JPS6250474B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new antibiotic and a method for producing the same, and more specifically, the present invention relates to a new antibiotic and a method for producing the same. This invention relates to a method for producing a new antibiotic SF-2132, which is characterized by collecting the antibiotic SF-2132 from a substance. The present inventors discovered that a substance that exhibits an antibacterial effect against bacteria is produced in the culture of a certain bacterial strain, isolated the effective substance from the culture, and investigated its properties. As a result, it was confirmed that this is a new antibiotic that is different from known substances, and this effective substance was named antibiotic SF-2132. The antibiotic SF-2132-producing bacteria of the present invention include:
enough antibiotics in the culture to harvest
Any strain may be used as long as it has the ability to produce SF-2132, but an example of such a strain is a strain newly isolated by the present inventors from soil in Kashihara City, Nara Prefecture. There are SF-2132 stocks. The mycological properties of this strain are as follows. Note that observations are mainly made using SHIRLING, EB, and
International Journal of GOTTLIEB, D.
Systmatic Bacteriology, 16, 313–340 (1966)
This was done according to the method described in. Morphology: The basal hyphae have a diameter of 0.4 to 0.6 microns, and in agar media, they simply branch and elongate in a wavy manner, but in liquid media, they divide irregularly as they are cultured. Aerial hyphae are abundantly formed with a diameter of 0.4 to 0.6 microns;
The hyphae are simply branched and the entire hyphae become a chain of spores. Spores usually form chains of 10 or more spores. The spores were oval to cylindrical in shape, 1.0 to 1.5 x 0.4 to 0.6 microns in size, with smooth surfaces and no motility. Other special structures such as sporangia, flagellated spores, and sclerotia were not observed. Growth status on various media The growth status of SF-2132 strain on various media is shown in the following table. Regarding the description of colors, the standards shown in [ ] are those of Container Corporation of
America (Container Corporation of
The one described in the "Color Harmony Manual" manufactured by America) was used. Observations were made after culturing for 14 to 21 days at 28°C.

【table】

[Table] Physiological properties (1) Growth temperature range: 15 to 40 on starch agar
It grows in a temperature range of 25°C to 37°C. (2) Liquefaction of gelatin: Positive (3) Hydrolysis of starch: Negative (4) Reduction of nitrate: Negative (5) Coagulation of skim milk: Positive Peptonization of skim milk: Positive (6) Production of melanin-like pigments: Positive (7) Salt tolerance: Slight growth at 7.0%, but 10.0
It will not grow above %. Utilization of carbon source Carbon source Growth D-glucose ++ D-xylose ++ D-fructose ++ D-mannitol + L-arabinose ++ L-rhamnose + i-inositol + sucrose ++ Ruffinose + No additives + ++ Good growth (availability :+) + Weak growth (Usability: -) Basic medium used Yeast extract (manufactured by Difco) 5g Calcium carbonate 1g Agar (manufactured by Difco) 15g Distilled water 1000ml Cell wall composition Diaminopimelic acid in whole cell hydrolyzate is the main In the meso form, xylose, majulose, and arabinose were not observed. Taking all the above properties into account, the basal hyphae are divided and the entire aerial hyphae form a chain of spores, and the whole cell hydrolyzate contains meso-type diaminopimelic acid and contains xylose. , Majulose, and arabinose belong to the genus Nocardiopsis among actinomycetes. (International Journal of Systematic
Bacteriology, 26, 487-493, 1976) Therefore, the present inventors identified the SF-2132 strain as Nocardiopsis sp. SF-2132.
(Nocardiopsis sp・SF-2132). science fiction
-2132 strain has been entrusted to the Institute of Microbial Technology as Microbiological Research Institute No. 6131. The present invention was completed based on the above findings, and is an antibiotic SF belonging to the genus Nocardiopsis.
- 2132-producing bacteria are cultured and antibiotics are extracted from the culture.
Antibiotics characterized by collecting SF-2132
The manufacturing method of SF-2132 and the new antibiotic SF-2132. Nocardiopsis sp. SF2132 strain is susceptible to changes in its properties, as seen in the case of other actinomycetes, and can be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, radiation, drugs, etc. Therefore, all Nocardiopsis bacteria having the ability to produce the antibiotic SF-2132, even such mutant strains, can be used in the method of the present invention. In the method of the present invention, strain SF-2132 is usually cultured in a medium containing nutrients that can be utilized by the microorganism. For example, glucose, sucrose, dextrin, starch, starch syrup, molasses, soybean oil, etc. can be used as the carbon source. Also, soybean flour, wheat germ, peptone,
Meat extract, yeast extract, cornstarch liquor, sodium nitrate, ammonium sulfate, etc. can be used. In addition, inorganic salts such as calcium carbonate, potassium chloride, and phosphates are added as needed, and antibiotic SF-2132 (hereinafter referred to as SF-
2132 substances) may be appropriately added. As for the culture method, any culture method under aerobic conditions may be applied, as is the case with general antibiotic production methods, but the deep culture method is most suitable. The temperature suitable for culturing is 20 to 35°C, but in most cases it is preferable to culture at around 26 to 32°C. Production of SF-2132 substance reaches its maximum accumulation in 2 to 7 days for both shaking culture method and tank culture method. For testing SF-2132 substances, use Staphylococcus aureus.
Use the paper disc/plate method using 209p as the test bacterium. In this assay, the SF-2132 substance
In the concentration range of 500 mcg/ml to 62.5 mcg/ml, the logarithm shows a linear relationship with the inhibition circle diameter, giving inhibition circles of 21 mm to 12 mm, respectively. The SF-2132 substance has the physical and chemical properties described below, so it can be extracted and purified according to its properties, but it can be efficiently extracted and purified by the following method.
Can be purified. In other words, the active ingredient is mainly contained in the culture filtrate, which is then added to the Diaion HP-
20 (manufactured by Mitsubishi Kasei), and the active ingredient is eluted with a mixed solvent of water and a water-soluble solvent such as acetone or methanol. This eluate was concentrated under reduced pressure, and further added to CM-Sephadex (manufactured by Pharmacia) and Diaion HP.
SF-2132 substance can be isolated as a white amorphous powder by appropriately combining column chromatography, etc. of -20. Since SF-2132 is a basic substance, it can be isolated as a free base or an acid addition salt. Examples of acid addition salts of SF-2132 substances include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid,
Salts with inorganic acids such as hydrobromic acid and the like, as well as salts with organic acids such as acetic acid, citric acid, tartaric acid, benzoic acid, ascorbic acid, etc. are possible, but salts that are practically acceptable are preferred. The physicochemical properties of the antibiotic SF-2132 thus obtained are as follows. (1) Color and shape of substance: White powder (2) Melting point: Foaming and decomposition begins at 180℃ (3) Specific rotation: [α] ²⁵ _D = -44° (Cl, water) (4) Molecular weight: 400 Above 1000 (by gel filtration method) (5) Elemental analysis: Actual value (%); C49.00, H7.21,
Measured value as N21.09 hydrochloride (%) C42.96, H6.93, N20.1, Cl9.76 In addition to the above elements, oxygen element (not measured) is included, but there are no other elements. (6) Ultraviolet absorption spectrum: As shown in Figure 1. λ ^H ₂ ^O _nax (E ¹ % ₁ cm) = 227 nm (232), λ ⁰ . ^{1N HCl} _nax (E ¹ % ₁ cm) = 227 nm (244
), λ ⁰ . ^{1N NaOH} _nax (E ¹ % ₁ cm) = 230nm (
shoulder) (198). (7) Infrared absorption spectrum: as shown in Figure 2
3350, 1700 (shoulder), 1640, 1530, 1460, 1410,
It exhibits absorption at 1340, 1240, 1170, 1050, 1010, 960, 900, and 860 cm ^-1 . (8) Nuclear magnetic resonance spectrum: As shown in Figure 3. (9) Thin layer chromatography: As shown below. Silica gel 60F-254 (manufactured by Merck) n-butanol-acetic acid-water
(2:1:1) Rf 0.14 n-propanol-pyridine-acetic acid-water
(15:10:3:12) Rf 0.59 Chloroform-methanol-4% ammonia
(2:1:1 upper layer) Rf 0.35 Cellulose F254 (manufactured by Merck) n-butanol-acetic acid-water
(2:1:1) Rf 0.64 n-propanol-pyridine-acetic acid-water
(15:10:3:12) Rf 0.71 (10) High-pressure filter paper electrophoresis: Using a high-pressure filter paper electrophoresis device (manufactured by Servant Instruments, high-voltage power supply HV5000A, electrophoresis tank model LT48A),
When electrophoresis is performed on Toyo Roshi No. 51 (manufactured by Toyo Roshi Co., Ltd.) at a constant voltage of 3500 V for 15 minutes using the buffer shown below, this substance moves to the cathode, and when the mobility of alanine is 1, this substance moves to the cathode. The mobility of the substance is 2.5. Buffer: 200 ml of pyridine and 8 ml of acetic acid were dissolved in purified water to a total volume of 3, and the pH at this time was 6.4. (11) Amino acid composition: When this substance is decomposed with 6N HCl at 105℃ for 18 hours, leucine, serine,
Equal moles of α, β-diaminobutyric acid and β-arginine are produced. (12) Color reaction: positive; Sakaguchi, ninhydrin, and Remieux. (13) Solubility: Easily soluble in water, slightly soluble in lower alcohols, insoluble in chloroform and n-hexane. The antibacterial spectrum of SF-2132 substance is shown in the table below. In addition, as a result of acute toxicity tests using mice, all mice survived after intravenous administration of 200 mg/Kg.

【table】

[Table] There are no known antibiotics that match the physical and chemical properties and biological properties of the SF-2132 substance described above, and the antibiotic SF-2132 substance of the present invention was recognized as a new substance. In addition to antibacterial activity, substance SF-2132 also has infection-preventing properties, and the experimental results are shown below. Method Test animals: JCL-ICR male mice, 4 weeks old, average weight 21.0 kg, 6 per group Test bacterial strain: Pseudomonas aeruginosa E2 inoculation solution; After culturing at 37℃ for 20 hours, the bacteria were collected, diluted with physiological saline to the required concentration, mixed with an equal volume of 5% mucin (Difco) solution, and inoculated. It was made into a bacterial solution. The inoculation capacity is 0.5
ml/mouse. Test method: SF obtained as a pure product in Example 2
Substance 2132 was dissolved in sterile distilled water to a certain concentration, and 0.2 ml of it was subcutaneously administered once to the thigh of a mouse.
After 4 days, the above-mentioned inoculum solution was intraperitoneally inoculated, and the survival of the mice was observed for 4 days. The results were as shown in the table below.

[Table] For controls, only distilled water was administered instead of SF-2132. As described above, SF-2132 substance clearly showed infection-preventing activity. SF having antibacterial and infection-preventing properties of the present invention
-2132 Substances are administered by suspending them in distilled water for injection, physiological saline, etc. The dosage form is parenteral, such as subcutaneous injection, intravenous or intramuscular injection, or oral, such as capsule. The dosage should be determined in consideration of the results of animal studies and various circumstances, and the total dosage should be administered continuously or intermittently within a range that does not exceed a certain amount. However, it goes without saying that the dosage may be changed as appropriate depending on the administration method, the patient's situation, such as age, body weight, sex, sensitivity, administration time, concomitant drugs, and the patient or the severity of the disease. The appropriate amount and frequency of administration under certain conditions are determined by an expert's appropriate dosage determination test based on the above guidelines. However, it is generally administered in a range of 200 to 3000 mg per day, for example 500 mg divided into 1 to 5 doses. The implementation instructions for the method for producing the SF-2132 substance are shown below, but it goes without saying that many modifications and modification methods not exemplified here may be used. Example 1 (Culture) Inoculum culture medium (glucose 1.0%, starch 1.0%, polypeptone 0.5%, meat extract 0.2%, yeast extract 0.3
%, soy flour 0.2%, calcium carbonate 0.2%, PH7.0)
Dispense 20 ml into a 100 ml Erlenmeyer flask and sterilize the Nocardiopsis sp. obtained from the slant culture.
3 bacterial bodies of SF-2132 strain (Feikoken Bacterium No. 6131)
Inoculate ~4 platinum loops and culture with shaking at 28°C for 2 days. 5 ml of this inoculum is inoculated into sterilized 500 ml Erlenmeyer flasks containing 80 ml of the above medium, and cultured with shaking at 28° C. for 24 hours. 80 ml of this seed culture was further inoculated with 40 ml each into two sterilized 5-volume Erlenmeyer flasks containing 600 ml of the above medium, and cultured with shaking at 28° C. for 20 hours. The resulting seed culture contains 600 ml of production medium
Inoculate into a 30-volume jar fermenter and stir at 28°C with aeration (aeration 20/min, stirring 350 rpm)
and cultured for 65 hours (using two jar fermenters). The composition of the production medium is as follows. Starch syrup 4.0% Calcium carbonate 0.2% Soybean oil 0.3% Silicone KM68-2F (antifoaming agent, manufactured by Shin-Etsu Chemical)
0.01% Soy flour 2.0% Sungrain (manufactured by Suntory) 0.5% Pharmamedia (manufactured by Traders Oil Mill)
1.0% Nickel chloride 0.0001% Cobalt chloride 0.0001% Ferrous sulfate (PH7.0 before sterilization) 0.001% After culturing, add filter aid and adjust pH to 5 with 6N hydrochloric acid.
The mixture was adjusted to 100% and then filtered to obtain filtrate 24. Example 2 (Extraction/purification) The culture filtrate 24 obtained in Example 1 was passed through column 2.5 of Diaion HP-20 (manufactured by Mitsubishi Kasei).
Adsorbed active ingredients. After washing this column with 3 portions of water, the active ingredient was eluted with 50% acetone water.
Combine the active fractions (3) and concentrate under reduced pressure,
Remove the acetone and convert the resulting aqueous solution to CM
- Passed through a 150 ml column of Cephadex C-25 (N _a ⁺ type) [manufactured by Pharmacia] to adsorb the active ingredient. This column was washed with 750 ml of water and then with 1500 ml of 0.05M sodium chloride solution, and then eluted with 0.2M sodium chloride solution, and 100 ml each was collected. Activity classification (Fraction No. 3 to Fraction No.
15) (1300ml) and add this to Diaion HP.
The active ingredients were adsorbed onto the resin through a -20 column (250ml). After washing the column with 150 ml of water, it was eluted with 25% acetone water. The active fraction was concentrated to dryness under reduced pressure.
1.76 g (purity: approximately 60%) of coarse powder (light brown) of SF-2132 substance was obtained. To obtain a pure SF-2132 substance, next, dissolve 800 mg of the above coarse powder in a small amount of water.
Place it on a column (60 ml) of CM-Seraph Adex C-25 (N _a ⁺ type). After washing the column with 300 ml of water and 600 ml of 0.05M sodium chloride solution,
Elution was carried out with 0.2M sodium chloride solution, and 15 g portions were collected. From Fraction No. 20 to Fraction No.
Since most of the active ingredients are collected in 50, this part is combined and passed through a Diaion HP-20 column (150ml) to adsorb the active ingredients. water 600
ml, then develop with 150 ml of 25% acetone water and separate out 15 g each. Fraction from Fraction No.11
No. 24 (water development category) was concentrated to dryness under reduced pressure, and SF
-115mg of pure substance 2132 was obtained as hydrochloride. Also, from Fraction No. 41 to Fraction No. 45 (25%
Acetone water development section) was concentrated to dryness under reduced pressure,
247 mg of pure SF-2132 substance was obtained as a free base.

[Brief explanation of the drawing]

Figure 1 shows an aqueous solution of antibiotic SF-2132 (solid line);
Ultraviolet absorption spectrum in 0.1N hydrochloric acid (dashed line) and 0.1N sodium hydroxide solution (dotted line), 2nd
The figure shows the infrared absorption spectrum of the same KBr tablet, and Figure 3 shows the proton nuclear magnetic resonance spectrum in D ₂ O with TMS as an external standard.

Claims (1)

[Claims] 1. A new water-soluble basic antibiotic SF-2132 and its salts having the following properties as a free base (1) Color and shape of the substance: White powder (2) Melting point: Foaming and decomposition starting from 180°C Starting (3) Specific rotation: [α] ²⁵ _D = -44゜ (Cl, water) (4) Molecular weight: 400 to 1000 (by gel filtration method) (5) Elemental analysis: Actual value (%); C49 .00，H7.21，
Measured value as N21.09 hydrochloride (%) C42.96, H6.93, N20.1, Cl9.76 In addition to the above elements, oxygen element (not measured) is included, but there are no other elements. (6) Ultraviolet absorption spectrum: As shown in Figure 1. λ ^H ₂ ^O _nax (E ¹ % ₁ cm) = 227 nm (232), λ ⁰ . ^{1N HCl} _nax (E ¹ % ₁ cm) = 227 nm (244
), λ ⁰ . ^{1N NaOH} _nax (E ¹ % ₁ cm) = 230nm (
shoulder) (198). (7) Infrared absorption spectrum: as shown in Figure 2
3350, 1700 (shoulder), 1640, 1530, 1460, 1410,
It exhibits absorption at 1340, 1240, 1170, 1050, 1010, 960, 900, and 860 cm ^-1 . (8) Nuclear magnetic resonance spectrum: As shown in Figure 3. (9) Thin layer chromatography: As shown below. Silica gel 60F-254 (manufactured by Merck) n-butanol-acetic acid-water
(2:1:1) Rf 0.14 n-propanol-pyridine-acetic acid-water
(15:10:3:12) Rf 0.59 Chloroform-methanol-4% ammonia
(2:1:1 upper layer) Rf 0.35 Cellulose F254 (manufactured by Merck) n-butanol-acetic acid-water
(2:1:1) Rf 0.64 n-propanol-pyridine-acetic acid-water
(15:10:3:12) Rf 0.71 (10) High-pressure filter paper electrophoresis: Using a high-pressure filter paper electrophoresis device (manufactured by Servant Instruments, high-voltage power supply HV5000A, electrophoresis tank model LT48A),
When electrophoresis is performed on Toyo Roshi No. 51 (manufactured by Toyo Roshi Co., Ltd.) at a constant voltage of 3500 V for 15 minutes using the buffer shown below, this substance moves to the cathode, and when the mobility of alanine is 1, this substance moves to the cathode. The mobility of the substance is 2.5. Buffer: 200 ml of pyridine and 8 ml of acetic acid were dissolved in purified water to a total volume of 3, and the pH at this time was 6.4. (11) Amino acid composition: When this substance is decomposed with 6N HCl at 105℃ for 18 hours, leucine, serine,
Equal moles of α, β-diaminobutyric acid and β-arginine are produced. (12) Color reaction: positive; Sakaguchi, ninhydrin, and Remieux. (13) Solubility: Easily soluble in water, slightly soluble in lower alcohols, insoluble in chloroform and n-hexane. 2 Antibiotic SF belonging to the genus Nocardiopsis
A method for producing a new antibiotic SF-2132, which comprises culturing -2132-producing bacteria in a medium, allowing the culture to produce and accumulate antibiotic SF-2132, and collecting the product. 3. The production method according to claim 2, wherein the antibiotic SF-2132-producing bacteria is Nocardiopsis.sp. SF-2132.