KR101749681B1 - A novel soil microorganism, a novel oxidoreductase seperated from the said soil microorganism, a gene encoding the said oxidoreductase and a methods for producing flavonoid aglycone using thereof - Google Patents

Abstract

The present invention relates to a novel Rhodobium sp. GIN611 ( Rhizobium sp. GIN611) KCTC11708BP, its cell extract, Rizobium sp. There is provided a method for producing a flavonoid aglycone using a recombinant strain comprising a GIN611-derived oxidative reductase, a gene encoding the same and a recombinant vector protein or an expression vector encoding the recombinant protein as a biocatalyst. The novel enzyme isolated from the novel microorganism of the present invention does not belong to the glucosidase group but belongs to the oxidoreductase group and has an activity of saccharifying the natural product. This novel oxidoreductase is able to produce diverse flavonoid amygdaloids by oxidizing and degrading sugars in various flavonoid glycosides.

Description

A novel redox enzyme isolated from a soil microorganism and the soil microorganism, a gene encoding the redox enzyme, and a method for producing a flavonoid antagonist using the same. {A NOVEL SOIL MICROORGANISM, A NOVEL OXIDOREDUCTASE SEPERATED FROM THE SAID SOIL MICROORGANISM, A GENE ENCODING THE SAID OXIDOREDUCTASE AND A METHODS FOR PRODUCING FLAVONOID AGLYCONE USING THEREOF}

The present invention relates to a novel microorganism isolated from soil, an oxidoreductase isolated from the microorganism, a method for degrading sugar of various flavonoid glycosides using the same, and a method for producing various flavonoid asparagines.

Ginseng (Panax ginseng C.A. Meyer) is one of the medicines traditionally used for the treatment and prevention of various diseases in Asian countries such as Korea, China and Japan. Ginseng saponin, which is called Jinsen-no-said, is a major active ingredient of ginseng and is known to have various physiological activities such as anti-aging, anti-inflammation, antioxidant activity in the central nervous system and cardiovascular and immune system, antidiabetic activity and antitumor activity.

It is known that ginsenosides are metabolized by intestinal microorganisms after ingestion, and their metabolites have various physiological activities. For example, representative protopanaxyl diol saponins Rb1, Rb2, and Rc are metabolized to CK by intestinal bacteria in humans. Re and Rg1, protopanaxate triol saponins, are metabolized by intestinal bacteria to Rh1 or F1 And exhibits various physiological activities. In the case of CK, it is known that PPD (S), which is a non-sugar chain, has a higher physiological activity effect than Rh2, which has a sugar attached thereto, to prevent tumor invasion and prevent tumor formation.

Therefore, research has been conducted to convert ginsenosides into a reduced sugar metabolite form. In addition to the enzymatic methods, weak acid hydrolysis and alkali decomposition have been reported. However, this method causes various side reactions such as epimerization, hydration, and hydroxylation, And intestinal microorganisms into active ginsenosides have been studied. However, some of the reported microorganisms are mostly anaerobic microorganisms as intestinal microorganisms, which limits industrial use. In addition, most of the enzymes have little activity to convert to asglycosides, and because of their specificity, they are limited to specific ginsenoside production.

To date, there have been many studies on the biotransformation from ginsenoside Rb1 to intestinally produced product CK, but production studies on aspergillosis are lacking. It has also been reported that ginsenosides, which have one sugar in the saponin skeleton, are no longer degraded by microbial enzymes. However, it is known that ginsenosides in the form of the aspirin are more easily absorbed through the bloodstream and act as active compounds. In addition, it can be a base technology that can specifically produce the desired form of ginsenoside through the production of the unglycoside, which is the skeleton of various ginsenosides. Therefore, it was necessary to search for enzymes involved in the production of antiserum of ginsenosides. In addition, it was necessary to investigate whether the enzyme that was searched produces non-saccharides of other components other than the nonsenoside of ginsenoside.

Accordingly, in order to solve the above problems, the present invention provides a novel microorganism and a method for producing various flavonoid antigens using the novel redox enzyme exhibiting a sugar-cleaving activity isolated from the microorganism, a gene encoding the same, and a recombinant protein.

The present invention relates to a process for the preparation of a medicament, The present invention provides a method for producing a flavonoid aglycone using GIN611 ( Rhizobium sp. GIN611) KCTC11708BP or a cell extract thereof as a biocatalyst.

In addition, A method for producing a flavonoid non-saccharide using an oxidative reductase derived from GIN611 as a biocatalyst.

The present invention also relates to a method for producing a cell extract comprising an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, a DNA encoding the oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, a cell extract comprising a host cell transformed with a recombinant DNA vector comprising a DNA encoding an oxidoreductase consisting of the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 3, A cell extract comprising a redox enzyme having 60% or more homology with the sequence and having a saccharide-degrading activity and an oxidoreductase having 60% or more homology with the amino acid sequence of SEQ ID NO: 3 and having a saccharide-degrading activity Lt; RTI ID = 0.0 > biochemical < / RTI > using one or more biocatalysts.

In the present invention, it was confirmed that the enzyme belonging to the oxidoreductase group which does not belong to the previously known glucosideases group had a sugar-degrading activity of the flavonoid glycosides. This novel redox enzyme is completely different from the sequence of the conventional enzymes of glucose degradation and has sequence similarity with that of the redox enzyme group and oxidizes sugar in the flavonoid glycoside to induce spontaneous glucose degradation to produce flavonoid glycoside .

Figure 1 shows the SDS polyacrylamide gel results comparing the protein expressed in the complete medium with the M9 / ginsenoside medium.
Figure 2 shows the results of a comparison of reactivity using proteins prepared from cells cultured in complete medium and M9 / ginsenoside medium.
Fig. 3 shows the results of a new soil microorganism, respisum sp., Grown using ginsenoside as a carbon source. 16S DNA sequence of GIN611.
Fig. The amino acid sequence of the oxidoreductase produced from GIN611 and the gene sequence encoding the amino acid sequence are shown.
Fig. 5 shows the result of measuring the sugar chain activity of Camellia side A and Camellia side B mixture.
Fig. 6 shows the result of measuring the sugar activity of icarin.

The present invention relates to a novel Rhodobium sp. A method of producing a flavonoid aglycone using GIN611 ( Rhizobium sp. GIN611) KCTC11708BP or a cell extract thereof as a biocatalyst. In the present invention, microorganisms growing by using various ginsenoside mixtures as carbon sources were selected, and it was confirmed that they have high reactivity as a result of observing the reactivity using ginsenoside CK of the selected microorganism as a substrate.

In addition, an embodiment of the present invention provides a method for sugar degradation of a flavonoid glycoside using the novel microorganism or cell extract thereof as a biocatalyst.

In addition, one embodiment of the present invention is a method of treating a disease selected from the group consisting of Rhizobium sp. A method for producing a flavonoid non-saccharide using an oxidative reductase derived from GIN611 as a biocatalyst.

In addition, one embodiment of the present invention is a method of treating a disease selected from the group consisting of Rhizobium sp. A method for sugar degradation of a flavonoid glycoside using an oxidative reducing enzyme derived from GIN611 as a biocatalyst.

In addition, one embodiment of the present invention is a cell extract comprising an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, a redox enzyme comprising the amino acid sequence of SEQ ID NO: A cell extract containing a host cell transformed with a recombinant DNA vector comprising a DNA encoding an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3; A cell extract comprising a redox enzyme having 60% or more homology with the amino acid sequence of SEQ ID NO: 3 and having a saccharide-degrading activity and an oxidoreductase having 60% or more homology with the amino acid sequence of SEQ ID NO: 3 and having a saccharide- A method of producing a flavonoid aspartate using one or more biocatalysts selected from the group consisting of to provide.

The DNA encoding the oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3 may be the sequence of SEQ ID NO: 2.

The redox enzyme having 60% or more homology with the amino acid sequence of SEQ ID NO: 3 and having a saccharide-degrading activity can be selected from the group consisting of Agrobacterium sp., Sphingobacterium sp. Or Stenotrophomonase sp. . ≪ / RTI >

A cell extract comprising the cell extract of Rhizobium sp. GIN611, a cell extract comprising an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, and a DNA encoding a redox enzyme comprising the amino acid sequence of SEQ ID NO: 3 A cell extract comprising a transformed host cell, a cell extract comprising a host cell transformed with a recombinant DNA vector comprising a DNA consisting of the sequence of SEQ ID NO: 2, an amino acid sequence having at least 60% homology with the amino acid sequence of SEQ ID NO: 3 A cell extract comprising a host cell transformed with a recombinant DNA vector comprising DNA encoding a protein having a glucose-degrading activity, or a cell extract having a homology of at least 60% to the amino acid sequence of SEQ ID NO: 3 and having a saccharide- May be prepared by inducing enzyme expression by adding ginsenoside.

The flavonoid asparagine can be produced by decomposing a sugar in a flavonoid glycoside. The sugar is decomposed by oxidizing the sugar. For example, in the case of glucose, the hydroxyl group (OH) at the 3-position of the glucose residue is oxidized Spontaneous sugar degradation occurs.

The sugar may be selected from the group consisting of glucose, galactose, rhamnose, arabinose, and xylose, though it is not particularly limited.

The flavonoid glycoside may be selected from the group consisting of, for example, icarin, camellia side A and camellia side B, although not particularly limited.

(1) Ginsenoside: active ingredient of ginseng with ginseng saponin

(2) Compound K (CK): 20- O- ? -D-glucopyranosyl-20 ( S ) -protopanaxadiol

(3) Ginsenoside Rh2: 3- O - beta -D- glycoside pyrazol nosil -20 (S) - Prototype wave incident diol (3- O -beta-D-glycopyranosyl -20 (S) -protopanaxadiol)

4, ginsenoside F2: 3- O - (beta -D- glucoside pyrazol-shi) -20- O - (beta -D- glucopyranosyl) -20 (S) protocol wave incident diol (3- O - ( beta-D-glucopyranosyl) -20- O- (beta-D-glucopyranosyl) -20 ( S ) protopanaxadiol)

(5) Ginsenoside Rb1: 3- O - [(beta-D-glucopyranosyl) (1,2) -beta-D-glucopyranosyl] -20- O - [(beta-D- glucopyranosyl ) (1,6) - beta -D- glucopyranosyl] -20 (S) Pro Coppa incident diol (3- O - [(beta- D-glucopyranosy) (1,2) -beta-D-glucopyranosyl] - O- [beta-D-glucopyranosyl] (1,6) -beta-D-glucopyranosyl] -20 ( S ) protopanaxadiol)

6, ginsenoside Rb2: 3- O-[(beta -D- glucoside pyrazol-shi) (1,2) - beta -D- glucopyranosyl] -20- O-[(alpha -L- arabino pyrazol nosil) (1,6) - beta -D- glucoside pyrazol nosil] -20 (S) protocol wave incident diol (3- O - [(beta- D-glucopyranosy) (1,2) -beta-D-glucopyranosyl] -20- O- [(alpha-L-arabinopyranosyl) (1,6) -beta-D-glucopyranosyl] -20 ( S ) protopanaxadiol)

(7) Ginsenoside Rc: 3- O - [(beta-D-glucopyranosyl) (1,2) -beta-D-glucopyranosyl] -20- O - [(alpha-L-arabinofura nosil) (1,6) - beta -D- glucoside pyrazol nosil] -20 (S) protocol wave incident diol (3- O - [(beta- D-glucopyranosy) (1,2) -beta-D-glucopyranosyl] -20- O- [(alpha-L-arabinofuranosyl) (1,6) -beta-D-glucopyranosyl] -20 ( S ) protopanaxadiol)

8 ginsenoside F1: 20- O - beta -D- glucopyranosyl -20 (S) - Prototype wave incident diol (20- O -beta-D-glucopyranosyl -20 (S) -protopanaxadiol)

(9) Ginsenoside Re: 6- O- [alpha-L-rhamnopyranosyl (1, 2) -beta-D-glucopyranosyl] -20- O- (beta-D- (S) - Prototype wave incident diol (6- O - [alpha-L -rhamnopyranosyl (1, 2) -beta-D- glucopyranosyl] -20- O - (beta-D-glucopyranosyl) -20 (S) -protopanaxatriol )

(10) PPD (S): 20 ( S ) -protopanaxadiol

(11) PPT (S): 20 ( S ) -protopanaxatriol

12 ginsenoside Rg2: 6- O - [alpha -L- ramno pyrazol nosil 1,2-beta -D- glucoside pyrazol nosil] -20 (S) - Prototype wave incident triol (6- O - [alpha] -L-rhamnopyranosyl (1,2) -beta-D-glucopyranosyl] -20 ( S ) -protopanaxatriol)

(13) Icariin: 3,4 ', 5,7-Tetrahydroxy-8-prenylflavone-4'-Me ether-3-O-alpha-L-rhamnopyranoside, 7-O-beta-D-glucopyranoside

(14) Camelliaside A: Camperol 3-O- (2-O-galactopyranosyl-6-O-ramaminopyranosyl) glycoxide (Kampferol 3-O- (2-O-galactopyranoyl -6-O-rhamnopyranosyl) glucopyranoside)

(15) Camelliaside B: Camperol 3-O- (2-O-giopyranosyl-6-O-ramaminopyranosyl) glycoside (Kampferol 3-O- (2-O-xylopyranosyl- 6-O-rhamnopyranosyl) glucopyranoside)

(16) Glycone: A variety of sugar molecules bound to the asparagus.

(17) Whole cell reaction: Whole cell whole reaction without breaking cells or separating enzyme

(18) Redox enzyme: An enzyme that catalyzes a redox reaction to supply energy required for a living body. The oxidation of organic compounds is mostly caused by dehydrogenation.

(19) Redox enzyme extract: Rhodobium sp. Containing redox enzyme. Cell extracts obtained by disrupting cells of GIN611 or a recombinant protein expressing an oxidoreductase

(20) MALDI-TOF mass spectrometer: Matrix Assisted Laser Desorption / Ionization -Time of flight

(21) HPLC: High Performance Liquid Chromatography

(22) PCR: Polymerase chain reaction - A method of specifically amplifying certain regions of DNA

(23) Cloning: A method of incorporating a DNA fragment into a recombinant DNA cloning vector and transforming a host cell using such a recombinant DNA

(24) bp: base pair

Hereinafter, the present invention will be described in detail with reference to examples of the present invention. It is to be understood by those skilled in the art that these embodiments are merely illustrative examples of the present invention in order to more particularly illustrate the present invention and that the scope of the present invention is not limited by these embodiments .

Example  One) Risibium sp . GIN611 Selection of

10 g of soil sample was added to 50 mL of phosphate buffered saline (PBS), and the mixture was stirred at room temperature for 2 hours. The suspended solution was filtered using a filter paper, and 0.2 mL of the filtered microorganism solution was added to the minimum amount of 10 mL of the following Table 1, followed by 3 times of incubation at 30 DEG C for 3 days. Then, 0.2 mL of the culture solution was added The cells were cultured at 30 ° C. for 24 hours in a solid minimal medium consisting of the liquid minimal medium and 1.5% agar. Each of the microorganisms was cultured in a liquid minimal medium in an amount of 3 mL each. The colonies were cultured in a colony of high activity for ginsenoside CK Was identified as Rhizobium species.

Example  2) Preparation of glucosease digestion enzyme extract

Cells cultured in a medium consisting of yeast extract (5 g / L), peptone (10 g / L) and sodium chloride (10 g / L) (hereinafter referred to as complete medium) were washed three times with PBS buffer The medium components outside the cells were removed. The recovered cells were suspended in a buffer solution consisting of 20 mM phosphate buffer, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethanesulfonylfluoride (PMSF), and 1 mM dithiothreitol (DTT) After the cells were disrupted using an ultrasonic disrupter, the cells were centrifuged at 13,000 rpm for 30 minutes, and the supernatant was recovered to produce a final saccharolytic enzyme extract.

Example  3) Gin Senocide  Induction of glucosease-induced expression of glucosease

Cells cultured using the liquid minimal medium (hereinafter referred to as M9 / ginsenoside medium) shown in Table 1 were washed three times with PBS buffer solution to remove the medium components outside the recovered cells. The recovered cells were suspended in a 5-fold volume of a disruption solution, and the cells were disrupted using an ultrasonic disrupter. After centrifugation at 13,000 rpm for 30 minutes, the supernatant was recovered, Cell extracts.

Example  4) With a complete badge M9 / Gin Senocide  Comparison of the reactivity and expression level of the protein produced in the medium

The amounts of the protein extracts of the cell extracts produced in Examples 2 and 3 were quantified, and then the reactivity to ginsenoside CK was compared using the same amount of protein. As a result of the comparison, it was confirmed that the cell extract produced in Example 3 showed higher reactivity than the cell extract produced in Example 2. This is shown in FIG. After reacting using the same amount of protein, the reactivity was compared according to the amount of each protein. As shown in the results, in the case of the protein prepared from the cell cultured in the complete medium of Example 2, when the protein of 500 or more was used, the ginsenoside CK was completely converted to PPD (S), whereas the M9 / ginsenoside culture medium of Example 3 Showed that the protein prepared from the cells cultured in the medium was converted to PPD (S) by a ginsenoside CK of 100 or more. The differences in the protein expression of the two extracts were compared by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and are shown in FIG. The arrows in FIG. 1 indicate proteins having different expression levels when the protein expression levels of the enzyme extracts in Examples 2 and 3 were compared.

Example  5) Isolation and purification of glucosease

Novel phosphoribum sp. The microorganisms were cultured using the liquid restriction medium of Table 1 to which 10 L ginsenoside was added to purify the enzyme that performs the glucose degradation reaction from GIN611. An enzyme extract was prepared from the cultured microorganism in the same manner as in Example 2. The prepared enzyme extract was prepared by 60-70% saturated ammonium sulfate precipitation fractionation. The prepared proteins were separated and purified using FPLC and various columns. The final purified proteins were finally reacted using Native-gel.

Example  6) N-terminal and internal Peptides  Sequencing

The sequence of the N-terminal amino acid of the oxidoreductase obtained by separation and purification in Example 5 was determined by 12% SDS-PAGE electrophoresis and then transferred to a PVDF membrane (BioRad), followed by a procise 491 sequencer Biosystems, Calif.). The sequence of the peptide fragments obtained after treatment with trypsin (promega) for sequencing was analyzed using an LTQ-orbitrap mass spectrometer and sequenced using the PEAKS program.

Example  7) Risibium sp . From GIN611  Whole cell DNA  Isolation

Cells cultured in complete medium were centrifuged at 4,000 rpm for 10 minutes at 4 ° C to precipitate the cells. The supernatant was removed and the cells were dissolved in 10 mL of lysis buffer (15% sucrose, 25 mM EDTA, 25 mM Tris buffer), and then 1.2 mL EDTA (0.5 M) and 0.13 mL pronase were added, Min. Then, 1 mL of 10% SDS was added, and the mixture was allowed to stand at 70 ° C for 10 minutes and then left in ice water for 10 minutes. Then, 2.5 mL of 5M potassium acetate was placed and allowed to react in ice water for 15 minutes. A phenol-chloroform mixture (50:50) in an amount equal to the amount of the solution was added and mixed for 30 minutes. Then, the supernatant was obtained by centrifugation at 4,000 rpm for 10 minutes at 4 ° C. The resulting solution was added with 0.5 times of chloroform, and the mixture was slowly mixed. The mixture was centrifuged at 4,000 rpm for 10 minutes at 4 ° C, and the supernatant was obtained. After the RNase treatment, the mixture was reacted at 37 ° C for 1 hour. After adding 0.8 times as much isopropanol, 2.5 times of 80% ethanol was added and gently shaken. Then, whole cell DNA was collected using a Pasteur pipette, transferred to a 1.5 mL microtube, dried and dissolved in sterilized water Respectively.

Example  8) PCR Gene sequence analysis of glucosease

A primer was prepared from the N-terminal amino acid sequence and the internal sequence obtained through Example 6, and the genomic DNA isolated through the procedure of Example 7 was used as a template to obtain a partial DNA fragment of an oxidoreductase. A primer specifically attached to the obtained DAN fragment was prepared and the remaining sequence was obtained using an inverse PCR method. For the template used for the inverse PCR, the genomic DNA fragment was digested with Hind III restriction enzyme, and the library was subjected to ligation and the self-ligated DNA library was used.

Example  9) recombination into an expression vector and transformation In E. coli  Expression

Gene sequences of the obtained oxidoreductase were digested with BamHI / SalI restriction enzyme, and the fractions were ligated to pETDuet-1 (Novagen), respectively, to generate recombinant plasmids. Transformed into Escherichia coli Rosetta-gami2 (DE3) . The resulting transformed E. coli was cultured in a medium containing ampicillin. When the cell suspension degree was 0.3 to 0.7, IPTG was added and the expression of the enzyme was induced by further culturing at 20 ° C for 15 hours.

Example  10) a mutant of three enzymes having a degree of similarity of 60% or more with the novel enzyme of SEQ ID NO: 3 Gin Senocide  And natural products Glycoside  Investigation of glucose degradation reactivity

Agrobacterium sp., Sphingobacterium sp., Stenotrophomonase sp., Agrobacterium sp. And an enzyme having an amino acid similarity of 60% or more to the resulting sequence number 3 was cloned to examine the glucose degradation reactivity to ginsenoside. It was confirmed that the enzyme derived from each microorganism was degraded to glucose by the oxidation reaction to the glucose residue of ginsenoside. In addition, it was confirmed that sugar chains were active using flavonoids such as Icarlin, Camelliaside A and Camelliaside B.

Example  11) Induced expression of the enzyme Antiserum  Production activity measurement

The expression-induced enzyme in Example 10 was prepared by the enzyme preparation method of Example 2, and the activity of producing an aspartic acid was measured using ginsenoside CK as a substrate. In addition, it was confirmed that sugar chains were active using flavonoid icarin, camellia side A and camellia side B.

Example  12) Various Antiserum  According to structure Sugar  Active measurement and On the glacier  Investigation of substrate specificity

Rizobium sp. An enzyme whose expression was induced from GIN611-derived oxidoreductase was reacted according to the above reaction method using flavonoid icarin, camellia side A, and camellia side B as substrates, and then activity was measured using a Mali mass spectrometer. As a result, The oxidation reaction and galactolytic activity of galactose on Camellia side A were confirmed. In addition, the oxidation reaction and the sugar chain activity of Xylose on Camellia side B were confirmed. In other words, it showed a sugar-soluble activity for sugar bound to a flavonoid-based non-sugar body structure, and it had a function of oxidizing galactose and xylose in addition to glucose and then decomposing sugar (Fig. 5). In addition, the oxidation reaction of glucose attached to icarin and the activity of sugar disruption were confirmed (Fig. 6).

Example  13) Glacial  Investigation of substrate specificity for binding

4-Nitrophenyl? -D-glucopyranoside, 4-Nitrophenyl? -D-glucopyranoside, 4-Nitrophenyl? -D-glucopyranoside, - Nitrophenol-Al

D-galactopyranoside, 4-Nitrophenyl-D-galactopyranoside, and 4-Nitrophenyl-D-galactopyranoside. And beta-linkage, the results showed that both alpha and beta were active. (Table 2)

Korea Biotechnology Research Institute KCTC11708BP 20100604

<110> AMOREPACIFIC CORPORATION <120> A NOVEL SOIL MICROORGANISM, A NOVEL OXIDOREDUCTASE SEPERATED FROM          THE SAID SOIL MICROORGANISM, A GENE ENCODING THE SAID          OXIDOREDUCTASE AND A METHODS FOR PRODUCING FLAVONOID AGLYCONE          USING THEREOF <130> 10P315IND <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 971 <212> DNA <213> Rhizobium sp. GIN611 <400> 1 gaatgcgagc ttaccatgca gtcgagcgcc ccgcaagggg agcggcagac gggtgagtaa 60 cgcgtgggaa tctaccgagc cctgcggaat agctccggga aactggaatt aataccgcat 120 acgccctacg ggggaaagat ttatcggggt ttgatgagcc cgcgttggat tagctagttg 180 gtggggtaaa ggcctaccaa ggcgacgatc catagctggt ctgagaggat gatcagccac 240 attgggactg agacacggcc caaactccta cgggaggcag cagtggggaa tattggacaa 300 tgggcgcaag cctgatccag ccatgccgcg tgagtgatga aggccctagg gttgtaaagc 360 tctttcaacg gtgaagataa tgacggtaac cgtagaagaa gccccggcta acttcgtgcc 420 agcagccgcg gtaatacgaa gggggctagc gttgttcgga attactgggc gtaaagcgca 480 cgtaggcgga tatttaagtc aggggtgaaa tcccggggct caacctcgga actgcctttg 540 atactgggta tcttgagtat ggaagaggta agtggaattg cgagtgtaga ggtgaaattc 600 gtagatattc gcaggaacac cagtggcgaa ggcggcttac tggtccatta ctgacgctga 660 ggtgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga 720 tgaatgttag ccgtcgggca gtatactgtt cggtggcgca gctaacgcat taaacattcc 780 gcctggggag tacggtcgca agattaaaac tcaaaggaat tgacgggggc ccgcacaagc 840 ggtggagcat gtggtttaat tcgaagcaac gcgcagaacc ttaccagctc ttgacattcg 900 gggtatgggc agtggagaca ttgtccttca gttaggctgg ccccagaaca ggtgctgcat 960 ggctgtcgtc a 971 <210> 2 <211> 1686 <212> DNA <213> Rhizobium sp. GIN611 <400> 2 atggcgaata atcattacga cgcgattgtt gtcggttcgg ggatcagcgg aggctgggcc 60 gcaaaagaac tcacacaaaa gggtctaaaa gttcttcttc ttgaacgtgg cagaaacatt 120 gaacacatca ccgattacca gaatgcagac aaggaagcgt gggactaccc tcaccgcaat 180 cgtgccacgc aggaaatgaa ggcaaagtat ccggttctga gccgcgatta tctgttggaa 240 gaagccacac tcggcatgtg ggctgacgaa caagaaacgc cttacgtcga agaaaaacgt 300 ttcgattggt tccgtgggta ccacgtgggt ggtcgttctc tcctttgggg ccgtcaaacc 360 tatcgatggt cacagaccga ttttgaggcc aatgcaaaag aaggcatcgc tgttgattgg 420 cctattcgtt accaggatgt tgcgccgtgg tacgactacg ttgaacggtt tgcgggcatt 480 tccggcagca aagaagggct cgatatcctt cctgatggtg aattccttcc accaatccct 540 ttgaactgcg tagaagaaga tgtggcgcgt cgtctgaagg acaggttcaa gggcacgcgt 600 cacctgatca attcccgctg cgccaacatc acacaggaac ttcctgacca ggatcgcaca 660 cgctgtcagt tcagaaacaa gtgtcggttg ggctgtccgt tcggcggtta cttcagcaca 720 caatcatcaa ccctgcctgc ggccgtcgcg accggcaatc tcaccctgcg gccgttctca 780 atcgtcaagg agatccttta cgacaaggac aagaagaagg cccgcggtgt cgagatcatc 840 gatgccgaaa ccaacatgac ctatgaatat accgcagaca ttatcttcct gaatgcctca 900 acgctgaatt cgacctgggt cctgatgaac tcagccaccg acgtgtggga agggggattg 960 ggaagcagtt ccggcgaact cggccacaat gtcatggacc atcatttccg catgggtgcg 1020 acgggtgagg tcgaaggatt tgacgagttc tatttcaagg gacgccgccc ggcaggtttc 1080 tacattcctc gcttccgcaa catcggcgat gaaaagcgta aatatctgcg tggttttggt 1140 tatcagggtt cggcaagccg ctcccgctgg gagcgcgaaa tcgccgagat gaatattgga 1200 gcagattata aagacgcgtt gaccgaacca ggcggctgga caatcggcat gacagccttt 1260 ggcgagatgc tgccctacca cgaaaatcgc gtgaagcttg accaaaacaa aaaggacaaa 1320 tgggggttgc cggtcctttc aatgaatgtc gagttgaaac aaaacgaact cgatatgcgt 1380 gaagacatgg tgaatgacgc tgtcgaaatg tttgaggccg tcggcatcaa gaacgtcaaa 1440 ccgacccgag gcagctacgc acccggtatg ggtattcacg aaatgggaac ggcgcgcatg 1500 ggccgcgatc caaagtcttc ggttctaaat ggcaacaacc aggtgtggga tgcccctaac 1560 gtgttcgtga cggatggtgc ctgcatgacg tctgctgcct gtgtaaatcc gtctctcacc 1620 tacatggcac tgacggcacg tgccgccgat tttgccgtgt cagagctcaa gaagggaaat 1680 ctgtaa 1686 <210> 3 <211> 561 <212> PRT <213> Rhizobium sp. GIN611 <400> 3 Met Ala Asn Asn His Tyr Asp Ala Ile Val Val Gly Ser Gly Ile Ser   1 5 10 15 Gly Gly Trp Ala Ala Lys Glu Leu Thr Gln Lys Gly Leu Lys Val Leu              20 25 30 Leu Leu Glu Arg Gly Arg Asn Ile Glu His Ile Thr Asp Tyr Gln Asn          35 40 45 Ala Asp Lys Glu Ala Trp Asp Tyr Pro His Arg Asn Arg Ala Thr Gln      50 55 60 Glu Met Lys Ala Lys Tyr Pro Val Leu Ser Arg Asp Tyr Leu Leu Glu  65 70 75 80 Glu Ala Thr Leu Gly Met Trp Ala Asp Glu Gln Glu Thr Pro Tyr Val                  85 90 95 Glu Glu Lys Arg Phe Asp Trp Phe Arg Gly Tyr His Val Gly Gly Arg             100 105 110 Ser Leu Leu Trp Gly Arg Gln Thr Tyr Arg Trp Ser Gln Thr Asp Phe         115 120 125 Glu Ala Asn Ala Lys Glu Gly Ile Ala Val Asp Trp Pro Ile Arg Tyr     130 135 140 Gln Asp Val Ala Pro Trp Tyr Asp Tyr Val Glu Arg Phe Ala Gly Ile 145 150 155 160 Ser Gly Ser Lys Glu Gly Leu Asp Ile Leu Pro Asp Gly Glu Phe Leu                 165 170 175 Pro Pro Ile Pro Leu Asn Cys Val Glu Glu Asp Val Ala Arg Arg Leu             180 185 190 Lys Asp Arg Phe Lys Gly Thr Arg His Leu Ile Asn Ser Arg Cys Ala         195 200 205 Asn Ile Thr Gln Glu Leu Pro Asp Gln Asp Arg Thr Arg Cys Gln Phe     210 215 220 Arg Asn Lys Cys Arg Leu Gly Cys Pro Phe Gly Gly Tyr Phe Ser Thr 225 230 235 240 Gln Ser Ser Thr Leu Pro Ala Ala Val Ala Thr Gly Asn Leu Thr Leu                 245 250 255 Arg Pro Phe Ser Ile Val Lys Glu Ile Leu Tyr Asp Lys Asp Lys Lys             260 265 270 Lys Ala Arg Gly Val Glu Ile Ile Asp Ala Glu Thr Asn Met Thr Tyr         275 280 285 Glu Tyr Thr Ala Asp Ile Phe Leu Asn Ala Ser Thr Leu Asn Ser     290 295 300 Thr Trp Val Leu Met Asn Ser Ala Thr Asp Val Trp Glu Gly Gly Leu 305 310 315 320 Gly Ser Ser Ser Gly Glu Leu Gly His Asn Val Met Asp His His Phe                 325 330 335 Arg Met Gly Ala Thr Gly Glu Val Glu Gly Phe Asp Glu Phe Tyr Phe             340 345 350 Lys Gly Arg Arg Pro Ala Gly Phe Tyr Ile Pro Arg Phe Arg Asn Ile         355 360 365 Gly Asp Glu Lys Arg Lys Tyr Leu Arg Gly Phe Gly Tyr Gln Gly Ser     370 375 380 Ala Ser Arg Ser Arg Trp Glu Arg Glu Ile Ala Glu Met Asn Ile Gly 385 390 395 400 Ala Asp Tyr Lys Asp Ala Leu Thr Glu Pro Gly Gly Trp Thr Ile Gly                 405 410 415 Met Thr Ala Phe Gly Glu Met Leu Pro Tyr His Glu Asn Arg Val Lys             420 425 430 Leu Asp Gln Asn Lys Lys Asp Lys Trp Gly Leu Pro Val Leu Ser Met         435 440 445 Asn Val Glu Leu Lys Gln Asn Glu Leu Asp Met Arg Glu Asp Met Val     450 455 460 Asn Asp Ala Val Glu Met Phe Glu Ala Val Gly Ile Lys Asn Val Lys 465 470 475 480 Pro Thr Arg Gly Ser Tyr Ala Pro Gly Met Gly Ile His Glu Met Gly                 485 490 495 Thr Ala Arg Met Gly Arg Asp Pro Lys Ser Ser Val Leu Asn Gly Asn             500 505 510 Asn Gln Val Trp Asp Ala Pro Asn Val Phe Val Thr Asp Gly Ala Cys         515 520 525 Met Thr Ser Ala Ala Cys Val Asn Pro Ser Leu Thr Tyr Met Ala Leu     530 535 540 Thr Ala Arg Ala Asp Phe Ala Val Ser Glu Leu Lys Lys Gly Asn 545 550 555 560 Leu    

Claims (9)

As a method for producing flavonoid aglycone,
The production method described above is a method for producing Ribium sp. GIN611 ( Rhizobium sp. GIN611) KCTC11708BP or a cell extract thereof was used as a biocatalyst,
The flavonoid asparagine is produced by decomposing sugar in a flavonoid glycoside,
Wherein the flavonoid glycosides are at least one selected from the group consisting of Icarin, Camelliaceide A and Camelliaceide B. delete A cell extract comprising an oxidoreductase consisting of the amino acid sequence of SEQ ID NO: 3; a recombinant DNA vector comprising a DNA encoding the oxidoreductase consisting of the amino acid sequence of SEQ ID NO: 3; , And a cell extract comprising a host cell transformed with a recombinant DNA vector comprising a DNA encoding an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3. A method for producing a flavonoid antagonist using a catalyst. The method of claim 3,
Wherein the flavonoid aspartate is produced by degrading a sugar in a flavonoid glycoside. 5. The method of claim 4,
Wherein the flavonoid glycoside is selected from the group consisting of icarin, camellia side A, and camellia side B. The method according to claim 1 or 4,
Wherein the saccharide is selected from the group consisting of glucose, galactose, rhamnose, arabinose and xylose. The method according to claim 1 or 4,
Wherein the sugar chain is decomposed by oxidizing the saccharide. delete The method according to claim 1 or 3,
Wherein the cell extract is prepared by adding ginsenoside to induce expression of the enzyme.

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