JPS5886087A - Novel antibiotic substance sf-2132 and its preparation - Google Patents

Abstract

PURPOSE:To prepare the novel antibiotic substance SF-2132 having antimicrobial activity and prophylactic activity, by culturing microorganisms belonging to Nocardiopsis genus. CONSTITUTION:Microorganisms belonging to Nocardiopsis genus and capable of producing the antibiotic substance SF-2132, e.g. Nocardiopsis sp. SF-2132 (FERM-P No.6131) are inoculated in a nutrient medium and cultured at 20- 35 deg.C for 2-7 days under aerobic conditions, and the antibiotic substance SF- 2132 is separated from the culture liquid.

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. The present invention relates to a method for producing a new antibiotic 5F-2132, which is characterized by culturing the antibiotic 5F-2132 and collecting the antibiotic 5F-2132 from the obtained culture.

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 the known substance was a new antibiotic, and this effective substance was named antibiotic 5F-2132.

The antibiotic 5F-2132-producing bacteria of the present invention contains a sufficient amount of antibiotic 5F-21 in its culture to be collected.
Any strain may be used as long as it has the ability to produce 32, but an example of such a strain is
There is a strain 5F-2132 newly isolated by the present inventors from the soil of Kashihara City, Nara Prefecture. The mycological properties of this strain are as follows.

In addition, observation is mainly 5HIRLING, E, and B.

and GOTTLIEB, D., International Journal of
SystmatLc BacLeriol.

16, 313-340 (1966).

(2) Morphology Substrate 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, and are simply branched, with the entire hyphae becoming a chain of spores. Spores usually form chains of 10 or more spores. The spores were oval to cylindrical in size, 1.0 to 1.5 x 0.4 to 0.6 microns, had a smooth surface, and no motility was observed. Other special structures such as sporangia, flagellated spores, and sclerotia were not observed.

■ Growth status on various media The growth status of the 5F1132 strain on various media is shown in the following table. Regarding the description of colors, the standards shown in [ ] are those of Container Corporation Kasumi Op America (Cont.
ainer Corporation, of
America) [Color Harmony? two,
Full (Color Harmony Manual) J
Use the one listed in. Observation was performed at 28° C. after 14 to 21 days of culture.

■ Physiological properties IL+ Growth temperature range: 15 to 15% on starch agar
It grows in a temperature range of 40°C and grows well at 25-37°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) Near salt tolerance, grows slightly at 0% but does not grow at 1080% or higher.

■ Utilization of carbon source Carbon source Growth D-glucose ++ D-xylose ++ D-fructose ++ D-mannitol + L-arabinose ++ L-rhamnose + i-inositol + sucrose ++ Raffinose + No additives Growth (Usability 20) 10 Weak Growth (Usability Knee) Basic medium used Yeast extract (manufactured by Difco) 5g Calcium carbonate 1g Agar (manufactured by Difco) 15g Distilled water 10100O! ■ Cell wall composition Diaminopimelic acid in the whole cell hydrolyzate was mainly of men type, and xylose, madinylose, and arabinose were not observed.

Combining the above properties, the substrate hyphae are divided, the entire aerial hyphae has the morphological characteristics of forming a chain of spores, and the whole cell hydrolyzate contains men-type diaminohimelic acid.
Xylose, Majinylose.

Those that do not have arabinose belong to the genus Nocardiopsis among actinomycetes. (
International Journal of
Systematic Bac ter i o Io
gy, 26. 487-493.1976) Therefore,
The present inventors used strain 5F-2132 as Nocardiopsis.
SP・5F-2132 (Nocardiopsis
sp, 5F-2132). 5F-213
The two strains were transferred to the Microbial Technology Research Institute, Kikoken Bacteria Collection No. 6131.
It has been entrusted as the No.

The present invention was completed based on the above findings, and is characterized by culturing antibiotic SF-2132-producing bacteria belonging to the genus Nocardiopsis, and collecting antibiotic 5F-2132 from the culture. A method for producing antibiotic 5F-2132 and antibiotic-resistant 5F-2132.

Nocardiopsis sp. 5F2132N is susceptible to changes in its properties, as seen in the case of other actinomycetes, and can be transformed by artificial transformation using ultraviolet rays, X-rays, radiation, chemicals, etc. Therefore, all Nocardiopsis bacteria capable of producing the antibiotic 5F-2132, even such mutant strains, can be used in the method of the present invention.

In the method of the present invention, strain 5F-2132 is typically cultured in a medium containing nutrients that can be utilized by the microorganism.

For example, seaweed using glucose, sucrose, dextrin, starch, starch syrup, sugar syrup, soybean oil, etc. as a carbon source.

Soy flour, wheat germ, and peptone are also used as nitrogen sources.

Meat extract, yeast extract, cornstarch liquor.

Sodium nitrate, ammonium sulfate, etc. can be used.

Calcium carbonate, potassium chloride, etc. as necessary
In addition to adding inorganic salts such as phosphates, organic and inorganic substances that aid the growth of bacteria and promote the production of antibiotic 5F-2132 (hereinafter referred to as 5F-2132 substance) can be appropriately added.

As a culture method, the burr method may be applied as long as it is a culture method under aerobic conditions, similar to the method for general antibiotic production, but the P4 culture method is suitable in terms of quantity. The suitable temperature for culturing is 20-35℃, but in many cases it is
It is preferable to culture in the vicinity. The production of 5F-2132 substance reaches its maximum accumulation in 2 to 7 days in both the shaking culture method and the tank culture method.

When testing the 5F-2132 substance, Staphylococcus aureus (Staphylococcus aureus) was used.
eus) 209p as the test bacterium.
Use the flat plate method. In this assay, the logarithm of the 5F-2132 substance shows a linear relationship with the diameter of the inhibition circle in the concentration range of 500 mc g/ml to 62.5 mc g/ml, and the inhibition circle of 21 to 12 cranes, respectively. give.

Since the 5F-2132 substance has the physical and chemical properties described below, it can be extracted and purified according to its properties, and can be efficiently extracted and purified by the following method. That is, the active ingredient is mainly contained in the culture filtrate, which is adsorbed onto a porous adsorption resin such as Diaion HP-20 (manufactured by Mitsubishi Kasei), and mixed with water and a water-soluble solvent such as acetone or methanol. The active ingredient is eluted with a mixed solvent. This eluate was concentrated under reduced pressure, and further mixed with CM-Sephadex (manufactured by Pharmacia) and Diaion HP-20.
By appropriately combining column chromatography and the like, 5F-2132 substance can be isolated as a white amorphous powder. Since the 5F-2132 substance is a basic substance, it can be isolated as a free base or an acid addition salt. Examples of acid addition salts of the 5F-2132 material include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, etc., as well as acetic acid.

Salts with organic acids such as 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 5F-2132 thus obtained are as follows.

(Color and shape of 11 substances: White powder (2) Melting point: Foaming decomposition begins at 180°C (3) Specific rotation: [α]g = -44° (C1, water) (4) Molecular weight: 400 or more 1000 The following (by gel filtration method) (5) Elemental analysis: Actual value (%) i C49,
00, H7, 21°N21.09 Actual value as hydrochloride (%) C42,96, H6,93, N20.1.
(ff19.76 Oxygen elements other than the above elements (not measured)
is included, but no other elements are present.

(6) Ultraviolet absorption spectrum: As shown in FIG. 2g (E:F,) = 227 nm (
232).

λ'By"(E:F,) = 227 nm (2
44).

LINNmllN λttrx (E: 5) = 230Im □l)
(198).

(7) Infrared absorption spectrum: 335 as shown in Figure 2
0, 1700 (shoulder), 1640.1530.14
60.1410. 1340°1240, 11?0.1
050.1010. 960. 900. and 860
Absorption is shown at cm-1.

(8) Nuclear magnetic resonance spectrum: As shown in FIG.

(9) Thin layer chromatography: η as shown below
be.

Silica gel 60F-254 (manufactured by Merck) n-butanol-acetic acid-water (2:1:1) Rf O,14
n-propanol-pyridine-acetic acid-water (15:10:
3:12) Rf O, 59 chloroform-methanol-4% ammonia (2:111 upper layer) Rfo
, 35 Cellulose F254 (manufactured by Merck & Co., Ltd.) n-butanol-acetic acid-water (2:1:1) Rf
O,64n-propanol-pyridine-acetic acid-water (
15:10:3:12) Rf O,710I
High-pressure filter paper electrophoresis: High-pressure filter paper electrophoresis device (manufactured by Serpent Instruments, high-voltage power supply HV5000A,
Using an electrophoresis tank (R-Dell LT48A), Toyo Roshi Na5
1 (manufactured by Toyo Roshi Co., Ltd.) using the following buffer solution at a constant voltage of 3.
When electrophoresis is performed at 500 V for 15 minutes, this substance moves to the cathode, and when the mobility of alanine is 1, the mobility of this substance is 2.5.

Buffer: Pyridine 2QQm# and acetic acid 8ml total volume 1I3
It is dissolved in purified water so that it becomes It, and at this time -
was 6.4.

(11) Amino acid composition Two substances with 6N Hα, 10
When decomposed f' at 5°C for 18 hours, equimolar amounts of leucine, serine, α, β-diaminobutyric acid and β-arginine are produced.

(12) Color reaction: positive; Sakaguchi, ninhydrin and Remini.

(13) Solubility: Easily soluble in water, poorly soluble in lower alcohols,
Insoluble in chloroform and n-hexane.

The antibacterial spectrum of the 5F1132 substance is shown in the table below. In addition, as a result of acute toxicity tests using mice, 200
All patients survived when administered mg/kg intravenously.

Table S Antibacterial spectrum of F-2132 substance * β-lactam antibiotic highly sensitive strain (method) a: Agar Godairyushi day method Nigelos Guilyushi day method (medium) l: Heart infugene agar medium 2 Nigel course , peptone medium.

3: Heart Infuge Nipross medium 4: Glycerol broth medium There is no known antibiotic that matches the physical and chemical properties and biological properties of the 5F-2132 substance described above, and the antibiotic 5F-2132 substance of the present invention is novel. It was recognized as a substance.

The 5F-2132 substance has an anti-infection effect in addition to its antibacterial activity, and the experimental results are shown below.

Method Test animal: JCL-ICR male mouse, 4 weeks old.

Average weight 21.0 g, 6 animals per group ge strain; Pseudomonas aeruginosa
aeruginosa) E2 inoculation solution: The above bacterial strain was smeared onto a Heart Infuge Sun Agar (manufactured by Dlf'co) plate at 37"c.

After culturing for 20 hours, the bacteria were collected, diluted with physiological saline to the required bacterial concentration, and added with an equal amount of 5% mucin (Difc).

(manufactured by Seiko Co., Ltd.) solution to obtain an inoculum solution. The inoculation capacity is 0.
It was set as 5mJ/mouse.

Test method: SF-213 obtained as a pure product in Example 2
Dissolve the two substances in sterile distilled water to a certain concentration, and (7) 0.
2mj! was subcutaneously administered once to the thigh of a mouse, and after 5 days, the above-mentioned inoculum solution was intraperitoneally inoculated, and the survival of the mouse was observed for 4 days.

The results were as shown in the table below.

Table (values indicate the number of survivors out of 6 animals per group) □ For controls, only distilled water was administered instead of 5F-2132.

As described above, the <5F-2132 substance clearly exhibited infection-preventing activity.

SF-213 having antibacterial and infection-preventing properties of the present invention
The two substances are administered by suspending them in distilled water for injection, physiological saline, etc. The dosage form is administered parenterally, such as by subcutaneous injection, intravenously, or intramuscularly, or orally, such as by 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, the dosage 1 depends on the administration method, the patient's situation, such as age, weight, sex, sensitivity, and administration time.

Of course, the administration can be changed as appropriate depending on the concomitant drugs, the patient, and the severity of the disease, and the appropriate dose and frequency of administration under certain conditions are determined by experts' tests to determine the appropriate dose based on the above guidelines. be done.

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 process of manufacturing 5F-2132 substance is shown below.
Of course, many modifications and modification means 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) 20m 12 to 100mj? Nocardiopsis sp. 5F-2132 strain obtained from sterile slant culture in an Erlenmeyer flask (Kikoken Bacterial Collection No. 6131
3 to 4 platinum loops of bacterial cells of No.) were inoculated and cultured with shaking at 28°C for 2 days.

Kono inoculum 5 m/80 mj of the above medium! 500 m
Dispense into sterilized 1N Erlenmeyer flasks and inoculate 2
Culture with shaking at 8°C for 24 hours. This seed culture is 80mj! Further, the above medium eoomIt was dispensed into two sterilized 51-volume Erlenmeyer flasks, 40 ml each was inoculated, and cultured with shaking at 2B'c for 20 hours.

600 mJ of the resulting seed culture containing 20 J of production medium
The seeds were inoculated into a 0.1 volume jar fermenter and cultured at 28°C for 65 hours with aeration (airing 20 J/min, stirring 350 rpm) (using 7-2 jar fermenters).

The composition of the production medium is as follows.

Starch syrup 4.0% calcium carbonate 0.2% soybean oil
0.3% silicon KM68-2F
0.01% (antifoaming agent, manufactured by Shin-Etsu Chemical) Soy flour 2.0% Sungrain (manufactured by Suntory) 0.5% Pharmamedia
1.0% (Traders Oil
M, i l l) nickel chloride
0.0001% cobalt chloride
0.000f% ferrous sulfate 0
．． 001% (pH 7, Q before sterilization) After culturing, add a filter aid, adjust the pH to 5 with 6N hydrochloric acid, filter, and filtrate 24I! , obtained.

Example 2 (Extraction/purification) 24It of the culture filtrate obtained in Example 1 was purified by Diaion H.
The active ingredients were adsorbed through column 2.51 of P-20 (manufactured by Mitsubishi Kasei). After washing this column with 3 It of water,
The active ingredient was eluted with % acetone water.

The active fractions were combined (31) and concentrated under reduced pressure to remove acetone, and the resulting aqueous solution of 21 was transferred to a 150 m# column of CM-Sephadex C-25 (N,' type) [manufactured by Pharmacia]. The active ingredients were adsorbed. This column was filled with 750ml of water and then 1500ml of 0.05M sodium chloride solution! , and then eluted with 0.2M sodium chloride solution, and aliquots of 100 mjl were collected.

1,300mj of the active section (Fluxy sun 3~Flaxy peeling magnetic 15) was combined and this was made into Diaion HP-2.
The active ingredients were adsorbed onto the resin through a 0 column (250 m,').

After washing the column with 150 mJ of water, it was eluted with 6% acetone water. The active fraction was concentrated to dryness under reduced pressure to obtain -3F-
A crude powder of substance 2132 (light brown color>1.76 g (purity: about 60%) was obtained.

In order to obtain a pure substance of 5F-2132, next, 800 mg of the above coarse powder was dissolved in a small amount of water, and this was dissolved in CM-2132.
Ceraphadex C-25 (Nt type) column (60 m
I put it on the rice. Then 300mJ of water and 600mA of 0.05M sodium chloride solution! After washing the column with 0.
Elution was performed with a 2M sodium chloride solution, and 15 g portions were collected. Since most of the active ingredients from Fluxi Nikmagai 20 are collected in Fluxi Nikmagai Co., Ltd., these parts are merged and this is transferred to a column of Diaion HP-20 (150m#).
pass through to absorb the active ingredients. Water 600mI! , then 25% acetone water] 5Q m j! 15g
Separate each portion. Concentrate and dry fraction 24 (water development section) from 1kll of Fraxyi under reduced pressure, and obtain 5F-2.
115 mg of pure substance 132 was obtained as a hydrochloride. Also, from Fraxi Sun 41 to Fraxi Sun 11h45 (25
% acetone water development section) was concentrated to dryness under reduced pressure to obtain 5F-
247 mg of pure substance 2132 was obtained as a free base.

[Brief explanation of the drawing]

Figure 1 shows an aqueous solution of antibiotic 5F-2132 (solid line). 0.IN hydrochloric acid (dashed line) and 0.IN hydrochloric acid (dashed line). Ultraviolet absorption spectrum in IN sodium hydroxide solution (dotted line), Figure 2 is infrared absorption spectrum in KBr tablet, and Figure 3 is proton nuclear magnetic resonance spectrum in D20 using TMS as an external standard. shows.

Claims (1)

[Claims] 1. New water-soluble basic antibiotic 5F-2132 and its salts having the following properties as a free base (11 Color and shape of substance: white powder (2) Melting point: foaming decomposition from 180°C Starting (3) Specific rotation: [α):' = -44° (CI, water) (4
) Molecular weight: 400 or more and 1000 or less (by gel filtration method) (5) Elemental analysis: Actual value (%) i C49,00,
H7,21N21.09 Actual value as hydrochloride (%
) C42,96, H6,93, N20.1. α9.76
In addition to the above elements, oxygen element (not measured) is included, but no other elements are present. (6) Ultraviolet absorption spectrum: As shown in FIG. λ”: (E: ordinary) -227 nm (232)
. λ (Ei?,) = 227 nm (244)
. λ';"'(E: t'm> = 230 nm (
shoulder) (198). (7) Infrared absorption spectrum: 335 as shown in Figure 2
0, 1700 (waste), 1640.1530.14
60.1410.1340°1240, 1170.10
50.1010. 960. 900. and 860 row 1 shows absorption. (8) Nuclear magnetic resonance spectrum: As shown in FIG. . (9) Thin layer chromatography: As shown below. Silica gel 60F-254 (manufactured by Merck) n-butanol-acetic acid-water (2:1:1) Rf O,14
n-propanol-pyridine-acetic acid-water (15:10:
3:12) RfO,59 chloroform-methanol-4% ammonia (2:1:1 upper layer)
RfO, 35 Cellulose F 254 (manufactured by Merck) n
-Butanol-acetic acid-water (2:1:1) Rf
O,64n-propanol-pyridine-acetic acid-water (1
5:10: 3712) Rf O, 71Ql High pressure filter paper electrophoresis: High pressure filter paper electrophoresis apparatus (servant
When performing electrophoresis for 15 minutes at a constant voltage of 3500 V using the following buffer on Toyo Roshi Gya 51 (manufactured by Toyo Roshi Co., Ltd.) using a high voltage power supply HV500QA (manufactured by Instrument Co., Ltd., electrophoresis tank model LT48A), The substance moves to the cathode, and when the mobility of alanine is 1, the mobility of this substance is 2.5
It is. Buffer: Pyridine 200mj! and 8 ml of acetic acid to a total volume of 3
It was dissolved in purified water so that the concentration was 1, and - at this time was 6.4. (11) Amino acid composition: This substance in 6N Hα, 10
Leucine when decomposed for 18 hours at 5°C. Equimolar moles of serine, α, β-diamino Mill, and β-arginine are produced. (12) Color reaction: positive; Sakaguchi, ninhydrin and Lemieux. (13) Solubility: 5 soluble in water, slightly soluble in lower alcohols,
Insoluble in chloroform and n-hexane. 2,7 Antibiotic 5F-21 belonging to the genus Caldeiopsis
32-producing bacteria were cultured in a medium, and the culture was treated with antibiotic 5F-
A method for producing a new antibiotic 5F-2132, which comprises producing and accumulating 2132 and collecting it. 3. The manufacturing method according to claim 2, wherein the antibiotic 5F-2132 producing bacterium is Nocardiopsis sp. SF 2132.