JPS5820960B2 - Antibiotics - Google Patents

Description

[Detailed description of the invention] The present invention relates to a novel antibiotic MX-A.

The present inventors have learned that an effective substance that exhibits a strong growth-inhibiting effect against bacteria of the genus Pseudomonas is produced and accumulated in cultures of certain strains belonging to the genus Bacillus, and investigated the properties of this effective substance. As a result, they discovered that it was a new antibiotic, named it antibiotic MX-A, and completed the present invention.

To produce the antibiotic MX-A of the present invention, use the antibiotic MX-A bacteria belonging to the genus Bacillus (hereinafter abbreviated as MX-A producing bacteria), for example, as described in JP-A-48-62993. Bacillus vit
ellinus ) &, Z-1159 strain and JP-A-4
Bacillus V-7 strain (this bacterium is currently identified as Bacillus circulans) described in Publication No. 9-13394 is used.

In addition, the Fermentation Research Institute, the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the American Type Culture Collection 7
reColle-ction), Bacillus vitellinus AZ-1159 strain is IF'0 13296, FAIKEN Bacteria No. 1203, ATCC31078, and Bacillus circulans A, V 7 strains are IFO13:3.
20, Microtechnical Research Institute No. 1480, and ATCC 21933, respectively.

Furthermore, MX-A used for producing the antibiotic of the present invention
The production amount of the antibiotic MX-A (hereinafter sometimes simply referred to as MX-A) can be increased by using the producing bacteria by a commonly used mutation treatment method such as X-rays, UV irradiation, or treatment with a mutagenic agent.

When MX-A producing bacteria are cultured to produce MX-A, various media used in normal fermentation are used as the culture medium.

That is, carbon sources include glycerin, glucose, maltose, starch, texturin, etc., and nitrogen sources include organic nitrogen compounds such as soybean flour, dried yeast, peptone, meat extract, urea, various amino acids, or ammonium sulfate, ammonium chloride, etc. Inorganic nitrogen compounds are used.

Furthermore, inorganic substances such as potassium, sodium, magnesium, calcium, iron, and phosphoric acid are used in combination.

If necessary, vitamins, yeast extract, organic acids, other growth promoting substances, and antifoaming agents are used in combination.

As a culture method, static culture may be used, but shaking culture or deep aeration agitation culture is suitable.

The appropriate culture temperature is 20° to 40° and the culture time is 24 to 90 hours.

The pH of the medium during cultivation is 5-9, preferably 6-8.

MX-A is mainly accumulated within the culture medium.

After removing the bacterial cells and other insoluble substances from the culture solution by methods such as r. filtration and centrifugation, the active ingredients in the culture solution are added to Amberlite IRC-50 (manufactured by Rohm & Barth), Amberly) CG-50 (Rohm & Barth), etc.・Manufactured by And...
It is adsorbed to the H type or NH4 type of cation exchange resins such as cation exchange resins, and is eluted with various alkaline solutions and mineral acids.

The eluate is adjusted to slightly alkaline (preferably about pH 1.0) and then treated with activated carbon.

At this time, the active ingredient is adsorbed on activated carbon and eluted with acid-containing alcohol.

This eluate is neutralized, concentrated under reduced pressure, extracted with n-butanol, and the extract is dried under reduced pressure to obtain a crude powder of MX-A.

In addition to MX-A, this coarse powder contains several MX-A-related substances that have a growth inhibiting effect on Pseudomonas bacteria, but the main component is MX-A.

Examples of MX-A related substances include evachloroform, methanol, 28% ammonia water, n-butanol, and water (
Examples include a substance that gives a spot to RfO, 33 and a substance that gives a spot to RfO945 in silica gel thin layer chromatography using 5:3:2:1:1 (lower layer).

The MX-AORf value under the same conditions is 0.23.

To isolate MX-A from the above crude MX-A, use Sephadex G-15 (manufactured by Pharmacia Fine Chemicals), CM-Sephadex (manufactured by Pharmacia Fine Chemicals), silica gel (manufactured by Merck & Co., Ltd.), Amberlite, etc. Chromatography using an adsorbent or adsorbent resin such as XAD-2 (manufactured by Rohm & Co., Ltd.) is effectively used.

For example, if an aqueous solution of the above crude powder is adsorbed on a silica gel column, eluted with chloroform-methanol-28% aqueous ammonia-water (1:8:2:1), and the active fraction is collected and concentrated under reduced pressure, MX-A A white powder is obtained.

The physicochemical properties of MX-A obtained by the method of Example 3 described below are as follows.

(1) Melting point: 236°C to 239°C (foaming decomposition) (2)
Solubility: Soluble in water, methanol, ethanol, propatool, butanol, but sparingly soluble or insoluble in acetone, benzene, ethyl acetate, chloroform, ethyl ether, etc.

(3) Thin layer chromatography: In silica gel thin layer chromatography, chloroform-methanol-2
8% ammonia water-n-butanol-water (5:3:2:
1:1 lower layer) RfO, 23, chloroform-methanol-28% ammonia water-water (1:8:2:1)R
f O, 53゜n-butanol-acetic acid-water (5:4:4
In thin layer chromatography of cellulose using RfO, 65° (4) specific optical rotation: [α] Show - 59° (c-1%, H
2O).

(5) The ultraviolet absorption curve shows no specific absorption and only terminal absorption.

(6) The optical external absorption curve (potassium bromide tablet) is as shown in FIG. 1, and acid amide bond absorption is clearly observed around 1650 cTL'.

(7) Color reaction: Positive for ninhydrin and Brave-Lieberg reagents, but negative for orcinol, ammoniacal silver nitrate, and Molish color reactions.

(8) Components: Dissolve ice crystals in 5N hydrochloric acid and heat to 110°C.
As a result of hydrolyzing for 48 hours and examining the constituent amino acids using cellulose thin layer chromatography and an amino acid analyzer, α/γ-diaminobutyric acid and leucine were detected.
The molar ratio of both was 4:3.

Dissolve ice crystal 40In9 in 4 ml of methanol and add 0.8
After adding ml of acetic anhydride, the mixture was left at room temperature for 1 hour.

Next, this is dried, dissolved in 2 ml of methanol, and 10 ml of ethyl ether is added to obtain a white precipitate of N-tetraacetylated MX-A.

This precipitate was collected and dissolved again in 2rrtl of methanol,
Add 10ml of ethyl ether and add N-tetraacetyl M.
X-A42m9 was obtained.

The elemental analysis values of N-tetraacetyl MX-A obtained in this way were actually measured C52, 70%, H8, 34%, N1
6.06%, 022.89% (difference), elemental analysis value C52 of N-tetraacetyl MX-A calculated with a molar ratio of α / γ-diaminobutyric acid and leucine of 4:3 and including 2 molecules of water. ,40%, H8,30%, N16.00%,
It is in good agreement with 023.30%.

The N-tetraacetyl MX-A obtained here is processed by the following method,
N-undecamethylated.

That is, 30 kg of sodium hydride was washed with dry ethyl ether, added to 1.5 rnl of dimethyl sulfoxide, and heated to 80°C.

N-tetraacetyl MX-15m9 was added to this, 1 ml of methyl iodide was added, and the reaction was allowed to proceed for 40 minutes at room temperature. The reaction product was extracted with chloroform, concentrated and dried.
Tetraacetyl, N-undecamethyl MX-A6~ was obtained.

An M+ peak was observed at m/e 1061 in the mass spectrum of the crystal.

Therefore, MX-A is leucine 3, α・γ-diaminobutyric acid 4
It is thought that it is a peptide consisting of molecules.

To date, many peptide antibiotics have been reported, but polymyxins (A,
B, C, D, E, M), colistin (A, B), circulin (A, B), polypebutin, cyanosine, and BN-7 described in JP-A-48-56895, JP-A-48
EM-49 (α, β, γ, δ
)It has been known.

The constituent amino acids of these antibiotics and MX-A are the first
As shown in the table.

*1 Antibiotics
cs: D, Gottliebu, P, D.

shaw) Volume 2, p. 255 (1967) *2 The Journal of Antibiotics
iotics: Y, Kimura, etc.) Volume 22, No. 9, Page 449 (1959) *3 Special Publication 16152-1973
Issue Publication *4 The Journal of Biochemistry (T
he J o n al of B iochem
istry: T, 5uzuki, etc.) Volume 54, No. 1, No. 2
5 pages (1963) *5 Experimenta (Exp.
erimentia: K, Hayashi, etc.) 24
Volume 656 (1968) *6 The Journal of Biological Chemistry
logical Chemistry: W, Hausm
an, etc.) Volume 198, page 405 (1952) *7 Japanese Patent Application Publication No. 48-56895 *8 Japanese Patent Application Publication No. 48-4688 *9 It is clear from Table 1 of Publication No. 46-42959 (, MX -A is distinguished from the polymyxin group, colistin group, circulin group, polypebutin, cyanosine, and BN~7 because it does not contain threonine, and EM-49 because it does not contain phenylalanine.
It is distinguished from γ and δ.

EM-49α and EM-49β have the same amino acid composition as MX-A, but the ratio of α/γ-diaminobutyric acid to leucine is 5:3 in the former, while it is 4:3 in the latter.
The difference between the two is obvious in that they are 3 pairs and the latter does not contain fatty acids.

Furthermore, the antibacterial activity of MX-A is similar to that of EM-49α and EM-4.
It is different from the antibacterial activity of 9β.

From the above points, MX-A was determined to be a new antibiotic different from known antibiotics.

The minimum inhibitory concentrations of antibiotic MX-A against representative bacterial strains are shown in Table 2.

Acute toxicity is LD5°50m9 after intravenous injection in mice.
/kg.

On the other hand, 100L of Pseudomonas aeruginosa N-18
D5° dose was injected intraperitoneally into mice, and MX was administered immediately after infection.
A biological test in which 1007 V/kg of -A was subcutaneously administered showed a mouse survival rate of 50%.

Survival of mice not given antibiotics was 0.

The antibiotic MX-A of the present invention is Pseudomonas aeruginosa.
It has antibacterial activity that is specifically effective against A. aeruginosa).

Therefore, the antibiotic MX-A or its physiologically acceptable salts can be used as an antibacterial agent.

For example, 0.3 to 1.0% of the antibiotic MX-A can be blended with a suitable carrier and used as a spray or liquid to disinfect instruments and the environment.

It is also applied to protect against infection by Pseudomonas aeruginosa in various animals.

For example, it can be used as a conventional topical cream or ointment containing 0.3-1.0% of the antibiotic MX-A, or as an injectable, usually 20-200~/kg per day.
can be administered.

Moreover, due to its specific antibacterial spectrum, it can be used as a detection reagent for Pseudomonas aeruginosa and is useful as a starting material for obtaining other antibiotics using chemical, biochemical or microbiological methods. be.

EXAMPLES The method for producing an antibiotic of the present invention will be explained in detail using Examples below, but the scope of the present invention is not limited by these Examples.

Example l MX-A cultured on glycerin broth agar slant
Living! Bacillus circulans strain AV-7 (IF”0
13320 Fine Technology Research Institute No. 1480 with polypeptone 3.0%, meat extract 1.0%, sodium chloride 0.5
% in a liquid medium (pH 7,2) containing
A seed culture solution was obtained by culturing with shaking for 24 hours.

The main fermentation medium (pH 7, 5) Inoculate 1001, 28℃, aeration 10
0% and a rotational speed of 200 revolutions per minute for 66 hours.

Solids were removed from the resulting culture solution by centrifugation, and the supernatant liquid 9
I got 0J.

The supernatant was adjusted to pH 7.0, and Amberlite IRC-
50 (NH4 type) (manufactured by Rohm and Haas) 51
The effective substances were adsorbed on the ion exchange resin.

After washing the resin with 20 J of water, the active fraction 81 in the eluate obtained by elution with 5% aqueous ammonia was passed through a column filled with activated carbon 21 for chromatography.

The activated carbon was washed with IOJ water and 20 J of 0.3N hydrochloric acid, and then eluted with methanol containing 5% hydrochloric acid.

Active fraction 61 in the eluate was concentrated under reduced pressure, neutralized with Amberlite IR-45 (OH type) (manufactured by Rohm & Co., Ltd.), and then concentrated under reduced pressure.

The pH of 500 rrll of the concentrated liquid was adjusted to 10.0 with caustic soda, and extracted with 500 T/l of n-butanol.

When the n-butanol layer was concentrated to dryness, the antibiotic MX-
A crude powder (purity 20%) 152 was obtained.

Example 2 MX-A cultured on glycerin broth agar slant medium
The producing bacterium Bacillus vitellinus AZ-1159 strain (
IFO13296 Microtech Research Institute No. 1203), polypeptone 1.0%, meat extract 0.7%, sodium chloride 0.
A liquid medium (pH 7.2) containing 5% was inoculated and cultured with shaking at 28° C. for 24 hours to obtain a seed culture.

The obtained seed culture solution was mixed with dextrin 2.0%, polypeptone 1.0%,
It was inoculated into 100 J of a fermentation medium (pH 7.5) containing 0.7% meat extract and 0.5% magnesium sulfate, and incubated at 28°C, 100% ventilation, and 200 revolutions per minute.
Cultured for 6 hours.

To the obtained culture solution was added 1 kg of Noiflo Super Cell (manufactured by Jones Manville Products) as an r-filtration aid, and the mixture was thoroughly filtered.

The obtained lachrymal fluid was adjusted to pH 7.0, treated in the same manner as in Example 1, and treated with antibiotic MX-A coarse powder (purity 15%).
:151 was obtained.

Example 3 Coarse powder obtained in Example 1 20. Of water 100rnl
3 cm in diameter and filled with Amberlite CG-50 (NH4 type) (manufactured by Mouss & Co., Ltd.).
When a concentration gradient elution from 0 to 3% was performed using aqueous ammonia adsorbed in a column with a height of 100 cm, MX-A was eluted at a concentration of aqueous ammonia between 1.5% and 2.0%.

Elution curves were determined using the paper disk method using Pseudomonas aeruginosa, and MX was determined using thin layer chromatography.
-A was detected.

The obtained MX-A fraction was concentrated under reduced pressure to give about 6 MX-A
6.52 of a coarse powder containing 0% was obtained.

This coarse powder 6.5S' was mixed with chloroform-methanol-
Diameter 2 filled with silica gel 60 (manufactured by Merck & Co., Ltd.) dissolved in a mixture of 28% aqueous ammonia-water (1:8:2:1).
crfL, was adsorbed in a column with a height of 150 crfL.

When elution was performed using a solution with the same composition as used for adsorption, MX-A was eluted between 550 rfl and 900 m#).

This fraction was concentrated under reduced pressure and freeze-dried to obtain 3 grams of white powder of antibiotic MX-A.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of antibiotic MX-A in potassium bromide tablets.

Claims (1)

[Claims] 1. Antibiotic MX-A, and MX-A has the following physicochemical properties. (1) Melting point: 236°C to 239°C (foaming decomposition) (2
) Solubility: Possibly soluble in water, methanol, ethanol, propatool, and butanol, but sparingly soluble or insoluble in acetone, benzene, ethyl acetate, chloroform, and ethyl ether. (3) Thin layer chromatography: In silica gel thin layer chromatography, chloroform-methanol-2
8% ammonia water-n-butanol-water (5:3:2:
1 Lower layer) RfO123, chloroform-methanol-28% ammonia water-water (1:8:2:1) Rf
O, 53°n-butanol-acetic acid-water (5:4:4
In thin layer chromatography of cellulose using RfO, 65° (4) Specific rotation: [α] -59° (c-1%, H
2O) (5) Ultraviolet absorption curve: No specific absorption is observed and only terminal absorption is shown. (6) Light external absorption curve (potassium bromide tablets): 1650°-1
Acid amide bond absorption is clearly observed in the vicinity. (7) Color reaction: Positive for ninhydrin and Brave-Lieberg reagents, but negative for orcinol, ammoniacal silver nitrate, and Molish color reactions.