JPH0799982A - New antibiotic mj885-mf8 and its production - Google Patents

Abstract

PURPOSE:To obtain the subject antibiotic useful as an antimicrobial agent for fishery, having antimicrobial activity against pathogenic bacteria of fish microb ism, having low toxicity to fish, by culturing a specific bacterium belonging to the genus Streptomyces. CONSTITUTION:A bacterium [e.g. Streptomyces sp.MJ885-mF8 strain (FERM P-13,863)]belonging to the genus Streptomyces, capable of producing antibiotic MJ885-mF8 is cultured in a nutrient medium and an antibiotic is collected from the culture mixture to give new antibiotic MJ885-mF8 which comprises a colorless, transparent, oily neutral substance, has a specific rotatory power [alpha]<23>D of-6 deg. (c 0.5, methanol), molecular formula of C18H22O6 and Rf value (TLC: silica gel) of 0.47 (chloroformm-methanol, 10:1) and 0.66 (toluene-acetone 1:1), is negative in ninhydrin reaction, Rydon-Smith reaction and ferric chloride reagent and positive in phosphomolybdic acid-sulfuric acid reaction and has excellent antimicrobial activity against pathogenic bacteria of fish microbism.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel antibiotic produced by a microorganism belonging to the genus Streptomyces and having an antibacterial activity, and in particular, Pastera which is a bacterium that causes nodular disease, which is a kind of bacterial infection of fish.・ Paste
urella piscicida), a novel antibiotic MJ885-mF8 showing a strong antibacterial activity, and a method for producing the same. The antibiotic MJ885-mF8 of the present invention is particularly useful as an antibacterial agent for fisheries.

[0002]

2. Description of the Related Art When an aquarium is transferred from a natural environment to an artificial environment and reared, various disorders (fish diseases) that are not normally found in the natural hydrosphere occur, and sometimes cause great damage on an industrial scale. In particular, cultured nodule diseases such as yellowtail are caused by infection with the bacterium Pasteurella piscicida, resulting in nodule-like small white spots on the kidney, spleen, and gills of the yellowtail.
Cause great damage. Amoxicillin, ampicillin, etc. are used as effective antibiotics for the fish disease, and aquaphene, thianphenicol, oxophosphoric acid, sulfisozole, etc. are used as synthetic drugs.

[0003]

[Problems to be Solved by the Invention] As the antibiotics and synthetic pharmaceuticals showing antibacterial activity against pathogenic bacteria such as nodule disease, which is a fish disease, the above-mentioned ones are widely used at present, but they are more powerful. Compounds having antibacterial activity are expected. In addition, the emergence of resistant bacteria is unavoidable, and thus new antibiotics are required. To date, no antibiotics effective for the treatment of nodular disease produced by microorganisms have been reported, and it is awaited to provide new antibiotics that are effective and have low toxicity.

[0004]

[Means for Solving the Problems] The present inventors have promoted research on the development and practical use of useful antibiotics, and the present invention also forms part of such research. The present inventors have found that the actinomycetes isolated from the soil by the present inventors include MJ885.
-Finding that the microorganism with the strain number of mF8 produces and accumulates an antibiotic having antibacterial activity in the culture,
We have successfully isolated this antibiotic. When the physicochemical properties of this antibiotic were examined, it was identified as a novel substance and was designated as antibiotic MJ885-mF8. Further, as described above, the novel antibiotic MJ885-m which has not been published in the conventional literature is
The present invention has been completed by finding that F8 has an antibacterial activity against Gram-positive bacteria, and particularly strongly inhibits the growth of the fish disease bacterium Pasteurella piscicida.

Therefore, according to the first aspect of the present invention, a novel antibiotic MJ885-mF8 is provided.

The novel antibiotic MJ8 according to the first invention
85-mF8 is a substance characterized by the following physicochemical and biological properties.

(1) Physicochemical properties of the antibiotic MJ885-mF8 A) Properties: neutral colorless oily substance B) Optical rotation: [α] _D ²³ -6 ° (c 0.5, MeOH) C) Molecular formula: C ₁₈ H ₂₂ O ₆ High resolution FAB mass spectrum 335.1477 (M + H) ⁺
positive D) FAB mass spectrum (m / z): 335 (M +
^{H) + positive 333 (M-} H) - negative E) UV absorption spectrum: λmax, nm (ε) 271 (12500) ( in methanol), .lambda.max shown in Figure 1 of the accompanying drawings, nm (ε) 270 (12600 ) (0.01N-HCl
-90% in methanol), λmax, nm (ε) 252 (8230) (0.01N-NaOH) shown in Figure 2 of the attached drawings.
-90% in methanol), as shown in Figure 3 of the accompanying drawings.

F) Infrared absorption spectrum (KBr tablet method): Shown in FIG. 4 of the accompanying drawings. The main absorption peaks are shown below: vmax: 2930, 1780, 1670, 1590, 1
440,1280 cm ⁻¹ G) ¹ H-NMR spectrum (in deuterated chloroform): shown in FIG. 5 of the attached drawing H) ¹³ C-NMR spectrum (in deuterated chloroform): I) Rf value (shown in FIG. 6 of the attached drawing) TLC; Merck silica gel Art. 57
15): 0.47 (chloroform-methanol, 10: 1) 0.66 (toluene-acetone, 1: 1) J) Color reaction: negative for ninhydrin, rydonsmith and ferric chloride reagents, and phosphomolybdic acid-sulfuric acid. Is positive.

(2) Biological properties of antibiotic MJ885-mF8 A) Antibacterial spectrum The antibiotic MJ885-mF8 according to the first aspect of the present invention has antibacterial activity against various bacteria. The minimum inhibitory concentrations for various bacteria and fungi on agar plates were determined by standard multiple dilution method. The results are shown in Table 1 below.

[0010]

[Table 1]

B) Acute toxicity As a result of testing the acute toxicity of MJ885-mF8, a novel antibiotic, to mice, the dose was 100 in the abdominal cavity of mice.
No abnormality was found in the mice even at mg / kg.

C) Fish Poison In a fish poison test using Himedaka, no toxicity to medaka was observed in water containing the antibiotic MJ885-mF8 at a concentration of 10 ppm, and it is recognized that the antibiotic is a safe drug.

The novel antibiotic MJ885-mF8 of the present invention
Can advantageously be produced according to the second inventive method.

That is, according to the second invention, the antibiotic MJ885-mF8-producing bacteria belonging to the genus Streptomyces is cultured in a nutrient medium, and the antibiotic MJ885 is collected from the culture. -A method of making mF8 is provided.

Antibiotic M which can be used in the method of the second invention.
An example of a J885-mF8 producing bacterium is October 1992,
At the Institute for Microbial Chemistry, there is an actinomycete isolated from soil in Suginami-ku, Tokyo, which has a strain number of MJ885-mF8.

The mycological properties of the MJ885-mF8 strain are described below. 1. Form MJ885-mF8 strain extends straight aerial hyphae from branched basal hyphae. No helix formation, limbal branch or sporangium is observed. The mature spore chain has a chain of more than 30 cylindrical spores, and the spore size is about 0.5 to 0.6 × 0.
It was 9 x 1.2 microns. The surface of the spores is wrinkled.

2. Regarding the description of colors in various media, the standard shown in [] is the Color Harmony Manual (Container Corporation of America) of the Continer Corporation of America.
Color harmony manual).

(1) Sucrose / nitrate agar medium (2
(7 ° C culture) On the colorless growth, aerial hyphae of gray [3dc, Natural] are slightly attached and no soluble pigment is observed. (2) Glucose-asparagine agar medium (27 ° C culture) For the development of light yellow [2ca, Lt Ivory], white to gray white [2d
c, Natural] aerial hyphae are slightly attached and no soluble pigment is observed. (3) Glycerin / asparagine agar medium (ISP-medium 5, 27 ° C. culture) light yellow [2ec, Biscuit] to gray yellow tea [2ie, Lt Musta
rd Tan] grows, and aerial hyphae of gray [3dc, Natural] to bright ash [3fe, Silver Gray] are settled. No soluble dye is found. (4) Starch / inorganic salt agar medium (ISP-medium 4, 2
Cultivation at 7 ℃) Light yellow [2ec, Biscuit] ~ Gray yellow tea [2ie, Lt Musta
[3fe, Silver Gray ~]
5fe, Ashes], and no soluble pigment is observed.

(5) Tyrosine agar medium (ISP-medium 7, 27 ° C. culture) light yellow [2ec, Biscuit] to light yellow tea [2lg, Mustard
On the growth of Tan], aerial hyphae of gray [3dc, Natural] to bright ash [3fe, Silver Gray] are settled. No soluble dye is found. (6) Nutrient agar medium (27 ° C. culture) White aerial mycelium slightly grows on the development of light yellow [3ec, Bisque]. Soluble dyes have a brownish tinge. (7) Yeast-malt agar medium (ISP-medium 2, 27
Cultivation) Light yellow [2ec, Biscuit] ~ Gray yellow tea [2ie, Lt Musta
[3fe, Silver Gray ~]
5fe, Ashes], and no soluble pigment is observed. (8) Oatmeal agar medium (ISP-medium 3, 27 ° C)
(Culture) On the growth of light yellow [2ec, Biscuit], bright ash [2f
e, Covert Gray to 3fe, Silver Gray] aerial mycelia are settled, and no soluble pigment is observed. (9) Glycerin / nitrate agar medium (27 ° C culture) On the development of light yellow [2cd, Ivory Tint], white aerial hyphae grew slightly and no soluble pigment was observed.

(10) Starch agar medium (27 ° C culture) On the development of light yellow [2ec, Biscuit], white aerial mycelia grow slightly, but a partially bright ash [2fe, Cover
t Gray]. No soluble dye is found. (11) Lime malate agar medium (27 ° C culture) Growth is colorless, no aerial hyphae do not grow, and no soluble pigment is observed. (12) Cellulose (synthetic solution with filter paper pieces added, cultured at 27 ° C.) It did not grow in 21 days of culture.

3. Physiological properties (1) Growth temperature range Using yeast / starch agar medium (soluble starch 1.0%, yeast extract 0.2%, string agar 3.0%, pH 7.0), 10 ℃, 20 ℃, 24 ℃, 27 ℃, 30 ° C, 37
As a result of testing at each temperature of 50 ° C and 50 ° C, it grows at any temperature except 50 ° C. Optimal growth temperature is 27 ℃
It seems to be around 30 ° C.

(2) Starch hydrolysis (starch / inorganic salt agar medium, ISP-medium 4 and starch agar medium,
Both cultures are 27 ° C.) In any medium, hydrolyzability was observed around the 3rd day after the culture, and the action was strong. (3) Formation of melanin-like pigment (tryptone, yeast,
Broth, ISP-medium 1; peptone yeast iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; 27 ° C culture) Positive in tryptone yeast broth and peptone yeast iron agar medium , The production of melanin on tyrosine agar is questionable.

(4) Utilization of carbon source (Pridham-Godlieve agar medium, ISP-medium 9, 27 ° C. culture) Grow with D-glucose, D-xylose, rhamnose, and lactose to grow L-arabinose, D -Fructose, sucrose, inositol, raffinose and D-mannitol are not used.

(5) Dissolution of lime malate (lime malate agar medium, 27 ° C. culture) Negative. (6) Decomposition of cellulose (synthesis solution with filter paper pieces added, culture at 27 ° C.) No decomposition is observed after 20 days of observation.

A summary of the above properties is MJ885-m.
Due to its morphology, the F8 strain extends straight aerial hyphae from basal hyphae, and no helix formation, limbal branch or sporangium is observed. 30 or more cylindrical spores are linked to the mature spore chain, and its surface is wrinkled. In various media, the growth showed a pale yellow to grayish yellow tea, the aerial mycelia exhibited a gray to bright ash, and no soluble pigment was observed. Optimum growth temperature is 27-30 ° C
It is in the vicinity. The formation of melanin-like pigment is positive, and the water degradability of starch is strong. In addition, 2,6 contained in the cell wall
-Diaminopimelic acid was the LL-form.

[0026] From these properties, the MJ885-mF8 strain, is considered to belong to Streptomyces (Streptomyce s) genus. Searching for closely related known species, Streptomyces zaomyceticus ,
Reference 1: Shirling, EB and D. Gottlieb "Internation
al Journal of Systematic Bacteriology ”Vol. 22, 3
74, 1972; Reference 2: Skerman, VBD, V. Mcg.
owan, and PHA Sneath `` International Journal of
Systematic Bacteriology "30: 406, 198
0 years) was given. Currently, a comparative study of the strains of the above-mentioned strains preserved by this laboratory and the MJ885-mF8 strain is under study. Therefore, at present, the MJ885-mF8 strain is designated as Streptomyces sp. MJ885.
-MF8.

The MJ885-mF8 strain was applied for deposit at the Institute of Biotechnology, Institute of Industrial Technology, Tsukuba City, Ibaraki Prefecture, and deposited on September 13, 1993, with the deposit number FERM P-13.
Commissioned as 863.

In carrying out the method of the second aspect of the present invention, the nutrient sources used for culturing the antibiotic MJ885-mF8 producing bacteria are those usually used as the nutrient sources for actinomycetes, such as carbon source and nitrogen. Sources, assimilable nutrient sources such as inorganic salts can be used. For example, dextrose, maltose, molasses,
Hydrocarbons such as dextrin, glycerin and starch, carbon sources such as soybean oil and peanut oil, and peptone,
Nitrogen sources such as meat extract, cottonseed flour, soybean flour, yeast extract, casein, corn steep liquor, NZ-amine, ammonium sulfate, ammonium nitrate, ammonium chloride, dipotassium phosphate, sodium phosphate, salt, calcium carbonate, magnesium sulfate, chloride An inorganic salt such as manganese can be used, and if necessary, trace metals such as cobalt and iron can be added. Other sources of nutrition include:
Any known nutritional source can be used as long as it can be utilized by the production strain used for producing the novel antibiotic MJ885-mF8.

The mixing ratio of the nutritional sources as described above is not particularly limited and can be varied over a wide range.
The composition and proportion of the optimum nutritional source depending on the antibiotic MJ885-mF8 producing bacterium to be used can be easily determined by those skilled in the art by a simple small-scale preliminary experiment.

A nutrient medium comprising the above nutrient sources is
It can be sterilized prior to culturing, and before or after this sterilization, it is advantageous to adjust the pH of the medium to the range 6-8, in particular the range pH 6.5-7.5.

Antibiotic MJ8885 in such nutrient medium
Cultivation of the mF8-producing bacterium can be performed according to a method usually used in the production of antibiotics by general actinomycetes. Usually, it is suitable to culture under aerobic conditions, and it can be usually performed with stirring and / or aeration. As the culture method, any of static culture, shaking culture, and submerged culture with aeration and stirring can be used, but liquid culture is suitable for mass production of the antibiotic MJ885-mF8.

The culture temperature that can be used is the antibiotic MJ885.
-There is no particular limitation as long as the growth of the mF8-producing bacterium is not substantially inhibited and the antibiotic can be produced, and it can be appropriately selected according to the producing bacterium to be used. Temperatures within the range of 25-30 ° C can be mentioned.

The culture is usually the antibiotic MJ885-mF8.
Can be continued until it fully accumulates in the culture. The culture time depends on the composition of the medium, the culture temperature, the use temperature,
The desired antibiotic can be obtained usually by culturing for 24-72 hours, although it varies depending on the production strain used.

The accumulated amount of the antibiotic MJ885-mF8 in the culture can be quantified by Pasteurella pisisida by the cylindrical plate method which is usually used for quantification of antibiotics.

Thus, in order to collect the novel antibiotic MJ885-mF8 accumulated in the culture from the culture, the bacterial cells were removed after culture by a known separation method such as filtration or centrifugation, if necessary. After that, the filtrate is subjected to solvent extraction using an organic solvent, particularly ethyl acetate, butyl acetate, a mixed solvent such as chloroform-methanol system, chromatography using adsorption or gel filtration, chromatography by countercurrent distribution, alone or , Can be used in combination to isolate and purify and collect.
The cells can be extracted with an organic solvent such as methanol or acetone, and isolated and purified from the extract containing the target antibiotic by the above separation and purification method. As a carrier for chromatography having adsorption or gel filtration, activated carbon, silica gel, porous polystyrene-divinylbenzene resin or various types of Sephadex can be used. Thus, a new antibiotic MJ having the above mentioned properties
885-mF8 is obtained.

Next, the present invention will be described more specifically with reference to examples.

[0037]

Example 1 Production of antibiotic MJ885-mF8 Galactose 2%, Dextrin 2%, Bact-Soyton 1%, Corn stapler 0.5%, Glycerin 1
%, Ammonium sulfate 0.2%, calcium carbonate 0.2%,
Liquid medium containing 1 drop of silicone oil (adjusted to pH 7.4) was dispensed into an Erlenmeyer flask in 110 ml aliquots.
Sterilized at 20 ° C. for 20 minutes. Then, against the liquid medium, Streptomyces sp. Grown on the agar slant medium was used. M
J885-mF8 strain (Ministry of Industrial Science, Microbial Research No. P-13863)
And incubate at 27 ° C for 1 day with rotary shaking (18 min / min).
(0 rotation). Using the obtained culture as a seed culture,
54 ml of an Erlenmeyer flask in which 110 ml of the same composition as the above was dispensed was inoculated in an amount of 2 ml each, and cultured at 27 ° C. for 2 days with rotary shaking (180 revolutions per minute).

The obtained culture solution was centrifuged and the supernatant was extracted with 5.8 l of ethyl acetate. Acetone 2
After extraction with 1 to remove acetone, water was added to make 2 l, followed by extraction with 2 l of ethyl acetate. Both extracts were combined and dried over anhydrous sodium sulfate. Removal of solvent gave 1.4 g of residue.

The residue was mixed with 20 ml of hexane and 2 ml of methanol.
After partitioning to 0 ml and concentrating the methanol layer to dryness, 1.2 g of residue was obtained, which was purified by silica gel 60 (Merck Ar Co.).
t. 7734) It was developed on a 100 ml column. Toluene (200 ml) as an elution solvent, toluene-acetone, 20: 1 (210 ml), 15: 1 (160 m)
l), 10: 1 (220 ml), 7: 1 (159 ml)
And 5: 1 (300 ml) each were used for successive elution.

TLC (Merck silica gel Art.571
According to 5), a fraction showing UV (254 nm) absorption at an Rf value of 0.47 (developing solvent: chloroform-methanol, 10: 1) was collected and concentrated to dryness to give a colorless transparent oil characterized by the above-mentioned physicochemical properties. Pure M
146 mg of J885-mF8 substance was obtained. 98 mg of MJ885-mF8 substance containing some impurities
Was obtained.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum curve diagram of antibiotic MJ885-mF8 in methanol.

FIG. 2 is a UV absorption spectrum curve diagram of the antibiotic MJ885-mF8 in 0.01 N-HCl-90% methanol.

FIG. 3 is a UV absorption spectrum curve diagram of the antibiotic MJ885-mF8 in 0.01 N-NaOH-90% methanol.

FIG. 4 is an infrared absorption spectrum curve diagram of the antibiotic MJ885-mF8.

FIG. 5 is a ¹ H-NMR spectrum curve diagram of the antibiotic MJ885-mF8.

FIG. 6 is a ¹³ C-NMR spectrum curve diagram of the antibiotic MJ885-mF8.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] November 9, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0040

[Correction method] Change

[Correction content]

TLC (Merck silica gel Art.571
According to 5), a fraction showing UV (254 nm) absorption at an Rf value of 0.47 (developing solvent: chloroform-methanol, 10: 1) was collected and concentrated to dryness to give a colorless transparent oil characterized by the above-mentioned physicochemical properties. Pure M
146 mg of J885-mF8 substance was obtained. 98 mg of MJ885-mF8 substance containing some impurities
Was obtained. The MJ885-mF8 substance of the present invention is
It was confirmed to be a compound represented by the structural formula shown below.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1: 465) (72) Inventor Ryuichi Sawa 4-6-7 Ayanishi, Ayase City, Kanagawa Prefecture (72 ) Inventor Yoshikazu Takahashi 3-2-3, Sakuragaoka, Tama-shi, Tokyo 3 (72) Inventor Hiroshi Naganawa 3-17, Denenchofuhonmachi, Ota-ku, Tokyo (72) Inventor Sawa Riki 4-6, Ayanishi, Ayase-shi, Kanagawa No. 7 (72) Inventor, Masaru Hamada, No. 405, Hidewa Residence No. 405 26, Naitocho, Shinjuku-ku, Tokyo

Claims (2)

[Claims] 1. The following physicochemical properties: -A) Property: Neutral substance of colorless transparent oil B) Optical rotation: [α] _D ²³ -6 ° (c 0.5, MeOH) C) Molecular formula: C ₁₈ H ₂₂ O ₆ High resolution FAB mass spectrum 335.1477 (M + H) ⁺
positive D) FAB mass spectrum (m / z): 335 (M +
^{H) + positive 333 (M-} H) - negative E) UV absorption spectrum: λmax, nm (ε) 271 (12500) ( in methanol), .lambda.max shown in Figure 1 of the accompanying drawings, nm (ε) 270 (12600 ) (0.01N-HCl
-90% in methanol), λmax, nm (ε) 252 (8230) (0.01N-NaOH) shown in Figure 2 of the attached drawings.
-90% in methanol), shown in FIG. 3 of the attached drawing F) Infrared absorption spectrum (KBr tablet method): shown in FIG. 4 of the attached drawing. The main absorption peaks are shown below: vmax: 2930, 1780, 1670, 1590, 1
440,1280 cm ⁻¹ G) ¹ H-NMR spectrum (in deuterated chloroform): shown in FIG. 5 of the attached drawing H) ¹³ C-NMR spectrum (in deuterated chloroform): I) Rf value (shown in FIG. 6 of the attached drawing) TLC; Merck silica gel Art. 57
15): 0.47 (chloroform-methanol, 10: 1) 0.66 (toluene-acetone, 1: 1) J) Color reaction: negative for ninhydrin, rydonsmith and ferric chloride reagents, and phosphomolybdic acid-sulfuric acid. Antibiotic MJ885-mF8 having antibacterial activity, characterized in that it is positive. 2. The method according to claim 1, which belongs to the genus Streptomyces.
The method for producing the antibiotic MJ885-mF8, which comprises culturing the bacteria producing the antibiotic MJ885-mF8 described in 1 above in a nutrient medium and collecting the antibiotic from the culture.