JPS5928481A - Antibiotic substance y-02039h and preparation thereof - Google Patents

Abstract

PURPOSE:To prepare a novel antibiotic substance Y-02039H capable of exhibiting an antimicrobial activity against various pathogenic germs, byh cultivating a microorganism belonging to the genus Bacillus. CONSTITUTION:A microorganism, e.g. Bacillus sp. Y-02039H (FERM-P No. 6621), belonging to the genus Bacillus, and having the ability to produce an antibiotic substance Y-02039H, is inoculated into a nutrient culture medium and cultivated at 25-37 deg.C and 6-8pH for about 3-4 days under aerobic conditions to collect the aimed antibiotic substance Y-02039H from the resultant culture.

Description

[Detailed description of the invention] The present invention relates to a novel antibiotic and a method for producing the same.

More specifically, two of the present inventions include Y-02039H, an antibiotic having physicochemical properties described below, and Panras (Baci
A method for producing Y-02039H, which comprises culturing an antibiotic Y-02039H-producing bacterium belonging to the genus P. llus ), accumulating Y-02039H in the culture, and collecting Y-02039H from the culture. .

Since the antibiotic Y-02039H of the present invention exhibits antibacterial activity against various pathogenic bacteria, it is effective in treating infectious diseases caused by these bacteria.

Next, the physicochemical properties of antibiotic Y-02039H are shown.

■ Elemental analysis: Carbon 51.78%, Hydrogen 8.24%.

Nitrogen 14.91% ■ Melting point = 223-227 ”c (decomposition) ■
Specific optical rotation: [α] 2o. =-25,5 (C=1.Mectu-L) ■ Infrared absorption spectrum (KBr tablet)
Figure 1 ■ Ultraviolet absorption spectrum (in methanol)
Figure 2■ Solubility: Easily soluble in methanol, soluble in water.

■Positive color reaction: Ninhidori / Negative reaction: Sakaguchi reaction ■When the constituent components are hydrolyzed in 6N-HC-ell0C/20 hours, αeγ-diaminobutyric acid, leucine, and Nenylalanino are produced. At the same time, Cl as an ether soluble component
82003 and β- with molecular formula CIOH2Oo30
Produces hydroxy fatty acids.

The approximate amino acid analysis ratio is α.r-diaminobutyric acid:leucine:7-phenylalanine=7:3:1.

(2) Thin layer chromatography [Silica gel (Merck & Co., Ltd.)] Developing solvent Rf value Developing solvent Y-02039H is a novel peptide antibiotic that is closely related to polymyxins based on physical properties higher than the Rf value.

Next Takumi 1 The antibacterial activity of the target substance of the present invention was measured by the agar plate dilution method.
Table 1 shows the comparison with B).

Table 1 Next, the method for producing the antibiotic Y-02039H of the present invention will be explained.

The target substance of the present invention is y-0203, which belongs to the genus Bacillus.
9Ha-producing bacteria were cultured in a nutrient medium to produce Y-02039.
Y-02039H can be obtained by accumulating Y-8 and collecting Y-02039H from the culture.

Microorganisms used in the production method of the present invention.

Microorganisms that belong to the genus Pacilrus and have the ability to produce Y-02039H can also be used at °C.

Examples of such microorganisms include:

Bacillus Sp. Y
-02039H.

This producing bacterium was isolated from the bottom of a river in Naka, Mito City, Ibaraki Prefecture, and was given the strain number Y-02039H.

The mycological properties are described below.

1) Morphological properties After 28-48 hours of culture at 33c on nutrient agar.

This product is from 05 to 1. OX A bacillus of 2.0 to 5 μm, with lateral flagella, and shows positive Durham staining. A single star-shaped spore is formed in the center or slightly at the end of the bacterial body. Durham staining does not necessarily show positive results when cultured for 72 hours.

2) Growth status on various media - Broth agar plate medium (33 tr, 2-7 days) Forms white to yellowish white, glossy, circular colonies that are rubbery or sticky. No pigment production is observed.

■Glucose broth agar plate medium (3311ml', 2~
7 days) Forms white to yellowish white, glossy, circular colonies that become extremely rubbery (and sticky) and gradually become opaque. No pigment production is observed.

■ Glucose broth liquid culture (30 t: 2-7 days) The culture solution becomes extremely viscous. The medium becomes cloudy throughout. Neither ring formation nor bacterial membrane formation was observed.

3) Physiological properties ■ Nitrate reduction 10 ■ VP test + ■ Methyl red test 10 Production of indole - ■ Production of hydrogen sulfide - ■ Hydrolysis of starch 10 ■ Utilization of citric acid (Simmons medium) No growth ■
Urease - ■ Oxidase + [phase] catalase + Decomposition of 0 tyrosine / - 〇 Deamination of phenylalanine - 〇
Milk culture Produces acid and coagulates ■ Liquefaction of gelatin Ten [phase] Growth temperature: Does not grow below 20C.

It grows at 25-40C, but not at 50C. Optimum growth temperature is 30-37C6 [Phase] It grows at pH 5.5-85. The optimum pH is 7
．． 0-8.0°O anaerobic culture + [phase] Growth on Sabouraud dextrose agar Growth on 10002% azide-added pro-pros - [phase]
Growth in 5% salt-added broth Growth in 17% salt-added broth - ■Usability of sugars Decomposes the following sugars to produce acids.

ri/l/=r-su, fusictose, xylose, mannitol.

No acid production was observed with the following sugars.

L-arabinose, L-rhamnose.

To summarize the above characteristics, strain Y-02039H is a (lateral flagellated) bacillus with Durham-positive motility.

It forms spores and is sticky and rubbery, especially on media containing 1% glucose. It is also facultatively anaerobic, has a positive VP test, a positive catalase reaction, and has nitrate reducing properties.
Grows at medium temperatures of ~40C. A fungal genus with such characteristics is listed in Bergey's Manual of Deter
When searched using Minative Bacteriology 8th edition, strain Y-02039H is BacNlus.
belongs to the genus. Furthermore, the genus Bacillus is classified into two groups: group 122 species and group 11 species.
26 types.

A total of 48 species have been described. Among these, Y-020
When searching for bacterial species similar to the 39H strain, Bacillus
uspolymyxa, Bacillus mace
rans, and Bacillus circulaus. The common characteristics of these three strains are that Durham staining is positive (~easily variable), that they form one spore in the center or slightly at the end of the cell, and that they contain arabinose.

Acid is generated from xylose and mannitol, and indole reaction, phenylalanine deamination reaction, and tyrodinolysis ability are negative.

The properties of strain Y-02039H are consistent with these, except that no acid production from arabinose is observed. However, the above Bergey's Manual of
Deter-minative Bacteriol
According to the description in ogy 8th edition.

Bacillus macerans is negative for casein degradability, does not form sticky or rubbery colonies in a medium containing liglucose, and Bacillus cir
culans is not necessarily positive for the decomposition of motile T-casein, growth in 5% sodium chloride prosthesis, etc., and does not appear sticky or rubbery in glucose-containing media.

It is different from the Y-02039H strain. On the other hand + Bacil
lus poly-myxa is different from Y-02039H in terms of bacterial morphology, growth status on various media, and various physiological properties.
It has many similarities with strains, but differs in the following properties.

From the above, Y-02039H strain is Bacillus
Although it is similar to the description of s poly-myxa, there are some points that do not match.

Therefore, this strain was called Bacjllus sp Y-020.
It was named 39H. Strain Y-02039H was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, with accession number 662.
No. 1 (FERM P-6621).

Bacillus nis p. Y-02039) 1 strain can be mutated by artificial mutation means using ultraviolet rays, Any strain capable of producing -02039H substance can be used in the present invention.

To obtain substance Y-02039H from the pressure of the present invention.

A variety of media are used for normal microbial fermentation. In other words, carbon sources such as glucose, D-
Xylose, fructose, ma/nitol, D-galactose, sucrose.

Dextri, starch, starch syrup, molasses, oils and fats (e.g. soybean oil, olive oil, etc.) are used as appropriate, and nitrogen sources include, for example, powdered bouillon, cornstarch liquor, gluten meal, cottonseed meal, meat extract, Soy flour, peptone, fish meal, dried yeast, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, and other organic or inorganic nitrogen sources are used. Examples of metal salts include Na, K, Mg, Ca, Zn, F
Sulfates, nitrates, chlorides, carbonates, phosphates, etc. may be added as necessary. Vitamins, nucleobases, amino acids, etc. may also be added as appropriate.

As for the culture method, it is common to culture under aerobic conditions, and the temperature is preferably in the range of 25 to 37C.1. Special [30
The optimum value is near U. It is preferable to maintain the pH of the medium in the range of 6 to 8. The culture period is generally about 3 to 4 days, and culture methods include liquid media such as shaking and 5 culture method and deep aeration agitation culture method. It is desirable to use it.

Isolation and purification of the target substance Y-02039H from the culture solution.

Separate and collect the usual fermentation products from the culture solution according to Step 1. For example, it can be isolated by a suitable combination of extraction methods using various organic solvents, chromatography using various adsorbents, or salt formation methods.

Next, the target compound of the present invention and the method for producing the same will be further explained with reference to Examples.

Example Glucose 1.0%, Chrycerol 1.0%, Sucrose 10%, Oatmeal 05%, Soybean flour 20%.

Dried yeast 10%, Casamino acid 0.5%, CaCO30
,1%.

After pouring 50 ml of the pH 7.0 medium into a 50° C. mZ Erlenmeyer flask and sterilizing it in the usual manner, it was grown on a Benet agar slant. C whitebait
Sp. Y-02039H strain is inoculated and cultured at 30C for 48 hours.

Separately, glucose 30% and dextrif 3.0%.

Soy flour 15%, K2HPO40.06%, K)12
PO40,025%.

CoCe2@6H200,0004%, pH7.0
Fill a 500 ml Erlenmeyer flask K60 with the following composition:
Dispense 18 bottles per ml, add 2 drops of ADEKA NOL (manufactured by Asahi Denka Co., Ltd.) as an antifoaming agent, sterilize as usual, then inoculate the above culture solution at a rate of 4%, and inoculate at 30 tZ'. Culture is carried out for 96 hours.

The pH value after the completion of culture is approximately 62. Culture solution 1.0
Add 81K hydrochloric acid to adjust the pH to 2, and stir at room temperature for 30 minutes. Next, add acetate 71.11 and let stand at room temperature for about 40 minutes. Add about 100 g of Radiolite≠600 (Showa Kagaku Kogyo KK) and filter. Collect the washing liquid and rinse with caustic soda.
Return to H7. After removing the generated precipitate, the P solution was subjected to IRC.
50 (NH4) 100 ml (ROHM &...
The column was filled with

Let it absorb. After washing the column with about 500 ml of water, the adsorbed active substance is eluted with 1N-NH4OH.

After concentrating the eluate under reduced pressure and adjusting the concentrated solution to pH 7, it is passed through a column packed with CG 50 (NH4) (Rohm & Haas) 25mZ for adsorption. After washing the column with water, elution was performed using a 0-1N-NH40H concentration gradient method π. The active fractions are collected, concentrated under reduced pressure, and then lyophilized to obtain 42 mg of crude Y-02039H. Further, the crude Y-02039H was subjected to preparative silica gel chromatography [developing solvent: chloroform:methanol:ammonia (20:15:8)]K, and the active fraction was scraped off and eluted with the same solvent. The eluate was concentrated, and the concentrated solution was again subjected to prevalent silica gel chromatography [developing solvent: chloroform: ethanol:
14% ammonia (4:2)], and the active fraction was scraped off and eluted with the same solvent.

The eluate was concentrated under reduced pressure and then lyophilized to yield Y-02039H.
76 mg of white powder is obtained.

[Brief explanation of drawings]

fil Figure 1 shows the infrared absorption spectrum of substance Y-02039H. (2) Figure 2 shows the ultraviolet absorption spectrum of substance Y-02039H. Patent applicant Yamanouchi Pharmaceutical Co., Ltd. Agent Akira Sasaki

Claims (1)

[Claims] 1. Antibiotic Y-020 having the following physical and chemical properties
39H (11 element analysis values: carbon '51.78%, hydrogen 824%
. Nitrogen 1491% (2) Melting point = 223-227 C (decomposition) (3)
Specific luminous intensity: [α] = -25,5 (C = 1. methanol) (4) Optical external absorption spectrum (KBr tablet):
Figure 1 (5) Ultraviolet absorption spectrum (in methanol):
Figure 2 (6) Solubility: Easily soluble in methanol, insoluble in water (7) Color reaction: Positive for di/hydrino reaction, negative for Sakaguchi reaction 2, antibiotic Y-02039H producing bacteria belonging to the genus Bacillus and Y- in the culture.
02039H and Y-02039 from the culture.
A method for producing Y-02039H, which comprises collecting H. 3. The production method according to claim 2, wherein the antibiotic Y-02039H-producing bacteria is Panrus sp. Y-02039H strain.