KR101392987B1 - Production of the rare ginsenosides compound Mc, compound Y, aglycon protopanaxadiol by a thermostable beta-glucosidase - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising aglycon protopanaxadiol, ginsenoside compound Y, ginsenoside compound G, and ginsenoside compound using a high-temperature β-glucosidase enzyme, compound Mc), and more particularly to a method for producing a high temperature resistant Dictyoglomus The present invention relates to a recombinant expression vector comprising turgidum- derived beta-glucosidase, a method for producing a large amount of beta-glucosidase by using the transformed microorganism and a method for producing the same, and a method for producing a recombinant expression vector containing a rare ginsenoside, Protopanaxadiol, compound wye, and compound MC.

Description

Production of the rare ginsenosides compound Mc, compound Y, aglycon protopanaxadiol by a thermostable beta-glucosidase}, a compound of the rare ginsenoside compound with high temperature beta-glucosidase, compound wye and aglycon

The present invention is the thermophilic beta-glucosidase (β-glucosidase) and rare binary relates to a ginsenoside, and more particularly ginsenoside substrate and thermophilic mousse tool gidum to Ogle dithiol Using Enzyme (Dictyoglomus glucuronidase enzyme which exhibits stable activity even at high temperature and which selectively hydrolyses only the glucose of Rb ₁ , Rb ₂ , Rc and Rd of ginsenosides at a high temperature using a high temperature β-glucosidase enzyme And to compositions and methods for preparing aglycon protopanaxadiol, Compound Y, and Compound Mc, which are rare ginsenosides that are not present in ginseng.

Ginsenoside uglycon protopanaxadiol, compound wye and compound MC, which are not present in ginseng, are produced in ginsenoside Rb ₁ and ginsenoside Rb ₂ ginsenoside Rc, respectively.

Ginsenoside Rb ₁ has four glucose units in the basic skeleton structure of uglycon protopanaxadiol, and selectively hydrolyzing the portion thereof can produce an aglycon protopanaxadiol (see Formula 1 below). 20 (S) -protopaxadiol-20- O- alpha-l-arabinopyranosyl 16-beta-di- glucopyranoside, -Protopanaxadiol-20- O- alpha-el-arabinofuranosyl 16-beta-di-glucopyranoside, see Formula 3 below) is an arabinopyranoside of ginsenoside Rb ₂ ) And arabinofuranoside of ginsenoside Rc can not be decomposed, and glucose of the third carbon can be hydrolyzed.

[Chemical Formula 1]

(2)

(3)

Until now, ginsenoside compounds have been known to have many excellent efficacies such as immunity enhancement, tumor angiogenesis inhibition, inhibition of cancer cell infiltration and inhibition of cancer cell proliferation, and thus a large amount of supply is required in the field of health food industry Therefore, there is a growing need for stable and efficient production.

Among them, the ginsenoside uglycon protopanaxadiol can be the major ginsenoside substance when the sugar is glycosylated. The ginsenoside compound material is produced using a mesophilic enzyme at a temperature in the range of 10 to 50 ° C. However, since the enzyme acts at a low reaction temperature, it is easily contaminated with microorganisms and the production yield is low.

In addition, the ginsenoside compounds Y (Y) and MC (Mc) are known to be effective for inflammatory diseases, but their production methods and activity have not been reported to date, and the yields in the natural field are considerably low There is not much research result using this.

Korean Patent Publication No. 1020030043168 discloses a method for producing a ginsenoside compound k using a cellulase derived from Aspergillus Niger or a lactose composition Y-AO, and the ginsenoside A method of producing compound k is a method of dissolving a diol based saponin contained in ginseng in a large amount and easy to separate into a solvent and reacting with a cellulase or lactase composition Y-AO isolated from Aspergillus niger to prepare a ginsenoside compound K is produced, eluted with an ion exchange resin or water-saturated butanol, and concentrated, followed by separation by column chromatography to produce a ginsenoside compound K,

As another related prior patent, Korean Patent Publication No. 1020060120932 discloses a method of reacting alpha -galactosidase (melibiase) with diol-based ginsenoside to produce ginsenoside (R), Rb1, Rb2, Rc, and Rd, which are diol based ginsenosides, are used alone or in combination as a mixture of aspergillus Aspergillus and Penicillium- derived alpha-galactosidase, an enzyme reaction is carried out to prepare ginsenoside F2 or compound K according to the reaction conditions.

Therefore, the present inventors have continued to study the conversion of rare ginsenosides, and as a result, they have found that a high temperature β-glucosidase gene is cloned in a thermophilic microorganism Dictyoglomus turgidum strain to produce a recombinant expression vector And a microorganism transformed with the transformed microorganism is produced, and a thermophilic β-glucosidase enzyme is produced using the transformed microorganism. Then, the glucose of the purely isolated ginsenoside Rb1 is decomposed and is passed through ginsenoside Rd, F ₂ , It was confirmed that the uglycon protopanaxadiol was prepared and the two compounds of glucose attached to the carbon number 3 of the ginsenosides Rb2 and Rc were selectively hydrolyzed to produce the compound wye and the mc in a high yield in a short time, .

As a result, the present invention relates to a pharmaceutical composition comprising aglycon protopanaxadiol, Compound Y and Compound Mc, which contain high temperature beta-glucosidase, And a manufacturing method thereof.

In order to achieve the above object, the present invention provides a composition for preparing rare ginsenosides containing beta-glucosidase as an active ingredient.

In one embodiment of the present invention, the rare ginsenoside is preferably aglycon protopanaxadiol, Compound Y, or Compound Mc, but is not limited thereto.

In another embodiment of the present invention,

Although the β-glucosidase preferably has the amino acid sequence of SEQ ID NO: 4, it can induce at least one substitution or deletion in the sequence of SEQ ID NO: 4, All mutants also fall within the scope of the enzymes of the present invention.

The present invention also provides a composition for preparing rare ginsenosides containing, as an active ingredient, a gene encoding beta-glucosidase.

In one embodiment of the present invention, the β-glucosidase-encoding gene preferably has the nucleotide sequence of SEQ ID NO: 3, but it is preferred that the nucleotide sequence of SEQ ID NO: All the mutants that can induce the desired effect in the present invention are also included in the gene range of the present invention.

The invention also provides a method for preparing rare ginsenosides by reacting beta-glucosidase with ginsenosides selected from the group consisting of ginsenosides Rb1, Rb2, Rc, and Rd, or mixtures thereof, or Moss extract .

Hereinafter, the present invention will be described in detail.

The present invention provides compositions for the preparation of rare ginsenosides containing a high temperature beta-glucosidase.

In the present invention, the rare ginsenoside is characterized by comprising Aglycon protopanaxadiol, Compound Y and Compound Mc or a mixture thereof.

Generally, beta-glucosidase is an enzyme which is variously existed in animals, plants, microorganisms, and the like and is an enzyme which generates sugar and uglycon by hydrolyzing glucoside bond of glycoside and small saccharide. Glucoside attached to aryl, amino, Disaccharide synthase, dinacyclohexane glucoside, and oligosaccharide or disaccharide. In addition, the enzyme plays a fundamental role in the regulation of metabolism, such as the production or breakdown of carbohydrates (Pack et al., (2007) J. Microbiol. Biotechnol. 17, 454-460). The optimal activity temperature of such a beta-glucosidase is 30 to 50 ° C.

However, the composition for preparing rare ginsenosides of the present invention contains a high temperature beta-glucosidase exhibiting an optimum enzyme activity at a higher temperature, and the optimum temperature for this high temperature beta-glucosidase is 70 to 95 ° C .

Further, the thermophilic beta-glucosidase of the present invention is derived from a microorganism belonging to the genus Ditioglomus, preferably a strain of Dictyoglomus turgidum , which is a thermophilic microorganism, and the nucleotide sequence of SEQ ID NO: 3 and / And having an amino acid sequence of SEQ ID NO: 4.

In general, differences in the composition of amino acids between the mesophilic beta-glucosidase and the beta-glucosidase derived from the strain of the dithioglomus strain of the present invention differ. The mesophilic beta glucosidase has a low content of cysteine (Cys) and a low ratio of arginine / lysine (Arg / Lys). This is a typical factor that can explain the thermostability of thermophilic enzymes in general (Themostable -galactosidase from the archaebacterium Sulfolobus solfataricus purification and properties. Eur. J. Biochem 187, 321-328 (1990)).

The dithioglormus tachyderm is a bacterial system; Dictyoglomi gate; Dictyoglomi River; Dictyoglomales tree; Dictyoglomaceae and Dictyoglomus . The microorganisms of the dithioglomo river grow in the hot spring water of the alkaline volcano of Kamchatka peninsula of Russia and exhibit optimal growth near the characteristic 80 ° C Svetlichnii, V. A, et al., Microbiology , 57, 435-441, 1988). In particular, dithioglomus tachidomis is known as an anaerobic gram-negative type showing optimal growth at 50 to 80 ° C and pH of 5.9 to 8.3 (Saiki, A. et al., International Journal of systematic Bacteriology , 35, 253-259, 1985).

To obtain the hyperthermal beta-glucosidase of the present invention from the dithioglomus toolus germ strains, it is preferable to 1) isolate and purify directly from the strain, or 2) clone the beta-glucosidase gene from the strain and express it in a recombinant expression vector And purifying it. The process of obtaining the enzyme from such microorganisms is by conventional methods in the art (Sambrook, J. and Russell, D.W. Molecular Cloning 3rd Ed., Cold Spring Harbor Laboratory, 2001).

The thus obtained hot β-glucosidase exhibits a relative activity of about 60% or more in the range of pH 4.8 to 6.5 when the enzyme activity is measured at 80 and a relative activity of about 80% or more in the range of pH 5.0 to 5.8 Activity and exhibits the maximum relative activity at around pH 5.5 (Fig. 1A). In addition, when measuring the enzymatic activity of the thermophilic β-glucosidase of the present invention at pH 5.5, it exhibits a relative activity of about 80% or more in the range of 70 to 87 ° C and a relative activity of about 90% or more in the range of 75 to 85 ° C , Exhibiting a maximum relative activity around 80 (Fig. 1B). In the measurement of the temperature stability of the enzyme activity of the thermophilic β-glucosidase of the present invention, the enzyme activity half-life was 2955 minutes at 70 ° C, 1402 minutes at 75 ° C, 682 minutes at 80 ° C, 112 minutes at 85 ° C, And 25 minutes at 90 ° C (FIG. 2).

In addition, the composition for preparing rare ginsenosides containing the thermophilic β-glucosidase of the present invention is characterized in that when it is reacted with ginsenosides Rb1, Rb2, Rc and Rd which are major dianhydrides of ginseng, And the reaction speed is rapidly controlled to exhibit the action effect that the ginsenoside uglycon protopanaxadiol, wai and MC are produced in a high yield in a short time.

The present invention provides a method for producing rare ginsenosides by reacting a mixture of ginsenosides Rb1, Rb2, Rc, and Rd selected from the group consisting of hot β-glucosidase as a substrate in a reaction solvent.

In the present invention, the thermophilic beta-glucosidase is a microorganism belonging to the thermophilic microorganism DIctyoglomus , preferably a microorganism belonging to the genus Dictyoglomus turgidum ) having the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4.

In a preferred embodiment of the present invention, a) a genomic DNA of dithioglormus thunbergii strain PCR was carried out with the primers of SEQ ID NOs: 1 and 2 to amplify a DNA fragment containing the thermophilic beta-glucosidase gene (SEQ ID NO: 3); B) DNA fragments containing the amplified thermophilic beta-glucosidase gene were treated with restriction enzymes NdeI and XhoI and cloned into plasmid vector pET-28a (+) to obtain recombinant expression vector pET-28a (+) / Glucosidase; C) transforming E. coli strain BL21 (DE3) ER2566 with a conventional transformation method; D) culturing Escherichia coli transformed with the thermophilic beta-glucosidase gene and inducing overexpression to produce thermophilic beta-glucosidase; And (e) isolating and expressing the expressed hyperthermal beta-glucosidase enzyme protein.

In the above, the β-glucosidase enzyme is produced by culturing E. coli transformed with the recombinant expression vector containing the β-glucosidase enzyme gene, inducing the expression of the β-glucosidase enzyme, It is preferable to use beta-glucosidase as a process of purification.

The process of separating the high temperature β-glucosidase enzyme protein expressed in step (d) comprises: (a) disrupting the microbial culture; (b) centrifuging the cell lysate to obtain a supernatant; (c) separating the enzyme solution by separating the supernatant liquid from the supernatant liquid, (d) separating the supernatant liquid by adsorption with affinity chromatography, and (e) eluting with an imidazole, And can be separated and separated.

In the step (a) of the present invention, the microbial culture solution is preferably pulverized using a sonicator by adding 50 mM phosphate buffer (50 mM phosphate buffer). In step (c) , And it is preferable that the filtrate is flowed at a flow rate of 1 mL / min using 250 mM imidazole in the step (e).

In addition, the beta-glucosidase is selected from diol-based saponins of ginsenosides Rb1, Rb2, Rc and Rd as a substrate and is selected and used when preparing ginsenoside uglycon protopanaxadiol, compound wye and compounded mc, May be used.

At this time, the reaction between the hyperbathic beta-glucosidase enzyme and the substrate of the buffer solution such as phosphate-citrate buffer is preferably carried out at pH 4.0 to 8.0, preferably pH 4.5 to 7.0, more preferably pH 5.5, preferably at a temperature of 70 to 98 DEG C, preferably 70 to 87 DEG C, more preferably at 80 DEG C in consideration of the temperature stability of the enzyme.

According to the method for producing rare ginsenosides using the thermophilic β-glucosidase enzyme of the present invention, when pure ginsenosides Rb1, Rb2, Rc, or a mixture thereof is used by using one enzyme stable at high temperatures The rare ginsenosides ginsenoside uglycon protopanaxadiol, wy, ech or mixtures thereof can be prepared.

The ginsenoside uglyconone protopanaxadiol containing the hypertonic beta-glucosidase enzyme of the present invention, the composition for the preparation of compound wye and compound MC, and the ginsenoside uglyconone prototype using the high temperature beta-glucosidase enzyme incident diol, and this compound and the compound according to the manufacturing method of the MC, tools mousse with thermophilic dt Ogle gidum (Dictyoglomus glucosidase derived from turgidum strain shows stable activity even at a high temperature and thus the reaction rate is accelerated. Due to the low substrate specificity for the remaining sugars except for glucose, the major ginsenosides Rb1, By converting Rb2, Rc, and Rd into different materials, it is possible to produce the uglycon protopanaxadiol, compound wye, and compounded MC rapidly and selectively, thus being industrially useful.

Fig. 1 shows the activity of the enzyme according to pH (a) and temperature (b) of the beta-glucosidase of the present invention.
Fig. 2 shows the results of the stability measurement of beta-glucosidase of the present invention against temperature.
FIG. 3 is a graph showing the results of analysis of high-pressure liquid chromatography (HPLC) of the ginsenoside material and the resulting ginsenoside material of beta-glucosidase used in the present invention and the results of purely separated ginsenosides Rb1, Rb2, Rc, (A and b) conversion of ginsenoside Rb1, (c and d) shows the conversion of ginsenoside Rb2 (E and f) is a conversion of ginsenoside Rc, and (g and h) is a conversion of ginsenoside Rd.
Figure 4 shows the production of rare ginsenosides by the high temperature beta-glucosidase of the present invention using the extract of Mishima as a substrate (Mc: ginsenoside compound MC; Y: ginsenoside compound w: K: Gin Senocide Compound K; APPD: Uglycone protopanaxadiol)
Figure 5 is a graphical representation of the ginsenosides converted by the beta-glucosidase of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1: High temperature beta- Glucosidase  Production of recombinant expression vectors and transforming microorganisms containing

In the present invention, in order to prepare a thermophilic β-glucosidase, a β-glucosidase gene derived from a strain of dithioglormus tool was isolated.

Specifically, a strain of dithioglomerus tumefaciens, in which a gene sequence and an amino acid sequence have already been identified, was selected and genomic DNA was extracted. Also, the nucleotide sequence of the beta-glucosidase gene of this strain [see sequence listing; (Genebank Accession No. YP_002352162)], two primers of the forward and reverse primers including the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, were prepared.

Then, PCR was performed using the genomic DNA and the primers to amplify the nucleotide sequence of the corresponding gene of Beta-glucosidase (SEQ ID NO: 3). 28a (+) (Novagen) using the restriction enzymes Nde I and Xho I amplified the above-mentioned β-glucosidase gene fragment by the above procedure, and the recombinant expression vector pET-28a ( +) / Beta-glucosidase.

Also, the recombinant expression vector prepared above was transformed into Escherichia coli BL21 (DE3) ER2566 strain by a conventional transformation method. The transformed recombinant Escherichia coli was transformed into E. coli BL21 (DE3) ER2566 pET-28a (+ / Beta-glucosidase strain. The transformed Escherichia coli was stored frozen before addition of 20% glycerine solution.

Example  2: High temperature beta- Glucosidase  Expression

In the present invention, in order to mass-produce beta-glucosidase, a frozen E. coli BL21 (DE3) ER2566 pET-28a (+) / beta-glucosidase strain is inoculated in a 250 ml flask containing 50 ml of LB medium And cultured with shaking in a shaking incubator at 37 DEG C until the absorbance at 600 nm became 2.0. Then, the culture was added again to a 2 L Erlenmeyer flask containing 500 ml of LB medium, and incubated until the absorbance at 600 nm reached 0.8. The stirring speed was 200 rpm and the incubation temperature was adjusted to 37 캜. Then, 0.1 mM IPTG (isopropyl-beta-thiogalactoside) was added thereto to induce the production of the overexpressed enzyme. The agitation speed was 150 rpm, and the incubation temperature was adjusted to 16 ° C.

In order to purify the high temperature β-glucosidase enzyme produced as described above, the culture of the transformed strain was centrifuged at 4,000 g for 30 minutes at 4 ° C., and the cells were suspended in 100 mM phosphate-citrate buffer buffer, pH 5.5), and the cell solution was disrupted with a sonicator. The cell lysate was further centrifuged at 13,000 x g for 20 minutes at 4 ° C. The supernatant was filtered through a 0.45 μm filter paper, adsorbed on a strap affinity chromatography column, imidazolylated, And was separated into an enzyme solution which can be used for production of ginsenoside.

Example  3: High temperature beta- Glucosidase pH  And activity according to temperature change

In the present invention, the effect of the hot β-glucosidase isolated in Example 1- (2) on ginsenosides Rb1, Rb2, Rc and Rd mainly present in ginseng was confirmed as follows.

end. High Temperature Beta- Glucosidase  Enzyme activity measurement

In the present invention, the activity of the thermophilic β-glucosidase enzyme was measured by using 4-nitrophenyl-β-D-glucopyranoside ( p NPGlc) as a substrate. Specifically, before the reaction, 1.0 mM 4-nitrophenyl-β-D-glucopyranoside, 50 mM phosphate-citric acid buffer solution (pH 5.5) and the thermophilic β-glucosidase enzyme of the present invention were diluted The reaction mixture was mixed at a total volume of 200 μl, and the reaction was allowed to proceed at 80 ° C for 10 minutes. The reaction was stopped by addition of 2M sodium carbonate again. The amount of 4-nitrophenyl-β-D-glucopyranoside used and the amount of 4-nitrophenol produced by the enzyme reaction were estimated by measuring the absorbance at 420 nm, and the enzyme unit was 1 μmol ( mole) of 4-nitrophenol. In addition, the protein concentration of the enzyme was estimated using the Bradford method based on bovine serum albumin.

I. High Temperature Beta- Glucosidase  About pH  Investigation of effects

First, to investigate the pH effect on the enzyme, the ginsenoside substrate Rd was dissolved in secondary distilled water, and a phosphate / citric acid buffer solution containing 0.4 units (units) / ml of enzyme was added at pH 4.0 to 8.0 Lt; / RTI > At this time, the reaction was carried out at a temperature of 80 ° C for 10 minutes. After the reaction was completed, n-butanol was added to terminate the reaction and the activity of the hot β-glucosidase enzyme was measured. As a result, it was confirmed that the optimum pH was 5.5 as shown in Fig.

All. High Temperature Beta- Glucosidase  Survey on temperature effect

In order to investigate the temperature effect on the enzyme, a phosphate / citric acid buffer solution (pH 5.5) containing the enzyme ginsenoside substrate Rd and 0.4 units (units) / ml in a temperature range of 70 to 90 ° C Respectively, for 10 minutes. After the reaction was completed, n-butanol was added to terminate the reaction and the activity of the hot β-glucosidase enzyme was measured. As a result, as shown in FIG. 1B, it was confirmed that the optimum temperature was 80 ° C.

Fig. 1 shows the activity of the enzyme according to the pH (A) and temperature (B) of the beta-glucosidase of the present invention.

la. High Temperature Beta- Glucosidase  Temperature stability investigation

In the present invention, in order to investigate the temperature stability of the thermophilic β-glucosidase, potassium pyrophosphate / citric acid containing the enzyme ginsenoside Rd and 0.4 units / ml in the temperature range of 70 to 90 ° C. The buffer solution (pH 5.5) was used to conduct the reaction until the enzymatic activity decreased to half. After the reaction was completed, the reaction was terminated by treating n-butanol and the activity of the hot β-glucosidase enzyme was measured.

Fig. 2 shows the result of measurement of the temperature stability of the beta-glucosidase of the present invention. In the graph of FIG. 2, the temperature is indicated by 70 ° C., 75 ° C., 80 ° C., 85 ° C. and 90 ° C., respectively.

As a result, as shown in FIG. 2, enzyme activity was reduced to half at 2955 minutes at 70 ° C, 1402 minutes at 75 ° C, 682 minutes at 80 ° C, 112 minutes at 85 ° C, and 25 minutes at 90 ° C I could confirm.

hemp. High Temperature Beta- Glucosidase  Purely-separated Gin Senocide Rb _One , Rb ₂ , Rc  And Rd Activity on

In order to investigate the effect of the thermophilic β-glucosidase enzyme of the present invention upon reacting purely isolated ginsenosides Rb ₁ , Rb ₂ , Rc and Rd, ginsenosides Rb1, Rb2, Rc And Rd were respectively used at 0.5 mg / ml. In addition, the enzyme was used at 1.6 unit (unit) / ml for Rd at 0.4 unit (unit) / ml, and the reaction was carried out at 80 ° C. for 6, 8, 8, and 12 hours, respectively. When the reaction was completed, n-butanol was added to terminate the reaction and the activity of the hot β-glucosidase enzyme was measured.

As a result, as shown in FIG. 3, ginsenoside Rb ₁ was converted to aglycon protopanaxadiol (see FIGS. 3A and B) via ginsenoside Rd, F ₂ , compound K, ginsenoside Rb ₂ is a compound Y (Y) (see Figure 3c and d), ginsenoside Rc are compounds with MC (Mc) (see Fig. 3e and f), ginsenoside Rd is compound K and compound K via the f ₂ (Fig. 3g and h), respectively.

Example  4: High temperature beta- Glucosidase  Enzyme-based rare Ginenoside  production

In the present invention, in order to develop a method for producing rare ginsenosides using the high temperature β-glucosidase enzyme, a temperature (80 ° C.) considering the optimal pH (pH 5.5) of the enzyme and the time during which the enzyme activity is reduced to half Rb ₁ , Rb ₂ , Rc, and Rd 0.4 units / ml and 1.6 units / ml, respectively, of the ginsenoside uglycon protopanaxadiol 0.25 mg / ml and 0.40 mg / / ml < / RTI > and 0.32 mg / ml of compound MC. The hourly production of ginsenoside uglycon protopanaxadiol, compounded wye and compound MC was measured with Mitsumon extract (concentration 10%) and enzyme 6.4 unit (unit) / ml.

An example of the production of rare ginsenosides to date is the beta-glycosidase of Sulfolobus acidocaldarius .

Sulfolobus beta acidocaldarius) - glycosidase is Rb _1, Rb _2, Rc, when reacting Rd each compound K 0.53 mg / ml (94%) , compound Y 0.56 mg / ml (80%) and compound MC 0.70, respectively ( 100%) mg / ml.

However, when the high temperature β-glucosidase according to the present invention was used, when 0.5 mg / ml of Rb1, Rb2, Rc and Rd was used, 0.25 mg / ml (95%) of uglycon protopanaxadiol, 0.40 mg / ml (99%) and compound MC 0.32 mg / ml (94%). The ginsenosides converted into Ms extract are capable of producing 1.17 mg / ml (60%) of uglycon protopanaxadiol, 0.76 mg / ml (93%) of compound wye and 0.88 mg / ml of compound MC (99% Furthermore, no single enzyme has been reported to produce ginsenoside uglycon protopanaxadiol, compound wye, and compound MC.

Therefore, it can be seen that beta-glucosidase isolated from dithiogromose of the present invention shows excellent effects on the production of rare ginsenoside.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (10)

A composition for the preparation of compound wye and compound MC, respectively, from ginsenosides Rb2 and Rc comprising beta-glucosidase consisting of the amino acid sequence of SEQ ID NO: 4 as an active ingredient. delete delete delete delete A composition for the preparation of compound wye and compound MC, respectively, from ginsenosides Rb2 and Rc comprising as an active ingredient a gene consisting of the nucleotide sequence of SEQ ID NO: 3 encoding beta-glucosidase. Comprising reacting a beta-glucosidase consisting of the amino acid sequence of SEQ ID NO: 4 with a ginsenoside selected from the group consisting of ginsenosides Rb2, and Rc, or a mixture thereof. delete delete 8. The method of claim 7,
Wherein the process is carried out at a temperature in the range of 70 to 95 < 0 > C.

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