JPH06239851A - New antibiotic and its production - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as an antimicrobial agent having anti-Staphylococcus aureus activity. CONSTITUTION:A compound of the formula. This compound is obtained by inoculating a bacterium belonging to the genus Streptomyces, capable of producing lactomycin, such as Streptomyces sp. YL01865P strain (FERM P-13,422) into a medium containing a nutritive source, culturing under an aerobic condition, centrifuging or filtering the prepared culture mixture, then extracting with an organic solvent (e.g. ethyl acetate) and purifying by fractionation. This new antimicrobial compound shows excellent action on Staphylococcus aureus, especially on Staphylococcus aureus of high multi-resistance such as MRSA. Physical and chemical properties of the compound of the formula. Color and property: block or transparent or translucent amorphous thick substance. classification of acidity, neutrality and basicity: neutral. Solubility: soluble in methanol, ethanol, acetone, ethyl acetate, benzene, etc., slightly soluble in water and hardly soluble in hexane.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel antibiotic having anti-staphylococcus aureus activity which is a compound useful as a medicine, especially an antibacterial agent, and a method for producing the antibiotic by a fermentation method.

[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, tobramycin and the like. 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 emerged in a large number in clinical settings, and has become a serious social problem.

[0003]

As is clear from such a situation, the creation of excellent novel antibiotics against Staphylococcus aureus containing MRSA is an extremely urgent and serious problem. Against the above background, the present inventors have been conducting research on substances produced by many naturally occurring microorganisms, and found that they are novel antibiotics belonging to the genus Streptomyces and showing excellent antibacterial activity against Staphylococcus aureus. It was discovered for the first time that a new antibiotic having a strong antibacterial action was produced in a medium by culturing a microorganism capable of producing the substance in the medium, and the present invention was completed by isolating this substance. did. It is also a finding obtained for the first time by the present invention that the novel antibiotic of the present invention has a strong antibacterial activity against Staphylococcus aureus including MRSA and has a completely new chemical structure. Therefore, the present invention provides a novel antibiotic having excellent antibacterial activity against Staphylococcus, and further, the present invention provides a novel production method for obtaining a novel antibiotic. is there.
Further, the present invention provides a new bacterial species producing the above antibiotic.

[0004]

That is, the present invention relates to a novel antibiotic lactomycin represented by the following chemical structural formula.

[0005]

[Chemical 2]

The present invention also relates to Streptomes.
It is a method for producing a novel antibiotic characterized by culturing a lactomycin- producing bacterium belonging to the genus yces ), producing and accumulating lactomycin in the culture, and collecting this substance from the culture. The compound of the present invention can be produced by culturing a novel lactomycin-producing bacterium, 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 such as optical isomers and geometric isomers exist, and the present invention also includes these various isomers. Examples of the strain producing the novel antibacterial substance lactomycin of the present invention include a microorganism Streptomyces sp. YL-01865P strain isolated from soil collected at Hoshisuna coast of Taketomi Island, Okinawa Prefecture. , Has the following mycological properties.

[0007] Streptomyc
es sp.) Microbiological properties of YL-01865P strain: This strain was isolated from soil collected at the Hoshisuna coast of Taketomi Island, Okinawa Prefecture, and the actinomycetes with the above strain number were given. As shown. (1) Morphology This strain grows well in various synthetic and organic media, and the color tone of growth is yellowish ash to yellowish brown. Aerial aerial hyphae are well formed on oatmeal agar medium, starch / inorganic salt agar medium, etc., and their shape is simple branching and shows a spiral shape with closed tips. The color of the aerial mycelium is gray series. Electron microscopic observation revealed that about 50 spores were linked, the spore surface was spiny, and the size was 0.8-1.0 x 0.5-
It is 0.7 μm. No special organs such as sporangia, sclerotium, and sulcus are observed. (2) Properties on various agar media Properties on various agar media are as follows. Unless otherwise specified, the cells were cultured at 27 ° C. for 21 days and observed according to a conventional method. The description of the color tone was based on the color standard (Japan Color Research Institute).

[0008]

[Table 1]

[0009]

[Table 2]

(3) Physiological properties

[Table 3] 1) Growth temperature range 15-40 ° C Optimal growth temperature 24-27 ° C 2) Liquefaction of gelatin Simple gelatin (20 ° C) positive glucose / peptone gelatin (27 ° C) negative 3) Coagulation of defatted milk positive 〃 peptonization positive 4) Nitrate reduction action 5) Starch hydrolysis action is positive 6) Formation of melanin pigment Tyrosine agar negative Peptone / Yeast / Iron agar negative 7) NaCl resistance <9% Note: The growth temperature depends on each temperature (5,10,15,20,24,27,30,33,37,4
0,45,50 ℃) 7 to 21 days, the effect on milk was observed at 37 ℃ 3 to 21 days, otherwise, unless otherwise indicated, after 2 weeks at 27 ℃. The observation results are shown.

(4) Assimilation of carbon source (Pridham-Godlieve agar medium, 27 ° C culture)

[Table 4] L-arabinose-mannose + D-xylose ± melibiose + D-glucose + lactose + D-fructose + D-galactose + sucrose + maltose + inositol + salicin-rhamnose-trehalose + raffinose + glycerin + dextmannitol. + Xanthine- Note: +; grows +-; doubtful growth-; does not grow

(5) Analysis of diaminopimelic acid (DAP) LECHVALIER et al. (LECHVALIER, MP. Et al; PP277-238)
in DIETZ, A et aled., Actinomycete Taxonomy, SIM Sp
According to ecial publication No. 6, 1980), an acid hydrolyzate of this strain was analyzed, and as a result, LL-diaminopimelic acid and glycine were detected.

A summary of the above properties is YL-0186.
In the 5P strain, the aerial hyphae are simple branches and the ends are closed spirals. A chain of about 50 spores was observed under an electron microscope, and the surface of the spores was spiny. The growth is yellowish grey to yellowish brown, and the aerial mycelium is gray, and no soluble pigment or melanin-like pigment is formed. In addition, LL-diaminopimelic acid and glycine were detected by analysis of the acid hydrolyzate of the bacterial cells.

From the above properties, this strain is considered to belong to the genus Streptomyces, and Streptomyces sp. YL-01.
It was named strain 865P. This strain has been deposited as FERMP-13422 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

(Manufacturing Method) In carrying out the method for manufacturing the novel antibiotic of the present invention, a strain producing the compound, Streptomyces sp. YL-01865P strain, is inoculated into a medium containing a nutrient source and aerobically. By developing,
A culture containing the novel target compound of the present invention is obtained. As the nutrient, those known as a nutrient source for actinomycetes may be used. For example, commercially available peptone, meat extract,
Corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, hydrolyzate of casein, fish meal, sodium nitrate, inorganic or organic nitrogen sources such as ammonium nitrate, commercially available sugar-concentrated, Carbon sources such as starch, carbohydrates such as dextrin, sucrose, glucose, maltose, fructose, xylose, rhamnose, mannitol and glycerin or fats can be used.

Further, as metal salts, Na, K, Mg, C
Sulfates such as a, Zn, Fe, etc., hydrochlorides, nitrates, phosphates, carbonates, etc. are added as required. If necessary, valine, leucine, isoleucine, threonine,
Phenylalanine, tryptophan, methionine, lysine, arginine, cysteine, cystine, and other commonly known amino acids, methyl oleate, lard oil, silicone oil, surfactants, and other antibiotic production promoters or defoamers Are used as appropriate. 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, and the culture method may be solid culture or liquid culture. In the case of liquid culture, any of static culture, shaking culture, stirring 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 40 ° C, which is the temperature at which the producing bacterium grows and can produce the compound of the present invention, but is preferably in the range of 25 ° C to 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 if possible. The culture time varies depending on various conditions and is 10 hours to 168 hours, but usually the target compound accumulated in the culture reaches the maximum titer in about 24 hours 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 the culture itself, or the culture filtrate obtained by centrifuging or adding a filter aid to the culture and filtering, ethyl acetate, chloroform, benzene,
Extract by adding an organic solvent immiscible with water such as toluene. 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. More specifically, the target compound is adsorbed by contacting it with a porous adsorption resin such as Amberlite XAD-2, Diaion HP20, Diaion CHP20P or Diaion SP900.

Then, the target substance is eluted using a mixed solution of water with an organic solvent such as methanol, ethanol, acetone, acetonitrile or the like. 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.
On the other hand, 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 target compound. Next, the fraction containing the target compound obtained by each of the above-mentioned operation methods was subjected to column chromatography using a conventional adsorption carrier such as activated carbon, alumina, silica gel, cellulose, or a silica gel-based ODS column. It can be further separated and purified by a conventional method such as high performance liquid chromatography.

[0019]

【Example】

Example 1. Glucose 1.0%, potato starch 2.
A seed medium (pH 7.0) containing 0%, yeast extract 0.5%, polypeptone 0.5%, and calcium carbonate 0.4% was prepared, and each 60 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.
Strains of PYL-01865P strain were scraped off and inoculated, and shake culture was carried out at 28 ° C. for 48 hours to obtain a seed culture solution. Next, as a production medium, the same medium as described above was dispensed by the same method, and 3% of the above seed culture solution was added to the sterilized medium.
The cells were inoculated at the ratio of, and cultured with shaking at 28 ° C. for 72 hours.

The culture broth of 1.4 l thus cultivated was centrifuged at 3,000 rpm for 10 minutes to obtain about 1.2 liter of centrifugation supernatant. 1.2 liters of this centrifugation supernatant (pH 7)
Ethyl acetate was added and shaken well to extract the target antibiotic, lactomycin. Next, this extract was dehydrated and then concentrated to dryness under reduced pressure to obtain 320 mg of YL-0186.
A crude extract of 5P was obtained. To further purify this, it was dissolved in a small amount of ethyl acetate, and a silica gel thin layer plate (Kieselgel 60F ₂₅₄ , manufactured by Merck, 20 mm x 20) was used.
(mm) 2 cm from the bottom, was charged in a strip shape and developed with a solvent of chloroform: methanol (9: 1). This thin layer plate is air-dried after development, and UV lamp (254nm)
After confirming the target band with, scrape off the target part and use the developing solvent (chloroform: methanol (9: 1)).
The desired product was eluted with. The eluted fraction was concentrated under reduced pressure and dried under vacuum to give 35.2 mg of almost pure lactomycin.

Example 2. Cultivation was carried out under the same seed culture medium, production medium and culture conditions as in Example 1, to obtain 1 culture solution of Streptomyces sp. YL-01865P strain.
9 l were obtained. A filter aid (Radiolite 600) was added to this culture solution.
・ Showa Kagaku Kogyo Co., Ltd.) and suction filtered.
5 l of culture filtrate was obtained. 15 l of ethyl acetate was added to this filtrate, and the mixture was shaken well to extract the target compound, lactomycin. The extract was dehydrated and then concentrated to dryness under reduced pressure to obtain 2.7 g of an oily crude lactomycin extract. In order to further purify the desired product from this extract, it was dissolved in a small amount of chloroform and the product was purified by silica gel (Wakogel C-200).
・ Column packed with Wako Pure Chemical Industries, Ltd. (φ2.
3 x 60 cm), chloroform: methanol (2
It was developed at 00: 1).

20 g of the eluate was collected, and each fraction was assayed with an assay plate of Staphylococcus aureus FDA209P to confirm the active fraction. As a result, the desired lactomycin was eluted in fraction numbers 38 to 48. The active fractions were collected and concentrated to dryness under reduced pressure to give 233 mg of crude lactomycin (purity 6
6.82%) was obtained. The purity at this time was quantified using liquid chromatography (HPLC).

At this time, lactomycin has a retention time of 13 minutes 3
It elutes in 3 seconds and the conditions are as follows. Column: STR-ODS (Shimadzu Techno Research Co., φ
4.6 × 250 mm) Elution solvent: Water: A mixture of acetonitrile (= 50: 50) Outflow rate: 1 ml / min Detection wavelength: 230 nm

In order to further purify the crude lactomycin thus obtained, liquid chromatography (HP
Further purification using LC). The method is φ2
A 0 × 250 mm STR-ODS column (manufactured by Shimadzu Techno Research Co.) was used, and a mixed solution of water: acetonitrile (= 50: 50) was made to flow out at a rate of 8 ml per minute as an elution solvent, and an ultraviolet ray of 230 nm Was detected, and the target lactomycin, which was eluted at a retention time of 37 minutes and 30 seconds, was collected. This was concentrated to dryness under reduced pressure to obtain 116 mg of pure lactomycin (purity 97.64%). At this time, the purity was measured under the same conditions as those of the quantitative system by liquid chromatography described above.

The extracted, separated and purified lactomycin has the following physicochemical properties. (1) Color and shape: colorless, transparent or translucent amorphous candy-like substance. (2) Classification of acidic, neutral and basic: neutral. (3) Solubility: It is soluble in methanol, ethanol, acetone, ethyl acetate, benzene, toluene, chloroform, etc., but it is hardly soluble in water and almost insoluble in hexane. (4) Melting point: The shape is amorphous and cannot be measured. (5) UV absorption spectrum: shows absorption only at the end,
In addition, it does not particularly absorb. The absorption curve is as shown in FIG. 1 (solvent: methanol).

(6) Infrared absorption spectrum (KBr):
As shown in FIG. (7) Molecular weight: 308.41792 (8) Mass spectrum (FAB-Mass):
308 [M ^+] (9) Molecular _{Formula: C 18 H 28 0 4 (} 10) Elemental _{analysis: C 18 H 28 0 4 ·} 0.25H 2 O as C (%) H (%) N (%) Found 69.07 9.17 0.00 Calculated value 69.09 9.19 0.00 (11) ¹ H-NMR spectrum (500 MHz, CDC
l ₃ ): The ¹ H-NMR spectrum of lactomycin is as shown in FIG. (12) ¹³ C-NMR spectrum (125 MHz, CDC
l _3): ¹³ C-NMR spectra of the lacto mycin is shown in the FIG. From the above physicochemical properties, the structural formula of lactomycin was determined as follows.

[0027]

[Chemical 3]

[0028]

INDUSTRIAL APPLICABILITY The present invention provides a novel and useful microorganism, and the novel antibacterial compound of the present invention has a strong activity against Staphylococcus aureus, and particularly against highly multidrug-resistant Staphylococcus such as MRSA. Shows excellent antibacterial action. These actions are shown below. In vitro antibacterial activity (MIC) The antibacterial activity of the compound of the present invention against various microorganisms was measured by an agar dilution plate culture method using a Mueller Hinton medium conventionally used for this purpose. Erythromycin was also used as a control.

[0029]

[Table 5] MIC (mcg / ml) Test bacteria (Organisms) Lactomycin Erythromycin (Erythromycin) Staphylococcus aureus CAY17701 (Staphylococcus aureus CAY17701) 1.56> 100 Staphylococcus aureus CAY18201 1.56> 100 Staphylococcus aureus (MRSA) (Staphylococcus aureus 3.1> MRSA)

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of lactomycin.

FIG. 2 shows an infrared absorption spectrum of lactomycin.

FIG. 3 shows a ¹ H-NMR spectrum of lactomycin.

FIG. 4 shows a ¹³ C-NMR spectrum of lactomycin.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kiyoshi Washizaki 1-11-30 Fujimi, Ageo City, Saitama Prefecture

Claims (3)

[Claims] 1. A novel antibacterial compound represented by the following structural formula: 2. A microorganism belonging to the genus Streptomyces and having the ability to produce the novel antibiotic according to claim 1, is cultured in a medium, and the novel antibacterial compound lactomycin is produced and accumulated in the culture. A novel method for producing an antibiotic, characterized by collecting the new target compound (Chemical Formula 1) produced and accumulated from the culture. 3. A Streptomyces (Streptomyces) strain belonging to the genus Streptomyces (Streptomyces) S. P. YL-01865P (FERM P-1342
The method according to claim 2, which is 2).