KR100824696B1 - Novel antibiotic compound derived from streptomyces sp - Google Patents

Abstract

A disaccharide compound is provided to treat and prevent plant diseases effectively generated by a Xanthomonas campestris pv. strain, thereby being useful for reducing harmful effects in an agricultural field generated by the strain. A disaccharide compound is represented by the formula(1), which is derived from Streptomyces KBJ-2-45(deposition no. KCCM 10455), and shows antibacterial activity on Xanthomonas campestris pv campestris. An anti-bacterial agent comprises the compound of the formula(1) as an effective ingredient. A method for preparing the compound of the formula(1) comprises a step of culturing the Streptomyces KBJ-2-45.

Description

Novel Antibiotic Compound Derived from Streptomyces sp.

Figure 1A shows the results for the cell growth curve of Streptomyces KBJ-2-44 and KBJ-2-45 [■: KBJ-2-44, ●: KBJ-2-45].

Figure 1B shows the result of monitoring the pH change during the incubation process of Streptomyces KBJ-2-44 and KBJ-2-45 [●: KBJ-2-44, ■: KBJ-2-45].

Figure 2 is a Streptomyces KBJ-2-44 and 2-45 KBJ-janto Pseudomonas Kam paste less paste-less hospital-type KAM (Xanthomonas of campestris pv . campestris ) shows the results of observing growth inhibition.

Figure 3 shows the comparison of the antimicrobial activity of KBJ-2-45 distilled water extract and Bordeaux solution for Xanthomonas Campestris pathogenic Campestris.

Figure 4 shows (2S, 4S, 5R) -3 (((2R, 4S, 5S) -2,4-dihydroxy-2,5-bis (hydroxymethyl) -tetrahydrofuran-3-siloxy) Schematic extraction of the separation of methyl) -5-((S) -1,2-dihydroxyethyl) -tetrahydrofuran-2,4-diol).

Figure 5 shows the dodge spectrum of Streptomyces KBJ-2-45.

The Gram-negative bacterium Xanthomonas is the most common plant pathogen and causes various diseases in various plants (Lenyns et al. al ., 1984). Especially Xanthomonas campestris) hospital type (pathovar, pv) Calm Fest lease (campestris) broccoli (Brassica spp.) and tomato (Lycopersicon spp.) the Black Root Rot in, and janto Monastir Calm Fest lease hospital-type Betsy Katori Ah (vesicatoria) is Pepper Necrosis and curling of V-types are caused at the edges of the leaves, and bacterial spot pattern disease occurs in which the coronary tissue is black. The yellowing and drying of the anterior lobe occurs as the disease progresses (Hayward et. al , 1993).

This disease is a problem that occurs sporadically in various places in the recent two to three years. The primary source of infection is pathogens on contaminated seeds, damage leaves, vines, seedlings, and grafting equipment. Transmission occurs due to inadvertent drops of care during the irrigation of seedlings or after planting, when leaves and vines are wet with dew. In recent years, the most prevalent factor is the increase in pollutant seeds caused by the use of seed disinfecting mercury and the sealing of the facility for a long time in order to save fuel for heating. Because. Insufficient sunlight causes severe damage to overgrown and overpack. It often occurs during plant cultivation. In general, the groundwater level is high, poor drainage, a lot of water droplets, insufficient insulation and ventilation, large numbers of irrigation, excessive fertilization of nitrogen and phosphoric acid, and not mulching on the surface. The resistance to the disease in breeds has not been developed yet.

Phytobacterial drugs are very limited in their kind, and antibiotics and copper hydrating agents are mostly used. For this reason, copper hydration has been used to prevent and treat these diseases for decades, but it has been reported that strains with resistant plasmids have resulted from excessive spraying of copper components (Bender, et. al . , 1990; Cooksey, et al . , 1990).

Therefore, efforts to develop novel antibiotics that effectively act against pathogenic microorganisms causing plant diseases are recognized as an important task in the field of antibiotic research. Representative bacteria producing bioactive natural products that can be used as pharmacological and agrochemical preparations include filamentous soil bacteria belonging to the genus Streptomyces. The microorganisms are used to produce about 75% of commercially valuable and medically useful antibiotics (Miyadoh et al , 1993), and 60% of antibiotics developed for agricultural use using Streptomyces spp. Produced (Tanaka and Mura, 1993).

Therefore, the present inventors have made efforts to develop antibiotics useful for the prevention and treatment of plant bacteria against substances produced in Streptomyces, Xanthomonas Campestris Hospital, which is produced in Streptomyces and the causative agent of plant bacteria A novel disaccharide compound having antimicrobial activity against type Campestris was extracted and purified, and the present invention was completed by confirming the antimicrobial activity against the strain.

An object of the present invention is to provide a (disaccharide) compound represented by [Formula I] derived from Streptomyces KBJ-2-45 (KCCM 10455).

Another object of the present invention to provide an antimicrobial agent comprising the compound as an active ingredient.

Still another object of the present invention is to provide a method for preparing [Formula I], which comprises culturing Streptomyces KBJ-2-45 (KCCM 10455).

In order to achieve the above object, an object of the present invention provides a novel compound represented by the following [Formula I].

[Formula I]

Another object of the present invention provides an antimicrobial agent comprising the compound represented by the above [Formula I] as an active ingredient.

Still another object of the present invention is to provide a method for preparing [Formula I], comprising culturing Streptomyces KBJ-2-45 (KCCM 10455).

Hereinafter, the present invention will be described in detail.

The present invention relates to a compound represented by the following [formula I] in one aspect.

[Formula I]

Compounds of the present invention are disaccharide compounds derived from Streptomyces KBJ-2-45 (KCCM 10455).

The inventors of the present invention have been more than 300 actinomycetes ( Actinomycetes) genus) was collected in Jeju Island to select strains for six plant pathogenic fungi and to find strains of other species with 10 antifungal activities (Kim et. al , 2001).

The identification of Streptomyces and other related microorganisms can be easily accomplished by a number of classification methods based on morphological and biochemical properties (Williams, et. al . , 1985), in the recent identification of new Streptomycetes , it has been found that 16S rRNA sequences are not useful in the strapomycetes classification (Mehling, et al . , 1995; Goodfellow, et al . , 1992). Overcoming these limitations, strains producing antibiotics against the Xanthomonas Campestris pathogenic strain were selected, which were identified as Streptomyces KBJ-2-44 and KBJ-2-45 ( Streptomyces). spp. KBJ-2-44 and KBJ-2-45), respectively.

The compounds of the present invention are Streptomyces KBJ-2-45 (Streptomyces spp. It was extracted and purified from the culture supernatant of KBJ-2-45) and identified as a disaccharide connected between H-3 of Sorbofuranose and H-2 of Altrofuranose, based on chemical structure (2S , 4S, 5R) -3 (((2R, 4S, 5S) -2,4-dihydroxy-2,5-bis (hydroxymethyl) -tetrahydrofuran-3-siloxy) methyl) -5- ((S) -1,2-dihydroxyethyl) -tetrahydrofuran-2,4-diol).

In addition, the compound of the present invention, when lyophilized, has a white powdery form, is soluble in water, but insoluble in alcohol and ethyl acetate.

The compounds of the invention are useful for treatment and prevention of black Root Rot caused by the microorganisms due to its antibacterial activity against Pseudomonas janto Kam paste less paste-less hospital-type KAM (Xanthomonas campestris pv. Campestris).

In another aspect, the present invention relates to an antimicrobial agent comprising the compound represented by the above [Formula I] as an active ingredient.

The compounds of the present invention are useful for controlling Xanthomonas Campestris pathogenic strains that cause mesenchymal disease, and are antibacterial by commonly used methods such as high-calen hydraulic spray, low-calen inlet spray, air spray, and air spray. It can be applied as a spray and a powder.

The amount of lime and the amount of action depends on the type of device used, the method and frequency of application required, and the progress of the disease. An effective amount is typically to use about 0.01 kg to 20 kg per hectare, preferably about 0.1 kg to 5 kg per hectare, and more preferably about 0.125 kg to 0.5 kg per hectare.

The compounds of the present invention can be used as seed protectants, soil antimicrobial agents and / or leaf antimicrobial agents. As a seed protectant, the compounds of the invention are coated at a dosage of about 10 mg to 20 mg per 50 kg of seed. As soil antimicrobial agents, the organic compounds of the present invention may be incorporated at dosages of about 0.5 kg to 20 kg, preferably about 1 kg to 5 kg, per hectare in or on the soil surface.

As leaf antimicrobial agents, the compounds of the present invention can be applied to growing plants at a dose of about 0.1 to 5 kg per hectare, preferably about 0.125 kg to 0.5 kg per hectare. As prepared for this purpose, it can be used in industrial or pure form as a solution or a combination, and can usually be absorbed or formulated in an extender to be suitable for use as an antimicrobial agent. For example, these organic compounds can be formulated as hydrating agents, dry powders, emulsifying concentrates, powders, granular blends, aerosols or flowable emulsion concentrates. In such formulations, these organic compounds expand with liquid or solid extenders and incorporate a suitable surfactant when dried.

Suitable wetting agents for spraying can be prepared by mixing the compounds of the present invention with finely divided solids such as clays, inorganic silicates and carbonates, and silica, and by mixing wetting agents, fixatives and / or dispersants in the mixture. The concentration of the active ingredient in such formulations is usually 20 to 98%, preferably 40 to 75%.

In particular, in the case of the leaf spray formulations, it is preferable to include auxiliaries such as wetting agents, diffusing agents, dispersing agents, fixatives, adhesives and the like according to the agricultural composition.

Powders are prepared by mixing the compounds, salts, and complexes thereof of the present invention with naturally pulverized inert solids that are organic or inorganic. Inerts useful for this purpose include vegetable powdered silica, silicates, carbonates and clays. One convenient way to prepare the powder is to dilute the hydrating agent using a fine grinding extender (carrier). Powder concentrates containing 20 to 80% of the active ingredient are mainly prepared and diluted to 1 to 10% when used.

The antimicrobial preparation of the present invention is not limited to the above examples. The amount of the antimicrobial composition of the present invention may vary depending on farmland, administration time, administration method, disease progression and crops.

Antimicrobials containing the compounds of the present invention can be prepared alone or in admixture with other antimicrobials. Examples can be prepared by mixing copper-based antimicrobials such as copper hydroxide, cuprous oxide, basic cuprous chloride, basic cuprous carbonate, copper terephthalate, copper naphtheneate and Bordeaux liquid.

In still another aspect, the present invention relates to a method for preparing a compound, comprising culturing Streptomyces KBJ-2-45 (KCCM 10455).

 In the method for producing a compound of the present invention, the culture process of Streptomyces can be made according to the appropriate medium and culture conditions known in the art. This culture process can be used by those skilled in the art can be easily adjusted according to the strain selected. Examples of such culture methods include, but are not limited to, batch, continuous and fed-batch cultures.

The method for separating and purifying the desired antimicrobial active material from the microbial culture may be separated and purified by conventional methods known in the art. In such a separation method, methods such as centrifugation, filtration, exchange chromatography, and crystallization may be used. For example, the supernatant obtained by removing the biomass by centrifugation of the microbial culture solution can be separated by ion exchange chromatography, but is not limited to these examples.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example  1: Screening of microorganisms with antimicrobial activity

In order to screen antibiotics, 316 strains were isolated from various soil samples taken from Jeju Island, and Bennett's agar (10 g of glucose, 1 g of yeast extract, 2 g of bactopeptone 2) was stored and selected as a medium for actinomycetes. g, beef extract 1 g / water 1 L). Xanthomonas Campestris Hospital type Besicataria (College of Agriculture and Life Sciences, Seoul National University), Xanthomonas Campestris (College of Agriculture and Life Sciences, Seoul National University) or Xanthomonas Campestris Hospital type duck ( X. campestris pv . Oryzae ) (College of Agriculture and Biological Sciences, Seoul National University) was incubated overnight at 27 ° C LB medium and used as a test strain for antimicrobial activity for the improved agar medium diffusion method (Okeke et al , 2001).

Sterile filter paper discs (8 mm) were absorbed in 20 μl of culture medium, dried at 37 ° C., then placed on LB agar medium, and the test strain contained in 0.9% agar was inoculated into the agar medium. The medium was incubated for 15 hours at 28 ℃, antimicrobial activity was evaluated by measuring the diameter of the antimicrobial zone. The strains with the highest antimicrobial activity were selected and streptomyces KBJ-2-44 and KBJ-2-45 ( Streptomyces). spp. KBJ-2-44 and KBJ-2-45), respectively.

Example 2: Culture of New Streptomyces

Selected Streptomyces KBJ-2-44 and KBJ-2-45 were produced in production medium (1.0% glucose, 0.3% yeast extract, 0.4% soybean meal, 1.0% corn steep liquor), 0.05% K ₂ HPO ₄ , 0.01% MgSO ₄ 7H ₂ O (pH 7.0)) was incubated at 28 ° C. and 160 rpm. The degree of cell growth was confirmed using the packed cell volume (PCV) method. The cultured strains were tested for antimicrobial activity at 8 hour intervals. As a result, in the case of KBJ-2-44, the cell growth started for about 48 hours, and after 2 days, the growth curve reached a steady state (Fig. 1A). During the incubation process, the pH of KBJ-2-44 was maintained in the range of 5.5 to 7.9, while the pH of KBJ-2-45 was maintained in the range of 6.5 to 7.6 (FIG. 1B).

The strains were pre-incubated for 24 hours in 500 ml of Elmmeyer flasks, followed by fermentation using 5 L of medium under 10% dissolved oxygen at 28 ° C using a 7 L fermentation tester (Korea Fermentation Co., Inchon, Korea). Was performed. The culture state was checked every 6 hours using the pH, PCV (%), antimicrobial activity standards, and after 36 hours of fermentation, only the culture supernatant was collected by filtration and mixing with culture medium and 300 g of celite.

Example 3: Identification of Antimicrobial Active Ingredients

The same amount of ethanol was mixed with the microbial culture supernatant of the three test strains to remove residual impurities by centrifugation at 2800g, which was used for the purification of the active ingredient. Concentrated using an evaporator (N-1000, EYELA, USA), and then finally dried extract was dissolved in distilled water and gently shaken. n-hexaine and ethyl acetate were added sequentially to the extract solution in a ratio of 1: 1 and separated using a separatory funnel. Each extract extracted with three solvents was measured for the antimicrobial activity against the test strain. As a result, it was found that the highest antimicrobial activity was obtained from the extract with distilled water. The solution was removed by evaporation at 40 ° C, dissolved in 20 ml of distilled water, and filtered. The obtained distilled water extract was injected into a polyacrylamide 2 (P2) gel column (610X 16 mm, Biorad, USA) by sterilizing distilled water at a flow rate of 0.5 ml / min, and subjected to gel filtration chromatography. The antimicrobial activity of each sample was confirmed by agar medium diffusion, and the highest antimicrobial activity was observed in the KBJ-2-45 strain (FIG. 2). Effective fractions of antibacterial activity was to have finally been selected, confirming thin layer chromatography results under UV _310, R _f 0.9, 0.7 and 0.53 of the three bands R _f 0.9 band material is antibacterial activity in water of through thin layer chromatography It turned out.

Example  4: Effect of Antimicrobial Active Ingredients

A sterilized filter paper disc (8 mm) was absorbed from the Streptomyces KBJ-2-45 strain culture supernatant as a stock solution, and the sterile filter paper discs (8 mm) were dried at 37 ° C, then placed on LB agar medium. , The test strain contained in 0.9% agar was inoculated on the agar medium. The medium was incubated for 15 hours at 28 ℃, antimicrobial activity was evaluated by measuring the diameter of the antimicrobial zone. Diluted commercially available Bordeaux liquid according to the method of use was tested in the same manner as the stock solution. Based on the antimicrobial diameter of Bordeaux solution, the antimicrobial activity was shown in relative percentages (FIG. 3).

Example  5: Extraction and Purification of Antimicrobial Active Ingredients

In order to extract the active fraction of the active fraction obtained in Example 3, acetonitrile (ACN), methanol and distilled water were added to a silica gel column (Kiselgel 60, 230-400 mesh, Merck, USA) at a volume ratio of 6: 1: 3. Chromatography was performed by mixing. After elution, the collected composition was analyzed by antimicrobial activity and thin layer chromatography, and the fraction was concentrated using an evaporator. The obtained sample (340 mg) was further re-fractionated through high-performance liquid chromatography (HPLC). This process was carried out using a SPECTRA system (UV1000, P2000; USA) with Phenomex Predigy IDS (250 × 10 mm) using a solvent degassed by HPLC. As a result, two A and B peaks were detected. ACN-distilled water was mixed at a volume ratio of 4: 1, injected at a flow rate of 3 ml / min, and eluted at 254 nm to confirm the antimicrobial activity of the substance corresponding to each peak. The obtained eluent was obtained from Xanthomonas Campestris Hospital. It was used for the antimicrobial activity test for type Campestris. Finally, the fraction with antimicrobial activity was concentrated using a freeze dryer Coldbag 50 (Coldvac 50, Hanil R & D, Korea) to obtain a final product having an antimicrobial activity of 103 mg (Fig. 4). This substance is white powdery and water soluble, but insoluble in alcohol and ethyl acetate.

Example  6: final antimicrobial activity Fraction  Chemical structure analysis

Nuclear magnetic resonance spectroscopy was used to characterize the active fractions A and B isolated from the microbial culture. The instrument used was a Bruker Avance 400 (400 MHz). Hydrogen Nuclear Magnetic Resonance Spectroscopy (1H-NMR), Carbon Nuclear Magnetic Resonance Spectroscopy (13C-NMR), DEPT (Distortionless Enhancement by Polarization Transfer), Cozy (COSY, Correlated Spectroscopy), HMQC of Fraction A , heteronuclear multiple quantum coherence (HMBC), heteronuclear multiple bonded connectivites (HMBC), TOCSY (total correlated spectroscopy), NOESY (nuclear overhauser exchanged spectroscopy), and HMQC-NOESY experiments were performed. However, fractions A and B were found to be mixtures as a result of performing diffusion-ordered spectroscopy (DOSY) experiments (FIG. 5).

Therefore, the data obtained from the results of nuclear magnetic resonance spectroscopy were analyzed and the connection of the two fractions was separated and analyzed. Mass spectrometry resulted in 381 = (180 + 180-18) + 39 (K). Existence is known. Since the fraction that is considered disaccharide of the two fractions was A, the structure of A was identified.

The results of nuclear magnetic resonance spectroscopy showed that all five monosaccharides exist, of which two monosaccharides are linked to each other, and it was found from the results of the TOCSY and HMQC-TOCSY experiments. The two sugars were interpreted as follows (Table 1).

Fraction 13C / ppm DEPT ^* 1H / ppm S-6 61.4 t 3.62 S-1 62.4 t 3.76 S-3 74.1 d 4.00 S-4 75.9 d 3.39 S-5 76.5 d 4.15 S-2 103.7 s A-6 60.2 t 3.75 A-5 69.3 t 3.41 A-2 74.2 d 3.18 A-3 75.8 d 3.43 A-4 81.4 d 3.83 A-1 95.9 d 4.57

* d = doublet, t = triplet, s = singlet

Since 12 peaks were observed in all, it was considered to be a disaccharide combined with two per six. From the analysis of the two-dimensional nuclear magnetic resonance spectroscopy results, it can be divided into two groups of peaks as follows. That is, a group with 61.4 / 3.62, 62.4 / 3.76, 74.1 / 4.00, 75.9 / 3.39, 76.5 / 4.15, 103.7ppm and 60.2 / 3.75, 69.3 / 3.41, 74.2 / 3.18, 75.8 / 3.43, 81.4 / 3.83, 95.9 / 4.57 ppm group. The former is the best match to the known L-sorbofuranoside, and the latter is the best match to D-altrofuranose.

In order to confirm the linkage between the two sugars, NOE was observed on the nod spectrum, and the c-linked peak was observed between H-3 of sorboburanoside and H-2 of altrofuranoside. As shown in Fig. 2, 3-OH of sorbofuranose and C-2 of altrofuranose were determined to be linked.

Fraction A is a natural product isolated from microorganisms, and the open field experiment was conducted to confirm that these two sugars do not exist, but exist as one compound. As a result, the two sugars were combined to form one compound. It became.

As a result, fraction A is (2S, 4S, 5R) -3-(((22,4S, 5S) -2,4-dihydroxy-2,5-bis (hydroxymethyl) -tetrahydrofuran-3-yloxy) methyl) -5 It was determined as-((S) -1,2-dihydroxyethyl) -tetrahydrofuran-2,4-diol.

The present invention is antimicrobial activity by culturing the compound showing antimicrobial activity against Xanthomonas campestris pv campestris , antimicrobial agent and Streptomyces KBJ-2-45 (KCCM 10455) containing the same It relates to a method for producing a compound having a compound. The compound according to the present invention is a phytopathogen Xanthomonas campestris pathotype ( Xanthomonas campestris pv.) Because it can effectively treat and prevent plant diseases caused by the strains, it is useful to reduce the damage caused by the current agricultural sector.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (5)

A (disaccharide) compound represented by the following [Formula I]. [Formula I]

The compound according to claim 1, which is derived from Streptomyces KBJ-2-45 (KCCM 10455). The method of claim 1, wherein the compound is Xanthomonas Campestris pathogenic Campestris ( Xanthomonas) campestris pv campestris ), characterized in that it exhibits antimicrobial activity. An antimicrobial agent comprising the compound represented by the following [Formula 1] as an active ingredient. [Formula I]

Streptomyces KBJ-2-45 (KCCM 10455) The method for producing a compound represented by the following [Formula I], characterized by culturing. [Formula I]

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