# Patent

## Title
Soil microorganism, novel oxidoreductase separated from the soil microorganism, gene encoding the oxidoreductase, and method for producing aglycones using the microorganism, the oxidoreductase and the gene

## Abstract

The present invention relates to the novel Rhizobium sp. GIN611 KCTC11708BP or to cell extracts thereof, to a novel oxidoreductase which exhibits a glycolytic activity, to a gene encoding the oxidoreductase, to a recombinant strain comprising recombinant vector proteins or to an expression vector encoding recombinant proteins, and to a method for the glycolysis of natural products using same as a biocatalyst. The present invention also relates to a method for producing aglycones from a variety of natural products using same. The novel oxidoreductase separated from the novel microorganism of the present invention does not belong to a glucosidase group but belongs to an oxidoreductase group, and has a glycolytic activity for natural products. The novel oxidoreductase oxidizes the sugar in the aglycones of natural products, thereby producing a variety of aglycones.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation of U.S. patent application Ser. No. 13/704,210, filed on Dec. 13, 2012, which claims priority to PCT Application No. PCT/KR2011/004361, filed on Jun. 14, 2011, which claims priority to Korean Patent Application No. 10-2010-0066307, filed on Jul. 9, 2010 and Korean Patent Application No. 10-2010-0055988, filed on Jun. 14, 2010, and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which in its entirety are herein incorporated by reference.


TECHNICAL FIELD
The present disclosure relates to a novel microorganism isolated from soil, an oxidoreductase isolated from the microorganism and a method for deglycosylating various plant glycosides and producing various aglycones using same.
BACKGROUND ART
Ginseng (Panax ginseng C. A. Meyer) is one of the herbs that have been traditionally used in Asian countries including Korea, China and Japan for treatment and prevention of a variety of diseases. Ginseng saponins also called ginsenosides are main active ingredients of ginseng and are known to have various physiological activities including anti-aging activity, anti-inflammatory activity, antioxidative activity in the central nervous system, cardiovascular system and immune system, anti-diabetic activity and antitumor activity.
Until now, more than 40 kinds of ginsenosides have been isolated and identified. The ginsenosides which are glycosides having a dammarane structure including aglycones may be largely classified into protopanaxadiols and protopanaxatriols. The ginsenosides belonging to the protopanaxadiol group are mainly Rb1, Rb2, Rc and Rd, and the ginsenosides belonging to the protopanaxatriol group are mainly Re and Rg1 (see FIG. 1 and FIG. 2).
After taken in, the ginsenosides are metabolized by intestinal microorganisms and the metabolic products are known to have various physiological activities. For example, the representative protopanaxadiol-based saponins Rb1, Rb2 and Rc are metabolized by human intestinal microorganisms to CK and the protopanaxatriol-based saponins Re and Rg1 are metabolized by intestinal microorganisms to Rh1 or F1, thereby exhibiting a variety of physiological activities. CK is known to induce anti-metastatic or anticancer effect of preventing invasion and formation of tumors. And, it is reported that its aglycone PPD(S) has higher physiological activity as compared to the sugar-attached counterpart Rh2.
Accordingly, studies have been made to transform ginsenosides into metabolites having less sugar. In addition to enzymatic methods, hydrolysis using weak acids, degradation using alkalis, or the like have been reported. However, since these methods induce several side reactions such as epimerization, hydration, hydroxylation, etc., methods of transforming into active ginsenosides using enzymes, intestinal microorganisms, and so forth are studied recently. But, most of the reported microorganisms are anaerobic intestinal microorganisms, there is a limit in industrial application. Also, since most enzymes lack the activity of transforming ginsenosides into aglycones and have their own specificity, they are applicable to production of specific ginsenosides only.
Although a lot of studies have been made thus far about the biotransformation of the metabolite of the ginsenoside Rb1 by intestinal microorganisms to CK, there are few researches about production of its aglycone. And, it is reported that the ginsenoside having one sugar on the saponin backbone is not degraded by the enzymes of microorganisms any more. Ginsenosides in aglycone form are known to be absorbed more easily into the bloodstream and act as active compounds. Also, the production of aglycones as backbones of various ginsenosides will make a base technology for specific production of desired types of ginsenoside. Accordingly, there is a need of exploring enzymes involved in the production of ginsenoside aglycones.
DISCLOSURE
Technical Problem
The present disclosure is directed to providing a novel microorganism, a novel oxidoreductase exhibiting deglycosylation activity isolated from the microorganism and a method for producing various ginsenoside aglycones, isoflavone aglycones or flavonoid aglycones using a gene and a recombinant protein encoding same.
Technical Solution
In one general aspect, there is provided novel Rhizobium sp. GIN611 (KCTC 11708BP) or a cell extract thereof.
In another general aspect, there is provided a method for deglycosylating a natural product using Rhizobium sp. GIN611 or a cell extract thereof as a biocatalyst.
In another general aspect, there is provided a method for producing aglycones from various glycosides using Rhizobium sp. GIN611 or a cell extract thereof as a biocatalyst.
In another general aspect, there is provided an oxidoreductase having an amino acid sequence of SEQ ID NO 3 or a cell extract including same.
In another general aspect, there is provided a DNA encoding an oxidoreductase having an amino acid sequence of SEQ ID NO 3, a DNA having a sequence of SEQ ID NO 2, a recombinant DNA vector including the DNA having a sequence of SEQ ID NO 2, a host cell transformed with the recombinant DNA vector including the DNA having a sequence of SEQ ID NO 2 or a cell extract including the host cell transformed with the recombinant DNA vector including the DNA having a sequence of SEQ ID NO 2
In another general aspect, there is provided an oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having a deglycosylation activity or a cell extract including the oxidoreductase.
In another general aspect, there is provided a method for deglycosylating a natural product using a biocatalyst selected from a group consisting of an oxidoreductase having an amino acid sequence of SEQ ID NO 3, a cell extract including the oxidoreductase having an amino acid sequence of SEQ ID NO 3, a host cell transformed with a recombinant DNA vector including a DNA encoding the oxidoreductase having an amino acid sequence of SEQ ID NO 3, a cell extract including the host cell transformed with a recombinant DNA vector including a DNA encoding the oxidoreductase having an amino acid sequence of SEQ ID NO 3, a host cell transformed with a recombinant DNA vector including a DNA having a sequence of SEQ ID NO 2, a cell extract of the host cell transformed with a recombinant DNA vector including a DNA having a sequence of SEQ ID NO 2, a host cell transformed with a recombinant DNA vector including a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, a cell extract including the host cell transformed with a recombinant DNA vector including a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, an oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, and a cell extract including the oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity.
In another general aspect, there is provided a method for producing aglycones from various glycosides using a biocatalyst selected from a group consisting of an oxidoreductase having an amino acid sequence of SEQ ID NO 3, a cell extract including the oxidoreductase having an amino acid sequence of SEQ ID NO 3, a host cell transformed with a recombinant DNA vector including a DNA encoding the oxidoreductase having an amino acid sequence of SEQ ID NO 3, a cell extract including the host cell transformed with a recombinant DNA vector including a DNA encoding the oxidoreductase having an amino acid sequence of SEQ ID NO 3, a host cell transformed with a recombinant DNA vector including a DNA having a sequence of SEQ ID NO 2, a cell extract of the host cell transformed with a recombinant DNA vector including a DNA having a sequence of SEQ ID NO 2, a host cell transformed with a recombinant DNA vector including a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, a cell extract including the host cell transformed with a recombinant DNA vector including a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, an oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, and a cell extract including the oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity.
In another general aspect, there is provided a method for preparing a cell extract of Rhizobium sp. GIN611, a cell extract including an oxidoreductase having an amino acid sequence of SEQ ID NO 3, a cell extract including a host cell transformed with a recombinant DNA vector including a DNA encoding an oxidoreductase having an amino acid sequence of SEQ ID NO 3, a cell extract including a host cell transformed with a recombinant DNA vector including a DNA having a sequence of SEQ ID NO 2, or a cell extract including an oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, including inducing enzymatic expression by adding a ginsenoside.
Advantageous Effects
In general, ginsenoside-deglycosylating enzymes are known to belong to the glucosidase family. The inventors of the present disclosure have found that an enzyme belonging to the oxidoreductase family, not to the glucosidase family, has a deglycosylation activity for ginsenosides. This novel oxidoreductase is entirely different from the previously known deglycosylating enzymes in sequence, has a sequence similarity with the enzymes in the oxidoreductase family, and induces spontaneous deglycosylation by oxidizing a sugar in a naturally occurring glycoside.



DESCRIPTION OF DRAWINGS
FIG. 1 shows a protopanaxadiol (PPD)-based ginsenoside.
FIG. 2 shows a protopanaxatriol (PPT)-based ginsenoside.
FIG. 3 describes a reaction whereby an aglycone PPD(S) is produced from a ginsenoside compound K (CK) by deglycosylation.
FIG. 4 shows a reactivity analysis result of a novel oxidoreductase. The asterisk indicates oxidized CK and the triangle indicates PPD(S). The chromatogram 3 shows a result of reaction for 3 hours and the chromatogram 4 shows a result of reaction for 12 hours. The amount of the substrate CK decreases with time, that of the intermediate, oxidized CK, increase and then decreases, and that of the final product PPD(S) increases consistently with time.
FIG. 5 shows a mass analysis result of oxidized CK analyzed in FIG. 4.
FIG. 6 compares SDS-polyacrylamide gel electrophoresis result of proteins expressed in a complete medium and an M9/ginsenoside medium.
FIG. 7 shows a result of comparing reactivity of proteins obtained from cells cultured in a complete medium and an M9/ginsenoside medium.
FIG. 8 shows a 16S DNA sequence of a novel soil microorganism Rhizobium sp. GIN611 growing with a ginsenoside as a carbon source.
FIG. 9 shows an amino acid sequence of an oxidoreductase produced by Rhizobium sp. GIN611 and a sequence of a gene encoding same.
FIG. 10 shows a reaction mechanism of a novel oxidoreductase.
FIG. 11 shows a reactivity analysis result of a novel oxidoreductase for various ginsenosides and isoflavone. It can be seen that the enzyme has specific reactivity for glucose.
FIG. 12 shows a result of measuring deglycosylation activity of a mixture of camelliaside A and camelliaside B.
FIG. 13 shows a result of measuring deglycosylation activity of icariin.



BEST MODE
The terms used in the present disclosure are those commonly used in the related art and may be easily understood by those skilled in the art. Some of them will be described briefly.

    (1) Ginsenoside: ginseng saponin; the active ingredient of ginseng. 
    (2) Compound K (CK): 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol.
    (3) Ginsenoside Rh2: 3-O-β-D-glycopyranosyl-20(S)-protopanaxadiol.
    (4) Ginsenoside F2: 3-O-(β-D-glucopyranosy)-20-O-(β-D-glucopyranosyl)-20(S)protopanaxadiol.
    (5) Ginsenoside Rb1: 3-O-[(β-D-glucopyranosy)(1,2)-β-D-glucopyranosyl]-20-O-[(β-D-glucopyranosyl)(1,6)-β-D-glucopyranosyl]-20(S)protopanaxadiol.
    (6) Ginsenoside Rb2: 3-O-[(β-D-glucopyranosy)(1,2)-β-D-glucopyranosyl]-20-O-[(α-L-arabinopyranosyl)(1,6)-β-D-glucopyranosyl)]-20(S)protopanaxadiol.
    (7) Ginsenoside Rc: 3-O-[(β-D-glucopyranosy)(1,2)-β-D-glucopyranosyl)]-20-O-[(α-L-arabinofuranosyl)(1,6) -β-D-glucopyranosyl)]-20(S)protopanaxadiol.
    (8) Ginsenoside Rb3: 3-O-[(β-D-glucopyranosy)(1,2)-β-D-glucopyranosyl]-20-O-[(β-D-xylopyranosyl)(1,6)-β-D-glucopyranosyl]-20(S)protopanaxadiol.
    (9) Ginsenoside F1: 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol.
    (10) Ginsenoside Re: 6-O-[(α-L-rhamnopyranosyl(1,2)-β-D-glucopyranosyl]-20-O-(β-D-glucopyranosyl)-20(S)-protopanaxatriol.
    (11) Daidzin: daidzein 7-O-β-D-glucoside.
    (12) PPD(S): 20(S)-protopanaxadiol.
    (13) Compound Y: 20-O-[(α-L-arabinopyranosyl)(1,6)-β-D-glucopyranosyl]-20(S)protopanaxadiol.
    (14) Compound Mc: 20-O-[(α-L-arabinofuranosyl)(1,6)-β-D-glucopyranosyl]-20(S)protopanaxadiol.
    (15) Compound Mx: 20-O-[(β-D-xylopyranosyl)(1,6)-β-D-glucopyranosyl]-20(S)protopanaxadiol.
    (16) PPT(S): 20(S)-protopanaxatriol.
    (17) Ginsenoside Rg2: 6-O-[(α-L-rhamnopyranosyl(1,2)-β-D-glucopyranosyl]-20(S)-protopanaxatriol.
    (18) Daidzein: 7-hydroxy-3-(4-hydroxyphenyl)chromen-4-one.
    (19) Icariin: 3,4′,5,7-tetrahydroxy-8-prenylflavone-4′-Me ether-3-O-alpha-L-rhamnopyranoside, 7-O-beta-D-glucopyranoside.
    (20) Camelliaside A: kaempferol 3-O-(2-O-galactopyranosyl-6-O-rhamnopyranosyl)glucopyranoside.
    (21) Camelliaside B: kaempferol 3-O-(2-O-xylopyranosyl-6-O-rhamnopyranosyl)glucopyranoside.
    (22) Glycone(s): sugar molecule(s) attached to a glycoside.
    (23) Whole-cell reaction: a reaction using whole cells without disrupting the cells or isolating enzymes.
    (24) Oxidoreductase: an enzyme that catalyzes oxidation and reduction reactions necessary for providing energy to an organism. Most of oxidation of organic compounds occurs by dehydrogenation.
    (25) Oxidoreductase extract: a cell extract comprising an oxidoreductase which is obtained disrupting the cells of Rhizobium sp. GIN611 or recombinant proteins expressing the oxidoreductase.
    (22) MALDI-TOF mass spectrometry: matrix-assisted laser desorption/Ionization time-of-flight mass spectrometry.
    (26) HPLC: high-performance liquid chromatography.
    (27) PCR: polymerase chain reaction; a technique of specifically amplifying a region of a DNA.
    (28) ORF: open reading frame; a sequence between an initiation codon and a stop codon.
    (29) Cloning: a technique of inserting a DNA fragment into a recombinant DNA cloning vector and transforming a host cell using the resulting recombinant DNA.
    (30) bp: base pair(s).


The present disclosure provides novel microorganism Rhizobium sp. GIN611 or a cell extract thereof. The inventors of the present disclosure have selected the microorganism which grows with a mixture of various ginsenosides as a carbon source and have confirmed that the microorganism has reactivity for ginsenoside CK as substrate.
In an embodiment, the present disclosure provides a method for deglycosylating a natural product using Rhizobium sp. GIN611 or a cell extract thereof as a biocatalyst.
The natural product may be a ginsenoside glycoside, an isoflavone glycoside or a flavonoid glycoside, but is not limited thereto.
In another embodiment, the present disclosure provides a method for producing various aglycones from glycosides using Rhizobium sp. GIN611 or a cell extract thereof as a biocatalyst.
The glycoside may be a ginsenoside glycoside, an isoflavone glycoside or a flavonoid glycoside, but is not limited thereto.
The aglycone may be a ginsenoside aglycone, an isoflavone aglycone or a flavonoid aglycone, but is not limited thereto.
The ginsenoside glycoside is not specially limited but may be selected, for example, from a group consisting of ginsenoside compound K (CK), ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rb3, ginsenoside F1, and ginsenoside Re. Specifically, it may be ginsenoside compound K (CK).
The ginsenoside aglycone is not specially limited but may be selected, for example, from a group consisting of ginsenoside PPD(S), ginsenoside compound Y, ginsenoside Mc, ginsenoside compound Mx, ginsenoside PPT(S) and ginsenoside Rg2. Specifically, it may be ginsenoside PPD(S).
The ginsenoside glycosides and ginsenoside aglycones are summarized in Table 1.










TABLE 1









Ginsenoside glycosides
Ginsenoside aglycones



Ginsenoside compound K (CK)
Ginsenoside PPD(S)



Ginsenoside Rh2
Ginsenoside PPD(S)



Ginsenoside F2
Ginsenoside PPD(S)



Ginsenoside Rb1
Ginsenoside PPD(S)



Ginsenoside Rb2
Ginsenoside compound Y



Ginsenoside Rc
Ginsenoside Mc



Ginsenoside Rb3
Ginsenoside compound Mx



Ginsenoside F1
Ginsenoside PPT(S)



Ginsenoside Re
Ginsenoside Rg2










The isoflavone glycoside is not specially limited but may be, for example, daidzin.
The isoflavone aglycone is not specially limited but may be, for example, daidzein.
The flavonoid glycoside is not specially limited but may be, for example, icariin, camelliaside A or camelliaside B.
The deglycosylation is accomplished by oxidizing a sugar of the glycoside of the natural product. For example, if the sugar is glucose, deglycosylation occurs spontaneously by oxidizing the 3-hydroxyl (OH) group of a glucose residue.
The sugar is not specially limited but may be selected from a group consisting of glucose, galactose, rhamnose, arabinose and xylose.
In another embodiment, the present disclosure provides an oxidoreductase comprising an amino acid sequence of SEQ ID NO 3 or a cell extract comprising the oxidoreductase. Specifically, the oxidoreductase may be isolated from Rhizobium sp. GIN611.
In another embodiment, the present disclosure provides a DNA sequence encoding an amino acid sequence of SEQ ID NO 3 or a DNA sequence encoding the oxidoreductase. Specifically, the sequence is SEQ ID NO 2. Specifically, the DNA may be a DNA encoding the oxidoreductase isolated from Rhizobium sp. GIN611.
In another embodiment, the present disclosure provides a DNA encoding a protein having a sequence identity of at least 60%, specifically at least 90%, more specifically at least 97%, further more specifically at least 99%, with a sequence of SEQ ID NO 3 and having a deglycosylation activity for a natural product. A sugar degraded by the protein may be selected from a group consisting of glucose, galactose, rhamnose, arabinose and xylose.
In another embodiment, the present disclosure provides a recombinant DNA vector comprising a DNA sequence encoding the amino acid sequence of SEQ ID NO 3, a DNA sequence encoding an oxidoreductase comprising the amino acid sequence of SEQ ID NO 3 or a DNA sequence comprising a sequence of SEQ ID NO 2.
In another embodiment, the present disclosure provides a host cell transformed with the recombinant DNA vector and a cell extract comprising the host cell.
In another embodiment, the present disclosure provides an oxidoreductase having a sequence identity of at least 60%, specifically at least 90%, more specifically at least 97%, further more specifically at least 99%, with a sequence of SEQ ID NO 3 and having a deglycosylation activity for a natural product and a cell extract comprising the oxidoreductase.
The oxidoreductase is not specially limited but may be derived from Agrobacterium sp., Sphingobacterium sp. or Stenotrophomonas sp.
In another embodiment, the present disclosure provides a method for deglycosylating a natural product using a biocatalyst selected from a group consisting of an oxidoreductase comprising an amino acid sequence of SEQ ID NO 3, a cell extract comprising the oxidoreductase comprising an amino acid sequence of SEQ ID NO 3, a host cell transformed with a recombinant DNA vector comprising a DNA encoding the oxidoreductase comprising an amino acid sequence of SEQ ID NO 3, a cell extract comprising the host cell transformed with a recombinant DNA vector comprising a DNA encoding the oxidoreductase comprising an amino acid sequence of SEQ ID NO 3, a host cell transformed with a recombinant DNA vector comprising a DNA comprising a sequence of SEQ ID NO 2, a cell extract of the host cell transformed with a recombinant DNA vector comprising a DNA comprising a sequence of SEQ ID NO 2, a host cell transformed with a recombinant DNA vector comprising a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, a cell extract comprising the host cell transformed with a recombinant DNA vector comprising a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, an oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, and a cell extract comprising the oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity.
In another embodiment, the present disclosure provides a method for producing various aglycones of a natural product using a biocatalyst selected from a group consisting of an oxidoreductase comprising an amino acid sequence of SEQ ID NO 3, a cell extract comprising the oxidoreductase comprising an amino acid sequence of SEQ ID NO 3, a host cell transformed with a recombinant DNA vector comprising a DNA encoding the oxidoreductase comprising an amino acid sequence of SEQ ID NO 3, a cell extract comprising the host cell transformed with a recombinant DNA vector comprising a DNA encoding the oxidoreductase comprising an amino acid sequence of SEQ ID NO 3, a host cell transformed with a recombinant DNA vector comprising a DNA comprising a sequence of SEQ ID NO 2, a cell extract of the host cell transformed with a recombinant DNA vector comprising a DNA comprising a sequence of SEQ ID NO 2, a host cell transformed with a recombinant DNA vector comprising a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, a cell extract comprising the host cell transformed with a recombinant DNA vector comprising a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, an oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, and a cell extract comprising the oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity. Specifically, the method is a method for producing aglycones from various glycosides using a biocatalyst, more specifically, a method for producing a ginsenoside aglycone, an isoflavone aglycone or a flavonoid aglycone from a ginsenoside glycoside, an isoflavone glycoside or a flavonoid glycoside.
In another embodiment, the present disclosure provides a method for preparing a cell extract of Rhizobium sp. GIN611, a cell extract comprising an oxidoreductase comprising an 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 comprising an amino acid sequence of SEQ ID NO 3, a cell extract comprising a host cell transformed with a recombinant DNA vector comprising a DNA comprising a sequence of SEQ ID NO 2, a cell extract comprising a host cell transformed with a recombinant DNA vector comprising a DNA encoding a protein having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, or a cell extract comprising an oxidoreductase having a sequence identity of at least 60% with a sequence of SEQ ID NO 3 and having deglycosylation activity, comprising inducing enzymatic expression by adding a ginsenoside.
Selection of Microorganism Comprising Enzyme Having Deglycosylation Activity for Ginsenoside
The inventors of the present disclosure have selected a soil microorganism comprising an enzyme having activity for ginsenoside CK from soil using a minimal medium comprising a mixture of ginsenosides as a carbon source, as described in Table 2. Table 2 shows the composition of the minimal medium comprising a mixture of ginsenosides as a carbon source.










TABLE 2







Ingredients
Contents




















Carbon source
Ginsenoside mixture
0.2%, w/v













(red ginseng extract)





Nitrogen source
NH4Cl
1
g/L



Buffer ingredient
64 g Na2HPO4•7H2O
12.8
g/L




15 g KH2HPO4
3
g/L



Trace ingredient
2.5 g NaCl
0.5
g/L




MgSO4
1
mM




CaCl2
0.1
mM











Solid ingredient
Agar
1-3%










The method of selecting the microorganism using the minimal medium is a simple method based on growth rate. During the culturing, the microorganisms having low deglycosylation activity are removed spontaneously and only the microorganism comprising the enzyme with high activity remains.
Thus selected microorganism is a novel one. The inventors have identified that it belongs to the genus Rhizobium based on the characteristic DNA sequence encoding the 16S rRNA sequence and named it Rhizobium sp. GIN611. They have deposited the microorganism in the Korean Collection for Type Cultures (KCTC) on Jun. 4, 2010 (KCTC 11708BP).
Investigation of the substrate specificity using a deglycosylating enzyme isolated from the selected Rhizobium sp. GIN611 revealed that the microorganism exhibits high activity for ginsenoside CK and has deglycosylation activity for other various ginsenosides. The inventors of the present disclosure have disrupted and centrifuged the microorganism and produced a cell extract comprising the active enzyme from a supernatant.
Production of Ginsenoside Aglycone Using Deglycosylating Enzyme Extract as Biocatalyst
Aglycone PPD(S) may be produced from a reaction solution comprising the microorganism or the deglycosylating enzyme extract and ginsenoside CK. The associated reaction is initiated by adding the microorganism or the enzyme extract to the reaction solution as a biocatalyst.
In addition to the ginsenoside CK, a PPD-based ginsenoside such as ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside F2 and ginsenoside Rh2 may be used as the substrate. As a PPT-based ginsenoside, ginsenoside Re or ginsenoside F1 may be used. Also, the isoflavone daidzin and the flavonoids icariin, camelliaside A or camelliaside B may be used.
Hereinafter, the present disclosure is described in further detail through examples. The examples are for illustrative purposes only. It will be appreciated by those of ordinary skill in the art that and are not intended to limit the scope of this disclosure.
Example 1
Selection of Rhizobium sp GIN611
A soil sample (10 g) was added to phosphate buffered saline (PBS; 50 mL) and stirred at room temperature for 2 hours. The resulting turbid mixture was passed through filter paper to remove suspending matter. The filtered microorganism solution (0.2 mL) was added to the minimal medium (10 mL) described in Table 2 and incubated at 30° C. for 3 days. After repeating this procedure 3 times, the culture solution (0.2 mL) was transferred to a solid minimal medium consisting of the liquid minimal medium and 1.5% agar and incubated at 30° C. for 24 hours. After incubating each 3 mL of microorganism in the liquid minimal medium and reacting, respectively, the colony exhibiting high activity for ginsenoside CK was identified as Rhizobium sp.
Example 2
Preparation of Deglycosylating Enzyme Extract
Cells cultured in a medium (hereinafter, a complete medium) consisting of yeast extract (5 g/L), peptone (10 g/L) and sodium chloride (10 g/L) were washed 3 times with PBS buffer (pH 7.0) and the medium components other than the cells were removed. The recovered cells were suspended in 5 cell volume equivalents of a buffer solution (hereinafter, a lysis buffer) consisting of 5 cell volume equivalents of 20 mM phosphate buffer, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT). After disrupting the cells using an ultrasonic homogenizer, followed by centrifugation at 13,000 rpm for 30 minutes, the supernatant was recovered and a deglycosylating enzyme extract was obtained.
Example 3
Inducement of Expression of Deglycosylating Enzyme by Addition of Ginsenoside
Cells cultured using the liquid minimal medium (hereinafter, M9/ginsenoside medium) described in Table 2 were washed 3 times with PBS buffer and the medium components other than the cells were removed. The recovered cells were suspended in a lysis buffer. After disrupting the cells using an ultrasonic homogenizer, followed by centrifugation at 13,000 rpm for 30 minutes, the supernatant was recovered and a cell extract comprising a deglycosylating enzyme which was induced to express in the minimal medium was obtained.
Example 4
Comparison of Reactivity and Expression Level of Proteins Prepared in Complete Medium and M9/Ginsenoside Medium
After quantitating the amount of proteins in the cell extracts prepared in Example 2 and Example 3, reactivity for ginsenoside CK was compared using the same amount of proteins. As a result, the cell extract prepared in Example 3 exhibited higher reactivity than the cell extract prepared in Example 2 (see FIG. 7). After reaction using the same amount of proteins, the reactivity was compared as per amount of proteins. Whereas the protein obtained from the cells cultured in the complete medium (Example 2) resulted in complete conversion from ginsenoside CK to PPD(S) when 500 or more of protein was used, the protein obtained from the cells cultured in the M9/ginsenoside medium (Example 3) resulted in conversion from ginsenoside CK to PPD(S) when 100 or more of protein was used. Also, expression level of the proteins from the two extracts was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The result is shown in FIG. 6. In FIG. 6, the arrows indicate that the proteins whose expression level is different in the enzyme extracts of Example 2 and Example 3.
Example 5
Isolation and Purification of Deglycosylating Enzyme
In order to isolate the enzyme catalyzing the reaction illustrated in FIG. 3 from the novel Rhizobium sp. GIN611, the microorganism was cultured in the liquid minimal medium described in Table 2 after adding a ginsenoside (10 L). Then, an enzyme extract was prepared from the cultured microorganism in the same manner as described in Example 2. The prepared enzyme extract was subjected to 60-70% saturated ammonium sulfate fractionation. Thus obtained proteins were isolated and purified by fast protein liquid chromatography (FPLC) using several columns. The reactivity of the purified protein was investigated by native gel electrophoresis.
Example 6
Activity of Purified Enzyme for Ginsenoside CK
The activity of the enzyme purified in Example 5 for ginsenoside CK was investigated. A reaction was performed by using about 100 μg enzyme solution, 0.1 mM CK and 50 mM phosphate buffer(pH 6.5), and adding ethyl acetate of the same volume as that of the solution. After the reaction is completed, HPLC analysis was performed by isocratic elution using 80% acetonitrile (ACN). The result is shown in FIG. 4. In the figure, the peak indicated by the triangle is that of PPD(S) and the peak indicated by the asterisk is that of oxidized CK. It can be seen that the amount of oxidized CK increase and then decreases with time and that of PPD(S) increases consistently. This suggests that the sugar is degraded by the novel enzyme through oxidation of ginsenoside CK. The reaction mechanism is illustrated in FIG. 10.
Example 7
Determination of N-Terminal and Internal Peptide Sequences of Novel Enzyme
The N-terminal amino acid sequence of the oxidoreductase purified in Example 5 was determined by Edman sequencing using the Procise 491 sequencer (Applied Biosystems, CA) using Edman analytic technique after performing 12% SDS-PAGE electrophoresis, transferring the fragments to PVDF membrane (Bio-Rad). The sequence of the internal peptide was determined using the PEAKS software after analyzing the sequence of the peptide fragments obtained by treating with trypsin for sequencing (Promega) using the LTQ-Orbitrap mass analyzer.
Example 8
Isolation of Total DNA from Rhizobium sp. GIN611
Cells cultured in the complete medium were centrifuged at 4° C. and 4,000 rpm for 10 minutes and precipitated. After removing the supernatant, the remaining cells were lysed in 10 mL of lysis buffer (15% sucrose, 25 mM EDTA, 25 mM Tris buffer) and left at 37° C. for 10 minutes after adding 1.2 mL of EDTA (0.5 M) and 0.13 mL of Pronase. Then, after adding 10% SDS (1 mL), the mixture was kept at 70° C. for 10 minutes and then in icy water for 10 minutes. Subsequently, reaction was performed for 15 minutes in icy water after adding 5 M potassium acetate (2.5 mL). After adding a phenol/chloroform mixture (50:50) of the same volume to the reaction solution and mixing for 30 minutes, centrifugation was performed at 4° C. and 4,000 rpm for 10 minutes and the supernatant was obtained. After adding 0.5 volume equivalent of chloroform to the resulting solution and mixing slowly, centrifugation was performed at 4° C. and 4,000 rpm for 10 minutes and the supernatant was obtained. Then, after treating with RNase until an amount of 50/mL, followed by incubation at 37° C. for 1 hour, 0.8 volume equivalent of isopropanol was added and then 2.5 volume equivalents of 80% ethanol was added. After gently shaking, total DNA was collected using a Pasteur pipette, transferred to a 1.5 mL microtube, dried and then dissolved in sterilized water for further use.
Example 9
Gene Sequencing of Deglycosylating Enzyme by PCR
After preparing primers using the N-terminal amino acid sequence and the internal sequence determined in Example 7, DNA fragments of the oxidoreductase were obtained using the genomic DNA obtained in Example 8 as a template. Primers binding specifically to the obtained DNA fragments were prepared and the remaining sequence was determined by inverse PCR. A self-ligated DNA obtained by cutting the genomic DNA fragments using the Hind III restriction enzyme and then treating with ligase was used as a template for inverse PCR.
Example 10
Recombination Expression Vector and Expression in Transformed E. coli 
The DNA sequence of the obtained oxidoreductase was digested with the BamHI/SalI restriction enzyme and the resulting fragments were ligated into pETDuet-1 (Novagen) to prepare a recombinant plasmid, which was then transformed E. coli for expression (Rosetta-gami2; DE3). The transformed E. coli was cultured in a medium containing ampicillin. When the optical density reached 0.3-0.7, IPTG was added and expression of the enzyme was induced by further incubating at 20° C. for 15 hours.
Example 11
Deglycosylation Reactivity of Three Enzymes Having Sequence Similarity of at Least 65% with Novel Enzyme of SEQ ID NO 3 for Ginsenoside and Glycoside Derived from Natural Product
Enzymes derived from Agrobacterium sp., Sphingobacterium sp. or Stenotrophomonas sp. and having amino acid sequence similarity of at least 60% with SEQ ID NO 3 were cloned and their deglycosylation reactivity for glucose of ginsenosides was investigated. The enzymes derived from the microorganisms were confirmed to degrade the glucose residue of ginsenosides by oxidation.
Example 12
Measurement of Aglycone Production Activity Using Expression-Induced Enzyme
After preparing the enzyme whose expression was induced in Example 11 by the method described in Example 2, aglycone production activity was measured using ginsenoside CK as a substrate.
Example 13
Measurement of Activity Using Various Ginsenosides and Isoflavone as Substrates
After reacting using ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rb3, ginsenoside F1, ginsenoside Re or the isoflavone daidzin as a substrate, followed by extraction by adding the same volume of ethyl acetate, the ethyl acetate layer was dried and then dissolved again in ethanol, which was subjected to activity measurement by MALDI mass spectrometry.
Example 14
Measurement of Deglycosylation Activity for Different Aglycone Structures and Specificity for Glycone Substrates
The oxidoreductase enzyme derived from Rhizobium sp. GIN611, whose expression was induced, was reacted with the flavonoid icariin, camelliaside A or camelliaside B as a substrate in the manner described above and activity was measured by MALDI mass spectrometry. Oxidation of galactose attached to camelliaside A and deglycosylation activity thereof were identified. Also, oxidation of xylose attached to camelliaside B and deglycosylation activity thereof were identified. That is to say, the enzyme was confirmed to show deglycosylation activity for sugars attached to the flavonoid-based aglycones and to capable of degrading not only glucose but also galactose and xylose by oxidizing them (see FIGS. 12 and 13).
Example 15
Substrate Specificity for Glycone Binding
Specificity of the enzyme for α-bonding and β-bonding was investigated using 4-nitrophenyl α-D-glucopyranoside, 4-nitrophenyl β-D-glucopyranoside, 4-nitrophenyl α-D-galactopyranoside and p-nitrophenyl β-D-galactopyranoside. The enzyme showed activity for both α-bonding and β-bonding (see Table 3).










TABLE 3







Substrates
Relative activity (%)









p-Nitrophenyl α-D-glucopyranoside
103



p-Nitrophenyl β-D-glucopyranoside
100



p-Nitrophenyl α-D-galactopyranoside
201



p-Nitrophenyl β-D-galactopyranoside
400










[Accession Number]

KCTC 11708BP

## Claims

1. A method of deglycosylating a ginsenoside glycoside, an isoflavone glycoside or a flavonoid glycoside using a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding an oxidoreductase having the amino acid sequence of SEQ ID NO 3, or a cell extract of the host cell transformed with the recombinant DNA vector as a biocatalyst.

2. The method according to claim 1,
wherein the ginsenoside glycoside is one or more selected from a group consisting of ginsenoside compound K (CK), ginsenoside Rh2, ginsenoside F2, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rb3, ginsenoside F1, ginsenoside Re,
wherein the isoflavone glycoside is daidzin, or
wherein the flavonoid glycoside one or more selected from a group consisting of icariin, camelliaside A and camelliaside B.

3. The method according to claim 1,
wherein the method is for deglycosylating a sugar in the ginsenoside glycoside, the isoflavone glycoside or the flavonoid glycoside, and
the sugar is one or more selected from a group consisting of glucose, galactose, rhamnose, arabinose and xylose.