KR20130142691A - MANUFACTURING METHOD FOR VARIOUS RARE GINSENOSIDES BY β-GLUCOSIDASE FROM PENICILLIUM ACULEATUM - Google Patents

Abstract

The present invention relates to a method for manufacturing diol based and triol based rare ginsenosides using β-glucosidase and, more specifically, to a method for manufacturing diol based and triol based rare ginsenosides using site specific decomposition activities of β-glucosidase driven from Penicilliumaculeatum and the β-glucosidase.

Description

{Manufacturing method for various rare ginsenosides by β-glucosidase from Penicillium aculeatum using β-glucosidase derived from penicillium aculeptum}

The present invention relates to a method for the preparation of Penicillium The present invention relates to the production of rare diastereoisomer and triazole-based ginsenosides using the site-specific degradation characteristics of β -glucosidase derived from aculeatum and, more particularly, Penicillium < RTI ID = 0.0 > aculeatum) beta represents the stable activity at high temperature to be derived from a strain-glucosidase die at a high temperature by using a location-specific characteristics of the enzyme olgye ginsenoside Rb _2, Rc, Rd and Rg ₃ and tri olgye Rf, (20- O- glycosides linked at the 20-position of Rg ₁ (except for the case of Rb ₁ , the 1-6 bond of the first glucose of the 20- O- glycosides of the 20- O- glycosides is cleaved but the second glucose The present invention relates to a composition for the preparation of rare ginsenosides and a process for their preparation which do not exist in ginseng produced by selectively hydrolyzing only glucose at other positions.

The ginsenoside compound wye, compound MC, compound k and uglycon protopanaxadiol, which are not present in ginseng, are produced from ginsenoside Rb ₂ , ginsenoside Rc, ginsenoside Rd and ginsenoside Rg ₃ , respectively. Senoside F ₁ and the aglycon protopanaxatriol are produced in ginsenoside Rg ₁ and ginsenoside Rf, respectively.

The substrates, which are diacylgeneenosides, are structures in which the basic skeletal structure of the aglycon protopanaxadiol is conjugated with glucoside, arabinofuranoside and arabinofuranoside, and Penicillium The β- glucosidase enzyme derived from aculeatum does not decompose the 20- O- glycosides linked to position 20 (except for Rb ₁ , which is the first of the glucose-glucose residues of 20- O- glycosides) 1-6 is a combination of Glucose did not break but the second Glucose is not cut off) selectively decomposing only glucose at a different location singer Rb ₂ O → → Compound (refer to formula 1 Compound Y), Rc → Compound Mc 1 → Compound Rd → F ₂ → Compound K (see Chemical Formula 3), and Rg ₃ → Rh ₂ → APPD (see Chemical Formula 4 below). The substrate, which is a triol-based ginsenoside, is a structure in which glucoside and rhamnose are bonded to the basic skeletal structure of the aglycone protopanaxatriol. The 20- O- glycosides connected to the 20-position are not decomposed, Only hydrolysis of the glucoside can produce Rg ₁ ? F ₁ (see Chemical Formula 5 below) and Rf? Rh ₁ ? APPT (see Chemical Formula 6 below).

[Ginsenoside Rb2, Compound O and Compound Y]

[Ginsenoside Rc, Compound Mc1 and Compound Mc]

[Ginsenoside Rd, Compound F2 and Compound K]

[Ginsenoside Rg3, Compound Rh2 and APPD]

[Ginsenoside Rg1 and F1]

[Ginsenoside Rf, Compound Rh1 and APPT]

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.

In addition, glycosylation of sugars in the ginsenoside uglycon protopanaxadiol (APPD) and the uglycon protopanaxatriol (APPT) can be a number of major ginsenoside substances. 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, as a conventional technique for manufacturing ginsenoside compound k, diol saponin is used as an enzyme, beta-glycosidase (Korean Patent Publication No. 2003-94757), and Naringin Ginseng or Asanase (Korean Patent Registration No. 418604) or the like, which has been separated from a disperser, is known.

By down-regulating the expression of Bcl-2 and Brn-3a from cancer cell proliferation inhibition, anticancer activity increasing action, allergy suppression and apoptosis by irradiation of ultraviolet B, It is known that HaCaT keratinocytes can be protected (Lee EH, et al., J. Invest. Dermatol. 121: 607-13, 2003).

(Hasegawa, H. et al., Planta Medica, 62: 453 (1996)), ginseng refined saponin as a water-based or buffered solution A method in which at least one of a naringinase and a pectinase is added to prepare a solution of a compound of formula (I) in a solvent or a mixture of an aqueous solvent such as water or a buffer solution and an organic solvent (Korean Patent Publication No. 2003-37005) Is known.

Recently, the method using intestinal bacteria has been interested in a manufacturing method using enzymes because of the problem that the reaction time is long and the yield of telephone 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. Also, neither Ginsenoside compound O (O) nor MC 1 (Mc ₁ ) has been reported to be active in production methods or substances, and the yield rate in the natural world is very low.

The present invention has been made in view of the above needs, and it is an object of the present invention to provide a rare ginsenoside preparation method such as Compound O (O) and MC 1 (Mc ₁ ).

Another object of the present invention is to provide a gene for producing the rare ginsenoside.

It is another object of the present invention to provide an enzyme for the production of the rare ginsenosides.

In order to accomplish the above object, the present invention provides a gene of SEQ ID NO: 4 which is used for production of a diol system and a triazole-based rare ginsenoside.

The present invention also provides a beta-glucosidase enzyme having an N-terminal sequence of SEQ ID NO: 1 which is encoded by the gene of the present invention.

The present invention also provides a composition for preparing ginsenoside comprising the gene of the present invention or the enzyme of the present invention as an active ingredient.

In one embodiment of the present invention, the rare ginsenoside is selected from the group consisting of Compound Y, Compound O, Compound Mc ₁ , Compound Mc, Compound K, ), Ugly cone protocol wave incident diol (aglycon protopanaxadiol), epi source (F _1), and ugly cone protocol wave incident triols (aglycon protopanaxatrio) as not exclusively a preferably a compound selected from the group consisting of in.

The invention is beta of the gene or the present invention of the present invention - glucosidase die olgye ginsenoside _{Rb 2, Rc, Rd, Rg} 3 and tri olgye ginsenoside Rg _1, Rf, and one or more mixed among them And reacting the compound with any one of the compounds in a reaction solvent to prepare a rare ginsenoside.

In one embodiment of the present invention, the reaction is preferably carried out in the pH range of 3.5 to 6.0,

In another embodiment of the present invention, the reaction is preferably performed at a temperature ranging from 60 to 78 ° C, but is not limited thereto.

Hereinafter, the present invention will be described.

As a result of continued research to develop the conversion of rare ginsenosides, the present inventors have found that fungus-derived eukaryotes Penicillium < RTI ID = 0.0 > glucuronidase was purified from a strain of aculeatum which was used to produce a beta-glucosidase enzyme. Then, purely isolated diol based ginsenosides Rb ₂ , Rc, Rd and Rg ₃ and tri- Senosides Rg ₁ and Rf are selectively degraded to form the rare ginsenoside compounds O, Wai, MC, MC ₁ , K and Uglycone protopanaxadiol, Ft ₂ and Ugly Cone protopanaxyl triol, and thus the present invention has been completed.

In the present invention, the beta-glucosidase is a fungus such as Penicillium aculeatum ). < / RTI > In addition, the beta-glucosidase may be characterized in that the optimal activation temperature is 60-78 ° C.

Hereinafter, the present invention will be described in detail.

It provides a die olgye and tri olgye rare ginsenosides (rare ginsenoside) for preparing a composition containing a glucosidase (β -glucosidase) - the present invention is mold-derived beta.

In the present invention, the rare ginsenosides die olgye true of Com pounds ginsenosides bit O (Compound O) compound Y (Compound Y), the compound MC-membered (Compound Mc _1), the compound MC (Compound Mc), Compound K (Compound K), and ugly cone protocol wave incident diol (Aglycon protopanaxadiol) or a mixture thereof and tri olgye ginsenoside of ginsenoside F1 (ginsenoside F ₁₎ and ugly cone protocol wave incident triols (Aglycon protopanaxatriol) thereof And 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 optimal enzyme activity at a temperature higher than this, and the optimum temperature for this high temperature beta-glucosidase is 60-78 ° C .

The beta-glucosidase of the present invention is also useful as a microorganism belonging to the genus Penicillium , preferably Penicillium aculeatum ) having the N-terminal sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 4.

The β-glucosidase of the present invention can be obtained from the above-mentioned Penicillium acullet strain by 1) isolating and purifying directly from the strain, or 2) cloning a β-glucosidase gene from the strain and expressing it in a recombinant expression vector, . 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 invention also beta of the present invention from Penny room Solarium ahkyul retyum strains were obtained through a salting out method is then isolated directly from the strain-glucosidase (β -glucosidase).

Thus, the composition for preparing rare ginsenosides containing beta -glucosidase of the present invention is characterized in that when the substrate is reacted in a mixture of ginsenoside single substrate and buffer solution, aqueous solvent and organic solvent, , Selectively hydrolyzing only a single substrate glucose, and converting the rare ginsenoside in a short time to produce a high yield.

The beta-glucosidase activity of the kinase in the (β -glucosidase) is preferably from 28 ℃ 5 ilgan culturing a penny room Solarium ahkyul retyum. The culture solution in the above procedure was filtered using Watman filter paper, and the filtrate was recovered, and crude microbial enzyme was obtained by salting-out method using ammonium sulfate. Thereafter, purification was carried out using an ion exchange chromatography method using a FPLC (fast protein liquid chromatography) apparatus to obtain beta-glucosidase.

The β-glucosidase thus obtained exhibits a relative activity of about 50% or more at pH 3.5 to 6.0 when the enzyme activity is measured at 75 ° C., and has a relative activity of about 80% or more at pH 4.0 to 5.0 And exhibits the maximum relative activity around pH 4.5 (Fig. 1A). In addition, when the enzymatic activity of the thermophilic β-glucosidase of the present invention is measured at pH 4.5, the relative activity is about 50% or more at 55 to 80 ° C, the relative activity is about 80% or more at 60 to 78 ° C , Exhibiting a maximum relative activity at around 75 DEG C (Fig. 1B). In measuring the temperature stability of the enzyme activity of the beta-glucosidase of the present invention, the half-life of the enzyme activity was found to be 136.8 hours at 60 ° C, 55 hours at 65 ° C, 9.6 hours at 70 ° C, and 0.96 hours at 75 ° C . (Fig. 2).

In addition, the composition for preparing rare ginsenoside containing beta-glucosidase of the present invention is characterized in that when it is reacted with ginsenosides Rb ₂ , Rc and Rd which are the main diol saponins of ginseng and Rf and Rg ₁ which are major triol saponins , The reaction speed is rapidly controlled at a high temperature of 60 to 78 占 폚 to exhibit an action effect of producing rare ginsenoside in a high yield in a short time.

The present invention relates to a method for preparing a mixture of a ginsenoside Rb ₁ , Rb ₂ , Rc, Rd, Rg ₃ , Rf, Rg ₁ , To provide a method for producing senosides.

In the present invention, the beta-glucosidase is a eucaryote belonging to the genus Peniciilium , preferably a fungus-derived acanthosis strain, preferably Penicillium aculeatum ) having the N-terminal sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4.

In a preferred embodiment of the present invention, a) a) culturing a strain of Penicillium acullet on a PDA plate culture medium at 25 to 28 degrees for 5 to 7 days; B) Inoculated on a rutin medium (Rutin 5 g / L) as a liquid medium and cultured at 25 to 28 degrees for 5 to 7 days, followed by induction of overexpression to produce beta-glucosidase.

The above-mentioned β-glucosidase enzyme is a process for culturing a fungus which overexpresses the β-glucosidase enzyme, inducing the expression of the β-glucosidase enzyme, separating and purifying the expressed enzyme protein, It is preferable to use azine.

The process for separating the beta-glucosidase enzyme protein expressed in the step b) comprises: (a) filtering the culture solution; (b) recovering the filtrate and obtaining the enzyme using salting-out method using ammonium sulfate; (c) adsorption with ion exchange chromatography and (d) separation of the active moiety with sodium chloride (NaCl).

In the step (a) of the present invention, it is preferable to filter the supernatant of the culture medium of the fungus into Watman filter paper. In step (b), 80% ammonium sulfate is preferably used. It is preferable to use Histrap Q and Resource Q which are chromatography columns. It is preferable to use a flow rate of 1 mL / min using 1M sodium chloride (NaCl) in the step (d) .

In addition, the beta-glucosidase is prepared by using ginsenosides Rb ₂ , Rc, Rd, and Rg ₃ , which are diol-based saponin, as substrates, and ginsenosides Rg ₁ and Rf, , Compound Wye, Compound MC One, Compound MC, Compound K, Uglycon Protopanax Diol, Ginsenoside Efone and Uglycone protopanax triol.

At this time, the reaction between the beta-glucosidase enzyme and the substrate of the buffer solution such as phosphate-citrate buffer is carried out at pH 3.5 to 6.0, preferably pH 4.0 to 5.0, more preferably pH 4.5 , Preferably at a temperature of 55 to 80 DEG C, preferably 60 to 78 DEG C, more preferably at 75 DEG C in consideration of the temperature stability of the enzyme.

According to the process for preparing a rare diastereoisomer and triazole-based ginsenoside using the beta-glucosidase enzyme of the present invention, pure diacylgeneenoside Rb2, Rc, Rd, Rg3 and tri Compounds W, Compounds W, Compound MC, Compound MC, Compound K, Uglycone protopanax diol, Ginsenoside Efon and Ugly, which are the rare ginsenosides, respectively, when using allergens Ginsenoside Rg1, Rf or mixtures thereof. Cone protopanaxyl triol, or mixtures thereof.

According to the present invention, there is provided a composition for preparing a rare-earth ginsenoside, a diol-based compound containing a beta-glucosidase enzyme derived from the fungus of the present invention, and a diol-based and triazole-based rare ginsenoside preparation method using a beta-glucosidase enzyme, Penicillium glucosidase derived from aculeatum strain exhibits stable activity at a high temperature and accelerates the reaction rate. Due to the substrate specificity to degrade only glucose, the major ginsenosides Rb ₂ , Rc, Rd and Rg ₃ And the triose-based ginsenosides Rg ₁ and Rf are converted into different substances, respectively, to rapidly and selectively produce a rare diastereoisomer and a triol-based ginsenoside, which can be industrially useful.

Figure 1 shows the degree of purification of the beta-glucosidase of the present invention according to SDS-PAGE (a) and tablets (b).
Fig. 2 shows the activity of the enzyme according to the pH (a) and the temperature (b) of the beta-glucosidase of the present invention.
Fig. 3 shows the results of the stability measurement of beta-glucosidase of the present invention against temperature.
FIG. 4 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 ginsenoside diol series Rb ₂ , Rc, Rd, Rg ₃ a to a substrate thermophilic beta of the present invention - glucosidase illustrates the production of rare ginsenosides by Ajay, (a) ginsenoside conversion of the side Rb _2, (b) conversion of Rb ₂ (D) conversion of Rc was analyzed by HPLC, (e) conversion of ginsenoside Rd, and (f) conversion of Rd was carried out by HPLC (G) shows conversion of ginsenoside Rg ₃ , and (h) shows conversion of Rg ₃ by HPLC.
FIG. 5 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 triol-based Rf and Rg ₁ (A) shows the conversion of ginsenoside Rf and (b) shows the conversion of Rf by HPLC. As a result, it was found that ( c) shows conversion of ginsenoside Rg ₁ , and (d) shows conversion of Rg ₁ by HPLC.
FIG. 6 is a graphical representation of the ginsenosides converted by the beta-glucosidase of the present invention, showing (a) the conversion of the ginsenoside diol family and (b) the ginsenoside triol family.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: beta- Glucosidase  Used rare Ginenoside  Produce

(1) beta-glucosidase method to obtain enzyme-glucosidase (β -glucosidase) in production conditions with beta

In the present invention, to prepare beta-glucosidase, first beta-glucosidase (β-glucosidase strainPenicillium aculeatum KCTC 6245 (National Life Research Resource Information Center, obtained from 52, Eeun-dong, Yuseong-gu, Daejeon, Korea) was plated on solid PDA (Potato dextrose broth) medium (potato 7g / L, glucose 20g / L) at 28 ℃ for 5 days. After the mycelia were collected and passaged with a liquid rutin medium (rutin 5g / L) and incubated for 5 to 7 days at 200rpm at 28 ℃.

In order to obtain the enzyme from the above culture filtrate, the present invention was purified as follows. The cultured tuberculosis cultured for 5 days is centrifuged at 13,000 x g for 20 minutes at 4 ° C after addition of ammonium sulfate of 561 g / l in which 80% protein can be salted. The ammonium sulfate was removed through a desalting process in which the precipitate was dissolved in a buffer solution such as sodium citrate or ginseng buffer, and the microorganisms were recovered.

The recovered crude microorganism was purified using an ion exchange chromatography method using FPLC (fast protein liquid chromatography) apparatus. Using a Histrap Q as the first column, a portion showing activity was obtained and a second column was obtained using Resource Q (Fig. 1).

       Figure 1 shows the degree of purification of the beta-glucosidase of the present invention according to SDS-PAGE (a) and tablets (b).

(2) Beta- Glucosidase  The temperature and pH  Investigation of effects

In the present invention, the effect of beta-glucosidase isolated in Example (1) on ginsenosides mainly present in ginseng was confirmed as follows.

end. 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- beta -D-glucopyranoside, 50 mM phosphate-citric acid buffer solution (pH 4.5) and the thermophilic beta-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 75 ° C for 10 minutes. The reaction was stopped by addition of 2M sodium carbonate again. The amount of 4-nitrophenyl - ? - D-glucopyranoside used and the amount of 4-nitrophenol produced by the enzyme reaction were estimated by measuring the absorbance at 420 nm. The enzyme unit was 1 micromolar ( lt; RTI ID = 0.0 > pmole) < / RTI > of 4-nitrophenol. In addition, the protein concentration of the enzyme was estimated using the Bradford method based on bovine serum albumin.

I. beta- Glucosidase  About pH  Investigation of effects

First, 4-nitrophenyl - ? - D-glucopyranoside was dissolved in secondary distilled water and then phosphate / citric acid buffer solution containing 37 units / l of enzyme The reaction was carried out in the range of pH 3.5 to 6.5. At this time, the reaction was allowed to proceed at a temperature of 75 占 폚 for 10 minutes. After the reaction was completed, the reaction was terminated by adding 2M sodium carbonate and the activity of the beta-glucosidase enzyme was measured. As a result, it was confirmed that the optimum pH was 4.5 as shown in Fig.

All. beta- Glucosidase  Survey on temperature effect

In order to investigate the temperature effect on the enzyme, a mixture of 4-nitrophenyl - ? - D-glucopyranoside and 37 units / liter of enzyme containing phosphate / And reacted with citric acid buffer solution (pH 4.5) for 10 minutes each. After the reaction was completed, the reaction was terminated by the addition of 2M sodium carbonate and the activity of the hot β-glucosidase enzyme was measured. As a result, as shown in FIG. 2B, it was confirmed that the optimum temperature was 75 ° C.

Fig. 2 shows the activity of the enzyme according to the pH (a) and the temperature (b) of the beta-glucosidase of the present invention.

la. beta- Glucosidase  Temperature stability investigation

In the present invention, in order to investigate the temperature stability of beta-glucosidase, 4-nitrophenyl - ? - D-glucopyranoside and 37 units (units) / liter of enzyme (PH 4.5) containing potassium phosphate / citric acid was used to conduct the reaction until the enzyme activity was reduced to half, respectively. After the reaction was completed, the reaction was terminated by treating with 2M sodium carbonate and the activity of the beta-glucosidase enzyme was measured.

FIG. 3 shows the result of measuring the temperature stability of the beta-glucosidase of the present invention. In the graph of FIG. 2, the temperature is indicated by 75 ° C., 70 ° C., 65 ° C. and 60 ° C., respectively.

As a result, as shown in FIG. 3, it was confirmed that the enzymatic activity was reduced to half at 60 ° C for 136.8 hours, 65 ° C for 55 hours, 70 ° C for 9.6 hours, and 75 ° C for 0.96 hours.

hemp. beta- Glucosidase  Purely-separated Diol system Gin Senocide Rb ₂ , Rc  And Rd and Triazol  Gin Senocide Rf , Rg _One Activity on

In order to investigate the effect observed when the beta-glucosidase enzyme of the present invention is reacted with purely isolated dihydrate ginsenosides Rb ₂ , Rc, Rd and Rg ₃ and triazole ginsenosides Rf and Rg ₁ , Rb ₂ , Rc, Rd, Rg ₃ , Rf and Rg ₁ of 0.5 mg / ml were used as substrates, respectively. The enzyme was used at 0.3, 0.3, 0.3, 3.7, 3.7 and 7.5 units / liter, respectively, and the reaction was carried out at a temperature of 70 DEG C for 6 hours. When the reaction was completed, n-butanol was added to terminate the reaction and the activity of the beta-glucosidase enzyme was measured.

As a result, as shown in FIGS. 4 and 5, the ginsenoside Rb ₂ is passed through the compound O to the compound Y (see FIG. 4A), the ginsenoside Rc is the compound Mc see _{Fig. 1)} through (Fig. 4c a compound MC), ginsenoside Rd is true through the ginsenoside F2 as compound K (compound K) (see Fig. 4e), ginsenoside Rg3 is ugly through the ginsenoside Rh ₂ Ginsenoside Rg1 to ginsenoside F1 (see Fig. 5c), ginsenoside Rf via ginsenoside Rh < ₁ > and aglycon protopanaxadiol (see Fig. 4g) (See FIG. 5A), respectively.

(3) Beta- Glucosidase  Enzyme-based rare Ginenoside  production

In the present invention, in order to develop a method for producing rare ginsenosides using the β-glucosidase enzyme, the optimum pH (pH 4.5) of the enzyme and the temperature at which the enzyme activity is reduced to half (70 ° C.) pure substrate, ginsenoside die olgye _{Rb 2, Rc, Rd, Rg} 3 0.3 units (unit) / ℓ, 0.3 units (unit) / ℓ, 0.3 units (unit) /ℓ3.7 unit (unit) / ℓ and Gene The compound of Senenoside Compound Wye, Compound MC, Compound K, and Uglycone protopanax Diol, Efon, and Uglycon proto are each having 3.7 units of Senoside triol Rf, Rg ₁ , and 7.5 units / Tricol.

Examples of rare ginsenosides that have been produced so far include beta-glycosidase from Sulfolobus acidocaldarius and beta-glycosidase from Dictyoglomus turgidum .

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. Beta-glycosidase of Dictyoglomus turgidum used 0.25 mg / ml (95%) of uglycon protopanaxadiol when using 0.5 mg / ml of Rb1, Rb2, Rc and Rd, respectively, 0.40 mg / ml (99%) and compound MC 0.32 mg / ml (94%).

However, beta according to the invention The glucosidase each _{Rb 2, Rc, Rd, Rg} 3 , when used with 0.5mg / ml and this compound 0.34mg / ml (98%), compound MC 0.35mg / ml (99 , Compound k 0.30 mg / ml (94%) and uglycon protopanaxadiol 0.06 mg / ml (45%). When using 0.5 mg / ml of Rf and Rg1, respectively, it is possible to produce F1 0.32 mg / ml (99%) and uglycon protopanaxatriol 0.28 mg / ml (89%).

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.

<110> Konkuk University Industrial Cooperation Corp. <120> Manufacturing method for various rare ginsenosides by          beta-glucosidase from Penicillium aculeatum <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 10 <212> PRT <213> Penicillium aculeatum <400> 1 Lys Ala Tyr Ser Pro Ile Ala Tyr Pro Ala   1 5 10 <210> 2 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ctcgagaaaa gaatgcggaa cagcttattg atttcgctt 39 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cgaattccta cttccgatgt tgagagcca 29 <210> 4 <211> 2850 <212> DNA <213> Penicillium aculeatum <400> 4 atgcggaaca gcttattgat ttcgcttgct gcggcagcac ttgccgaggg caaagtgagt 60 ttccccgtca attgattctc atccattctt atattataac ctgaactaac tgttgtgtgc 120 ctacatcagg cctactcacc tcccgcgtat cctgctccct gggccagtgg tgccggggaa 180 tgggctcaag cccatgagag agctgtcgag ttcgtctcgc agttgacctt ggccgaaaag 240 ataaatttga cgactggtgt tgggtatgtc gagagatcat tgaacaaata tctacgacac 300 tggactgaca attggtagat gggagggtgg acaatgtgtc ggtaacactg gaagtattcc 360 ccgtttggga ttccgcagcc tctgcatgca ggattcacca ctcggtgtga gagacagtat 420 gtcgtaccct tttacctcct acctcttatt tccgcttctt gaaactggtg taatgcatat 480 agctaactgc tcttttttca gctgattata atactgcctt ccctgccggt gtcaatgtcg 540 ccgctacctg ggatctcgat cttgcctacc ggcgcggtat agccatggcc gaagaacacc 600 gtggcaaagg tgtggatgtg cagcttggtc ccgttgctgg tccactagga agagtaccag 660 agggcggtcg taactgggaa ggtttcgcgc cggaccctgt gctgaccggc cggatgatgg 720 caagcactat ccaaggaatg cagaataccg gtgtgattgc ttgcgcaaag cactatatcg 780 gaaacgaaca agagcacttc cgtcagggct cccaggaaga cttcaccgtt gctgatgcta 840 tcagctcgaa catcgatgac gttactttgc acgaattgta cctatggccg tttgccgatg 900 cggtcagggc aggtgtcggt tccgtcatgt gctcttacaa tcaattgaac aacagctacg 960 cctgcggcaa cagctacagt ttgaaccaca tcctcaaggg tgagctcgac tttcaaggct 1020 ttgtcatgac agattggagt gctcaacact ccggtgttgg cgatgctcta gccggagctg 1080 acatggatat gcctggtgat gtggctttcg acagtggaac tgctttttgg ggtaccaact 1140 tgacaattgc cgtgctcaat ggcactgttc ccgaatggcg tattgatgac atggctgttc 1200 gtatcatgtc tgctttctac aaagttggtc gtgatcgcac tcaagtcccc gtcaactttg 1260 ctagctggac tttggatacc tatggcaacg aatactacta cgccggcgag ggatacaagg 1320 aaatcaacca gcacgttgat gttcgtggtg accacgccaa agttgtccgt gaaattggca 1380 gtgccagcat tgttctcctc aaaaatgttg atggtgctct gccattgact ggctcagaga 1440 ggtttgtcgc agtgttcgga gaggatgctg gctccaatcc tgacggtgtc aatggttgtt 1500 ctgaccgtgg ctgtgataac ggtaccttgg ctatgggatg gggtagtggt actgccaact 1560 tcccttactt ggtcacccct gaacaagcta tccaagccga agtcttgaag aatggcggga 1620 tgtttactgc tattaccgac agcggcgcca ccaataccac agccacgact gtggctgctc 1680 aagcttcgta agtactggtc gtaatcagat gagagactaa tggttttcag atgactgact 1740 tggttttgat agggcttgcc tagtgtttgc caacgcagac tccggagaag gatacatcaa 1800 cgttgacggc aatcagggag atcgtaagaa cttgacatta tggcagaacg gtgaagctat 1860 gatctcggcc gtcgcaggta actgcaacaa caccattgtc gttcttcaca ctgttggacc 1920 cgttcttgtc gaagattggg tgaatcatcc taacatcacc gctgttttgt gggctggttt 1980 gcccggagag cagagcggta actccttggt tgatgttctc tatggtagcg tcaaccctgg 2040 aggcaaaact ccattcactg gggcaagcaa cgttctgact ggggagttga tgtcatctat 2100 gaacccaaca acggagatgg tgctcctcag caggacttca ccgagggtat cttcattgat 2160 tatcgacact ttgacaaata caacattact cccacttacg aatttggtta tggtcttagt 2220 tacagcacat tctcattctc aaacctcaag gtgactcctc tcgctgctcc tccttaccaa 2280 ccagccaagg gccagagcgg tccggcacct gtgctaggca aggttttgaa cgccacggcc 2340 tatctgttcc cagactacat caaacgcatt gaagcgttca tttacccatg gctcaactct 2400 actgatctga agacttcctc cggtgatcca aactacggct ggcctacttc tcagtacgtg 2460 cctgacggcg ctcaagacgg gtctccacaa cctgtcaatc ccgctggcgg tgctcctggt 2520 ggtaaccctg ctctatatga ccctgttgcc gaaattagtg tgactatcaa aaacaccgga 2580 aaggtcgctg gtattgaagt gcctcaactc tatgtctcgc tcggtggtcc ctccgatgca 2640 cccaaagttc ttcgtggctt tggccgactt tctctcggcg ctggcgggga ggctcagtgg 2700 actgccactt tgaccaggcg tgacgtatct aactgggacc ccgtcagcca gaactgggtt 2760 gtcacaaact acaccaagac tgtctatgtt ggcaactctt ctcgcaactt gccgctccag 2820 cagactttgg ctctcaacat cggaaagtag 2850

Claims (8)

The gene of SEQ ID NO: 4 used for producing diol-based and triol-based rare ginsenosides. A β-glucosidase enzyme having an N-terminal sequence of SEQ ID NO: 1 encoded by the gene of claim 1. A composition for preparing ginsenoside comprising the gene of claim 1 or the enzyme of claim 2 as an active ingredient. The method of claim 3, wherein the rare ginsenosides are Compound Y, Compound O, Compound Mc ₁ , Compound Mc, Compound K, Ugly A composition for preparing rare ginsenosides, characterized in that the compound is selected from the group consisting of aglycon protopanaxadiol, epione (F ₁ ), and aglycon protopanaxatrio. The beta-glucosidase of claim ₁ , wherein the beta-glucosidase of claim ₂ is selected from any one of diol-based ginsenosides Rb ₂ , Rc, Rd, Rg ₃ and triol-based ginsenosides Rg ₁ , Rf and a mixture of one or more thereof. A method of preparing a rare ginsenoside by reacting a compound with a reaction solvent. The method of claim 5, wherein the rare ginsenoside is Compound Y, Compound O, Compound Mc ₁ , Compound Mc, Compound K, Ugly A method for producing rare ginsenosides, characterized in that the compound is selected from the group consisting of aglycon protopanaxadiol, epione (F ₁ ), and aglycon protopanaxatrio. 6. The method of claim 5, wherein the reaction is in the range of pH 3.5 to 6.0. 6. The method of claim 5, wherein the reaction is carried out at a temperature in the range of 60-78 &lt; 0 &gt; C.

Images