JPH021837B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic AM-5344-A ₁ and a method for producing the same. In the process of searching for antibiotics produced by actinomycetes, the present inventors discovered that an actinomycete AM-5344-
By discovering that the M81 strain produces an antibiotic, investigating the mycological properties of this strain, and after isolating this antibiotic, we were able to investigate its physicochemical and biological properties. The invention has been completed. Antibiotic M-5344 obtained by the present invention
A ₁ has the following physical and chemical properties. (1) Elemental analysis value {Contains carbon, hydrogen, nitrogen, and oxygen, and does not contain sulfur or halogen. The elemental analysis values of this substance are shown below. C64.51%, H4.19%, N2.46% (2) Molecular weight: In mass spectrum (EI mass spectrum and FD mass spectrum) analysis,
A large peak was observed at mass number 529. (3) Melting point: No clear melting point is shown. It turns brown at around 240℃, and turns blackish brown at 275℃. (4) Specific rotation: [α] ²³ _D = -92° (C0.05, chloroform) (5) Ultraviolet absorption spectrum: The maximum absorption in chloroform (E1% 1cm) is
303nm (516), 376nm (219), and 385nm (214) are shown. As shown in Figure 1. (6) Infrared absorption spectrum: KBr method, ν ^KBr _nax cm ^-1 3550, 3370, 2980, 1660, 1639, 1618,
1558, 1500, 1460, 1445, 1425 As shown in Figure 2. (7) Solubility in solvents: Soluble in chloroform, sparingly soluble in organic solvents such as acetone, ethyl acetate, benzene, and lower alcohols, and soluble in n-hexane, ethyl ether,
Insoluble in water. (8) Color reaction: Positive for ferric chloride reaction, negative for Dragendorff reaction and ninhydrin reaction. (9) Classification of basic, neutral, and acidic: It is a neutral substance. (10) Color of substance: Yellow (11) R _f value: R _f when silica gel thin layer chromatography (Merck TLC aluminum sheet, silica gel 60F ₂₅₄ , thickness 0.2 mm) is performed in the usual manner. The values are: R _f value (a) Chloroform: methanol (40:1) 0.39 (b) Benzene: acetone (1:1) 0.69 (c) Benzene: methanol (4:1) 0.63 (d) Ethyl acetate 0.27 (e) n- Butanol:acetic acid:water (4:1:1)
0.67 The biological properties of the antibiotic AM-5344-A ₁ are as follows. (1) Antibacterial properties The minimum inhibitory concentration (MIC) determined by the agar dilution method is
As shown in Table 1. Table 1 Test Bacterial MIC (mcg/ml) Staphylococcus aureus TPR23
(Staphylococcus aureus) 0.20 Streptococcus fuecalis ATCC8043
(Streptococcus faecalis) 0.78 Microlococcus flavus IFO3242
(Micrococcus flavus) 0.39 Sarcina lutea ATCC9341 (Sarcina lutea)
25 Bacillus subtilis ATCC6633
subtilis) 0.20 Bacillus cereus IF03466 (Bacillus cereus)
＞100 Esierisia coli NIHJ Jc-2
(Escherichia coli) >100 Salmonella typhimurium (Salmonella
typhimurium) ＞100 Klebsiella pneumoniae IFO3512
(Klebsiella pneumoniae) >100 Enterobacter aerogenes IFO5467
(Enterobacter aerognes) >100 Proteus vulgaris A33 (Proteus
vulgaris) ＞100 Pseudomonas energinosa IAM1054
(Pseudomonas aeruginosa) >100 Clostridium perfringens
ATCC1312 (Clostridium perfringens) 0.012 Eubacterium lentum ATCC25559
(Eubacterium lentum) 0.012 Bifidobacterium bifidium
ATCC11146 (Bifidobacterium bifidum) 0.098 Peptococcus pleboci ATCC9321
(Peptococcus prevotii) 0.098 Lactobacillus acidophilus IFO3205
(Lactobacillus acidophilus) 0.049 Bacteroides fragilis ATCC23745
(Bacteroides fragilis) 0.049 (2) Anti-trichomoniasis activity Minimum inhibitory concentration (MIC) by liquid medium dilution method
is as shown below. Note that Trichosel broth (manufactured by BBL) was used as the liquid medium. MIC (mcg/ml) Trichomonas
0.4 As mentioned above, this antibiotic exhibits antibacterial and antiprotozoal activity against Gram-positive bacteria, anaerobic bacteria, and trichomonads _.
is considered to be used as an antibacterial agent, and appears to be particularly effective in the prevention and treatment of anaerobic bacterial infections. Furthermore, this substance is highly expected to be used as a veterinary drug due to its antibacterial spectrum. As a result of comparing the above physicochemical and biological properties with those of known antibiotics, antibiotic AM-
5344-A ₁ was found to be a new substance. In other words, it is neutral, fat-soluble, and has a UV absorption spectrum.
It has absorption maximum near 303nm, 376nm, and 385nm,
Similar antibiotics effective against Gram-positive bacteria include, for example, Albofungin, Antibiotiki 4 , 10-13 (1959);
Chloralbofungin
Dokl.Akad, Nauk, SSSR 207 , 1347―1350
(1972)]. However, the nitrogen content (5.3%) and specific optical rotation (-670°) of albohuangin are higher than those of the antibiotic AM-5344-A ₁ based on elemental analysis values.
It is clearly different from the nitrogen content (2.46%) from the elemental analysis value. Chloralbophaazine contains chlorine based on elemental analysis.
Antibiotic M-5344-A ₁ is clearly different as it does not contain chlorine. As mentioned above, antibiotic AM
-5344-A ₁ is novel because it is clearly distinguishable from known antibiotics. A practical example of the microorganism used to produce the antibiotic AM-5344- _A1 of the present invention is Streptomyces spp. SP AM
5344-M81 is mentioned. (1) Morphological properties Vegetative hyphae develop well in both natural and synthetic media and usually do not have septa. Aerial mycelium is
It adheres moderately on glycerose/asparagine agar, tyrosine agar, etc., but it adheres poorly or does not adhere on other media. Aerial mycelium is yellow to gray in color and powdery or velvety. When observed under a microscope, the sporophyte was linear or loop-shaped, and no spores were observed on glycerol/asparagine agar or tyrosine agar, and chains of 10 to 50 spores were observed on starch inorganic salt agar. Ru. The surface of the spore is smooth.
No sclerotia, sporangia and zoospores are found. (2) Properties on various media E.B. Shearing et al. (lnt.J.Syst.
Bacteriol, Vol. 16, p. 313, 1966), in conjunction with known media and experimental methods. The results are shown in Table 2. The color tones were determined as standard colors using the Color Harmony Manual, 4th edition (Container Corporation of America, Chicago, 1958), and the code is written in parentheses along with the color chart name. The following are observations of each culture medium at 27°C for 2 weeks unless otherwise specified.

【table】

【table】

[Table] (3) Physiological properties 1 Formation of melanin pigment (a) Tyrosine agar negative (b) Peptone/yeast iron agar negative (c) Glucose/peptone/gelatin medium puncture (21-23℃) Negative (d) Tryptone yeast solution Negative 2 Tyrosinase reaction Negative 3 Hydrogen sulfide production Negative 4 Nitrate reduction Negative 5 Liquefaction of gelatin (21-23°C) (glucose pebtone gelatin medium) Negative 6 Hydrolysis of starch Positive 7 Coagulation of skim milk (36℃) Negative 8 Peptonization of skim milk (36℃) Positive 9 Decomposition of cellulose Negative 10 Growth temperature range 6-36℃ 11 Usability of carbon sources Frequently used: - glucose, D-mannitol, D-fructose, LL-arabinose, i-inositol, rhamnose, raffinose, maltose Slightly used: D-xylose Not used: sucrose (4) Cell wall composition Diaminopimelic acid is LL-type, and arabinose and galactose are not observed. The mycological properties of this bacterium can be summarized as follows. The cell wall composition has LL-diaminopimelic acid. In addition, the spores form a linear or loop-shaped sporophore, and the surface of the spores is smooth. In terms of culture properties, vegetative mycelium exhibits a yellow to dark brown color that does not change with changes in pH;
Aerial mycelia are yellow to gray in color. Produces a yellow soluble pigment in sucrose/nitrate agar, glucose/asparagine agar, etc. Does not produce melanin pigment. These results indicate that this bacterial strain belongs to the genus Streptomyces, and is also classified according to Primodham and Strener's classification (Burgess Manual of Determinative Bacteriology No. 8).
It is considered to be a bacterial species belonging to the Yellow or Gray series according to the Japanese edition (Ed., 1974, pp. 748-829). This strain is Streptomyces sp.
AM―5344―M81 (Streptomyces sp.AM―5344
-M81) has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology. (Feikokenboku No. 6006). Streptomyces sp. strain M-5344-M81 is a specific example of the bacteria used in the present invention.
Mutant strains obtained through artificial mutation using radiation, chemical agents, etc. are, of course, microorganisms belonging to the Streptomyces genus, and are used in antibiotic AM-
Any product capable of producing 5344-A ₁ can be used in the present invention. In the present invention, as a medium for culturing the microorganism that produces the antibiotic AM-5344-A ₁ , a wide variety of media containing a carbon source, a nitrogen source, inorganic substances, etc. that are commonly used for culturing microorganisms can be used. The carbon source for the medium may be any assimilable carbon compound, such as glucose, maltose, lactose, sucrose, starch, dextrin, glycerin, and molasses. In addition, as a nitrogen source for the culture medium,
Any available nitrogen compound may be used, such as soybean flour, corn steep liquor, cottonseed flour, peptone, meat extract, yeast extract, dried yeast, casein hydrolyzate, ammonium salts, nitrates, and the like. In addition, salts such as phosphate, magnesium, potassium, calcium, sodium, iron, and manganese are used as necessary in preparing the medium. Cultivation is usually carried out aerobically, and aerated agitation culture is preferred. The culture temperature can be changed as appropriate within a range that allows the microorganisms to grow and produce the antibiotic AM-5344-A ₁ , but is particularly preferably 25 to 30°C. PH is preferably 6-7. The culture time varies depending on various conditions, but is usually about 50 to 100 hours, and the culture is terminated at an appropriate time, taking into consideration the time when the antibiotic AM-5344-A ₁ reaches its maximum titer. The antibiotic AM-5344-A ₁ belonging to Streptomyces thus obtained is collected from the culture of the microorganism producing the antibiotic AM-5344-A ₁ . These methods can be selected and combined as appropriate.
An example of a means for separating and purifying the antibiotic AM-5344-A ₁ from a culture is as follows. That is, the culture is separated into bacterial cells and liquid, and the bacterial cells are extracted with acetone, ethyl acetate, etc., or the bacterial cells are treated with methanol and then extracted with an organic solvent such as ethyl acetate, chloroform, etc. The solution is separated from water such as ethyl acetate and benzene, extracted with an organic solvent that dissolves the antibiotic AM-5344-A ₁ , and then extracted using a known method commonly used in the purification of fat-soluble substances. 5344
-Collect A ₁ . For example, the extract (ethyl acetate layer) is concentrated under reduced pressure, the resulting precipitate is separated,
After washing this with n-hexane, the antibiotic AM-5344-A ₁ is isolated by silica gel column chromatography using a mixed solvent system of chloroform and methanol as an elution solvent. The produced antibiotic AM-5344-A ₁ was detected and quantified using silica gel thin layer chromatography (Silica gel 60F ₂₅₄ manufactured by Merck, thickness 0.2
mm, developing solvent; chloroform: methanol = 40:
1. Antibiotic AM-5344-A ₁ R _f value 0.39) and Acholeplasma leideruii (Acholeplasma leideruii)
A biological assay using PG-8 was used. Next, an example of the antibiotic AM-5344-A ₁ of the present invention will be shown, but this example is merely an example and does not limit the present invention. Example 1 Streptomyces sp. AM-5344-M81
One platinum loop from a slant culture of Streptomyces sp. The seed culture was inoculated at a ratio of 1% into a jar fermentor, and cultured with aeration and stirring at 27°C for 89 hours. The medium is glucose for seed culture.
1.0%, starch 2.0%, yeast extract 0.5%, peptone 0.5%, calcium carbonate 0.4%.For main culture, glycerin 2.0%, cinchona flour 2.0%, salt 0.3%.
After adjusting the pH to 7.0, heat at 121℃.
The one used was sterilized for 15 minutes. After adding 10 parts of ethyl acetate to 30 parts of the culture containing bacterial cells and stirring well, the ethyl acetate layer was separated by centrifugation. This ethyl acetate extract was heated to 500 ml under reduced pressure.
The mixture was concentrated to 1 ml, and 300 ml of n-hexane was added to remove oily substances to obtain 3.0 g of brown powder. Add this to a silica gel column (Merck Kieselgel 60, 120g)
After placing it on the plate, it was developed with chloroform 1.5, and then a mixed solvent of chloroform:methanol (50:1) was added.
Active fraction obtained by elution with 5.0 (fraction number 130
550, 12 ml/tube) was concentrated to dryness under reduced pressure to obtain 500 mg of brown powder. This brown powder was subjected to preparative thin-layer chromatography (R _f value) using a mixed solvent of chlororum:methanol (40:1).
0.39) to obtain 25 mg of orange powder of antibiotic AM-5344-A ₁ . The properties of this product were consistent with the physicochemical and biological properties described above. Example 2 Streptomyces sp. AM-5344-
One platinum loop from a slant culture of M81 (Streptomyces sp.
The seed culture solution was inoculated at a rate of 5% into a 100-volume jar fermenter containing the main culture medium, and culture was normally performed with stirring at 28°C for 65 hours. For seed culture, the medium is
Glucose 1.0%, Starch 2.0%, Yeast Extract 0.5%,
Peptone 0.5%, CaCO ₃ 0.4%, for main culture, glycerin 2.0%, cinchona flour 2.0%, NaCl 0.3%
After adjusting the pH to 7.0, heat at 121℃.
The one used was sterilized for 30 minutes. To the bacterial cells obtained by filtering the culture solution 54 through a drum filter, 30 methanol was added and stirred thoroughly, and then filtered again through a large Buchner funnel. Chloroform 22 was added to the obtained bacterial cells and extracted with stirring.
The chloroform layer obtained was separated and concentrated under reduced pressure to a candy-like consistency, 1 portion of n-hexane was added thereto, and the resulting precipitate was separated and dried to obtain 23 g of crude material. A silica gel column (Wako Gel C manufactured by Wako Pure Chemical Industries, Ltd.) filled with 10 g of this crude substance was packed with chloroform.
200, 400g), developed with chloroform 2, and then developed and eluted with chloroform:methanol (50:1) mixed solvent 3. Active fraction No.
22-36, 100ml/fractiontube) to 250ml under reduced pressure.
Add 20ml of acetonitrile and cool.
The obtained precipitate was separated and dried to obtain 2.5 g of an orange-yellow powder of antibiotic AM-5344-A ₁ . The properties of this product were consistent with the physicochemical and biological properties described above.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum (measured in chloroform) of the antibiotic AM-5344-A ₁ , and Figure 2 shows the infrared absorption spectrum (KBr method).

Claims (1)

[Claims] 1. Antibiotic AM having the following physical and chemical properties:
5344—A ₁ . Elemental analysis: C64.51%, H4.19%, N2.46% Molecular weight: M ⁺ , m/z529 (EI mass spectrum) Specific optical rotation: [α] ²³ _D = -92° (C0.05, chloroform) Ultraviolet absorption spectrum: Maximum absorption in chloroform (E1% 1cm) 303nm (516), 376nm (219), 385nm (214) Infrared absorption spectrum: KBr method, υ ^KBr _nax cm ^-1 3550, 3370, 2980, 1660, 1639, 1618,
1558, 1500, 1460, 1445, 1425 Solubility in solvents: Soluble in chloroform, sparingly soluble in organic solvents such as acetone, ethyl acetate, benzene, and lower alcohols, soluble in n-hexane, ethyl ether,
Insoluble in water. Color reaction: Positive for ferric chloride reaction, negative for Dragendorff reaction and ninhydrin reaction. Classification of basic, neutral, and acidic: Neutral Substance color: Yellow Rf value: Silica gel thin layer chromatography (Merck TLC aluminum sheet, silica gel 60F ₂₅₄ , thickness 0.2 mm) was performed in the usual manner. The Rf value at the time of Rf value (a) Chloroform: methanol (40:1) 0.39 (b) Benzene: acetone (1:1) 0.69 (c) Benzene: methanol (4:1) 0.63 (d) Ethyl acetate 0.27 (v) n-butanol : Acetic acid: Water (4:1:1)
0.67 2 Belongs to the genus Streptomyces and is an antibiotic AM-
A bacterial strain capable of producing 5344-A ₁ is cultured aerobically in a medium, and the antibiotic AM-5344-A ₁ having the following physicochemical properties is accumulated in the culture,
Antibiotic AM characterized by collecting this
5344-A ₁ manufacturing method. Elemental analysis: C64.51%, H4.19%, N2.46% Molecular weight: M ⁺ , m/z529 (EI mass spectrum) Specific optical rotation: [α] ²³ _D = -92° (C0.05, chloroform) Ultraviolet absorption spectrum: Maximum absorption in chloroform (E1% 1cm) 303nm (516), 376nm (219), 385nm (214) Infrared absorption spectrum: KBr method, ν ^KBr _nax cm ^-1 3550, 3370, 2980, 1660, 1639, 1618,
1558, 1500, 1460, 1445, 1425 Solubility in solvents: Soluble in chloroform, sparingly soluble in organic solvents such as acetone, ethyl acetate, benzene, and lower alcohols, soluble in n-hexane, ethyl ether,
Insoluble in water. Color reaction: Positive for ferric chloride reaction, negative for Dragendorff reaction and ninhydrin reaction. Classification of basic, neutral, and acidic: Neutral Substance color: Yellow Rf value: Silica gel thin layer chromatography (Merck TLC aluminum sheet, silica gel 60F ₂₅₄ , thickness 0.2 mm) was performed in the usual manner. The Rf value at the time of Rf value (a) Chloroform:methanol (40:1)
0.39 (b) Benzene: Acetone (1:1) 0.69 (c) Benzene: methanol (4:1) 0.63 (d) Ethyl acetate 0.27 (e) n-butanol: acetic acid: water (4:1:
1) 0.67 3 The microorganism is Streptomyces sp. AM
-5344-M81 The manufacturing method according to claim 2.