KR101127163B1 - A novel bacterium KME-002 and use thereof - Google Patents

Abstract

The present invention relates to a novel bacterial strain KME-002 and its use, and more specifically, it has been found that the new bacterial strain by analyzing the gene morphology and biochemical properties of marine bacterial strain KME-002 isolated from the sea urchin. Produces 7-ketodexoycholic acid and 3-Oxodeoxycholic acid in cholic acid derivatives, which are the main components of bile, And it has been found that the causative agent causing diseases such as rash and new strain KME-002, its genome and its components can be useful for mass production of choline acid derivatives, and prevention and treatment of bruise disease.

Bacterial strain KME-002, choline acid derivatives, 7-ketodeoxycholine acid, 3-oxodeoxycholine acid, bruising disease.

Description

A novel bacterium KME-0002 and use about

The present invention relates to a novel bacterial strain KME-002 and its use, and more particularly to a novel bacterial strain KME-002 isolated from sea urchin, a method for producing a deoxycholic acid derivative using the strain, and the strain It relates to a screening method for preventing and treating bruises such as granulomatosis and rash.

In the human body, bile acid, the main component of bile, is synthesized by cholesterol in the liver, and bile acid secreted by the liver is a major component of bile bile, which is the digestion and digestion of foods, especially fats, carotenoids and vitamins. Plays an important role in However, insufficient bile acid secretion can cause digestive disorders as well as hepatic diseases such as hepatitis or cirrhosis. In recent years, bile acids are active in the reduction of blood cholesterol levels, treatment of fatty liver, polysaccharides and diabetes, and their importance is increasing day by day (Korean Patent 2003-0092063).

Cholic acid and its derivatives are mainly produced in eukaryotes and are components of bile acids in animals. Cholic acid easily binds to taurine and glycine in vivo to produce taurocholic acid and glycocholic acid, and other derivatives, ursodeoxychoic acid acid, glycodeoxycholic acid, Taurusodeoxycholic acid, Glycoursodeoxycholic acid and the like have been reported (Batta AK et al. J. Lipid Res . 1981, 22 , 712-714). Deoxycholic acid derivatives, such as 7-ketodeoxycholic acid and 3-oxodeoxycholic acid, are known to dissolve gallstones and mitigate cholelithiasis (Kuroki, S. et al. J. Lipid Res . 26 , 1205-1211, 1985).

Cholic acid and derivatives thereof are currently synthesized for industrial use by chemical synthesis (Cravotto G. et al. Steroids . 2005, 70, 77-83) and by separation of bovine gallbladder in the form of a bile acid mixture (Maneerat S. et al. . Appl. Microbiol. Biotechnol. 2005 , 67, 679-683) are prepared. Industrial use of microorganisms is known in the production of industrially useful steroid compounds such as choline acid by converting the structures of various steroidal substances by enzymatic action of microorganisms (Mahato, SB et al. Biochem. J. 1988, 255 , 769-774; Ambrus, G. et al. Steroids 1995, 60 , 621-625; Miyamoto, Y. et al. Biochim. Biophys. Acta. 1995, 1588 , 139-148), to date through pure culture Microorganisms producing choline acid derivatives are only a few species of microorganisms, including three marine bacteria belonging to the genus Myroides (Maneerat S. et al. Appl. Microbiol. Biotechnol. 2005, 67 , 679-683). (Kim et al. J. Microbiol. Biotechnol. 2007, 17 , 403-407). Moreover, no microorganisms producing ketodeoxycholic acid, such as Compound A and Compound B, have been reported to date. The production of cholic acid derivatives by microorganisms is not only easier to operate than conventional manufacturing methods, but also expected to cost less, which is expected to be suitable for industrial mass production.

Sea urchin is a favorite food of Korea, Japan's Hokkaido region and some Asian countries. Recently, sea urchin has been found to contain large amounts of natural anticancer and antibiotic substances in addition to bioactive substances such as collagen, vanadium, dietary fiber and chondroitin. Demand is increasing (Azumi K. et al. Biochemistry 1990, 29, 159-165; Mayer and Custafson, Int. J. Cancer. 2003, 105, 291-299).

However, since the late 1970s, farmed sea urchins are known to have caused massive mortality due to sea urchin diseases such as runny nose and granulosis, with mortality rates of up to 80% of annual farming and annual damages of about 20 to 40 billion won. (Dragwater canal, 2007). The mass mortality of these farmed sea urchins is not only a huge direct economic loss to the domestic sea urchin farming industry, but also a major obstacle to the export of domestically produced sea urchins.

The external characteristic of mass-cultured sea urchin is so-called turbidity, which softens the cysts surrounding the surface of the sea urchins, increases the non-ideal mucus, and causes the internal tissues to leak out due to necrosis or perforation of the cysts. do. Another characteristic of our death is called granulosis, and the cysts of cultured sea urchins are attached to Suhayeon (垂下 延), but the flesh part flows down, and the cysts break down. Investigate environmental factors (water temperature, salinity, dissolved oxygen, pH, chlorophyll α content, etc.) to identify the causes of these diseases (Na et al . , Korean Fish. Soc. 1991, 24, 52-58; 1998, 218-230; Hong et al. Journal of Aquaculture 2000, 13, 285-293; Shin et al. Journal of Aquaculture 2007, 20, 226-231), physiological factors (growth of sea urchins per mountain), bacteriological investigation (digestion) Total number of bacteria on board) (Donghae Fisheries Research Institute Business Report 1998, 218-230), and molecular genetic approach (Cho, Thesis, Pusan National University, Thesis, 2007) Since the cause is not clearly identified, prevention and countermeasures are insufficient.

Accordingly, the present inventors were the first to discover a new bacterial strain KME-002, while searching for the causative agent of the sea urchin disease in which cultured sea urchins are carried, and the bacterial strain KME-002 is 7-ketodexoycholic acid. And antibiotics for preventing or treating the disease of sea urchins by producing 3-oxodeoxycholic acid, which can be used for the mass production of deoxycholine acid derivatives, and confirming that it is the causative agent of causing disease in cultured sea urchins. The present invention has been completed by discovering that it can be used for development.

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel bacterial strain KME-002, a method for producing deoxycholic acid using the strain, and a method for screening and preventing the treatment of bruise disease of granulomas or rashes using the strain. will be.

In order to achieve the above object, the present invention provides a novel bacterial strain KME-002 deposited with accession number KFCC11437P.

The present invention also provides 16S rDNA of the novel bacterial strain KME-002 described as SEQ ID NO: 1.

The present invention also provides a method for producing a deoxycholic acid derivative using the novel bacterial strain KME-002.

In addition,

1) treating the test substance in the culture solution of the new bacterial strain KME-002;

2) measuring the number of live bacteria of the strain in the culture medium; And

3) It provides a screening method for preventing and treating bruise disease of granulomas or rashes, comprising the step of selecting a test substance that significantly reduces the number of viable cells compared to the control group not treated with the test substance.

In addition,

1) treating the test substance to the new bacterial strain KME-002 cells;

2) measuring the cell viability of the strain cells; And

3) It provides a screening method for preventing and treating bruise disease of granulomas or rashes, comprising the step of selecting a test substance that significantly reduces the cell viability compared to the control group not treated with the test substance.

The novel bacterial strain KME-002 of the present invention produces 3-oxodeoxycholic acid and 7-ketodeoxy cholic acid in choline acid derivatives which are the main components of bile, As it is found that the cultured sea urchin is a microbe causing disease, it can be usefully used for mass production of deoxycholic acid derivatives, and prevention and treatment of sea urchin diseases such as granulomas and rashes.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel bacterial strain KME-002 deposited with accession number KFCC11437P.

Strain KME-002 of the present invention is preferably derived from sea urchin, but is not limited thereto.

Strain KME-002 of the present invention preferably 16S rDNA having a nucleotide sequence set forth in SEQ ID NO: 1, but is not limited thereto.

The present inventors carried out species analysis by analyzing 16S rDNA sequences of single strains isolated from the inlet and outlet tissues of sea urchin, and strain KME-002 was obtained from Dinoroseobacter shibae and Pseudoruria By confirming the low homology of 94.7% and 94.6% to Pseudoruegeria aquimaris , the bacterial strain KME-002 of the present invention was isolated from the new microorganism genus of Rhodobacteraceae family. It was confirmed that it is a new species.

The inventors have found that strain KME-002 forms red colonies in A1C (A1 + C) medium, is cultured under aerobic culture conditions, and exhibits Gram negative, so that strain KME-002 is aerobic Gram negative cocci. (See FIGS. 1 and 2).

The strain KME-002 of the present invention has a DNA G + C content of 71.60 mol%, a main menaquinone of Q-10, a main fatty acid of C _{18: 1} ω7с , and a physiological pattern of enzyme production. It can be seen that the chemical properties (see Table 4).

Accordingly, the present inventors compared the genes and biochemical data of strain KME-002 with data of previously reported strains, and identified them as new strains and named them KME-002. Deposited on the 24th of February (Accession No .: KFCC11437P).

The strain KME-002 of the present invention is 7-ketodeoxy cholic acid represented by the following [Formula 1] (3-oxodeoxycholic acid) and 3-oxodeoxycholic acid represented by the following [Formula 2]. Preferably produces, but is not limited to:

The present inventors added the adsorption resin to the culture medium of strain KME-002, and left it for 1 hour, followed by filtration to take the adsorption resin. The acetone extract prepared by adding acetone to the adsorption resin was dried under reduced pressure, and the extract obtained was separated into four fractions using silica gel column chromatography. Elution conditions were using ethyl acetate-methanol (20: 1, 10: 1, and 5: 1) mixed solvent, and then 100% methanol was used as final solvent. The obtained fractions were analyzed by principal liquid chromatography using high-performance liquid chromatography. As a result, a choline acid derivative was identified in the ethyl acetate-methanol (5: 1) fraction. The ethyl acetate-methanol (5: 1) fraction was started with 30% acetonitrile / water and increased to 70% acetonitrile / water for 40 minutes using a gradient gradient elution condition through reverse phase liquid chromatography. Purification gave two white powdery deoxycholic acids.

The present inventors analyzed chemical structures of the two compounds by analyzing high-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance spectroscopy (NMR) data, respectively. Ketodeoxycholine acid and 3-oxodeoxycholine acid represented by [Formula 2] were identified.

The present inventors confirmed through thin film chromatography (TLC) analysis that there is no cholesterol constituting the basic chemical skeleton of choline acid in A1C (A1 + C) pure medium used for culturing the strain KME-002, It was confirmed that the novel bacteria alone synthesized the two compounds.

Therefore, it can be seen that the strain KME-002 of the present invention can be usefully used for mass production of deoxycholine acid derivatives such as 3-oxo deoxycholic acid and 7-ketodeoxycholic acid. Can be.

Strain KME-002 of the present invention is preferably a pathogenic microorganism that causes turbidity or granulomas in the sea urchin, but is not limited thereto.

The present inventors treated cells and culture medium obtained by liquid culture of strain KME-002 for 5 days in a tank containing a sea urchin, so that all the sea urchins that had been in the tank cracked the cyst in two weeks, so that the cyst and the meat part were separated. By observing a representative disease pattern of the dripping sea urchin, it was found that it is a microorganism causing caustic disease (see FIGS. 3 and 4).

Therefore, it can be seen that the strain KME-002 itself, the genome or the components thereof of the present invention can be used for biomarkers, microbial growth inhibitory devices, gene therapy and antibiotic development for the prevention and treatment of bruising disease.

The present invention also provides a method for producing a deoxycholic acid derivative using the novel bacterial strain KME-002.

The deoxycholic acid derivative is preferably 7-ketodeoxycholic acid represented by the above [Formula 1] and 3-oxodeoxycholic acid represented by the [Formula 2], but is not limited thereto.

Specifically, the method for producing a deoxycholic acid derivative using the novel bacterial strain KME-002 of the present invention is preferably a method comprising the following steps, but is not limited thereto:

1) inoculating the new bacterial strain KME-002 into a liquid medium, followed by preincubation, and culturing the precultured liquid in the liquid medium;

2) adding the organic solvent to the culture solution by adsorbing the adsorption resin and then adding the organic solvent, or directly adding the organic solvent to the culture solution and extracting it;

3) drying the extract under reduced pressure, and then obtaining a fraction by column chromatography; And

4) separating and purifying 7-ketodeoxycholic acid or 3-oxodeoxycholic acid from the fraction further using column chromatography.

In the above method, the liquid medium of step 1) is preferably A1C (A1 + C) liquid medium, but is not limited thereto. The pre-culture is preferably cultured for 3 days to 7 days, and more preferably for 4 days, but is not limited thereto. The culture is preferably cultured 4 days to 14 days, more preferably 4 to 5 days culture is not limited thereto.

In the above method, the adsorption resin of step 2) is preferably HP20 or XAD resin, and after adding 5 g to 20 g per liter, it is preferable to leave the culture solution and the resin for 1 to 3 hours and then filter it. It is not limited to this. The organic solvent is preferably extracted using acetone, ethanol or methanol, but is not limited thereto. The extraction is preferably performed at room temperature, but is not limited thereto. In addition, an extract may be prepared by directly adding an organic solvent such as ethyl acetate, methylene chloride or ether, without using an adsorptive resin to the culture.

The extraction method may be replaced by a method using an extraction apparatus such as supercritical extraction, high pressure extraction or ultrasonic extraction, and a method of preparing an extract by directly drying the culture solution after removing the bacterial cells by filtration or centrifugation. It doesn't work.

In the above method, the reduced pressure drying of step 3) preferably uses a rotary vacuum evaporator, lyophilization or spray drying, but is not limited thereto. When drying under reduced pressure, the temperature is preferably 20 to 40 ° C., more preferably 30 ° C., but is not limited thereto.

In the method, the column chromatography of step 2) or step 3) is selected from the group consisting of silica gel, Sephadex, silica-based reverse phase resin, polymer-based reverse phase resin, polyamide, HP20, Toyopearl and XAD resin. Column chromatography using any one of the fillers can be performed to isolate and purify the active compound, but is not limited thereto. Column chromatography can be carried out several times by selecting the appropriate filler as needed, and from the group consisting of chloroform, methylene chloride, hexane, petroleum ether, ether, ethyl acetate, acetone, methanol, ethanol, acetonitrile and water as effluent solvents. Any one selected may be used, but the present invention is not limited thereto.

In addition, the present invention provides a method of screening for the prevention and treatment of bruise disease using a novel bacterial strain KME-002.

The bruise disease is preferably, but not limited to, mud flat or granny.

Specifically, the screening method for preventing and treating seaweed diseases using the novel bacterial strain KME-002 of the present invention is preferably, but not limited to, a method comprising the following steps:

1) treating the test substance to the culture medium in which the new bacterial strain KME-002 was cultured;

2) measuring the number of live bacteria of the strain in the culture medium; And

3) selecting a test substance that significantly reduces the number of viable cells compared to a control which is not treated with the test substance.

In the above method, the culturing of step 1) is preferably cultivated in A1C (A1 + C) liquid medium, preferably for 4 to 14 days, and more preferably for 4 to 5 days. It is not limited to this.

In the method, the test substance of step 1) is preferably any one selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma. It is not limited.

In the above method, the viable cell count of step 2) is preferably a colony count method or a Most Probable Nuber (MPN) method, but is not limited thereto.

In addition, the method of screening for the prevention and treatment of bruise disease using the novel bacterial strain KME-002 of the present invention is preferably, but not limited to, a method comprising the following steps:

1) treating the test substance to the new bacterial strain KME-002 cells;

2) measuring the cell viability of the strain cells; And

3) selecting a test substance that significantly reduces cell viability compared to a control that is not treated with the test substance.

In the method, the test substance of step 1) is preferably any one selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma. It is not limited.

In the above method, the cell viability of step 2) is preferably measured using an MTT assay, a trypan blue exclusion method, or an Eosin Y dye exclusion method. It is not limited.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following Examples illustrate the present invention in detail, and the content of the present invention is not limited by the following Examples.

Example 1 Microbial Separation for Screening of Novel Microbes

The present inventors collected the natural sea urchin from the coast of Gangneung-si, Gangwon-do, and finely chopped the tissue of the inlet and outlet of the sea urchin with sterilized scissors, and then the sterilized seawater (50 times 10 times) with respect to the volume of the fine tissue (5 cm2). Ml) was added and diluted. The diluent was heated at 55 ° C. for 6 minutes and then 100 microliters were taken and plated on A1-C (A1 + C) agar plates. While culturing the agar medium at 25 ° C. for 14 days, a single strain was isolated to obtain seven strains that produce colonies of different forms. Each strain was purely cultured by inoculating each of the isolated microbial colonies on the A1 + C agar plate.

Example 2 Genetic, Morphological and Physiological Characterization of New Microorganisms

In order to analyze the species of the obtained strains, the present inventors obtained the G-spin ^TM Genomic DNA Extraction Kit (Intron) after culturing strain KME-002 at 25 ° C. and 200 rpm for 5 days in A1C (A1 + C) liquid medium. Genomic DNA was extracted according to the instructions. The polymerase chain reaction (PCR) method was used to obtain 16S rRNA gene sequences, which are used as molecular markers most useful for studying the evolutionary strain of microorganisms from the extracted DNA. First, the reagents of Table 1 were sequentially placed in 0.5 ml microtubes, and then PCR was performed under the conditions of Table 3.

Reagents for PCR Kinds content 10 X reaction buffer 5 μl dNTP mixture (10 mM) 1 μl PCR primer 27F (100 pmol) 2 μl 1492R (100pmol) 2 μl Template DNA 1 μl Distilled water 38.75 μl Taq DNA Polymerase (5 U / μl) 0.25 μl system 50 μl

Primers for PCR Kinds order location Primer 27F 5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 2) 8-27 Primer 1492R 5'-GGCTACCTTGTTACGACTT-3 '(SEQ ID NO: 3) 1510-1492

PCR conditions Kinds Temperature and time Preheat 94 ℃, 5min PCR 35 cycles Heat denaturation  94 ℃, 30sec Annealing  55 ℃, 30sec kidney       72 ℃, 2min Add       72 ℃, 7min Cooling 4 ℃

Thereafter, in order to remove primer dimers, which are byproducts generated during PCR, PCR products were purified according to instructions using Solgent PCR Purification Kit (Solgent). In order to determine the nucleotide sequence of the 16S rRNA gene obtained by PCR, a sequencing reaction was performed with a Fluorescent dye terminators method (ABI prism ^TM Bigdye ^TM terminator cycle sequencing ready reaction Kit V3.1). The sequence was determined using an ABI 3730XL capillary DNA Sequencer (50 cm capillary) (Solgent).

As a result, it has been named the strain showed 94.7% respectively and the low homology of 94.6% with respect to a Dino to Seo bakteo swibe (Dinoroseobacter shibae) and the shoe bases Ria Accu Maris (Pseudoruegeria aquimaris) to 'KME-002'. It was confirmed that the strain KME-002 is a novel bacterial species corresponding to the new microorganism genus of Rhodobacteraceae family.

The strain KME-002 formed red colonies in A1C (A1 + C) agar medium (10 g Starch, 4 g Peptone, 2 g Yeast Extract, 1 g Calcium Carbonate in 1 L of Sea water), and was cultured in aerobic culture conditions. Gram staining (Suβmuth, R., Eberspacher, J., Haag, R. & Springer, W. (1987) .Biochemischmikrobiologisches Praktikum. Thieme Verlag, Stuttgart.) And spore staining (Merck Microbiology Manual 12 ^th Edition, p.642) Experiments showed that aerobic Gram-negative bacteria (FIGS. 1 and 2).

To determine the DNA guanine + cytosine (G + C) content of the strain KME-002, the following method (Mesbah, M. et al. Int. J. Syst. Bacteriol. 1989, 39 , 159-167). Strain KME-002 DNA solution was taken into a tube and centrifuged (14000 rpm, 5 minutes), and then 100 μl of the supernatant was transferred to a new tube. The supernatant was heated at 100 ° C. for 5 minutes for DNA denaturation, cooled at room temperature, 10 μl of P1 nuclease (1 unit / μl) was added and reacted at 50 ° C. for about 3 hours, followed by alkaline phosphatase. (10 µL of buffer and 5 µl of Phosphatase) were added thereto, and left at 37 ° C for 12 hours. The mixed solution was centrifuged (14000 rpm, 5 minutes) and analyzed using high performance liquid chromatography. The high performance liquid chromatography analysis conditions were as follows: [Instrument: Younglin SP930D; Elution rate: 0.5 mL / min; Eluent: 0.02 M ammonium phosphate (NH 4 H 2 PO 4) -acetonitrile (20: 1); Column: Waters Spherisorb 5 μm ODS2 4.6 mm × 250 mm; Detector: ultraviolet detector (270 nm). As a result, the DNA guanine + cytosine (G + C) content of strain KME-002 was found to be 71.60 mol%.

In order to determine the menaquinone of the strain KME-002, it was analyzed using the following method (Park Jin Sook, microbial classification identification method, World Science). After lyophilizing only the cells of the strain KME-002 culture solution, chloroform-methanol (2: 1) solution was added and extracted by shaking for 3-4 hours. Impurities were removed using filter paper, and the filtered solution was concentrated. The concentrate was dissolved in 100 μl of chloroform-methanol (8.5: 1.5), followed by centrifugation (14000 rpm, 5 minutes) and analyzed using high performance liquid chromatography. The analysis conditions are as follows. [Device: Younglin SP930D; Eluent: methanol-isopropyl ether (4: 1); Column: Waters Spherisorb 5 μm ODS2 4.6 mm × 250 mm; Detector: ultraviolet detector (254 nm). As a result, it was found that the menaquinone of strain KME-002 was Q-10.

In order to determine the main fatty acid (fatty acid) of the strain KME-002, the following method (Miller, LT J. Clin. Microbiol. 1982, 18, 861-867; Yang, P. et al. Syst Appl Microbiol 1993 , 16, 47-71). After culturing about 40 mg of cells of strain KME-002 were transferred to a tube, 1 mL of a solution in which 15% sodium hydroxide was added to 50% methanol was added, and heated at 100 ° C. for 30 minutes to cool at room temperature. To this was added 2 mL of a hydrogen chloride-methanol mixed solution (mixed solution of 6.0 N hydrogen chloride and 275 mL of methanol) and heated at 80 ° C. for 10 minutes, followed by quenching, followed by 1.25 ml of hexane- (methyl-tert-butyl Ether) (1: 1) and mixed well for 10 minutes. After standing at room temperature, the reaction solution was separated into two layers. Only the lower liquid was removed and 3 ml of diluted sodium hydroxide (10.8 g sodium hydroxide / 900 mL distilled water) was added and mixed well for 10 minutes. About 2/3 of the supernatant was transferred to a glass bottle and used as a sample. Gas chromatography (Agilent technologies 6890) was used for the analysis of the prepared samples, and the separation column was a cross-linked methyl siloxane column (HP-1, A30 m X 0.320 mm X 0.25 µm). Analysis was performed using the program (Sherlock MIS Software), and the peaks were identified, retention time, peak area and area ratio by comparison with standard calibration solution. As a result, the main fatty acid (fatty acid) of strain KME-002 cells was found to be C _{18: 1} ω7с .

In order to determine the specific enzyme production of the strain KME-002, it was carried out according to the instructions using the API Kit (ZYM). As a result, as shown in Table 4, strain KME-002 is esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, β- It was found that it produces enzymes of galactosidase, β-gluronidase, α-glucosidase, β-glucosidase and N-acetyl-β-glucosaminidase (Table 4).

Enzyme Production of New Bacterial Strain KME-002  Enzyme entry Enzyme Production Esterase (C4) U Esterase lipase (C8) U Alkaline phosphatase radish Lipase (C14) U Cystine arylamidase radish Trypsin radish Leucine Arylamidase U Acid phosphatase radish α-chymotrypsin radish α-galactosidase radish β-galactosidase U β-gluronidase U β-glucosidase U α-glucosidase U N-acetyl-β-glucosaminidase U α-mannosidase radish α-fucosidase radish Naphthol-AS-BI-phosphohydrolase U Valine arylamidase radish

Therefore, as a result of analyzing the gene morphology and biochemical properties of the strain KME-002, it was confirmed that it is a new bacterial strain, and the strain KME-002 was deposited on December 24, 2008 at the 'Korea Microorganism Conservation Center' (Accession No .: KFCC11437P).

Example 3 Mass Liquid Culture of Novel Bacterial Strain KME-002

In order to mass cultivate the novel bacterial strain KME-002, the present inventors first inoculated one colony of the strain into 25 mL of A1C (A1 + C) liquid medium and incubated in a shake incubator at 25 ° C. for 4 days. Thereafter, a portion of the culture solution was inoculated with 500 mL of A1C (A1 + C) liquid medium and shake-cultured at 25 ° C. for 4 days, and then all the 500 mL cultures were inoculated in 5 L of liquid medium and shaken at 25 ° C. for 5 days. . In addition, the high-performance liquid chromatography method was used to analyze the production pattern of the compound from the culture. The analysis conditions of the high performance liquid chromatography were as follows: [Instrument: Agilent 1100 LC / MS system; Elution rate: 0.7 mL / min; Eluent: increasing the composition of acetonitrile for 30 minutes from 10% acetonitrile / water to 100% acetonitrile; Column: Phenomenex Luna 5u C18 (2) 4.6 × 150 mm; Detector: DAD ultraviolet detector (254 nm).

As a result of the analysis, as shown in Figure 5, the main components of strain KME-002 were observed at retention times of 16.6, 17.1, 22.7 and 23.1 minutes in a culture medium in which the new bacterial strain KME-002 was monocultured for 5 days (Figure 5). .

Example 4 Separation and Purification of 7-Ketodeoxycholic Acid and 3-Oxodeoxycholic Acid

The present inventors added Amberlite XAD7 resin to the culture solution obtained in Example 3, left for 1 hour, and filtered to obtain the resin. Acetone was added to the XAD7 resin obtained from the culture, and then left for 1 hour and then filtered to remove the XAD7 resin. The acetone extract was dried under reduced pressure at 30 ° C. using a rotary vacuum evaporator to prepare an extract (980 mg), and the extract was divided into four fractions using silica gel column chromatography. Elution conditions were using ethyl acetate-methanol (20: 1, 10: 1, and 5: 1) mixed solvent, and then 100% methanol was used as the final solvent. As a result of analyzing the main components of the fractions obtained by high performance liquid chromatography, ethyl acetate-methanol (5: 1) fractions (31 mg) were identified as collic acid derivatives. The concentration gradient elution condition is started to increase the content of acetonitrile starting with 30% acetonitrile / water containing 0.02% TFA additionally to the ethyl acetate-methanol (5: 1) fraction for 40 minutes. Purification via reverse phase liquid chromatography gave 7-ketodeoxycholic acid (1 mg) at a retention time of 16.5 minutes and 3-oxodeoxycholic acid (1.5 mg) at a retention time of 32 minutes in the form of a white powder. The conditions of the reverse phase chromatography were as follows: [Equipment: Gilson Preparative HPLC system; Elution rate: 12 mL / min; Eluent: concentration gradient starting with 30% acetonitrile / water with 0.02% TFA to 70% acetonitrile / water for 40 minutes; Column: Phenomenex Luna 15u C18 (2) 100A 250 × 21.20 mm, 15 micron; Detector: refractive index detector].

The present inventors analyzed the MS data and nuclear magnetic spectroscopy data of the 7-ketodeoxycholine acid and 3-oxodeoxycholine acid (FIGS. 6 and 7), respectively, and the chemical structures of [Formula 1] and [ It was found that it is determined by the formula (2).

[Formula 1]

[Formula 2]

Example 5 Evaluation of the Effect of Novel Strain KME-002 on the Seaweed Disease

The inventors were shaken for 5 days in a novel bacterial strain KME-002 isolated from the sea squirt in A1C (A1 + C) liquid medium, and then the culture was directly treated in the sea squirt tank. The water temperature was kept constant at 13 ° C., and three cultured sea urchins were raised in a water tank containing 14 L of seawater to treat microorganisms, and compared with the sea urchins treated with untreated microorganisms and A1C nutrition medium.

As a result, no abnormal signs were found in the strain KME-002 untreated group (control), while the mucus material increased abnormally in the strain KME-002 treated group (experimental), and within 2 weeks after treatment of the microorganisms. As the cysts of all the sea urchins in the tank cracked, all of them died in a form in which the cysts and the flesh were separated (see FIGS. 3 and 4).

<Example 6> A presence of cholesterol in A1C (A1 + C) pure medium

In order to confirm that cholesterol which constitutes the basic chemical skeleton of choline acid does not exist in A1C (A1 + C) pure medium, a thin layer using a hexane-ethyl acetate (2: 1) mixed solvent as a developing solvent Chromatography (TLC) was used to compare the A1C (A1 + C) medium and the target cholesterol.

As a result, it was confirmed that cholesterol was not contained in the A1C (A1 + C) medium. Therefore, it can be seen that the novel bacterial strain KME-002 biosynthesizes a 7-ketodeoxycholine acid represented by the above [Formula 1] and a cholic acid derivative which is 3-oxodeoxycholine acid represented by the following [Formula 2]. there was.

The novel bacterial strain KME-002 of the present invention can be usefully used for mass production of 7-ketodeoxy cholic acid or 3-oxodeoxycholic acid using microbial mass culture. In addition, by studying the biosynthesis mechanism of choline acid derivatives by the strain can be used in the mass production using microorganisms of other useful choline acid derivatives in addition to deoxycholine acid derivatives, one of the microorganisms causing the mass death of cultured sea urchin As it can be found, it can be usefully used for research and development of the bruise farming industry to prevent mass mortality caused by the bruise of granulomas and rashes.

1 is a photograph showing the morphology of A1C (A1 + C) agar medium of the novel bacterial strain KME-002.

2 is a micrograph showing the gram stained form of the novel bacterial strain KME-002.

Figure 3 is a photograph showing the form of the dead seam 5 days after treatment of the culture of the new bacterial strain KME-002 in the tank containing the sea urchin.

Figure 4 is a photograph showing the dead form of the cysts and flesh parts of the dead sea urchin separated by the culture of the novel bacterial strain KME-002.

Figure 5 is a photograph showing a chromatogram of the new bacterial strain KME-002 analyzed by high-performance liquid chromatography of the culture medium in a single culture for 5 days.

6 is a photograph showing a hydrogen nuclear magnetic spectroscopy spectrum (500 MHz) of 7-ketodeoxycholine acid represented by the above <Formula 1> (solvent CD3OD).

7 is a photograph showing a hydrogen nuclear magnetic spectroscopy spectrum (500 MHz) of 3-oxodeoxycholine acid represented by the above <Formula 2> (solvent CD3OD).

<110> Korea Institute of Science and Technology <120> A novel bacterium KME-002 and use <130> 8p-12-60 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1429 <212> DNA <213> 16S rDNA of novel bacterium KME-002 <400> 1 agagtttgat catggctcag aacgaacgct ggcggcaggc ttaatacatg caagtcgagc 60 gcaccttcgg gtgagcggcg gacgggtgag taacgcgtgg gaatgtgccc tttggtacgg 120 aatagccttg ggaaaccgag agtaataccg tatgtgccct tcgggggaaa gatttatcgc 180 caaaggatca gcccgcgttg gattaggtag ttggtggggt aatggcctac caagccgacg 240 atccatagct ggtttgagag gatgatcagc cacactggga ctgagacacg gcccagactc 300 ctacgggagg cagcagtagg gaatcttaga caatgggcga aagcctgatc tagccatgcc 360 gcgtgtgtga cgacggccct agggtcgtaa agcactttcg ccagggatga taatgacagt 420 acctggtaaa gaaaccccgg ctaactccgt gccagcagcc gcggtaatac ggagggggtt 480 agcgttgttc ggaattactg ggcgtaaagc gcgcgtaggc ggactaccca gtcagaggtg 540 aaatcccagg gctcaaccct ggaactgcct ttgatactag tagtcttgag ttcgagagag 600 gtgagtggaa ttccgagtgt agaggtgaaa ttcgtagata ttcggaggaa caccagtggc 660 gaaggcggct cactggctcg atactgacgc tgaggtgcga aagcgtgggg agcaaacagg 720 attagatacc ctggtagtcc acgccgtaaa cgatgaatgc cagacgtcgg gcagcatgct 780 gttcggtgtc acacctaacg gattaagcat tccgcctggg gagtacggcc gcaaggttaa 840 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 900 aacgcgcaga accttaccaa cccttgacat cctgatcgcg gtttctcgag agagattcct 960 tcagttcggc tggatcagtg acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020 gttcggttaa gtccggcaac gagcgcaacc cacatcccta gttgccagca gttcggctgg 1080 gcactctatg gaaactgccc gtgataagcg ggaggaaggt gtggatgacg tcaagtcctc 1140 atggccctta cgggttgggc tacacacgtg ctacaatggc atctacagtg ggttaatccc 1200 caaaagatgt ctcagttcgg attggggtct gcaactcgac cccatgaagt cggaatcgct 1260 agtaatcgcg taacagcatg acgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1320 tcacaccatg ggagttggtt ctacccgacg acggtgcgct aaccttcggg aggcagccgg 1380 ccacggtagg atcagcgact ggggtgaagt cgtaacaagg tagccgtaa 1429 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 27F <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer 1492R <400> 3 ggctaccttg ttacgactt 19  

Claims (12)

New bacterial strain KME-002 deposited with accession number KFCC11437P. The novel bacterial strain KME-002 of claim 1, wherein the strain has a nucleotide sequence of 16S rDNA as set forth in SEQ ID NO: 1. According to claim 1, wherein the strain is DNA G + C content of 71.60 mol%, the main menaquinone (menaquinone) is Q-10, the main fatty acid (fatty acid) is characterized in that C _{18: 1} ω 7с , New Bacterial Strain KME-002. The method of claim 1, wherein the strain is 7-ketodexoycholic acid represented by the following [Formula 1] (7-ketodexoycholic acid) and 3-oxodeoxycholic acid represented by the formula [2] (3-Oxodeoxycholic acid Novel bacterial strain KME-002, characterized by producing a deoxycholic acid derivative) [Formula 1]

[Formula 2]

The novel bacterial strain KME-002 according to claim 1, wherein the strain is a pathogenic microorganism that causes bruising or sputum in the sea urchin. 16S rDNA of a novel bacterial strain KME-002 having the nucleotide sequence set forth in SEQ ID NO: 1. 1) culturing the novel bacterial strain KME-002 of claim 1 to obtain a culture solution; And 2) 3-oxodeoxycholine acid or 7-ketodeoxycholine acid, characterized by separating the 3-oxodeoxycholine acid or 7-ketodeoxycholine acid of claim 4 from the culture solution. Production method. The method of claim 7, wherein 1) inoculating the new bacterial strain KME-002 of claim 1 in a liquid medium and culturing the preculture obtained in the pre-culture in a liquid medium to obtain a culture medium; 2) adsorbing the culture solution by adding an adsorptive resin, and then adding an organic solvent, or directly adding and extracting an organic solvent to the culture solution to obtain an extract; 3) drying the extract under reduced pressure, and then obtaining a fraction by column chromatography; And 4) separating and purifying the 3-oxodeoxycholic acid or 7-ketodeoxycholic acid of claim 4 using column chromatography further from the fractions. Method of producing choline acid or 7-ketodeoxycholine acid. 3-oxodeoxycholine acid or 7-ketodeoxycholine, comprising the step of culturing the gene of the novel bacterial strain KME-002, which is the nucleotide sequence set forth in SEQ ID NO: 1, into another aeromicrobial strain or E. coli Acid production method. 1) treating the test substance to the culture solution incubated with the strain of claim 1; 2) measuring the number of live bacteria of the strain in the culture medium; And 3) A screening method for preventing and treating bruising disease of mumps or granules, comprising screening a test substance which significantly reduces the number of living cells compared to a control which is not treated with the test substance. 1) treating the test substance to the strain cell of claim 1; 2) measuring the cell viability of the strain cells; And 3) A screening method for preventing and treating bruising disease of mumps or granules, comprising screening a test substance which significantly reduces cell viability compared to a control which is not treated with the test substance. The method according to claim 10 or 11, wherein the test substance of step 1) is any one selected from the group consisting of peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma. A method for screening for a prophylactic or therapeutic drug for the treatment of rash or granule bruise.

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