No Hydrogen sulfide formation: No Starch hydrolysis: Positive Citric acid usage: Yes Nitrate usage: Yes Pigments Production: None Urease: None Cytochrome oxidase: Yes Casein decomposition: Yes Gelatin decomposition: Yes Growth PH: PH5-9 Growth possible, optimum PH7 Growth temperature: 15-45℃, optimum temperature 27-37℃ Against oxygen Attitude: Aerobic, does not grow anaerobically in sweetened broth Sodium chloride tolerance: Does not grow at 10%.

O-F test: Acid is produced fermentatively from glucose.

Catalase: Positive Tyrosine degradation: None D Presence or absence of acid production from sugars (*) L-arabinose + D-xylose + D-glucose + D-mannose + D-fructose + D-galactose + maltose + sucrose + lactose - Trehalose + D-Sorbit ± D-Mannite + Inosyte + Glycerin + Starch - (*) Basal medium composition (NH ₄ ) ₂ HPO ₄ 1g, potassium chloride 0.2g, magnesium sulfate 0.2g, yeast extract 0.2g, various sugars 5g, Agar 15g, Bromcresol Purple 0.008g, Distilled Water 1000g
ml, the bacteriological properties of the AF-8 strain were compared to the description in Virgie's Manual of Determinative Bacteriology (8th edition), and the culture properties, physiological properties, and production of acid from sugars were found. Based on the characteristics, strain AF-8 is recognized as a strain belonging to the species Bacillus subtilis. This strain is Bacillus subtilis AF-8 (Bacillus subtilis AF-8).
-8) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the microorganism accession number 4850.

Bacteria used in the present invention include not only the above-mentioned Bacillus subtillus AF-8 and its mutant strains, but also those belonging to the genus Bacillus and the antibiotic AF-8.
Any bacteria that produces can be used.

The carbon source and nitrogen source used in the culture medium may be any type that can be used by the strain used. That is, as a carbon source, for example, glucose,
Various carbohydrates can be used, such as glycerol, fructose, maltose, galactose, lactose, starch or its hydrolyzate. Its concentration is preferably 0.1 to 5.0% relative to the normal medium. Also,
Various organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, and various amino acids such as glycine, glutamic acid, and alanine can also be used.

Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, urea, peptone, NZ amine, meat extract, dried yeast, corn steep liquor, casein hydrolyzate, fruit meal or Nitrogen-containing organic substances such as digested products and pupal hydrolysates, various amino acids such as glycine, glutamic acid, and alanine can be used.

As inorganic substances, various phosphates, magnesium sulfate, and other trace amounts of heavy metal salts are used, but their addition is not necessarily required in a medium containing natural substances.

Furthermore, when using a mutant strain that exhibits nutritional requirements, a substance that satisfies the nutritional requirements must be added to the medium.

Although the culture may be allowed to stand still, it is usually preferable to carry out the culture under aerobic conditions such as shaking or aerated agitation culture. When antifoaming is required during culture, use NITSUKOR BO-
Antifoaming agents such as 2 (manufactured by Nikko Chemicals Co., Ltd.), silicone resin, soybean oil, and Adekanol (manufactured by Asahi Denka Co., Ltd.) can be used, but among them, the antifoaming agent Adekanol LG-126 is the most effective. be. The pH of the medium is 5.0 to 9.0, and the culture temperature is 15 to 45.
℃, and the culture period is usually 2 to 7 days. The optimum culture conditions may be selected depending on the characteristics of the antibiotic AF-8 producing microorganism used.

The antibiotic AF-8 substance is mainly produced and accumulated in the culture solution. Collection and purification of the antibiotic AF-8 substance from the culture can be performed according to the methods used for collecting and purifying general antibiotics. For example, from a culture solution in which bacterial cells and other substances have been removed, the antibiotic is adsorbed on activated carbon, etc., eluted with aqueous acetone, etc., and the eluate is concentrated.
It can be extracted with n-butanol. Also,
The antibiotic can also be extracted from the culture solution using an organic solvent such as n-butanol. After concentrating the extract thus obtained, the antibiotic can be isolated using silica gel column chromatography, Sephadex LH20 column chromatography, or the like.

Next, a production example of the novel antibiotic AF-8 of the present invention will be shown, but this example is not intended to limit the present invention in any way.

Example 1 Soy flour 1.0%, monosodium glutamate 1.0%,
100ml medium containing 1.0% glucose (PH7.0)
Dispense into a 500 ml Sakaguchi flask and sterilize at 120°C for 20 minutes. This includes Bacillus subtilis AF
-8 (Feikoken Bacteria No. 4850) was inoculated and heated to 48°C at 27°C.
Culture with reciprocating shaking at 120 revolutions per minute. This culture was grown for 30 min with a medium of the same composition as above.
Transplanted at a rate of 1% into a container fermenter at a temperature of 27°C and a stirring rate of 250 rpm at 10 min.
sterile air was supplied and aeration stirring culture was carried out for 44 hours to obtain a culture containing the antibiotic AF-8 substance.

The bacterial cells and medium residue were removed from this culture by centrifugation to obtain a culture solution 15. 220 g of activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd.) was added to this culture solution, and the mixture was stirred at room temperature for about 30 minutes. The activated carbon was recovered from the culture medium by centrifugation, and the active substance was eluted twice from the activated carbon with 70% aqueous acetone 2. This eluate was concentrated under reduced pressure to a concentration of about 1,000 ml, and the active substance was extracted twice with 500 ml of n-butanol from this aqueous solution. Add n-butanol extract to 500ml of 0.1N.
After sequentially washing with hydrochloric acid and 500 ml of water, the n-butanol layer was concentrated to dryness under reduced pressure to obtain 22 g of yellowish brown powder. This was dissolved in chloroform, and 800 g of silica gel (Kieselgel 60, manufactured by Merck & Co., Ltd.) was placed on a column previously filled with chloroform, and then eluted with a mixed solvent of chloroform:methanol (7:1). The antibacterial activity of the eluted fractions (approximately 15 ml each) was determined by measuring the circle of inhibition by the paper disc method using agar plates containing Alternaria quictiana. The active fractions (from the 200th fraction to the 450th fraction) were combined (approximately 3700 ml) and concentrated to dryness under reduced pressure to obtain approximately 4 g of yellow powder. Transfer 1 g of this yellow powder to a Cephadex LH-20 column (bed capacity approx. 600).
80 mg of pure AF-8 material was obtained as a white powder giving a single spot on thin layer chromatography.

Example 2 Glucose 0.5%, L-glutamic acid 1.0%, citric acid 0.1%, primary potassium phosphate 0.05%, dibasic potassium phosphate 0.05%, magnesium sulfate heptahydrate 0.02
%, manganese sulfate tetrahydrate 0.001%, ferrous sulfate 7
500ml of 100ml of medium containing 0.001% water salt (PH6.8)
Dispense into ml Sakaguchi flasks and sterilize at 120°C for 20 minutes. In addition, Bacillus subtilis AF-8
(Feikoken Bacteria No. 4850) and cultured with reciprocating shaking at 120 revolutions per minute at 27°C for 48 hours. This culture was transferred at a rate of 1% to a 30-volume jar fermenter containing 20 medium of the same composition as above, and the temperature
At 27°C and with a stirring rate of 250 rpm, sterile air was supplied at a rate of 10 per minute for 72 hours with aeration and agitation.
A culture containing -8 was obtained.

Bacterial cells were removed from this culture by centrifugation to obtain culture solution 15. From this culture, the active substance was extracted twice with 3 portions of n-butanol.
The n-butanol extract was concentrated under reduced pressure to obtain about 20 g of a yellow syrupy substance. This yellow syrupy substance was subjected to silica gel column chromatography and Sephadex LH-20 column chromatography in the same manner as described in Example 1, and 100 mg of the white powder of the antibiotic AF-8 substance was added. Obtained.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the antibiotic AF-8 in methanol, and Figure 2 shows the same substance's KBr absorption spectrum.
Fig. 3 shows the proton nuclear magnetic resonance spectrum of the same substance in deuterated chloroform, and Fig. 4 shows the C-13 nuclear magnetic resonance spectrum of the same substance in deuterated chloroform.

Claims (1)

[Claims] 1. A novel antibiotic having the following physicochemical properties:
AF-8. (1) Elemental analysis: C58.52%, H8.64%, N9.04% (2) Melting point: 145-147℃ (3) Specific rotation: [α] ²⁵ _D = +12.0° (C = 1 , chloroform) (4) Ultraviolet absorption spectrum: Shown in Figure 1 (methanol solvent). (5) Infrared absorption spectrum: shown in Figure 2 (KBr
law). (6) Solubility in solvents: Soluble in benzene, ethyl acetate, chloroform, and butanol, poorly soluble in methanol, ethanol, and ether,
Insoluble in n-hexane, acetone, and water. (7) Color reaction: Positive for Dragendorff and Lydon-Smith reactions, negative for ninhydrin and para-anisidine reactions. (8) Distinction between acidic, neutral, and basic: Acidic (9) Color of substance: White (10) Proton nuclear magnetic resonance spectrum: As shown in Figure 3 (measured in deuterium chloroform). (11) C-13 nuclear magnetic resonance spectrum: As shown in Figure 4 (measured in deuterated chloroform). (12) Rf value: Silica gel thin layer chromatography (Merck Kieselgel G, thickness 300μ)
The Rf value when carried out in the usual manner is as follows. Rf value (a) n-butanol: acetone: water (4:
5:1) 0.40 (b) Chloroform: methanol: acetic acid (15:1:1) 0.50 (c) n-butanol: acetic acid: water (3:1:
1) 0.72 (13) Acid hydrolysis: When hydrolyzed with 6N hydrochloric acid for 16 hours, it is a substance containing leucine, valine, glutamic acid, and aspartic acid in a molar ratio of 4:1:1:1. 2 A microorganism belonging to the genus Bacillus and having the ability to produce the new antibiotic AF-8 is aerobically cultured in a medium to produce and accumulate the new antibiotic AF-8,
A new antibiotic characterized by collecting this
Manufacturing method of AF-8. 3. The method for producing the novel antibiotic AF-8 according to claim 2, wherein the microorganism belonging to the genus Bacillus is Bacillus subtillus AF-8.