JPH06340651A - Antibiotic and its production - Google Patents

Abstract

PURPOSE: To provide new antibiotics having an antibacterial activity, particularly an anti-MRSA activity, which are useful as medicines.

CONSTITUTION: An antibiotic YL-02905S-A of formula I and an antibiotic YL-02905S-B of formula II are provided. The antibiotics belonging to genus Cytophaga are obtained by culturing microorganisms such as Cytophaga SP- YL-02905S in a medium to produce the compd. in the culture, accumulating the compd. and removing the produced and accumulated compd. from the culture.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

The present invention relates to methicillin-resistant Staphylococcus aureus (M) which is a compound useful as a medicine, especially as an antibacterial agent.
And an anti-staphylococcus aureus antibiotic containing RSA) and a method for producing the antibiotic by fermentation.

[0002]

2. Description of the Related Art Conventionally, various antibiotics produced by microorganisms include β-lactam antibiotics such as penicillin, cephalosporin and carbapenem, macrolide antibiotics such as erythromycin, josamycin and rokitamycin, kanamycin, gentamicin and tobramycin. Aminoglycoside antibiotics are well known. Many of these antibiotics and compounds obtained by improving them by chemical synthetic methods have been effectively used as clinically very useful antibacterial agents. However, in recent years, methicillin-resistant Staphylococcus aureus (MRSA), which has a high degree of resistance to these known antibiotics, has appeared in the clinical field in a large number and is becoming a serious social problem.

[0003]

Under these circumstances, the present inventors have been studying substances produced by many naturally occurring microorganisms and found that they belong to the genus Cytoferga and are Gram-positive. By culturing a microorganism having the ability to produce a substance having antibacterial activity against fungi in a medium, a new anti-Gram-positive bacterial antibiotic having an antibacterial action is produced in the medium. The present invention has been completed. Further, the novel antibiotic of the present invention is MRS
It was discovered that the substance has antibacterial activity against Gram-positive bacteria including A and has a completely new chemical structure. Therefore, the object of the present invention is to provide a novel antibiotic having useful antibacterial activity. Another object of the present invention is to provide a novel production method for obtaining a novel antibiotic, and further to provide a novel microorganism producing the above antibiotic.

[0004]

That is, the present invention provides a novel antibiotic YL-02905S represented by the following chemical structural formula.
-A and YL-02905S-B.

[0005]

[Chemical 2]

The present invention also relates to a Cytofer
haga) YL-02905S-A and YL belonging to the genus
-02905SB-B-producing bacterium was cultivated, and YL-
YL-02905S-A and YL-02905S-B characterized by accumulating 02905S-A and YL-02905S-B and isolating and collecting this material from the culture.
Manufacturing method. The compound of the present invention is a novel YL-029.
It can be produced by culturing 05S-A and YL-02905S-B producing bacteria, producing and accumulating the compound in the culture, and collecting the compound from the culture. Furthermore, since the compound of the present invention has an asymmetric carbon and a double bond, various isomers exist. YL-02905S-A and YL-0290 having novel antibacterial activity of the present invention
As a 5S-B-producing strain, for example, a microorganism Cytophagas sp. (Cytophagas sp.) Isolated from soil collected in Kalimantan Island, Indonesia
p. ) YL-02905S strain can be mentioned. The mycological properties will be described below.

Cytophaga s
p.) Microbiological properties of YL-02905S strain 1) Morphology Cells cultured on broth agar at 28 ° C for 5 days
Gram stain is negative and motile with 0.7-1.2 × 3.5-12.5 μm bacilli. In addition, formation of spores and microcysts is not observed. 2) Growth conditions on various media On transfection agar media, translucent, pale yellow-brown colonies are formed. The area around the colony is raised and gliding. In addition, formation of soluble pigment is not observed. In broth stab culture, the upper part of the medium is suspended and forms a film. Cultured in litmus milk shows weak acidity. 3) Physiological properties

[0008]

[Table 1] Nitrate reduction Positive Denitrification reaction Negative MR test Negative VP test Negative Indole formation Negative hydrogen sulfide formation Negative Starch hydrolysis Positive Utilization of citric acid Negative pigment formation Negative urease Positive oxidase Positive catalase positive Growth temperature range 15-40 ° C Optimal growth temperature 24-37 ° C Growth pH range pH6-9 Optimum growth pH pH6-8 Growth under anaerobic conditions Negative OF test Non-degradable arginine degradation reaction positive Negative poly-β-hydroxybutyrate intracellular accumulation Carotenoid production Growth in broth medium with negative NaCl growth occurs at 2% but not at 3% 4) Availability of carbon source

[0009]

[Table 2] Utilization of sugar (degradability) L-arabinose + D-xylose + D-glucose + D-mannose + D-fructose + sucrose + inosit + rhamnose + raffinose ± D-mannitol + D-galactose + maltose + trehalose + lactose + D-sorbit + salicin + salicin. Meribiose + Glycerin + Starch + Xanthine + Chitin- +: Positive, ±: False positive,-: Negative

5) Biochemical properties It has MK-7 as a cell isoprenoid quinone. Summarizing the above microbiological properties, this strain is a gram-negative aerobic bacillus that is motile and gliding on agar medium. Growth temperature range is 15-40 ℃, oxidase test, catalase test, nitrate reducibility, gelatin liquefaction, starch degradability, acid production in glucose medium is positive, and OF test result is non-degradation It is a type. On the other hand, the production of gas in glucose medium, the production of hydrogen sulfide, the production of indole, and the VP test result are negative.
In addition, intracellular accumulation of poly-β-hydroxybutyrate was not observed. As cell isoprenoid quinone,
It has MK-7. Bacteria having such properties are used in the Bergey's Manual of Systematic Bacteriolo.
gy, 1989) and other literatures, the strain was judged to be a bacterium belonging to the genus Cytophaga.

Further, based on the above-mentioned properties, the present strain was compared and examined with various known literatures of Cytoferga genus, but no similar bacterial species was found. Therefore,
This strain was judged to be a new species of the genus Cytoferga, and Cytophagasp YL-02905S
I named it. This strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science, on January 12, 1993.

(Manufacturing Method) In carrying out the method for manufacturing a novel antibiotic of the present invention, a strain producing the compound, Cytoferga sp. YL-02905S strain, is inoculated into a medium containing a nutrient source and aerobically developed. By doing so, a culture containing the novel target compound of the present invention can be obtained. As the nutrients, those known as a nutrient source for bacteria may be used. For example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, casein hydrolyzate, fish meal, sodium nitrate, ammonium nitrate and other inorganic or organic substances. Nitrogen sources, commercially available molasses, starch, dextrin, sucrose, glucose, maltose, fructose, xylose, rhamnose, mannitol, glycerol, etc., or carbon sources such as fat can be used.

Further, as metal salts, Na, K, Mg and Ca are used.
, Zn, Fe and other sulfates, hydrochlorides, nitrates, phosphates,
Carbonate etc. are added as needed. If necessary, valine, leucine, isoleucine, threonine, phenylalanine, tryptophan, methionine, lysine,
In addition to arginine, cysteine, cystine, etc., commonly known amino acids, and antibiotic production promoters or antifoaming agents such as methyl oleate, lard oil, silicone oil, and surfactants are appropriately used. In addition to these, any can be used as long as it can be used by the producing bacterium and is useful for the production of the novel antibiotic of the present invention. The culture method may be the same as a general antibiotic production method.
The culture method may be solid culture or liquid culture. In the case of liquid culture, any of static culture, stirring culture, shaking culture and the like may be carried out, but aeration stirring culture is particularly preferable. The culturing temperature can be appropriately changed within the range of 15 ° C to 37 ° C, that is, the temperature at which the producing bacterium grows to produce the compound of the present invention, but is preferably about 32 ° C.

The pH of the medium can be appropriately changed within the range of 4 to 9, but pH 6 to 8 is preferable. The culture time varies depending on various conditions and is 10 hours to 168 hours, but usually 2
The target compound accumulated in the culture reaches the maximum titer in about 4 to 120 hours. In order to collect the target compound from the culture, the usual means for extraction, separation and purification used for metabolites produced by microorganisms are appropriately used. The target compound in the culture is an organic compound that is immiscible with water such as ethyl acetate, chloroform, benzene, and toluene in the culture filtrate obtained by filtering the culture as it is or by centrifuging or adding a filter aid to the culture. Extract by adding a solvent. Further, the target compound can be extracted by bringing the culture filtrate into contact with an appropriate carrier to adsorb the target compound in the filtrate and then eluting with a suitable solvent. For example, Amber Light XA
D-2, Diaion HP20, Diaion CHP2
The target compound is adsorbed by contacting it with a porous adsorption resin such as 0P or Diaion SP900.

Then, the desired product is eluted using a mixed solution of water with an organic solvent such as methanol, ethanol, acetone or acetonitrile. Needless to say, the mixing ratio of the solvent at this time is set to a value at which the target compound can be most efficiently eluted. That is, by increasing the solvent ratio stepwise or continuously from a low concentration to a high concentration, it is possible to obtain a fraction having a higher ratio of the target compound.
When extracting with an organic solvent such as ethyl acetate or chloroform, add these solvents to the culture filtrate and shake well to extract the target compound. Next, the fraction containing the target compound obtained by each of the above-mentioned operation methods is subjected to column chromatography using a conventional adsorption carrier such as activated carbon, alumina, silica gel, or cellulose, or a silica gel-based ODS reverse phase carrier. It can be further purified and separated by a conventional method such as high performance liquid chromatography using a column.

[0016]

【Example】

Example 1 YL-02905S-A substance Glucose 1.0%, potato starch 2.0%, yeast extract 0.5%, polypeptone 0.5%, calcium carbonate 0.4% seed medium (pH 7.0). Create 60 each
Each ml was dispensed into a 500 ml Erlenmeyer flask. This medium was sterilized at 121 ° C. for 20 minutes, and then grown well on Bennett's agar, Cytoferga SP YL-02.
The strain 905S strain was scraped and inoculated, and shake culture was performed at 28 ° C. for 72 hours to obtain a seed culture solution. Then, as a production medium, the same medium as described above was dispensed by the same method, and the sterilized medium was inoculated with the seed culture solution at a rate of 3%.
The cells were cultured with shaking at 72 ° C for 72 hours.

An equal amount of acetone was added to 2.5 l of the culture solution thus cultivated, and the mixture was shaken for 10 minutes. This acetone extract was centrifuged at 6,000 rpm for 15 minutes. The centrifugation supernatant was concentrated under reduced pressure to remove acetone. The obtained concentrated liquid was adjusted to pH 7.0 and extracted with 3 l of ethyl acetate. Glauber's salt was added to the organic layer to remove water, and then ethyl acetate was concentrated to dryness under reduced pressure to obtain YL-02905S-A and YL-.
A crude extract of 02905S-B was obtained. This crude extract 2.
Column chromatography (φ21 × 85) of 64 g of silica gel (Wakogel C-200, manufactured by Wako Pure Chemical Industries, Ltd.)
0 mm), chloroform-methanol (= 98:
2) Developed with solution. The eluate was fractionated into 19 g fractions.

Staphylococcus aureus FDA
When the antibacterial activity against 209P was examined as an index, the target substance was eluted in fraction numbers 99 to 152. This fraction was collected and concentrated to dryness to obtain 294 mg of crude purified product. Next, the crude product was subjected to preparative liquid chromatography to collect the active peak eluting at a retention time of 28 minutes prior to the YL-02905S-B substance, extracted with ethyl acetate, and concentrated to dryness. 30 mg of YL-0290
5S-A material was obtained.

The conditions of liquid chromatography (HPLC) are as follows. Column: STR-ODS (Shimadzu Techno Research Co., φ
20 × 250 mm Elution solvent: Water: Methanol: Acetonitrile: Tetrahydrofuran (= 45: 30: 10: 15) mixed solution Outflow rate: 8 ml / min Detection wavelength: 280 nm

The above-mentioned extracted, separated and purified YL-029
The 05S-A substance has the following physicochemical properties. (1) Color and shape: white or slightly yellow amorphous semitransparent solid. (2) Classification of acidic, neutral and basic: neutral. (3) Solubility: Soluble in methanol but hardly in water, chloroform and benzene, and almost insoluble in hexane. (4) Ultraviolet absorption spectrum: 277.6, 285.
It shows the absorption maximum at 6,299,313.4 nm and is as shown in Fig. 1 (solvent: methanol).

(5) Molecular weight: 540.741 (6) Mass spectrum (FAB-Mass): 541
[M + H] ⁺ , 563 [M + Na] ⁺ , 539 [M-
^H] - (7) Molecular _{Formula: C 33 H 48 0 6 (} 8) 1 H-NMR spectrum (500 MHz, CDC
l ₃ ): ¹ H-NMR spectrum of YL-02905S-A substance is as shown in FIG. (9) ¹³ C-NMR spectrum (125 MHz, CDC
l _3): ¹³ C-NMR spectrum of YL-02905S-A substance is as Figure 3. From the above physicochemical properties, the chemical structural formula of the YL-02905S-A substance was determined as follows.

[0022]

[Chemical 3]

Example 2 YL-02905S-B substance YL-02905S crude extract 294 obtained in Example 1
mg was collected using preparative liquid chromatography under the same conditions as in Example 1. At this time YL-029
The eluted fraction of YL-02905S-B substance (holding time 38 minutes), which elutes after the 05S-A substance (holding time 28 minutes), was extracted with ethyl acetate and concentrated to dryness to 70
Obtained mg of YL-02905S-B material. The extracted, separated and purified YL-02905S-B substance has the following physicochemical properties. (1) Color and shape: white or slightly yellow amorphous semitransparent solid. (2) Categorization of acidic, neutral and basic: Neutral (3) Solubility: Soluble in methanol but hardly soluble in water, chloroform and benzene, hardly soluble in hexane. (4) Ultraviolet absorption spectrum: Absorption maximum at 278.4 nm, as shown in FIG. 4 (solvent: methanol).

(5) Molecular weight: 540.741 (6) Mass spectrum (FAB-Mass): 563
[M + Na] ⁺ , 539 [MH] ^- (7) Molecular formula: C ₃₃ H ₄₈ O ₆ (8) ¹ H-NMR spectrum (500 MHz, CDC
l _3): ¹ H-NMR spectrum of the YL-02905S-B material is as Figure 5. (9) ¹³ C-NMR spectrum (125 MHz, CDC
l _3): ¹³ C-NMR spectrum of YL-02905S-B material is as Figure 6.

From the above physicochemical properties, YL-0290
The chemical structural formula of the 5SB substance was determined as follows.

[0026]

[Chemical 4]

[0027]

INDUSTRIAL APPLICABILITY The present invention provides a novel and useful microorganism and has an antibacterial action against Staphylococcus which is active against various Gram-positive bacteria and has a high multidrug resistance such as MRSA among others. These actions are shown below. Antibacterial activity in vitro The antibacterial activity of the YL-02905S-B substance against various microorganisms was measured by the paper disk (TOYOROSHI, diameter 10 mm) method. Incidentally, YL-0290
The 5S-B substance was measured by impregnating a paper disc with 100 μg / ml methanol solution.

[0028]

[Table 3] Test bacteria inhibition circle diameter (mm) Staphylococcus aureus FDA209P (Staphylococcus aureus FMR209) 12.5 Staphylococcus aureus (MRSA) 16.0

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a YL-02905S-A substance.

FIG. 2 shows a ¹ H-NMR spectrum of YL-02905S-A substance.

[3] ¹³ C-NMR of the YL-02905S-A substance
The spectrum is shown.

FIG. 4 shows an ultraviolet absorption spectrum of YL-02905S-B substance.

FIG. 5 shows a ¹ H-NMR spectrum of YL-02905S-B substance.

FIG. 6 ¹³ C-NMR of YL-02905S-B substance
The spectrum is shown.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasuyo Shimizu 2-1-30 Fujimidai, Kunitachi, Tokyo Metropolitan Government 2-102 (72) Inventor Takeo Sugawara 1-4-30 Otaniba, Urawa City, Saitama Prefecture Roomy Urawa No. 208 (72) Inventor Tatsusuke Tokunaga 2-5-1-702 Inodai, Toride-shi, Ibaraki (72) Inventor Benjamin Steawan Indonesia Jakarta 10310 Jalan Lengbang NO. 11 (72) Inventor Ratna Mulni Lanti Atomojo Indonesia Jakarta 11510 Aulti 008 Arvie 009 Complex Greenville Block Oh Number 12

Claims (3)

3 [Claims] 1. A novel antibacterial compound represented by the following structural formula: 2. Cytophaga
A microorganism belonging to the genus and capable of producing the novel antibacterial compound according to claim 1 is cultured in a medium, and the novel antibacterial compounds YL-02905S-A and YL-02905 are contained in the culture.
A method for producing a novel antibacterial compound, which comprises producing and accumulating SB, and collecting the produced and accumulated novel target compound from a culture. 3. Cytophaga
Strains belonging to the genus Cytophag (Cytophag
The production method according to claim 2, which is a) SP YL-02905S (deposited on January 12, 1993, at the Institute of Life Science and Technology).