KR101749680B1 - A novel soil microorganism, a novel oxidoreductase seperated from the said soil microorganism, a gene encoding the said oxidoreductase and a methods for producing effective ginsenosides using thereof - Google Patents

Abstract

The present invention relates to novel Rebium sp. There is provided a recombinant strain comprising GIN611 ( Rhizobium sp. GIN611) KCTC11708BP or a cell extract thereof, a novel redox enzyme exhibiting glycosylation activity, a gene encoding it and a recombinant vector protein, or an expression vector encoding a recombinant protein, And a method of sugar-digesting natural products using these as a biocatalyst. Also provided are methods of using these to produce various ginsenoside antiserum. 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 the activity of degrading glycosenoside. This novel redox enzyme enables the production of various aspartates by oxidizing glucose residues in natural product glycosides.

Description

TECHNICAL FIELD The present invention relates to a novel redox enzyme isolated from a soil microorganism and the soil microorganism, a gene encoding the oxidoreductase, and a method for producing various ginsenosides using the same. BACKGROUND OF THE INVENTION 1. Field of the Invention [0002] A GENE ENCODING THE SAID OXIDOREDUCTASE AND METHODS FOR PRODUCING EFFECTIVE GINSENOSIDES USING THEREOF}

The present invention relates to a novel microorganism isolated from a soil, an oxidoreductase isolated from the microorganism, and a method for producing various ginsenoside antigens and a sugar degradation reaction of various plant glycosides using the same.

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.

To date, about 40 kinds of ginsenosides have been isolated and identified. Ginsenoside, a glycoside containing a non-saccharide, which is a dammarane structure, includes protopanaxadiol, protopanaxatriol, (protopanaxatriol) system can be roughly divided into. The ginsenosides belonging to the protopanaxadiol type saponin are mainly Rb1, Rb2, Rc and Rd, and the ginsenosides belonging to the protoquinol triol saponin are mainly Re and Rg1. (See Figs. 1 and 2)

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.

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

The present invention relates to novel Rebium sp. GIN611 ( Rhizobium sp. GIN611) KCTC11708BP or a cell extract thereof.

The present invention also provides a method for sugarysis of a natural product using Rizbium sp. GIN611 or a cell extract thereof as a biocatalyst.

The present invention also provides a method for producing a product from the substrate of Table 1 using Rizbium sp. GIN611 or a cell extract thereof as a biocatalyst.

The present invention also provides an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3 or a cell extract containing the same.

The present invention also provides a DNA encoding an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, a DNA comprising the sequence of SEQ ID NO: 2, a recombinant DNA vector comprising the DNA, a host cell transformed with the recombinant DNA vector, And a cell extract comprising the host cell.

In addition, the present invention provides an oxidoreductase having a sugar chain activity that is at least 60% homologous to the sequence of SEQ ID NO: 3, or a cell extract comprising the oxidoreductase.

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 DNA encoding an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, a sequence selected from the group consisting of SEQ ID NO: 3 And an oxidoreductase cell extract having 60% or more homology with the sequence of SEQ. ID. NO. 3 and having a sugar activity of 60% or more homology with the sequence of SEQ ID NO. 3, To provide a method for sugar disintegration of natural products.

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: 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 cell extract comprising a host cell transformed with the sequence of SEQ ID NO: 2 , 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, a cell extract comprising a host cell transformed with the sequence of SEQ ID NO: 3 by 60% , An oxidoreductase having a sugar activity with a homology of at least 60% with the sequence of SEQ ID NO: 3 A method for producing a product from a substrate of the following Table 1 using a biocatalyst selected from the group consisting of an oxidoreductase cell extract having a sugar activity.

The present invention also relates to a cell extract of Rizbium sp. GIN611, a cell extract comprising an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, a recombinant DNA comprising a DNA encoding the oxidoreductase comprising the amino acid sequence of SEQ ID NO: A cell extract comprising a host cell transformed with a DNA vector, 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, or a cell extract comprising a sequence of SEQ ID NO: A method for producing a cell extract comprising an oxidoreductase having a homogeneous activity and a sugar activity, the method comprising the step of inducing the expression of an enzyme by adding ginsenoside.

Generally, the enzyme for degrading ginsenoside is known as an enzyme belonging to the glucosidase group. In the present invention, it was confirmed that the enzyme belonging to the oxidoreductase group not belonging to the previously known glucosideases group had a saccharide-decomposing activity of ginsenoside. This novel redox enzyme is completely different from the sequence of the conventional enzymes of glucose degradation related enzymes and has sequence similarity with the redox enzyme group and oxidizes the glucose residue in the natural glycoside to induce spontaneous glucose degradation .

Figure 1 shows protopanaxadiol (PPD) ginsenosides.
Figure 2 shows protopanaxatriol (PPT) ginsenosides.
FIG. 3 is an objective reaction for producing an aspartic acid PPD (S) from a ginsenoside Compound K (CK) through sugarysis.
Figure 4 shows the results of the reactivity analysis with a novel redox enzyme. The asterisk indicates oxidized CK and the triangle indicates PPD (S). Line 3 is the reaction result for 3 hours, and 4 is the reaction result for 12 hours. The substrate CK decreases with time, the oxidized CK corresponding to the intermediate tends to increase while decreasing, and the final product, PPD (S), shows a steady increase over time.
FIG. 5 shows mass analysis results of the oxidized CK analyzed in FIG.
Figure 6 is a SDS polyacrylamide gel result comparing the protein expressed in the complete medium with the M9 / ginsenoside medium.
FIG. 7 shows the results of a comparison of reactivity using proteins prepared from cells cultured in complete medium and M9 / ginsenoside medium.
Fig. 8 shows the results of a new soil microorganism, respisum sp. 16S DNA sequence of GIN611.
FIG. An amino acid sequence of an oxidoreductase produced from GIN611, and a gene sequence encoding the amino acid sequence.
10 is a reaction mechanism of a novel redox enzyme.
Figure 11 shows that the enzyme is specifically reactive to glucose as a result of the reactivity of the novel redox enzyme to various ginsenosides and isoflavones.

The present invention relates to novel Rebium sp. GIN611 ( Rhizobium sp. GIN611) microorganism or a cell extract thereof. 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 sugarysis of a natural product using the novel microorganism or a cell extract thereof as a biocatalyst. The sugar may be glucose, rhamnose, arabinose or xylose. In addition, the above-described sugar disintegration method is a method of decomposing glucose of a natural glycocide, and is preferably a method of decomposing glucose of ginsenoside. The ginsenosides are preferably, but not particularly limited to, ginsenoside compound K (CK), ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, Side Rb3, ginsenoside F1, ginsenoside Re, and daidzin. The above-described method of saccharification is a method of spontaneously dissolving sugar by oxidizing the hydroxyl group (OH) at position 3 of the glucose residue.

In addition, one embodiment of the present invention provides a method for producing various ginsenoside antigens using Rizbium sp. GIN611 or a cell extract thereof as a biocatalyst. The ginsenoside antagonist is not limited, but is the product of Table 1, produced, for example, by the degradation of glucose from the substrate of Table 1. Specifically, the substrate is a ginsenoside compound K and the product is ginsenoside PPD (S).

temperament product Ginsenoside compound K (CK) Ginsenoside PPD (S) Ginsenoside Rh2 Ginsenoside PPD (S) Gin Senocide F2 Ginsenoside PPD (S) Ginsenoside Rb1 Ginsenoside PPD (S) Ginsenoside Rb2 Ginsenoside Compound Y Gin Senocide Rc Gin Senoide Mc Ginsenoside Rb3 Ginsenoside Compound Mx Gin Senocide F1 Ginsenoside PPT (S) Gin Senocide Re Ginsenoside Rg2 Daidzin Daidzein

The term used in the present invention is commonly used in the art and can be understood by anyone skilled in the art. Hereinafter, a brief description will be given herein.

(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 Rb3: 3- O - [(beta -D-glucopyranosy) (1,2) - beta -D-glucopyranosyl] -20- O - [ ( beta -D-xylopyranosyl) (1, 6 ) beta -D- glucopyranosyl] -20 (S) protocol wave incident diol (3- O - [(beta- D-glucopyranosy) (1,2) -beta-D-glucopyranosyl] -20- O - [( beta-D-xylopyranosyl) (1,6) -beta-D-glucopyranosyl] -20 ( S ) protopanaxadiol)

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

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

11 daidzin (Daidzin): daidzein 7- O - beta -D- glucoside (daidzein 7- O -beta-D- glucoside)

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

13. Compound Y: 20- O - [(alpha -L- arabino pyrazol nosil) (1,6) - beta -D- glucoside pyrazol nosil] -20 (S) protocol wave incident diol (20- O - [ (alpha-L-arabinopyranosyl) (1,6) -beta-D-glucopyranosyl] -20 (S) protopanaxadiol)

14 Compound Mc: 20- O - [(alpha -L- arabinofuranosyl) (1,6) - beta -D- glucopyranosyl] -20 (S) protocol wave incident diol (20- O - [ (alpha-L-arabinofuranosyl) (1,6) -beta-D-glucopyranosyl] -20 ( S ) protopanaxadiol)

15. Compound Mx: 20- O - [(beta -D- xylene in pyrazol nosil) (1,6) - beta -D- glucoside pyrazol nosil] -20 (S) protocol wave incident diol (20- O - [ (beta-D-xylopyranosyl) (1,6) -beta-D-glucopyranosyl] -20 ( S ) protopanaxadiol)

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

17 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)

(18) Daidzein: 7-hydroxy-3- (4-hydroxyphenyl) chromen-4-one

(19) Whole-cell reaction: Whole cell-free reaction without breaking cells or separating enzyme

(20) 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.

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

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

(23) HPLC: High Performance Liquid Chromatography

(24) PCR: Polymerase chain reaction - A method to specifically amplify certain regions of DNA

(25) ORF: Open reading frame, an array from initiation codon to stop codon

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

(27) bp: base pair

An embodiment of the present invention provides an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3 or a cell extract comprising the oxidoreductase. The above-mentioned redox enzyme may preferably be obtained by separation and purification from Rizobium sp GIN611.

One embodiment of the present invention provides a DNA sequence encoding the amino acid sequence of SEQ ID NO: 3 or a DNA sequence encoding the redox enzyme. The sequence is specifically SEQ ID NO: 2. The DNA is preferably a DNA encoding an oxidoreductase obtained by isolating and purifying from Rizobium sp GIN611.

In addition, one embodiment of the present invention is a polypeptide having a homology of not less than 60%, preferably not less than 90%, more preferably not less than 97%, still more preferably not less than 99% Provides DNA encoding the protein. At this time, the sugar may be glucose, rhamnose, arabinose or xylose, preferably glucose.

An embodiment of the present invention is a recombinant DNA comprising a DNA sequence encoding the amino acid sequence of SEQ ID NO: 3, a DNA sequence encoding the oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, or a DNA sequence comprising the DNA sequence of SEQ ID NO: DNA vector.

One embodiment of the present invention provides a host cell transformed with the recombinant DNA vector and a cell extract comprising the host cell.

One embodiment of the present invention relates to a protein having a sugar chain activity which has 60% or more, preferably 90% or more, more preferably 97% or more, and even more preferably 99% or more homology with the sequence of SEQ ID NO: 3, An enzyme and a cell extract comprising the redox enzyme. The oxidoreductase can be, but is not limited to, Agrobacterium sp., Sphingobacterium sp. Or from Stenotrophomonase sp. ≪ / RTI >

The sugar may be glucose, rhamnose, arabinose or xylose. In addition, the above-mentioned redox enzyme decomposes the glucose of the natural glycoside, and preferably decomposes the glucose of the ginsenoside. The ginsenosides are not particularly limited, but preferably include ginsenoside compound K, ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rb3, Ginsenoside F1, ginsenoside Re, and daidzin. The oxidoreductase oxidizes the hydroxyl group (OH) at the 3-position of the glucose residue to spontaneously cause sugar disintegration.

The redox enzyme produces a variety of ginsenoside antigens. For example, the substrate in Table 1 decomposes glucose to produce a product. Specifically, the substrate is a ginsenoside compound K and the product is ginsenoside PPD (S).

An 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 cell extract containing the oxidoreductase comprising the amino acid sequence of SEQ ID NO: A cell extract comprising a host cell transformed with a recombinant DNA vector comprising DNA, 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 cell extract comprising a host cell transformed with a recombinant DNA vector comprising a DNA consisting of the sequence of SEQ ID NO: 2, a cell extract comprising a sequence of SEQ ID NO: 3, A recombinant DNA vector comprising a DNA encoding a protein having a sugar activity of at least 60% A transformed host cell, a cell extract comprising a host cell transformed with a recombinant DNA vector comprising a DNA encoding a protein having a homology of at least 60% with the sequence of SEQ ID NO: 3 and having a sugar-active activity, A redox enzyme having 60% or more homology with the sequence of SEQ ID NO: 3 and having sugar activity and a redox enzyme cell extract having 60% or more homology with the sequence of SEQ ID NO: 3 and having sugar activity One method for producing various ginsenoside antisera using biocatalysts is provided. For example, a method of producing a product from the substrate of Table 1 above is provided. For example, the substrate is a ginsenoside compound K and the product is a ginsenoside PPD (S).

An embodiment of the present invention includes a cell extract of Rizobium sp. GIN611, a cell extract containing an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3, and a DNA encoding the oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3 A cell extract comprising a host cell transformed with the recombinant DNA vector, a cell extract comprising a host cell transformed with a recombinant DNA vector comprising DNA consisting of the sequence of SEQ ID NO: 2, a cell extract comprising 60% A cell extract comprising a host cell transformed with a recombinant DNA vector comprising a DNA encoding a protein having the above homology and having a sugar activity and having a homology of not less than 60% with the sequence of SEQ ID NO: 3, The present invention relates to a method for producing a cell extract containing an oxidoreductase, which comprises adding ginsenoside to induce enzyme expression It provides a cell extract preparation, characterized in that.

Gin Senocide Sugar  Selection of microorganisms containing enzymes with activity

For the selection of soil microorganisms, the present inventors constructed a minimal medium containing a mixture of ginsenosides having the compositional components shown in the following Table 2 as a carbon source, and used the enzyme as an enzyme having activity to ginsenoside CK Were selected. Table 2 below is the minimum medium containing the ginsenoside mixture as a carbon source.

The selection method using the minimal medium of the present invention is a simple method of selecting microorganisms according to the growth rate. Since microorganisms having low sugar activity are spontaneously removed at the culture stage, highly active microorganisms including enzymes having high activity can be selected.

The microorganisms selected in the present invention are novel ones, and the inventors identified them as Rhizobium species through the characteristic of the DNA sequence encoding the 16s r RNA partial sequence thereof, GIN611. Also, it was deposited in KCTC (Korean Collection for Type Cultures) on June 4, 2010, and the deposit number is KCTC11708BP.

The selected < RTI ID = 0.0 > As a result of confirming the substrate specificity using a glycosidase isolated from GIN611, the microorganism showed high activity on ginsenoside CK and was found to have sugar activity on various ginsenosides in addition to ginsenoside CK. The present inventors also disrupted the microorganism, centrifuged it, and produced a cell extract containing the active enzyme from the supernatant of the centrifugation liquid.

Sugar  Enzyme extract With biocatalyst  Used Gin Senocide Antiserum  Production method

A non-saccharide PPD (S) is produced from a reaction solution composed of the microorganism or the resulting saccharogenic enzyme extract, ginsenoside CK. The microorganism or produced enzyme extract of the present invention is added to the reaction solution as a biocatalyst to initiate the reaction.

In addition to the ginsenoside CK, the substrate usable in the present invention may contain, in addition to ginsenoside CK, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside F2, Side Rh2, and ginsenoside Re and ginsenoside F1 belonging to the PPT system. There is also the isoflavone-based Daidzin.

Hereinafter, the present invention will be described in detail with reference to examples of the present invention. It will be apparent to those skilled in the art that these embodiments are provided by way of illustration only for the purpose of more particularly illustrating the present invention and that the scope of the present invention is not limited by these embodiments .

Example  One) Risibium sp . GIN611  Selection

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 suspension was filtered with a filter paper to remove suspended solids, and 0.2 mL of the filtered microorganism solution was added to 10 mL of the minimum medium shown in Table 2 and incubated at 30 DEG C for 3 days. 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 shredder, the supernatant was recovered by centrifugation at 13,000 rpm for 30 minutes, and the final sugar solution enzyme extract was produced.

Example  3) Gin Senocide  By addition Sugar  Induction of enzyme expression

Cells cultured using the liquid minimal medium (hereinafter referred to as M9 / ginsenoside medium) shown in Table 2 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, and the supernatant was recovered. Cell extracts.

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

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 protein expression between the two extracts were compared using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and are shown in FIG. The arrows in FIG. 6 indicate proteins having different expression levels when the protein expression levels of the enzyme extracts in Examples 2 and 3 were compared.

Example 5) Separation of glycosylase

New respiration sp. The microorganisms were cultured from the GIN611 using the liquid restriction medium of Table 2 supplemented with 10 L of ginsenoside to purify the enzyme performing the reaction shown in Fig. An enzyme extract was prepared by the same method as in Example 2 for the cultured microorganism. 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 protein was finally reacted using Native-gel.

Example 6: Investigation of activity against ginsenoside CK using purified enzyme

The activity against the ginsenoside CK was examined using the enzyme purified in Example 5. About 100 μg of the enzyme solution and 0.1 mM CK, 50 mM phosphate buffer solution (pH 6.5) were added to the reaction volume in the same amount of ethyl acetate, and the reaction was terminated and analyzed by HPLC. The conditions used for the analysis were 80% acetonitrile (ACN). The results are shown in FIG. 4, the peaks indicated by triangles correspond to PPD (S), and the peaks indicated with an asterisk indicate oxidized CK, the CK oxidized with time increases and decreases, and PPD (S ) Were found to increase continuously. Therefore, it was found that the sugar was decomposed through the oxidation process of ginsenoside CK by the newly discovered enzyme. This is shown in Fig.

Example 7) Determination of N-terminus and internal peptide sequence of a novel enzyme

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 8) Rhizobium sp. Whole cell DNA isolation from GIN611

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) 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. Chloroform was added to the obtained solution, and the mixture was slowly mixed. The mixture was centrifuged at 4,000 rpm for 10 minutes at 4 ° C, and the supernatant was collected. After the RNase treatment, the reaction 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 9) Gene sequencing of glucosease by PCR

A primer was prepared from the N-terminal amino acid sequence and the internal sequence obtained through Example 7, and the genomic DNA isolated through the procedure of Example 8 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 10) Recombination into expression vector and expression in transformed E. coli

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 11) Glucose degradation activity of ginsenosides and natural product-derived glycosides of three enzymes having a degree of similarity of 65% or more to the novel enzyme of SEQ ID NO: 3

Agrobacterium sp. Sphingobacterium sp., Stenotrophomonase sp. ≪ RTI ID = 0.0 > And an enzyme having 60% or more amino acid similarity to SEQ ID NO: 3 was cloned to investigate the glucose degradation reactivity to ginsenoside. It was confirmed that the enzymes derived from each microorganism were sugar-soluble through an oxidation reaction to the glucose residue of ginsenoside.

Example 12: Measurement of non-saccharide production activity using an inducible expressed enzyme

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.

Example 13 Measurement of activity of various ginsenosides and isoflavones as substrates

The present invention relates to a pharmaceutical composition comprising at least one compound selected from the group consisting of ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rb3, ginsenoside F1, ginsenoside Re and isoflavone glycoside daidzin ) Was used as a substrate. After the reaction, the same amount of ethyl acetate was added to the reaction mixture. The ethyl acetate layer was dried and then dissolved in ethanol. The activity was measured using a Maldi mass spectrometer.

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 METHODS FOR PRODUCING EFFECTIVE GINSENOSIDES          USING THEREOF <130> 10P265IND <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 (28)

An oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3. A cell extract comprising the oxidoreductase of claim 1. A DNA encoding the oxidoreductase of claim 1. 4. The DNA of claim 3, comprising the sequence of SEQ ID NO: 2. A recombinant DNA vector comprising the DNA of claim 3. A recombinant DNA vector comprising the DNA of claim 4. A host cell transformed with the recombinant DNA vector of claim 5. A host cell transformed with the recombinant DNA vector of claim 6. A cell extract comprising the host cell of claim 7. A cell extract comprising the host cell of claim 8. The redox enzyme according to claim 1, wherein the oxidoreductase degrades glucose of the ginsenoside or isoflavone glycoside. 12. The composition of claim 11, wherein the ginsenoside is selected from the group consisting of ginsenoside K (CK), ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rb3, Wherein the isoflavone-based glycoside is at least one selected from the group consisting of ginsenoside F1 and ginsenoside Re, and the isoflavone-based glycoside is daidzin. 2. The redox enzyme according to claim 1, wherein the oxidoreductase produces a product from the substrate of Table 1 below:

. A method for producing a recombinant vector, which comprises using an oxidoreductase of claim 1, a cell extract of claim 2, a host cell of claim 7, a host cell of claim 8, a cell extract of claim 9, and a cell extract of claim 10, A method of glycosylation of ginsenosides or isoflavone glycosides. 15. The method according to claim 14, wherein the sugar disaggregation method decomposes glucose of the ginsenoside or isoflavone glycoside. 15. The method of claim 14, wherein the ginsenoside is selected from the group consisting of ginsenoside K, ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rb3, F1, and ginsenoside Re, and wherein the isoflavone-based glycoside is daidzin. A cell extract of claim 1, a host cell of claim 7, a host cell of claim 8, a cell extract of claim 9, and a cell extract of claim 10 as a biocatalyst, Methods for producing the product from the substrate of Table 1:

. 18. The method of claim 17, wherein the substrate is a ginsenoside compound K and the product is a ginsenoside PPD (S). A novel recombinant sp. Producing an oxidoreductase comprising the amino acid sequence of SEQ ID NO: 3 according to claim 1. GIN611 ( Rhizobium sp. GIN611) KCTC11708BP. A method according to claim 19, Cell extracts of GIN611. A method according to claim 19, A method for sugar digestion of a ginsenoside or isoflavone glycoside using the cell extract of GIN 611 or 20 as a biocatalyst. 22. The method according to claim 21, wherein the sugar disintegration method decomposes glucose in a ginsenoside or isoflavone glycoside. 23. The composition of claim 22, wherein the ginsenoside is selected from the group consisting of ginsenoside K (CK), ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rb3, Wherein the isoflavone-based glycoside is at least one selected from the group consisting of ginsenoside F1 and ginsenoside Re, and the isoflavone-based glycoside is daidzin. 22. The method according to claim 21, wherein the method of saccharification resolves the hydroxyl group (OH) at position 3 of the glucose residue. A production method for producing a product from the substrate of the following Table 1 using the Rizbium sp. GIN 611 of claim 19 or the cell extract of claim 20 as a biocatalyst:

. 26. The method of claim 25, wherein the substrate is a ginsenoside compound K and the product is ginsenoside PPD (S). A method for producing a cell extract according to claim 2 or a cell extract according to claim 20, wherein ginsenoside is added to induce the expression of an oxidoreductase. The method for producing a cell extract according to claim 9 or 10, wherein ginsenoside is added to induce the expression of an oxidoreductase.

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