JP3117294B2 - New antibiotics and their production - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to pharmaceuticals, particularly to antimicrobial Staphylococcus aureus antibiotics, including methicillin-resistant Staphylococcus aureus, which are useful as antibacterial agents, and to a process for producing such antibiotics 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 rokitamicin, kanamycin, gentamicin and tobramycin. Aminoglycoside antibiotics are well known. Many of these antibiotics and compounds obtained by modifying them by chemical synthesis 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 very frequently in clinical settings and is becoming a serious social problem.

[0003]

Under such circumstances, the present inventors have been studying substances produced by many naturally occurring microorganisms, and found that they belong to the genus Arthrobacter and belong to the genus Gram-positive. A new anti-gram-positive antibiotic with MRSA resistance is produced in the medium by culturing a microorganism capable of producing a substance having antibacterial activity against the microorganism, and this substance is isolated. Thus, the present invention has been completed. In addition, the present inventors have found that the novel antibiotic of the present invention has antibacterial activity against Gram-positive bacteria including MRSA and is a compound having a completely novel chemical structure. Accordingly, it is an object of the present invention to provide new antibiotics. Another object of the present invention is to provide a method for producing an antibiotic produced by a fermentation method using a microorganism belonging to the genus Arthrobacter. It is a further object of the present invention to provide a novel microorganism belonging to the genus Arthrobacter.

[0004]

That is, the present invention relates to a novel antibiotic or a salt thereof having the following physicochemical properties.

Physicochemical properties of new antibiotics Molecular weight: 299.413 Mass spectrum (FAB-Mass): 300
[M + H] 298 [M-H] Molecular Formula: C ₁₉ H ₂₅ N0 ₂ ultraviolet absorption spectrum: 253,344Nm to show an absorption maximum, as ¹ H-NMR spectrum of Figure 1: ¹ YL-02729S substance
The H-NMR spectrum is as shown in FIG. 2. Next, the YL-02729S substance can form a salt with an acid. The target compound of the present invention includes these salts. Suitable salts include mineral salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate and the like, formate, acetate, propionate, oxalate, malonic acid. Salt, succinate, fumarate, maleate, lactate, malate, citrate, tartrate, carbonate, picrate, methanesulfonate, ethanesulfonate,
Organic acid salts such as glutamate can be mentioned.

According to the production method of the present invention, a microorganism capable of producing a novel antibiotic YL-02729S belonging to the genus Arthrobacter can be cultured in a medium, and the microorganism can be produced and accumulated in the culture. A method for producing a novel anti-Staphylococcus aureus antibiotic, comprising culturing a microorganism capable of producing the above-mentioned novel antibiotic in a medium, producing the compound in a culture, and then collecting the compound. It is.
The strain producing the novel compound having antibacterial activity of the present invention is a microorganism isolated from soil collected on Kalimantan Island, Indonesia and has the following mycological properties.

Microbiological Properties 1) Morphology Cells cultured on a broth agar medium at 33 ° C. for 5 days show polybacteria to cocci (polyplasia), and motile cells may be observed. Gram stain is indeterminate and no spores are observed. 2) Cultural properties On the broth agar medium, milky white colonies are observed, and no gliding properties are observed. After culture for 24 hours in a heart infusion liquid medium, the upper part of the medium was observed to be turbid, and after 5 days of culture, the entire medium was turbid and a film was also observed. In the medium of litmus milk, the medium became acidic from about the fourth day.

3) Physiological properties Oxidase: Negative Catalase: Positive urease: Positive Indole generation: Negative hydrogen sulfide generation: Negative Arginine resolution: Positive intracellular accumulation of poly-β-hydroxybutyrate: Negative OF test: O-type NaCl addition Growth on medium: grows at 3% but does not grow above 4% Citric acid availability: negative Sodium nitrate availability: positive Coagulation of skim milk: negative Peptoneization of skim milk: positive Nutritional requirement: none Growth Temperature: 15 to 37 ° C Growth pH: 5 to 8.5 Anaerobic culture: negative Litmus milk: peptone without coagulation Liquefaction of gelatin: positive 4) Availability of carbon source

[0009]

[Table 2] Utilization of sugar Generation of acid Ｌ L-arabinose ± NT D-xylose ++ D-glucose + + D-mannose + NT D-fructose + NT D-galactose + NT maltose + ± sucrose--lactose-+ trehalose + NT D-mannitol ± ± glycerin + NT melibiose-NT ninitol + NT inositol Salicin-NTL-raffinose-NTL-rhamnose-NT Starch + NT +: Positive ±: False positive-: Negative NT: Not tested

(5) GC content of DNA (by HPLC method) G + C (mol%) = 67.4 To summarize the above microbiological properties, this strain has an indeterminate Gram-staining property, and is characterized by bacilli to cocci in various media. Has polymorphism and motility. The growth temperature range is 15 ~ 37 ℃,
The oxidase test is negative, the catalase test is positive, and the test results for hydrogen sulfide production and indole production are negative. The GC content of the DNA is 67.4 mol% (HPLC method).

[0011] Bacteria having such properties are
Baergey's Manual of Determinative Bacter 8th edition (Baergey's Manual of Determinative Bacter
iology, 8th ed., 1975), and the Birdies Manual of Systematic Bacteriology (Be
rgey's Manual of Systematic Bacteriology, 1984),
And other literatures, the strain was determined to be a bacterium belonging to the genus Arthrobacter. Next, the properties of this strain were compared with those of known strains belonging to the genus Arthrobacter according to the above-mentioned literatures, but no bacterial strain was found that matched the physiological properties, sugar availability, and the like.

Therefore, this strain was named Arthrobacter sp. YL-02729S. This strain was sent to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology
Deposited as 2).

(Production method) In carrying out the method for producing the novel antibiotic of the present invention, a medium containing a nutrient source of the strain producing the compound, Arthrobacter sp. YL-02729S, is inoculated to grow aerobically. By letting
A culture containing the novel compound of the present invention is obtained. As nutrients, those known as bacterial nutrient sources may be used. For example, commercially available peptone, meat extract,
Inorganic or organic nitrogen sources such as corn steep liquor, cottonseed flour, peanut flour, soy flour, yeast extract, NZ-amine, casein hydrolyzate, fish meal, sodium nitrate, ammonium nitrate, commercially available molasses, starch , Dextrin, sucrose, glucose, maltose, fructose, xylose, rhamnose, mannitol, glycerin and other carbohydrates or carbon sources such as fats.

As metal salts, Na, K, Mg, C
Sulfates, hydrochlorides, nitrates, phosphates, carbonates and the like such as a, Zn, and Fe are added as needed. If necessary, valine, leucine, isoleucine, threonine,
In addition to phenylalanine, tryptophan, methionine, lysine, arginine, cysteine, cystine, etc., commonly known amino acids, and antibiotic generation promoting substances such as methyl oleate, lard oil, silicone oil, surfactants and defoamers Is appropriately used. Other than these, any can be used as long as it is utilized by the producing bacteria and is useful for producing the novel antibiotic of the present invention. The culture may be performed in the same manner as a general antibiotic production method, and the culture method may be solid culture or liquid culture.

In the case of liquid culture, any of stationary culture, stirring culture, shaking culture and the like may be performed, but aeration and stirring culture is particularly preferable. The cultivation temperature can be appropriately changed within the temperature at which the producing bacteria grow and produce the compound of the present invention, that is, in the range of 15 ° C to 37 ° C. The pH of the medium can be appropriately changed within the range of 4 to 9,
8 is preferred. The cultivation time depends on various conditions.
10 hours to 168 hours, but usually 24 hours to 120 hours
The target compound that accumulates in the culture in about an hour reaches the highest titer. In order to collect the target compound from the culture, the usual extraction, separation, and purification means used for metabolites produced by microorganisms are appropriately used. The target compound in the culture can be used as it is, or as a culture filtrate obtained by centrifugation or by adding a filter aid to the culture and filtering, and adding an organic compound that is immiscible with water such as ethyl acetate, chloroform, benzene, and toluene. Add solvent and extract.

The target compound can be extracted by bringing the culture filtrate into contact with an appropriate carrier, adsorbing the target compound in the filtrate, and then eluting with a suitable solvent. More specifically, for example, Amberlite XAD-2,
The target compound is adsorbed by bringing it into contact with a porous adsorption resin such as Diaion HP20, Diaion CHP20P or Diaion SP900. Then methanol,
The target substance is eluted using a mixture of water and an organic solvent such as ethanol, acetone, or acetonitrile. Needless to say, the mixing ratio of the solvent at this time is a value at which the target compound can be eluted most efficiently. That is, by increasing the solvent ratio stepwise or continuously from a low concentration to a high concentration, a fraction having a higher ratio of the target compound can be obtained.

When extracting with an organic solvent such as ethyl acetate or chloroform, these solvents are added to the culture filtrate and shaken well to extract the desired compound. Next, the fraction containing the target compound obtained by each of the above-mentioned procedures is used for column chromatography using a conventional adsorption carrier, for example, activated carbon, alumina, silica gel, cellulose or the like, or silica gel ODS reverse phase carrier. Further separation and purification can be achieved by conventional methods such as high performance liquid chromatography using a column.

[0018]

【Example】

Example 1 A seed medium (pH 7.0) containing 1.0% glucose, 2.0% potato starch, 0.5% yeast extract, 0.5% polypeptone, and 0.4% calcium carbonate was prepared, and each of the seed medium was prepared to have a pH of 60%.
Aliquots of 500 ml were dispensed into 500 ml Erlenmeyer flasks. This medium was sterilized at 120 ° C. for 20 minutes, and then grown well on Bennett agar, Arthrobacter sp. YL-0.
The cells of the 2729S strain were scraped off and inoculated.
Shaking culture was performed for a time to obtain a seed culture solution. Next, as a production medium, the same medium as described above was dispensed in exactly the same way.
Inoculate the above-mentioned seed culture at a rate of 3% into a sterilized one.
The cells were cultured at 8 ° C for 72 hours.

[0019] The culture medium of the thus-3 was cultured l 6
The mixture was centrifuged at 000 rpm for 10 minutes to obtain about 2.5 l of a centrifuged supernatant. To this centrifuged supernatant (pH 7), 3 l of ethyl acetate was added, and the mixture was shaken well to extract the desired YL-02729S substance. Next, the ethyl acetate extract was thoroughly dehydrated with sodium sulfate and concentrated to dryness under reduced pressure to obtain 963.3 mg of YL-
A 02729S crude extract was obtained. For further purification, this was dissolved in about 5 ml of ethyl acetate, and 10 silica gel thin-layer plates (Kieselgel 60F254, Merc
(Company k, 20 cm × 20 cm) was charged into a band at a position 2 cm from the bottom, and developed with a solvent of benzene: acetone (6: 4).

This thin layer plate is air-dried after being developed, and after confirming the band of the target substance with a UV lamp (254 nm), the portion is scraped off and ethyl acetate: methanol (9: 1).
Eluted the desired product. The eluted fraction was concentrated under reduced pressure and dried under vacuum to obtain 32.6 mg of crude YL-02729S substance. For further purification, about 0.3
was dissolved in ml of ethyl acetate, one silica gel thin layer plates (Kieselgel 60F254, Merck Co., 2
(0 cm × 20 cm), charged in a band shape from the bottom to 2 cm, chloroform: methanol: ammonia (9:
1: 0.1).

The thin layer plate was air-dried after development, and after confirming the band of the target substance with a UV lamp (254 nm), the portion was scraped off and the target substance was eluted with benzene: acetone (6: 4). The eluate was concentrated to dryness under reduced pressure and then dried in a vacuum desiccator overnight to obtain pure YL-026.
11.0 mg of 32S material was obtained.

The above-mentioned extracted, separated and purified YL-026
The 32S material has the following physicochemical properties. Molecular weight: 299.413 Mass spectrum (FAB-Mass): 300 [M
+ H] 298 [M-H] Molecular Formula: C ₁₉ H ₂₅ N0 ₂ ultraviolet absorption spectrum: 253,344Nm to show an absorption maximum, is as Figure 1.

¹ H-NMR spectrum: YL-0272
The ¹ H-NMR spectrum of the 9S substance is as shown in FIG. ¹³ C-NMR spectrum: ^{13 of} YL-02729S substance
The C-NMR spectrum is as shown in FIG. Color and shape: white or slightly yellow amorphous translucent solid. Acid, neutral, basic classification: neutral. Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate, chloroform, etc., but hardly soluble in water, and hardly soluble in hexane. From the above physicochemical properties, the following A or B is suggested as the chemical structure of the YL-02729S substance.

[0024]

Embedded image

Example 2 A seed culture medium and a production medium were prepared in exactly the same manner as in Example 1, and cultured in the same manner. 5 l of the culture solution thus cultured was centrifuged at 6000 rpm for 10 minutes to obtain about 3.5 l of a centrifuged supernatant. The obtained centrifugal supernatant (p
H7) was extracted with 4 l of ethyl acetate, and the organic layer was concentrated.
A crude extract of L-02729S material was obtained. Then, the mixture was subjected to silica column chromatography (Wakogel C-300, manufactured by Wako Pure Chemical Industries, Ltd.), and benzene: acetone (= 9:
It was developed in 1) and fractionated into 5 ml fractions.
As a result, the target object is 1 from fraction number 87.
09 eluted.

The fractions were collected, concentrated to dryness, and dried under vacuum overnight to obtain 102.6 mg of the desired product. Then, it was subjected to ODS-HPLC (STR-ODS, manufactured by Shimadzu Techno-Research) to give a 0.05 M phosphate buffer (pH 4.3).
5): Tetrahydrofuran: methanol (= 30: 5:
The peak having activity developed in step 65) was collected. As a result, 15.7 mg of pure YL-02729S substance was obtained.

[0027]

Industrial Applicability The compound of the present invention provides a novel and useful microorganism and has activity against various Gram-positive bacteria.
Among them, it also exhibits antibacterial activity against staphylococci including highly multidrug resistance such as MRSA. These effects will be described below. Antimicrobial activity in vitro (MIC) The antimicrobial activity of YL-02729S substance against various microorganisms is measured. This measurement was performed by an agar dilution plate culture method using Mueller Hinton medium.

[0028]

[Table 3]

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a YL-02729S substance.

FIG. 2 shows a 1H-NMR spectrum of a YL-02729S substance.

FIG. 3 shows a 13 C-NMR spectrum of a YL-02729S substance.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 1/20 C12R 1:06) (C12P 17/12 C12R 1:06) (72) Inventor Shigeru Miyazaki Fujimi, Konosu City, Saitama Prefecture 1-25, Machi (72) Inventor Yasuhito Suzuki Casa de Familia 2F, 1-12-5 Yanagibashi, Taito-ku, Tokyo (72) Inventor Tatsusuke Tokunaga 2-5-1-702 Inodai, Toride-shi, Ibaraki Pref. 72) Inventor Bud Rumanau Indonesia Jakarta Jakarta Selatan Artee 004 Aruvy 006 Caribata Indah Block Dee 18 (72) Inventor Ratna Murni Lantiatomojo Indonesia Jakarta Jakarta 11510 Artee 008 Art Vee 009 Complex Greenville Block O Number 12 (72) 58) Field surveyed (Int. Cl. ⁷ , DB name) C07D 215/60 C12P 17/12 CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] (1) The following formula : Antibiotic YL-02729S substance or a salt thereof shown. 2. A belongs to Arthrobacter (Arthrobacter) genus, culturing a microorganism having an ability to produce antibiotic YL-02729S material of claim 1 to the culture medium, it was produced and accumulated in the culture anti preparation of Y L-02729S material you and collecting the raw material YL-02729S material. 3. The method according to claim 2, wherein the microorganism belonging to the genus Arthrobacter is Arthrobacter sp. YL-02729S (FERM BP-6326) . 4. The YL-02729S substance according to claim 1.
Can be produced, Arthrobacter
ter) SP YL-02729S (FERM BP-6
326).