KR20160031245A

KR20160031245A - A COMPOSITION FOR PRODUCING GINSENOSIDE COMPOUND K AND A PREPARATION METHOD OF GINSENOSIDE COMPOUND K USING THE MIXED ENZYMES OF THERMOSTABLE β-GLYCOSIDASE AND α-L-ARABINOFURANOSIDASE

Info

Publication number: KR20160031245A
Application number: KR1020140121023A
Authority: KR
Inventors: 오덕근; 신경철
Original assignee: 건국대학교 산학협력단
Priority date: 2014-09-12
Filing date: 2014-09-12
Publication date: 2016-03-22
Also published as: WO2016039575A1

Abstract

The present invention relates to a composition and a method for preparing ginsenoside compound K from an extract of red ginseng or fine roots of ginseng using thermostable beta-glycosidase (β-glycosidase) and alpha-L-arabinofuranosidase (α-L-arabinofuranosidase) enzymes. More specifically, the present invention relates to a composition and a method for preparing ginsenoside compound K, by using thermostable beta-glycosidase and alpha-L-arabinofuranosidase enzymes, which are derived from thermostable Sulfolobus solfataricus and Caldicellulosiruptor saccharolyticus strains, and exhibit stable activity even at high temperatures, so as to confirm the optimum concentration ratio between the two enzymes for raising the productivity of ginsenoside compound K, and rapidly regulate the reaction rate at high temperatures, thereby mass-producing the ginsenoside compound K from the extracts of red ginseng and fine roots of ginseng in a short time, and therefore can be favorably used for industrial purposes.

Description

[0001] The present invention relates to a composition for producing ginsenoside compound k and a preparation method thereof using a high temperature beta-glycosidase and an alpha-l-arabinofuranosidase mixed enzyme. mixed enzymes of thermostable β-glycosidase and α-L-arabinofuranosidase}

The present invention relates to a composition for the production of ginsenoside compound K using a high temperature β-glycosidase and an α-L-arabinofuranosidase enzyme, more specifically it relates to a method of manufacture, the thermophilic Setting Tari prisoner bus solpa Syracuse (Sulfolobus solfataricus) and emitter syrup to kaldi cellulite Saccharomyces Rollei Tea Syracuse (Caldicellulosiruptor saccharolyticus ), and the reaction rate is rapidly controlled at a high temperature by using the high temperature beta-glycosidase and alpha-el-arabinofuranocidase enzyme which exhibits stable activity even at a high temperature. As a result, The present invention relates to a composition for the production of ginsenoside compound k, which can be industrially useful because it can mass-produce ginsenoside compound k in a short time from an extract.

The ginsenoside compound K (20 (S) -proctopanaxadiol-20-O-beta-di-glucopyranoside, see the following formula 1) is a ginseng saponin component intestinal bacterial metabolite, The glucosinide Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd, which are dianhydrides, are produced by hydrolysis of the glucose moiety.

[Chemical Formula 1]

So far, ginsenoside compound k is known to have various excellent effects such as immunity enhancement, tumor angiogenesis inhibition, inhibition of cancer cell infiltration and inhibition of cancer cell proliferation, Therefore, there is a growing need for stable and efficient production.

As a conventional technique for manufacturing such ginsenoside compound k, there has been known a method in which a diol-based saponin is reacted with an enzyme beta-glycosidase (Korean Patent Publication No. 2003-94757), a cellulase isolated from a penicillium genus microorganism, Beta] -galactosidase (Korean Patent No. 377546), pectinase isolated from Penicillium or pectinase isolated from Aspergillus (Korean Patent No. 418604), and the like A method of manufacturing compound k is known.

As described above, the ginsenoside compound K is produced using a mesophilic enzyme at a temperature ranging from 10 to 50 ° C. However, since the enzyme acts at a low reaction temperature, it is likely to be contaminated with microorganisms and yield is low.

In order to produce ginsenoside compound k in high yield, several enzymes must be mixed and used. Enzyme mixtures showed the highest productivity in the production of ginsenoside compound k. This method was found to be the most effective in the production of ginsenoside compound k. This method was performed using beta - glucosidase (6.3 mg / ml) A solution of about 5 mg / ml of protopanaxadiol was prepared by the method of using a suspension of alpha-el-arabinofuranosidase (1.4 mg / ml) and beta-galactosidase (1.4 mg / ml) Saponin-containing extracts of mossam (2.9 mg / ml) were produced in 20 hours (Kyung-Chul Shin et al, 2013, J. Biotechnol. 167, 33-40, 2013). However, this method has the inconvenience of using three enzymes and needs to be further improved in productivity.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a composition for producing a high-yield ginsenoside compound k in a short time.

Another object of the present invention is to provide a method for manufacturing a large amount of high-yield ginsenoside compound k in a short time.

In order to accomplish the above object, the present invention provides a ginsenoside compound K (hereinafter referred to as " ginsenoside ") containing an active ingredient of? -Glycosidase and? -L-arabinofuranosidase compound K).

In one embodiment of the present invention, the β-glycosidase is preferably composed of the amino acid sequence set forth in SEQ ID NO: 1, but it is preferable that the β-glycosidase mutant has one or more substitutions or deletions in the sequence, All mutants that achieve the purpose are also included in the scope of the present invention.

In another embodiment of the invention, the beta-glycosidase is selected from the group consisting of Sulfolobus solfataricus , and the β-glycosidase preferably has an optimum activity temperature of 70 to 95 ° C, but is not limited thereto.

In another embodiment of the present invention, the α-L-arabinofuranosidase is preferably composed of the amino acid sequence set forth in SEQ ID NO: 2, but it is preferable that the α-L-arabinofuranosidase has one or more substitutions, All mutants which cause mutation to achieve the purpose of the present invention are also included in the scope of the present invention.

In one embodiment of the present invention, alpha -L-arabinofuranosidase is preferably derived from Caldicellulosiruptor saccharolyticus strain,

The α-L-arabinofuranosidase preferably has an optimal activity temperature of 70 to 95 ° C, but is not limited thereto.

In a preferred embodiment of the present invention, the composition ratio of beta-glycosidase to alpha-el-arabinofuranosidase of the composition is preferably 1: 9 to 9: 1 (w / v) No.

The present invention also relates to a gene encoding a β-glycosidase and a gene coding for α-L-arabinofuranosidase as a ginsenoside compound To provide a composition for producing K (compound K).

In one embodiment of the present invention, the gene coding for the β-glycosidase is preferably composed of the nucleotide sequence shown in SEQ ID NO: 3, and the α-L-arabinofuranosidase ( α-L-arabinofuranosidase) is preferably composed of the nucleotide sequence shown in SEQ ID NO: 4, but all the mutants that achieve the purpose of the present invention by causing mutations such as one or more substitutions or deletions in the nucleotide sequence are also included in the present invention .

The present invention also provides a method for preparing ginsenoside compound ke by treating beta-glycosidase and alpha-el-arabinofuranosidase to a substrate.

In one embodiment of the present invention, the substrate is preferably a red ginseng extract, a ginseng extract or a protopanaxadiol saponin. However, the ginseng extract may be selected from the group consisting of Korean ginseng, Korean ginseng, American ginseng, Ginseng saponin mixture or extract obtained by extracting and extracting from Himalayan ginseng root or Vietnamese root ginseng root, but not limited thereto.

Hereinafter, the present invention will be described.

The present invention produced a ginsenoside compound k with high concentration and high productivity from protopanaxadiol saponin using two high temperature enzymes.

The present inventors have continued to develop a new method for producing ginsenoside compound k. As a result, the present inventors have found that a combination of a high temperature beta-glycosidase derived from a thermophilic microorganism sulfolabus solfataricus strain and a calf cellulosic saccharose Alpha-L-arabinofuranosidase derived from Tikus strain was cloned to prepare a recombinant expression vector and a microorganism transformed with the recombinant expression vector, and using the same, high temperature beta-glycosidase and alpha-el-arabinofuranos It was confirmed that a large amount of ginsenoside compound k was prepared and reacted with red ginseng and mushroom extract in high yield after confirming the optimum ratio of two enzyme after producing shidase enzyme.

Hereinafter, the present invention will be described in detail.

To obtain the thermophilic beta-glycosidase and alpha-el-arabinofuranocidase of the present invention from the thermophilic microorganisms of the present invention, Sulfolobus solfataricus and Caldicellulose syrups saccharolyticus strain, 1) direct isolation from the strain, or 2) a beta-galactosidase gene is cloned from the strain and expressed in a recombinant expression vector and purified. 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).

When β-glycosidase thus obtained was treated with red ginseng extract or licorice extract, ginsenoside Rc and compound MC in the protopanaxadiol saponin remained, and the production yield of ginsenoside compound k Therefore, in the present invention, there is a method of simultaneously converting alpha-L-arabinofuranosidase into protopanaxadiol-based saponins in red ginseng extract or mint extract to compound k to provide.

The β-glycosidase and α-L-arabinofuranosidase of the present invention, when the ginsenoside Rc was used as a substrate, Indicating the maximum generation of compound K (Figure 1).

When red ginseng extract and mint extract were used as substrates, beta - glycosidase and alpha - el - arabinofuranosidase showed the maximum production of ginsenoside compound k at 4: 1 and 8: 3 ratios, respectively .

Thus, the composition for the preparation of ginsenoside compound ke containing the high temperature beta-glycosidase and alpha-el-arabinofuranosidase of the present invention is characterized in that ginsenosides Rb1 and Rb2, which are the main diol-based saponins in red ginseng extract or ginseng extract, , Rc, and Rd, buffer solution, and aqueous solvent, the reaction rate is rapidly controlled at 85 ° C at a high temperature to produce a high yield of ginsenoside compound k in a short time using a low enzyme concentration .

In a preferred embodiment of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: a) genomic DNA of Sulforo busesulphatricus and Caldicellulose syrup saccharolyticus, PCR was carried out with the primers to amplify DNA fragments containing the thermophilic beta-glycosidase and alpha-el-arabinofuranosidase genes, respectively; B) Each DNA fragment containing the amplified thermophilic beta-glycosidase and alpha-el-arabinofuranosidase gene was treated with restriction enzymes and cloned into plasmid vector pET-24a (+) to obtain recombinant expression Vector pET-24a (+) / beta-glycosidase and pET-24a (+) / alpha-el-arabinofuranosidase; C) transforming E. coli strain BL21 (DE3) ER2566 with a conventional transformation method; D) culturing each Escherichia coli transformed with the thermophilic beta-glycosidase gene and the alpha-el-arabinofuranosidase gene; E) inducing the expression of the gene during culture to produce a thermophilic beta-glycosidase and alpha-el-arabinofuranosidase enzyme; And f) isolating the expressed hot β-glycosidase and alpha-el-arabinofuranosidase enzyme protein.

The process of separating the high temperature beta-galactosidase and the alpha -el-arabinofuranocidase enzyme protein expressed in the above (a) process comprises: (a) disrupting the microorganisms in a culture medium; (b) centrifuging the cell lysate to obtain a supernatant; (c) centrifuging again by heat treatment at high temperature; And (d) filtering the supernatant obtained therefrom; And separating the enzyme solution as a process.

In step (a), cells are preferably disrupted at a pressure of about 15,000 lb / in ² using a device such as a French press. In step (c), the cell supernatant is heat- In step (d), it is preferable to perform filtration using a filter paper having a pore size of about 0.45 탆 or the like.

In addition, the substrate may be ginsenosides Rb1, Rb2, Rc, Rd, which are diol-based saponins in red ginseng extract or ginseng extract, and may be used as a mixture in the production of ginsenoside compound k, and the reaction solvent is McIlvaine, Buffer solutions such as buffers may be used.

The reaction between the thermophilic beta-glycosidase and the alpha-el-arabinofuranosidase enzyme and the substrate in the reaction solvent is preferably performed at pH 6.0 and preferably at 80 ° C.

In addition, when the ginsenoside Rc was used as a substrate, the optimal ratio of the concentration of beta-glycosidase and alpha-l-arabinofuranosidase enzyme was 3: 2 (0.6 mg / ml: 0.4 mg / ml) (2 mg / ml: 0.5 mg / ml) was preferable when the extract was used as a substrate and 8: 3 (2 mg / ml: 0.75 mg / ml) desirable.

According to the method for producing ginsenoside compound ke using the high temperature beta-glycosidase and alpha-el-arabinofuranosidase enzyme of the present invention, it is possible to produce a ginsenoside compound from a high temperature resistant Sulfolobus solfataricus strain One high temperature beta-glycosidase and a high temperature caldic cellulosic syrup sucarolite ticus ( Caldicellulosiruptor saccharolyticus ) shows stable activity even at high temperature, so that the reaction speed is increased, and thus, a large amount of ginsenoside compound k is produced in a short time and exhibits a high yield and thus can be industrially useful.

The composition of the present invention for preparing ginsenoside compound ke containing a high temperature beta-glycosidase and an alpha-el-arabinofuranosidase enzyme, and a composition comprising a high-temperature beta-glycosidase and alpha-el-arabinofuranosidase, According to the process for producing a ginsenoside compound ke using an enzyme, a high temperature beta-glycoprotein derived from a high-temperature Sulfolobus solfataricus and a Caldicellulosiruptor saccharolyticus strain, As a result, the activity of Shidase and Alpha-L-arabinofuranosidase is stable at high temperature, so that the reaction speed is increased and thus a large amount of ginsenoside compound k is produced in a short time and exhibits a high yield. Can be usefully used.

Fig. 1 shows the production of compound K according to the concentration ratio of the thermophilic beta-glycosidase and alpha-l-arabinofuranosidase enzyme when ginsenoside Rc is used as a substrate.
FIG. 2 is a graph showing the results obtained when (A) the red ginseng extract was used as a substrate and (B) when the extract was used as a substrate and when the concentration of the thermophilic β-glycosidase was fixed at 2 mg / The concentration of Ranocidase enzyme was varied to show the decrease of compound MC.
FIG. 3 shows the production of ginsenoside compound k by 2 mg / ml of hot β-glycosidase when red ginseng extract was used as a substrate (A) and mycorrhizae extract was used as a substrate (B).
4 (A) shows the production of ginsenoside compound k by 2 mg / ml beta-glycosidase and 0.5 mg / ml alpha-elabinofuranosidase using red ginseng extract as a substrate, FIG. 4 (B) shows the production of ginsenoside compound k by 2 mg / ml beta-glycosidase and 0.75 mg / ml alpha-elabinofuranosidase, respectively, using Moss extract as a substrate.

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-glycosidase or high temperature alpha- Arabinofuranosidase Production of recombinant expression vectors and transforming microorganisms containing

In the present invention, in order to prepare a high-temperature beta-glycosidase, a beta-glycosidase gene derived from a strain of Sulfolobus solfataricus was first isolated, and a high temperature alpha-elabinofuranosidase was prepared , The alpha-el-arabinofuranosidase gene derived from the sialiculolyticus strain of Caldelcellulose was isolated.

Specifically, Sulfolobus solfataricus strain and Caldicellulose syrup saccharolyticus strain, in which the nucleotide sequence and the amino acid sequence have already been identified, were selected and each genomic DNA was extracted. In addition, the nucleotide sequence of the beta-glycosidase gene of Sulforovus solfataricus strain (Genebank Accession No. M34696) and the alpha-L-ara of Saccharomyces cerevisiae Saccharolyticus strain (SEQ ID NO: 5), SSbglR (5'-GCGAATCTCGAGTTAGTGCCTTAATGGCTTTAC (SEQ ID NO: 5)) on the basis of the nucleotide sequence of the vinofuranosidase gene (Genebank Accession No. CP000679 3 '(SEQ ID NO: 6), CSabfF (5'-TTTGGATCCATGAAAAAAGCAAAAGTCATCTA-3' (SEQ ID NO: 7), CSabfR (5'-TTTCTGCAGTTAATTTTCTCTCTTCTTCAATCTG- PCR was carried out using primers to amplify the nucleotide sequences of the corresponding genes. A large amount of each gene was obtained in the above procedure, and then inserted into plasmid vectors pET-24a (+) and pTrc-99a The recombinant expression vectors pET-24a (+) / beta-glycosidase and pTrc-99a / alpha-elabinofuranosidase of the present invention were prepared.

The transformed recombinant E. coli was transformed into E. coli BL21 (DE3) ER2566 pET-24a (+) by transforming E. coli BL21 (DE3) strain ER2566 by a conventional transformation method. / Beta-glycosidase strain and E. coli BL21 (DE3) ER2566 pTrc-99a / alpha-l-arabinofuranosidase. The transformed Escherichia coli was stored frozen before addition of 20% glycerine solution.

Example 2: High temperature beta-glycosidase and high temperature alpha-el- Arabinofuranosidase Expression and purification

In the present invention, in order to mass-produce beta-glycosidase and alpha-el-arabinofuranosidase, a cryopreserved E. coli BL21 (DE3) ER2566 pET-24a (+) / beta-glycosidase strain and Escherichia coli BL21 (DE3) ER2566 pTrc-99a / alpha-el-arabinofuranosidase strain was inoculated into a 250 ml flask containing 50 ml of each LB medium until the absorbance at 600 nm reached 2.0 And shake-cultured in a shaking incubator at 37 ° C. Then, the culture was added again to a 2-liter 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 ° C . To this, 0.1 mM IPTG (isopropyl-beta-thiogalactoside) was added to induce the production of the over-expressed enzyme. The stirring speed was 150 rpm and the incubation temperature was adjusted to 16 ° C.

Also, in order to purify the high temperature beta-glycosidase and alpha-el-arabinofuranocidase enzyme produced as described above, the culture of the transformed strains was centrifuged at 4,000 x g for 30 minutes at 4 ° C The cell solution was then ruptured at 15,000 lb / in ² with French fries. The cell lysate was again centrifuged at 13,000 x g for 20 minutes at 4 ° C, heat-treated at 75 ° C for 10 minutes, and centrifuged again at 13,000 x g for 20 minutes at 13,000 x g Respectively. The resulting supernatant was filtered through 0.45 ㎛ filter paper and separated into enzyme solution which can be used for the production of ginsenoside compound k.

Example 3: High temperature beta-glycosidase and high temperature alpha-el- Arabinofuranosidase Optimal rate investigation

When the high-temperature beta-glycosidase isolated in Example 2 was treated with red ginseng extract or licorice extract, the ginsenoside Rc and the compound MC remained in the protopanaxadiol saponin to produce ginsenoside compound k It was confirmed that the yield was limited. In order to convert remaining ginsenoside Rc and compound MC into compound K, alpha-el-arabinofuranosidase was added and treated with beta-glycosidase. The ratio of two enzyme concentrations to compound k Respectively.

In the present invention, the optimum ratio of the thermophilic beta-glycosidase and alpha-el-arabinofuranocidase enzyme isolated in Example 2 was confirmed as follows. Under various substrate conditions, the two enzymes were reacted with the substrate at different concentration ratios and the degree of compound k production was compared.

end. Gin Senocide Rc Was used as a substrate, high temperature beta - glycosidase and alpha - Arabinofuranosidase Determination of optimum ratio of enzyme concentration

In the present invention, ginsenoside Rc was used as a substrate in order to confirm the enzyme concentration ratio of the high temperature beta-glycosidase and alpha-el-arabinofuranosidase. Specifically, before the reaction, 0.4 mg / ml ginsenoside Rc, 50 mM Mcilvaine buffer (pH 6.0) and two enzymes were treated at a concentration of 1 ml in total volume, Min. &Lt; / RTI > After the reaction was completed, n-butanol was added to terminate the reaction and the amount of Compound K was measured. At this time, the concentration of beta-glycosidase was increased from 0.0 to 1.0 mg / ml by 0.1 mg / ml, and the concentration of alpha-el-arabinofuranosidase was decreased from 1.0 to 0.0 mg / ml by 0.1 mg / ml , And the mixture of two enzymes so that the sum of the two enzymes was 1 mg / ml was used as the enzyme solution. As a result, as shown in Fig. 1, it was confirmed that the highest compound k production was obtained when 0.6 mg / ml beta-glycosidase and 0.4 mg / ml alpha-elabinofuranosidase were mixed.

I. When red ginseng extract was used as substrate, high temperature beta - glycosidase and alpha - Arabinofuranosidase Determination of optimum ratio of enzyme concentration

When red ginseng extract was used as a substrate, red ginseng containing about 6.5 mg / ml of protopanaxadiol saponin was used to confirm the enzyme concentration ratio of beta-glycosidase and alpha-l-arabinofuranosidase. Extract, a 50 mM Mcilvaine buffer (pH 6.0) and a mixture of two enzymes. When 2 mg / ml of beta-glycosidase alone was treated with the red ginseng extract as a substrate, it was confirmed that most of the ginsenoside Rd disappeared at 12 hours as shown in FIG. 2a, and the concentration of beta-glycosidase Was fixed at 2 mg / ml and the concentration of alpha-el-arabinofuranosidase was varied to confirm the concentration of alpha-el-arabinofuranocidase, which is all converted to compound MC.

Based on the 6: 4 ratio identified in Example 3 above, the concentration of alpha-l-arabinofuranosidase was reduced from 1.33 to 0.125 mg / ml, resulting in a reduction in the concentration of beta-glucosidase of 2 mg / ml, it was confirmed that the compound MC was completely converted by treating with alpha-el-arabinofuranosidase at a concentration of 0.5 mg / ml or more.

All. Mitham extract As the substrate, the high temperature beta-glycosidase and alpha-L- Arabinofuranosidase Determination of optimum ratio of enzyme concentration

In order to determine the enzyme concentration ratio of beta-glycosidase and alpha-l-arabinofuranosidase when Mitham extract was used as substrate, about 5 mg / ml of protopanaxadiol saponin Extract, a 50 mM Mcilvaine buffer (pH 6.0) and a mixture of two enzymes. When 2 mg / ml of beta-glucosidase alone was treated with MYCAM extract, as shown in FIG. 2B, most of the ginsenoside Rd disappeared at 12 hours, and the concentration of beta-glycosidase at 12 hours Was fixed at 2 mg / ml and the concentration of alpha-el-arabinofuranosidase was varied to confirm the concentration of alpha-el-arabinofuranocidase, which is all converted to compound MC.

As in Example 3 above, the concentration of alpha-l-arabinofuranosidase was reduced from 1.33 to 0.125 mg / ml, and as a result, when beta-glycosidase was fixed at 2 mg / ml, - Treatment of arabinofuranosidase more than 0.75 mg / ml confirmed that all compound MC was converted.

Example 4: High temperature beta-glycosidase and high temperature alpha-EL- Arabinofuranosidase Used Gin Senocide Compound K's production

In the present invention, in order to develop a method for producing ginsenoside compound kk using the above-mentioned high-temperature beta-glycosidase and alpha-el-arabinofuranosidase, the optimal ratio of enzyme concentration in each substrate The amount of ginsenoside compound k was measured by ginseng extract and Minshang extract.

FIG. 4A shows the effect of the 2.0 mg / ml beta-glycosidase of the present invention and 0.5 mg / ml alpha-el-arabinofuranoside in red ginseng extract containing protopanaxadiol saponin at about 6.5 mg / ml as a substrate It was confirmed that graphene, which indicates the amount of ginsenoside compound k produced by azela, produced 4.6 mg / ml of senoside compound k, which was completely converted in 12 hours. Fig. 4B also shows that the extract of the present invention containing 2.0 mg / ml of beta-glycosidase and 0.75 mg / ml of alpha-el-arabinofue of the present invention, which contains about 5 mg / ml protopanaxadiol saponin as a substrate, The graph showing the production of ginsenoside compound k by Ranocidae was confirmed to produce 3.3 mg / ml of senoside compound k, which was completely converted in 12 hours.

To date, the highest productivity of the Ginsenoside compound k is in the production of beta-glucosidase (6.3 mg / ml) derived from Sulforo bus ash Caldarius and alpha-glucosidase derived from Caldicellulose syrups saccharolyticus (1.4 mg / ml), beta-galactosidase (1.4 mg / ml), and was used to prepare a suspension containing about 5 mg / ml protopanaxadiol saponin In the present study, it was reported that the extracts of Mitsam extracts produced 2.9 mg / ml in 20 hours (Kyung-Chul Shin et al., 2013, J. Biotechnol., 167, 33-40, 2013).

In comparison, when using the thermophilic beta-glycosidase and alpha-el-arabinofuranocidase of the present invention, the total enzyme concentration was 3.6 times lower than that of the three enzymes when the extract was used And the productivity was increased to about 1.9 times. When the red ginseng extract was used, the total enzyme concentration was 3.3 times lower, and the productivity was increased to about 2.7 times, indicating that the productivity of the present invention was much better.

<110> Konkuk University Industrial Cooperation Corp <120> A Composition for producing ginsenoside compound K and a preparation method of ginsenoside compound K using the mixed enzymes of thermostable beta-glycosidase and alpha-L-arabinofuranosidase <130> HY140960 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 489 <212> PRT <213> Sulfolobus solfataricus <400> 1 Met Tyr Ser Phe Pro Asn Ser Phe Arg Phe Gly Trp Ser Gln Ala Gly 1 5 10 15 Phe Gln Ser Glu Met Gly Thr Pro Gly Ser Glu Asp Pro Asn Thr Asp 20 25 30 Trp Tyr Lys Trp Val His Asp Pro Glu Asn Met Ala Ala Gly Leu Val 35 40 45 Ser Gly Asp Leu Pro Glu Asn Gly Pro Gly Tyr Trp Gly Asn Tyr Lys 50 55 60 Thr Phe His Asp Asn Ala Gln Lys Met Gly Leu Lys Ile Ala Arg Leu 65 70 75 80 Asn Val Glu Trp Ser Arg Ile Phe Pro Asn Pro Leu Pro Arg Pro Gln 85 90 95 Asn Phe Asp Glu Ser Lys Gln Asp Val Thr Glu Val Glu Ile Asn Glu 100 105 110 Asn Glu Leu Lys Arg Leu Asp Glu Tyr Ala Asn Lys Asp Ala Leu Asn 115 120 125 His Tyr Arg Glu Ile Phe Lys Asp Leu Lys Ser Arg Gly Leu Tyr Phe 130 135 140 Ile Leu Asn Met Tyr His Trp Pro Leu Pro Leu Trp Leu His Asp Pro 145 150 155 160 Ile Arg Val Arg Arg Gly Asp Phe Thr Gly Pro Ser Gly Trp Leu Ser 165 170 175 Thr Arg Thr Val Tyr Glu Phe Ala Arg Phe Ser Ala Tyr Ile Ala Trp 180 185 190 Lys Phe Asp Asp Leu Val Asp Glu Tyr Ser Thr Met Asn Glu Pro Asn 195 200 205 Val Val Gly Gly Leu Gly Tyr Val Gly Val Lys Ser Gly Phe Pro Pro 210 215 220 Gly Tyr Leu Ser Phe Glu Leu Ser Arg Arg Ala Met Tyr Asn Ile Ile 225 230 235 240 Gln Ala His Ala Arg Ala Tyr Asp Gly Ile Lys Ser Val Ser Lys Lys 245 250 255 Pro Val Gly Ile Ile Tyr Ala Asn Ser Ser Phe Gln Pro Leu Thr Asp 260 265 270 Lys Asp Met Glu Ala Val Glu Met Ala Glu Asn Asp Asn Arg Trp Trp 275 280 285 Phe Phe Asp Ala Ile Ile Arg Gly Glu Ile Thr Arg Gly Asn Glu Lys 290 295 300 Ile Val Arg Asp Leu Lys Gly Arg Leu Asp Trp Ile Gly Val Asn 305 310 315 320 Tyr Tyr Thr Arg Thr Val Val Lys Arg Thr Glu Lys Gly Tyr Val Ser 325 330 335 Leu Gly Gly Tyr Gly His Gly Cys Glu Arg Asn Ser Val Ser Leu Ala 340 345 350 Gly Leu Pro Thr Ser Asp Phe Gly Trp Glu Phe Phe Pro Glu Gly Leu 355 360 365 Tyr Asp Val Leu Thr Lys Tyr Trp Asn Arg Tyr His Leu Tyr Met Tyr 370 375 380 Val Thr Glu Asn Gly Ile Ala Asp Asp Ala Asp Tyr Gln Arg Pro Tyr 385 390 395 400 Tyr Leu Val Ser His Val Tyr Gln Val His Arg Ala Ile Asn Ser Gly 405 410 415 Ala Asp Val Arg Gly Tyr Leu His Trp Ser Leu Ala Asp Asn Tyr Glu 420 425 430 Trp Ala Ser Gly Phe Ser Met Arg Phe Gly Leu Leu Lys Val Asp Tyr 435 440 445 Asn Thr Lys Arg Leu Tyr Trp Arg Pro Ser Ala Leu Val Tyr Arg Glu 450 455 460 Ile Ala Thr Asn Gly Ala Ile Thr Asp Glu Ile Glu His Leu Asn Ser 465 470 475 480 Val Pro Pro Val Lys Pro Leu Arg His 485 <210> 2 <211> 1467 <212> DNA <213> Sulfolobus solfataricus <400> 2 atgtactcat ttccaaatag ctttaggttt ggttggtccc aggccggatt tcaatcagaa 60 atgggaacac cagggtcaga agatccaaat actgactggt ataaatgggt tcatgatcca 120 gaaaacatgg cagcgggatt agtaagtgga gatctaccag aaaatgggcc aggctactgg 180 ggaaactata agacatttca cgataatgca caaaaaatgg gattaaaaat agctagacta 240 aatgtggaat ggtctaggat atttcctaat ccattaccaa ggccacaaaa ctttgatgaa 300 tcaaaacaag atgtgacaga ggttgagata aacgaaaacg agttaaagag acttgacgag 360 tacgctaata aagacgcatt aaaccattac agggaaatat tcaaggatct taaaagtaga 420 ggactttact ttatactaaa catgtatcat tggccattac ctctatggtt acacgaccca 480 ataagagtaa gaagaggaga ttttactgga ccaagtggtt ggctaagtac tagaacagtt 540 tacgaattcg ctagattctc agcttatata gcttggaaat tcgatgatct agtggatgag 600 tactcaacaa tgaatgaacc taacgttgtt ggaggtttag gatacgttgg tgttaagtcc 660 ggttttcccc caggatacct aagctttgaa ctttcccgta ggcatatgta taacatcatt 720 caagctcacg caagagcgta tgatgggata aagagtgttt ctaaaaaacc agttggaatt 780 atttacgcta atagctcatt ccagccgtta acggataaag atatggaagc ggtagagatg 840 gctgaaaatg ataatagatg gtggttcttt gatgctataa taagaggtga gatcaccaga 900 ggaaacgaga agattgtaag agatgaccta aagggtagat tggattggat tggagttaat 960 tattacacta ggactgttgt gaagaggact gaaaagggat acgttagctt aggaggttac 1020 ggtcacggat gtgagaggaa ttctgtaagt ttagcgggat taccaaccag cgacttcggc 1080 tgggagttct tcccagaagg tttatatgac gttttgacga aatactggaa tagatatcat 1140 ctctatatgt acgttactga aaatggtatt gcggatgatg ccgattatca aaggccctat 1200 tatttagtat ctcacgttta tcaagttcat agagcaataa atagtggtgc agatgttaga 1260 gggtatttac attggtctct agctgataat tacgaatggg cttcaggatt ctctatgagg 1320 tttggtctgt taaaggtcga ttacaacact aagagactat actggagacc ctcagcacta 1380 gtatataggg aaatcgccac aaatggcgca ataactgatg aaatagagca cttaaatagc 1440 gtacctccag taaagccatt aaggcac 1467 <210> 3 <211> 505 <212> PRT <213> Caldicellulosiruptor saccharolyticus <400> 3 Met Lys Lys Ala Lys Val Ile Tyr Asp Lys Glu Phe Val Ile Gly Gln 1 5 10 15 Ile Asp Lys Arg Ile Tyr Gly Ser Phe Leu Glu His Met Gly Arg Ala 20 25 30 Ile Tyr Thr Gly Ile Tyr Glu Pro Asp His Pro Gln Ala Asp Glu Met 35 40 45 Gly Phe Arg Lys Asp Val Leu Glu Leu Val Arg Lys Leu Asn Val Pro 50 55 60 Ile Val Arg Tyr Pro Gly Gly Asn Phe Val Ser Gly Tyr Asn Trp Glu 65 70 75 80 Asp Gly Val Gly Pro Lys Glu Lys Arg Pro Arg Arg Leu Glu Leu Ala 85 90 95 Trp Arg Ala Ile Glu Thr Asn Glu Val Gly Ile Asn Glu Phe Val Glu 100 105 110 Trp Ala Lys Arg Ala Asn Thr Ser Val Met Met Thr Val Asn Leu Gly 115 120 125 Thr Arg Gly Ile Asp Ala Ala Arg Asn Leu Val Glu Tyr Cys Asn Phe 130 135 140 Pro Gly Gly Thr Tyr Tyr Ser Asp Leu Arg Arg Gln His Gly Tyr Glu 145 150 155 160 Gln Pro His Asn Ile Lys Val Trp Cys Leu Gly Asn Glu Met Asp Gly 165 170 175 Asp Trp Gln Ile Gly His Lys Thr Ala Tyr Glu Tyr Gly Arg Leu Ala 180 185 190 Arg Glu Ala Ala Lys Val Met Lys Trp Val Asp Pro Ser Ile Glu Leu 195 200 205 Val Ala Gly Ser Ser Gly Pro Lys Met Pro Thr Phe Pro Glu Trp 210 215 220 Glu Ala Ile Val Leu Asp His Thr Tyr Asp Leu Val Asp Tyr Val Ser 225 230 235 240 Leu His Val Tyr Tyr Gly Asn Pro Glu Lys Asp Thr Lys Asn Phe Val 245 250 255 Ala Lys Ser Leu Glu Met Glu Glu Phe Ile Lys Thr Val Ile Ser Thr 260 265 270 Ile Asp Tyr Val Lys Ala Lys Lys Arg Ser Lys Lys Val Val Asn Ile 275 280 285 Ser Phe Asp Glu Trp Asn Val Trp Tyr His Ala His Leu Glu Gly Lys 290 295 300 Asp Glu Lys Ala Gln Pro Trp Ala Arg Ile Arg Ala Ile Ala Glu Glu 305 310 315 320 Asp Tyr Val Phe Glu Asp Ala Ile Leu Val Gly Cys Met Leu Ile Ala 325 330 335 Leu Leu Lys His Cys Asp Arg Val Ile Ala Cys Met Ala Gln Leu 340 345 350 Val Asn Val Ile Ala Pro Ile Thr Thr Val Lys Gly Gly Ile Ala Tyr 355 360 365 Arg Gln Val Ile Tyr Tyr Pro Phe Met His Ala Ala Asn Tyr Gly His 370 375 380 Gly Val Ala Leu Leu Pro Lys Val Asn Ser Pro Lys Tyr Asp Ser Lys 385 390 395 400 Asp Phe Thr Asp Val Tyr Ile Glu Thr Val Ala Thr Tyr Asn Glu 405 410 415 Glu Lys Gly Glu Ile Thr Val Phe Ala Val Asn Arg Asp Leu Glu Glu 420 425 430 Glu Met Gln Val Glu Phe Lys Leu Asp Gly Phe Glu Gly Phe Glu Val 435 440 445 Val Glu His Ile Val Tyr Glu Ser Asp Asp Ile Tyr Lys Gly Asn Thr 450 455 460 Gln Asp Lys Pro Asp Asn Ala Val Pro His Lys Gly Gly Ser Ser Lys 465 470 475 480 Ile Glu Gly Asn Ile Leu Thr Ser Ile Leu Pro Lys Phe Ser Trp Asn 485 490 495 Val Ile Arg Leu Lys Lys Arg Glu Asn 500 505 <210> 4 <211> 1515 <212> DNA <213> Caldicellulosiruptor saccharolyticus <400> 4 atgaaaaaag caaaagtcat ctacgataag gagtttgtga ttgggcaaat agacaaaaga 60 atctatggtt catttttaga gcacatggga agagcaatat acacaggaat ctatgaaccc 120 gaccatccac aggctgatga aatgggattt agaaaagatg ttttagagct tgttcgaaag 180 cttaatgttc ctattgtaag atatcctggc ggcaattttg tgtcggggta taactgggaa 240 gagggagttg gcccaaaaga aaaaaggcca agaagacttg agcttgcgtg gagagcaatt 300 gagacaaatg aggtcggtat aaacgaattt gttgaatggg caaaaagagc aaacacctct 360 gttatgatga cagtaaacct tggcacgcgt ggaattgatg ctgcaagaaa cttagttgag 420 tattgcaact ttccaggtgg cacatattac agtgacctgc gtcgtcagca cggctatgag 480 cagccacaca atataaaggt gtggtgcttg ggcaatgaga tggatggtga ctggcagatt 540 ggccacaaaa ctgcatatga gtatggaagg cttgcaagag aagctgcaaa agttatgaaa 600 tgggtagacc caagtattga gcttgttgca gctggaagct cagggcccaa aatgcccaca 660 tttcctgagt gggaagcaat tgttttggac cacacatatg atcttgtaga ttatgtttct 720 ctacatgtgt actatggaaa tcctgaaaag gacacaaaga attttgttgc aaaatcactt 780 gaaatggaag agtttataaa gacagtaatt tccacaattg attatgtaaa ggctaaaaag 840 aggagtaaaa aggttgtcaa tatctcattt gacgaatgga atgtatggta ccacgcacat 900 cttgagggaa aagacgaaaa agcacagccc tgggcacgaa ttcgtgctat tgctgaagaa 960 gattatgtgt tcgaagatgc aattttggtg ggatgtatgc taattgcact cttaaagcat 1020 tgtgacaggg tcaggatagc atgtatggca caacttgtta atgtcattgc cccaattacc 1080 actgtaaaag gtggaattgc ttacagacag gtaatctatt atcctttcat gcatgctgca 1140 aactatggtc atggggttgc actgcttcct aaggtaaatt cccctaaata tgattcaaaa 1200 gactttactg atgttccata tattgaaaca gtcgcaacat acaatgagga aaagggtgaa 1260 ataacagttt ttgccgtcaa cagagattta gaagaggaga tgcaagttga atttaaactt 1320 gatggttttg aaggatttga ggttgtggag cacattgtat atgaaagtga tgatatttac 1380 aaaggaaaca ctcaagataa gcctgacaat gccgtgcccc acaaaggtgg aagttcaaaa 1440 atagaaggca atatcttaac atccatattg cctaaattct catggaatgt aatcagattg 1500 aagaagagag aaaat 1515 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcgtctgcat atgtactcat ttccaaatag c 31 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gcgaatctcg agttagtgcc ttaatggctt tac 33 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tttggatcca tgaaaaaagc aaaagtcatc ta 32 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tttctgcagt taattttctc tcttcttcaa tctg 34

Claims

A composition for the preparation of ginsenoside compound K, which contains, as an active ingredient, beta-glycosidase and alpha-L-arabinofuranosidase.

2. The composition of claim 1, wherein the beta-glycosidase comprises an amino acid sequence as set forth in SEQ ID NO: 1.

The method of claim 1, wherein the beta-glycosidase is selected from the group consisting of Sulfolobus solfataricus ). < / RTI >

The composition according to claim 1, wherein the β-glycosidase has an optimal activity temperature of 70 to 95 ° C.

2. The composition of claim 1, wherein the alpha -L-arabinofuranosidase comprises the amino acid sequence set forth in SEQ ID NO: 2.

The composition according to claim 1, wherein the alpha -L-arabinofuranosidase is derived from Caldicellulosiruptor saccharolyticus strain.

The composition of claim 1, wherein the alpha-el-arabinofuranosidase has an optimal activity temperature of 70-95 ° C.

The composition of claim 1, wherein the composition ratio of beta-glycosidase to alpha-el-arabinofuranosidase is from 1: 9 to 9: 1 (w / v).

A gene encoding a beta-glycosidase and a gene encoding an alpha-L-arabinofuranosidase as an active ingredient, and a compound K ).

10. The composition according to claim 9, wherein the gene coding for beta-glycosidase is a nucleotide sequence as set forth in SEQ ID NO: 3.

10. The composition according to claim 9, wherein the gene encoding the alpha -L-arabinofuranosidase comprises the nucleotide sequence set forth in SEQ ID NO: 4.

A process for preparing ginsenoside compound K by treating beta-glycosidase and alpha-L-arabinofuranosidase to a substrate.

13. The method of claim 12, wherein the beta-glycosidase is derived from a Sulfolobus solfataricus strain.

13. The method according to claim 12, wherein the alpha-L-arabinofuranosidase is Caldicellulosiluptus Saccharolyticus ). < / RTI >

13. The method according to claim 12, wherein the substrate is red ginseng extract, licorice extract, or protopanaxadiol saponin.

13. The method of claim 12, wherein the ratio of the treating beta-glycosidase to alpha-l-arabinofuranosidase is from 1: 9 to 9: 1 (w / v).

[16] The method according to claim 15, wherein the ginseng extract is a ginseng saponin mixture or extract obtained by extracting ginseng, ginseng, American ginseng, mustard ginseng, Himalaya ginseng, or Vietnamese ginseng roots or ground parts.