COMPOSITION FOR PRODUCTION OF GINSENOSIDE COMPOUND K COMPRISING HIGH TEMPERATURE alpha-L-ARABINOFURANOSIDASE, AND METHOD FOR PREPARING GINSENDOSIDE COMPOUND K

TECHNICAL FIELD

Disclosed are a composition for production of ginsenoside compound K using a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase, and a method for preparing ginsenoside compound K.

BACKGROUND ART

Ginsenoside compound K (20(S)-protopanaxadiol-20-O-β-D-glucopyranoside; see the following Formula 1) is an intestinal bacterial metabolite of ginseng saponin components. It is produced by hydrolysis of glucose, arabinopyranose and arabinofuranose moieties in ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd, which are protopanaxadiol-type saponins.

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Until now, ginsenoside compound K has been known to have many excellent effects such as immunity enhancement, inhibition of tumor angiogenesis, inhibition of cancer cell infiltration and inhibition of cancer cell proliferation. Accordingly, there is an increasing demand for mass supply of the compound in the field of health foods and cosmetics. Therefore, there is a growing need for producing it stably and efficiently.

The prior art for production of ginsenoside compound K includes methods for preparing compound K by treating diol-type saponins with enzymes such as β-glycosidase (Korean Patent Laid-Open No. 2003-94757), cellulase isolated from a microorganism of the genus Penicillium or β-galactosidase isolated from the genus Aspergillus (Korean Patent No. 377546), naringinase isolated from the genus Penicillium, or pectinase isolated from the genus Aspergillus (Korean Patent No. 418604), etc.

As described above, ginsenoside compound K is mostly produced using mesophilic enzymes active at a temperature in the range of 10 to 50° C. However, since these enzymes act at a low reaction temperature, they are likely to be contaminated with microorganisms and have a low production yield.

In some cases, ginsenoside compound K is produced using high temperature enzymes. However, α-L-arabinofuranosidase shows poor expression and activity, and thus has difficulty in converting ginsenoside Rc to compound K.

Therefore, in order to solve these problems, there is an urgent need to develop enzymes industrially useful for production of ginsenoside compound K and a preparation method using the same.

SUMMARY OF INVENTION
Technical Problem

Thus, the present inventors have continuously studied to develop a new method for preparing ginsenoside compound K. An object of the present invention is to provide a composition for production of ginsenoside compound K comprising a high temperature-β-glycosidase derived from a high temperature microorganism, Sulfolobus solfataricus, and an α-L-arabinofuranosidase derived from Thermotoga petrophila and a method for producing ginsenoside compound K using the same.

In one aspect of the present invention, these enzymes are cloned from the high temperature microorganisms to produce recombinant expression vectors and microorganisms transformed with the same. Then, a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase are produced by enhancing the expression of an α-L-arabinofuranosidase derived from Thermotoga petrophila, which had a low expression level, and the optimum ratio of these two enzymes are determined. The present inventors have found that when the resultant is reacted with red ginseng extract, a large quantity of ginsenoside compound K is produced in a short time, resulting in a high yield, and thereby completed the present invention. Thus, an object of the present invention is to provide a composition for production of ginsenoside compound K comprising a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase.

In another aspect, an object of the present invention is to provide a preparation method for converting all the protopanaxadiol-type ginsenosides in red ginseng extract into ginsenoside compound K by using a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase.

Solution to Problem

In one aspect, the present invention provides a composition for production of ginsenoside compound K comprising a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase.

In one aspect, the present invention may provide the use of a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase for production of ginsenoside compound K.

In one aspect of the present invention, the high temperature-β-glycosidase may be a β-glycosidase of Sulfolobus solfataricus, and the high temperature-αL-arabinofuranosidase may be an α-L-arabinofuranosidase of Thermotoga petrophila.

In one aspect of the present invention, the content of the high temperature-α-L-arabinofuranosidase may be 1 part by weight or more based on 100 parts by weight of the high temperature-β-glycosidase.

In one aspect of the present invention, the high temperature-α-L-arabinofuranosidase may be 2.5 parts by weight or more based on 100 parts by weight of the high temperature-β-glycosidase.

In one aspect of the present invention, the high temperature-β-glycosidase may be an enzyme consisting of the amino acid sequence of SEQ ID NO: 2, and the high temperature-α-L-arabinofuranosidase may be an enzyme consisting of the amino acid sequence of SEQ ID NO: 4.

In one aspect of the invention, the method may be a method for preparing a composition for production of ginsenoside compound K, comprising expression in E. coli transformed with a vector comprising the base sequence of SEQ ID NO: 3; and a vector comprising the base sequences of SEQ ID NO: 13 and SEQ ID NO: 14.

In another aspect, the present invention provides a method for preparing ginsenoside compound K, comprising the step of fermenting a saponin-containing material comprising at least one of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, and ginsenoside Rd with a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase.

In another aspect of the present invention, the step of fermentation may be fermentation using the composition for production of ginsenoside compound K according to any one of the aspects of the present invention.

In another aspect of the invention, the step of fermentation may be applying each of a high temperature-β-glycosidase and a high temperature-aα-L-arabinofuranosidase.

In another aspect of the present invention, the high temperature-β-glycosidase may be a β-glycosidase of Sulfolobus solfataricus, and the high temperature-α-L-arabinofuranosidase may be an α-L-arabinofuranosidase of Thermotoga petrophila.

In another aspect of the present invention, the high temperature-α-L-arabinofuranosidase may be applied in an amount of 1 part by weight or more based on 100 parts by weight of the high temperature-β-glycosidase.

In another aspect of the present invention, the saponin-containing material may be red ginseng extract.

In another aspect of the present invention, the fermentation may be fermentation at a temperature of 70° C. to 95° C.

In another aspect of the present invention, the fermentation may be fermentation at a temperature of 80° C. to 90° C.

Advantageous Effects of Invention

The composition for production of ginsenoside compound K and the method for preparing ginsenoside compound K according to one aspect of the present invention allow high temperature-β-glycosidase and high temperature-α-L-arabinofuranosidase to exhibit stable activity even at high temperatures, thereby increasing a reaction rate.

The composition for production of ginsenoside compound K and the method for preparing ginsenoside compound K according to one aspect of the present invention allow a large quantity of ginsenoside compound K to be produced in a short time, thereby exhibiting an effect of producing a high yield, and thus can be utilized industrially.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of Test Example 1 regarding α-L-arabinofuranosidases in cell debris, enzyme suspension and purified enzyme liquid when α-L-arabinofuranosidases derived from Thermotoga petrophila were expressed in various host strains and coexpressed with chaperone.

FIG. 2 shows the decrease of compound Mc when the concentration of α-L-arabinofuranosidase was varied while the concentration of high temperature-β-glycosidase was fixed at 2 mg/ml and red ginseng extract was used as a substrate.

FIG. 3 shows the production of ginsenoside compound K by 2 mg/ml of high temperature-β-glycosidase when red ginseng extract was used as a substrate.

FIG. 4 shows the production of ginsenoside compound K by 2 mg/ml of β-glycosidase and 0.05 mg/ml of α-L-arabinofuranosidase when red ginseng extract was used as a substrate.

DESCRIPTION OF EMBODIMENTS
Embodiments

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a composition for production of ginsenoside compound K comprising a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase.

As used herein, the term “high temperature” enzyme refers to an enzyme that exhibits optimum activity at a high temperature of 70-95° C., rather than an intermediate temperature of 10-50° C., which is the optimum temperature for enzyme activity.

In one aspect of the present invention, the high temperature-β-glycosidase may be a β-glycosidase of Sulfolobus solfataricus, and the high temperature-α-L-arabinofuranosidase may be an α-L-arabinofuranosidase of Thermotoga petrophila.

In one aspect of the present invention, the high temperature-β-glycosidase and the high temperature-α-L-arabinofuranosidase of the present invention are obtained from Sulfolobus solfataricus and Thermotoga petrophila, which are high temperature organisms, by 1) directly isolating them from these strains and purifying them or 2) cloning the genes of each of the enzymes from the strains, expressing them in a recombinant expression vector, and purifying them. The method for obtaining the enzymes from microorganisms is a conventional method in the art (Sambrook, J. and Russell, D. W. Molecular Cloning 3rd Ed. Cold Spring Harbor Laboratory, 2001).

When the β-glycosidase obtained by a conventional method is applied to red ginseng extract, ginsenoside Rc and compound Mc among protopanaxadiol-type saponins are left, which limits the production yield of ginsenoside compound K (FIG. 3). Thus, in one aspect, the present invention provides a method for converting all the protopanaxadiol-type saponins in red ginseng extract or tiny-sized ginseng extract to compound K by applying α-L-arabinofuranosidase simultaneously.

In one aspect of the present invention, α-L-arabinofuranosidase derived from Thermotoga petrophila exhibited about 17 times higher activity than α-L-arabinofuranosidase derived from Caldicellulosiruptor saccharolyticus, which has been conventionally used in the production of ginsenoside compound K. Also, its expression pattern was enhanced by host cell selection and the introduction of chaperone (FIG. 1).

Specifically, the content of the temperature-α-L-arabinofuranosidase may be 1 part by weight or more, 1.5 parts by weight or more, 2.0 parts by weight or more, 2.1 parts by weight or more, 2.2 parts by weight or more, 2.3 parts by weight or more, 2.4 parts by weight or more, 2.5 parts by weight or more, 2.6 parts by weight or more, 2.7 parts by weight or more, 2.8 parts by weight or more, or 3.0 parts by weight or more based on 100 parts by weight of the high temperature-β-glycosidase. Also, the content of the high temperature-α-L-arabinofuranosidase may be 5.0 parts by weight or less, 4.5 parts by weight or less, or 4.0 parts by weight or less based on 100 parts by weight of the high temperature-β-glycosidase.

The high temperature-α-L-arabinofuranosidase can achieve the maximum generation of compound K economically while minimizing the concentration of the enzyme, when the weight ratio of the high temperature-β-glycosidase is within the above range.

Preferably, the content of the temperature-α-L-arabinofuranosidase may be 2 parts by weight or more based on 100 parts by weight of the high temperature-β-glycosidase.

More preferably, the content of the high temperature-α-L-arabinofuranosidase may be 2.5 parts by weight or more based on 100 parts by weight of the high temperature-β-glycosidase. In one aspect of the present invention, when red ginseng extract is used as a substrate, all of the remaining compounds Mc are converted to compounds K at a concentration ratio of β-glycosidase derived from Thermotoga petrophila and α-L-arabinofuranosidase derived from Sulfolobus solfataricus of 40:1 (FIG. 2).

As described above, the composition for production of ginsenoside compound K comprising a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase according to one aspect of the present invention controls the reaction rate rapidly at a high temperature of 85° C. and thereby achieves the effect of producing ginsenoside compound K in a short time at a high yield and using a low enzyme concentration, when reacted with a mixture of ginsenosides Rb1, Rb2, Rc, and Rd, which are major diol-type saponins in red ginseng extract, in a mixed solution of a buffer solution and an aqueous solvent.

In another aspect of the invention, the step of fermentation may be applying each of a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase. In another aspect of the present invention, the high temperature-β-glycosidase may be a β-glycosidase of Sulfolobus solfataricus, and the high temperature-α-L-arabinofuranosidase may be an α-L-arabinofuranosidase of Thermotoga petrophila. In another aspect of the present invention, the high temperature-α-L-arabinofuranosidase may be applied in an amount of 1 part by weight or more based on 100 parts by weight of the high temperature-β-glycosidase. Specifically, the content of the high temperature-α-L-arabinofuranosidase may be 1 part by weight or more, 1.5 parts by weight or more, 2.0 parts by weight or more, 2.1 parts by weight or more, 2.2 parts by weight or more, 2.3 parts by weight or more, 2.4 parts by weight or more, 2.5 parts by weight or more, 2.6 parts by weight or more, 2.7 parts by weight or more, 2.8 parts by weight or more, or 3.0 parts by weight or more based on 100 parts by weight of the high temperature-β-glycosidase. Also, the content of the high temperature-α-L-arabinofuranosidase may be 5.0 parts by weight or less, 4.5 parts by weight or less, or 4.0 parts by weight or less based on 100 parts by weight of the high temperature-β-glycosidase.

In one embodiment of the present invention, a) PCR is performed with genomic DNA of Sulfolobus solfataricus and Thermotoga petrophila and their respective primers to amplify the DNA fragments comprising each of high temperature-β-glycosidase and high temperature-α-L-arabinofuranosidase genes; b) the amplified DNA fragments comprising each of high temperature-β-glycosidase and high temperature-α-L-arabinofuranosidase gene are treated with restriction enzymes and each of them is cloned into plasmid vectors pET-24a(+) and pET-21a(+) to construct recombinant expression vectors pET-24a(+)/β-glycosidase and pET-21a(+)/α-L-arabinofuranosidase; c) E. coli ER2566 is transformed with the vectors according to a conventional transformation method; d) E. coli transformed with each of high temperature-β-glycosidase genes and high temperature α-L-arabinofuranosidase genes is cultured; e) gene expression is induced during culture to produce a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase; and f) the expressed high temperature-β-glycosidase and high temperature-α-L-arabinofuranosidase proteins are isolated and obtained.

The pET-21a(+)/α-L-arabinofuranosidase in the above step c) may be transformed together with the chaperone vector pGro7 into BL21(DE3), which shows the highest expression among various strains such as E. coli ER2566, BL21(DE3), JM109 and Origami B, as a host.

The process of isolating the high temperature (β-glucosidase and high temperature-α-L-arabinofuranosidase proteins expressed in the above step f) may consist of the steps of: (a) lysing the culture solution of microorganisms; (b) centrifuging the cell lysate to obtain a supernatant; (c) subjecting the supernatant to heat treatment at a high temperature and centrifuging the resultant; and (d) filtering the thus-obtained supernatant to isolate an enzyme liquid.

In the above step (a), preferably, cells are lysed at a pressure of about 15,000 lb/in²using a device such as a French press. In the above step (c), preferably, the cell supernatant is subjected to heat treatment at a temperature of 75° C. for about 10 minutes. In the above step (d), preferably, the filtration is performed using a filter paper of about 0.45 μm.

Also, the substrate may be ginsenosides Rb1, Rb2, Rc, and Rd, which are diol-type saponins in red ginseng extract, and may be used as a mixture in the preparation of ginsenoside compound K. The reaction solvent may be a buffer solution such as Mcllvaine buffer.

As described above, the reaction between the high temperature-β-glycosidase and high temperature-α-L-arabinofuranosidase and the substrate in the reaction solvent is performed preferably at a pH of 5.0 to 7.0 and a temperature of 70 to 95° C., more preferably at a pH of 6.0 and a temperature of 85° C.

The method for preparing ginsenoside compound K using a high temperature-β-glycosidase and a high temperature-α-L-arabinofuranosidase according to the present invention allows a high temperature-β-glycosidase derived from Sulfolobus solfataricus and a high temperature-α-L-arabinofuranosidase derived from Thermotoga petrophila to exhibit stable activity even at high temperatures, thereby increasing a reaction rate. As a result, it allows a large quantity of ginsenoside compound k to be produced in a short time, thereby exhibiting an effect of producing a high yield, and thus can be utilized industrially.

In another aspect of the present invention, the saponin-containing material may be red ginseng extract.

In another aspect of the present invention, the fermentation may be performed at a temperature of 70° C. to 95° C. Specifically, the fermentation temperature may be 70° C. or more, 72° C. or more, 74° C. or more, 76° C. or more, 78° C. or more, 80° C. or more, 82° C. or more, or 84° C. or more. Also, the fermentation temperature may be 95° C. or less, 93° C. or less, 91° C. or less, 90° C. or less, 88° C. or less, 86° C. or less, or 84° C. or less. When the temperature is within the above range, the production yield of ginsenoside K is excellent.

Hereinafter, preferred examples of the present invention will be described to facilitate understanding of the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and the scope of the present invention is not limited thereto.

Example 1

Preparation of a Recombinant Expression Vector Comprising a High Temperature-α-glycosidase Coding Base Sequence or a High Temperature-α-L-arabinofuranosidase Coding Base Sequence, and a Transformed Microorganism

In order to prepare a high temperature-β-glycosidase, a β-glycosidase gene derived from Sulfolobus solfataricus was isolated. Also, in order to prepare a high temperature-α-L-arabinofuranosidase, an α-L-arabinofuranosidase gene derived from Thermotoga petrophila was isolated.

Specifically, Sulfolobus solfataricus and Thermotoga petrophila, whose base sequence and amino acid sequence are already specified, were selected and the genomic DNA of each was extracted. The Sulfolobus solfataricus used was DSM 1617 purchased from the DSMZ (Germany), and the Thermotoga petrophila used was DSM 13995 purchased from the DSMZ (Germany).

Also, primers were prepared using the base sequence of the β-glycosidase gene of Sulfolobus solfataricus (GenBank Accession No. M34696) and the base sequence of the α-L-arabinofuranosidase gene of Thermotoga petrophila (GenBank Accession No. ABQ46651, respectively.

The DNA base sequence of the β-glycosidase of Sulfolobus solfataricus was as shown in SEQ ID NO: 1, and the amino acid sequence thereof was as shown in SEQ ID NO: 2.

The DNA base sequence of the α-L-arabinofuranosidase of Thermotoga petrophila was as shown in SEQ ID NO: 3, and the amino acid sequence thereof was as shown in SEQ ID NO: 4.

The forward and reverse primers for the β-glycosidase of Sulfolobus solfataricus were as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

In addition, the forward and reverse primers for α-L-arabinofuranosidase of Thermotoga petrophila were as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

Polymerase chain reaction (PCR) was performed using the genomic DNA and primers to amplify the base sequences of the corresponding genes. After the respective genes were obtained in large quantities by the above procedure, they were inserted into plasmid vectors pET-24a(+) and pET-21a to prepare recombinant expression vectors pET-24a(+)/β-glycosidase and pET-21a/α-L-arabinofuranosidase.

The plasmid vector pET-24a(+) was as shown in SEQ ID NO: 9.

The plasmid vector pET-21a was as shown in SEQ ID NO: 10.

The recombinant expression vector pET-24a(+)/β-glycosidase was as shown in SEQ ID NO: 11.

The recombinant expression vector pET-21a/α-L-arabinofuranosidase was as shown in SEQ ID NO: 12.

Also, the thus-prepared recombinant expression vectors were transformed into E. coli strain ER2566 by a conventional transformation method. pET-21a/α-L-arabinofuranosidase was also transformed into E. coli strains BL21(DE3), JM109 and Origami B.

The E. coli strains ER2566 and BL21(DE3) were purchased from New England Biolabs (NEB).

The E. coli strain JM109 was purchased from Takara.

The E. coli strain Origami B was purchased from Novagen.

BL21(DE3), which among them exhibited the highest expression, as a host was transformed with pET-21a/α-L-arabinofuranosidase and the chaperone vector pGro7, which was a commercial chaperone vector purchased from Takara. The chaperone vector pGro7, which was an independent plasmid, was co-transformed with the pET-21a/α-L-arabinofuranosidase vector into the strain BL21(DE3).

The chaperone vector pGro7 was a vector that simultaneously expresses GroEL and GroES genes. The GroEL gene was as shown in SEQ ID NO: 13, and the GroES gene was as shown in SEQ ID NO: 14. A schematic diagram of the chaperon pGro7 vector is shown in FIG. 5.

The transformed recombinant E. coli is referred to as E. coli strain ER2566 pET-24a(+)/β-glycosidase, E. coli strains ER2566, BL21(DE3), JM109, and Origami B pET-21a/α-L-arabinofuranosidase, and E. coli strain BL21(DE3) pET-21a/α-L-arabinofuranosidase-pGro7.

The transformed E. coli was added with 20% glycerine solution and stored frozen before culture.

Example 2
Expression and Purification of a High Temperature-β-Glycosidase and a High Temperature-α-L-Arabinofuranosidase

In order to mass produce β-glycosidase and α-L-arabinofuranosidase, the frozen E. coli strain ER2566 pET-24a(+)/β-glycosidase, E. coli strains ER2566, BL21(DE3), JM109, and Origami B pET-21a/α-L-arabinofuranosidase, and E. coli strain BL21(DE3) pET-21a/α-L-arabinofuranosidase-pGro7 each were seeded into a 250 ml flask containing 50 ml of LB medium, and then subjected to shaking culture in a shaking incubator at 37° C. until the absorbance at 600 nm reached 2.0. Then, the culture solution was added to a 21 Erlenmeyer flask containing 500 ml of LB medium and cultured until the absorbance at 600 nm reached 0.8. During the process, the stirring speed was 200 rpm and the culture temperature was 37° C. The resultant was added with 0.1 mM IPTG (isopropyl-beta-thiogalactoside) to induce production of the overexpressed enzyme. The stirring speed was adjusted to 150 rpm and the culture temperature was adjusted to 16° C.

In order to purify the thus-obtained high temperature-β-glycosidase and high temperature-α-L-arabinofuranosidase, the cultures of the transformed strains were centrifuged at 4,000×g for 4 to 30 minutes. Then, the cell solutions were lysed using a French press at 15,000 lb/in². The cell lysates were centrifuged again at 13,000×g for 4 to 20 minutes and subjected to heat treatment at a high temperature of 75° C. for 10 minutes. The thus-obtained heat-treated product was centrifuged again at 13,000×g for 4 to 20 minutes. The resultant supernatant was filtered with a 0.45 μm filter paper and isolated as an enzyme liquid which can be used for the production of ginsenoside compound K.

Test Example 1
Determination of the Expression Level of α-L-Arabinofuranosidase

The expression levels of the α-L-arabinofuranosidase enzyme liquids isolated from various host strains, the enzyme suspensions before subjected to heat treatment, and the cell debris obtained by centrifugation according to Example 2 were qualitatively compared through SDS-PAGE analysis.

As a result, as shown in FIG. 1, it was found from the cell debris that α-L-arabinofuranosidase expressed in the E. coli strain BL21(DE3) (well No. 2) was most expressed. Also, it was found from the purified enzyme liquid and the enzyme suspension that in the case of coexpression using chaperone pGro7 in the E. coli strain BL21(DE3) (well No. 4), α-L-arabinofuranosidase reached the highest concentration, and the expression of α-L-arabinofuranosidase of relatively high solubility was enhanced.

Test Example 2
Experiment on the Optimum Ratio of High Temperature-α-Glycosidase and High Temperature-α-L-Arabinofuranosidase

It was found that when the high temperature-β-glycosidase isolated in Example 2 was applied to red ginseng extract, ginsenoside Rc and compound Mc among protopanaxadiol-type saponins were left, which limited the production yield of ginsenoside compound K (FIG. 3).

In order to convert the residual ginsenoside Rc and compound Mc into compound K, α-L-arabinofuranosidase was added for co-treatment with β-glycosidase, and then the compound K production was compared.

The high temperature-β-glycosidase isolated in Example 2 was added with varying concentration of α-L-arabinofuranosidase, which was confirmed to have enhanced expression in Test Example 1, and the optimum ratio of the enzymes was determined in the following manner. The two enzymes were reacted with red ginseng extract and compared for the degree of compound K production.

In order to determine the optimum concentration ratio of β-glycosidase and α-L-arabinofuranosidase, red ginseng extract containing about 7.5 mg/ml of protopanaxadiol-type saponins, 50 mM Mcilvaine buffer solution (pH 6.0), and a mixture of the two enzymes were applied.

When 2 mg/ml of β-glycosidase alone was applied to red ginseng extract as a substrate, it was found that most of ginsenosides Rd disappeared after 12 hours as shown in FIG. 3.

The concentration of α-L-arabinofuranosidase at which all of compounds Mc (C-Mc) are converted was determined by varying the concentration of α-L-arabinofuranosidase with the concentration of β-glycosidase fixed at 2 mg/ml. Specifically, the concentration of α-L-arabinofuranosidase was decreased from 0.1 mg/ml to 0.0032 mg/ml. As a result, as shown in FIG. 2, it was found that when α-L-arabinofuranosidase at a concentration of 0.05 mg/ml or more was applied with the concentration of β-glycosidase fixed at 2 mg/ml, all of the compounds Mc were converted.

Example 3

Production of Ginsenoside Compound K using High Temperature-β-Glycosidase and High Temperature α-L-Arabinofuranosidase

In order to develop a method for preparing ginsenoside compound K using the high temperature-β-glycosidase of Example 2 and the α-L-arabinofuranosidase with enhanced expression in Test Example 1, the production of ginsenoside compound K over time was measured using red ginseng extract and tiny-sized ginseng extract at an optimum ratio of the enzymes in each substrate as determined above.

The test results are shown in FIG. 4. FIG. 4 is a graph showing the production of ginsenoside compound K by 2.0 mg/ml of β-glycosidase and 0.05 mg/ml of α-L-arabinofuranosidase of the present invention in red ginseng extract containing about 7.5 mg/ml of protopanaxadiol-type saponins as a substrate. FIG. 4 shows that after 12 hours, all of the materials were converted to produce 4.2 mg/ml of ginsenoside compound K (C-K).

Until now, a suspension of β-glycosidase (2.3 mg/ml) from Sulfolobus solfataricus and α-L-arabinofuranosidase (0.39 mg/ml) from Thermotoga petrophila has been found to achieve the highest productivity in production of ginsenoside compound K. It has been reported that the use of the suspension in red ginseng extract containing about 7.5 mg/ml of protopanaxadiol-type saponins resulted in production of 4.2 mg/ml of ginsenoside compound K for 12 hours (Kyung-Chul Shin et al. 2015, Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus. PLoS One. 28;10(12):e0145876.).

Upon comparing the above case and the present invention, in the case of using the high temperature-β-glycosidase and the high temperature-α-L-arabinofuranosidase according to one aspect of the present invention, the total enzyme concentration was about 1.3 times lower than in the case of using the two enzymes, and the concentration of α-L-arabinofuranosidase among them was 8 times lower than the above case, and the productivity increased by about 1.2 times. Thus, it was confirmed that the productivity per enzyme concentration in this experiment was 1.3 times higher than the above case.

Sequence Listing Free Text

SEQ ID NO: 1

ggatcaatac taggaggagt agcatataat tacgttacac aattttataa cccaatatat
60

tcaatagacc ttatgcttat cctatcctct attctaagat tctcggtatc tcccctattc
120

ttgaccataa aagatactcg ctcaaagctt aaataatatt aatcataaat aaagtcatgt
180

actcatttcc aaatagcttt aggtttggtt ggtcccaggc cggatttcaa tcagaaatgg
240

gaacaccagg gtcagaagat ccaaatactg actggtataa atgggttcat gatccagaaa
300

acatggcagc gggattagta agtggagatc taccagaaaa tgggccaggc tactggggaa
360

actataagac atttcacgat aatgcacaaa aaatgggatt aaaaatagct agactaaatg
420

tggaatggtc taggatattt cctaatccat taccaaggcc acaaaacttt gatgaatcaa
480

aacaagatgt gacagaggtt gagataaacg aaaacgagtt aaagagactt gacgagtacg
540

ctaataaaga cgcattaaac cattacaggg aaatattcaa ggatcttaaa agtagaggac
600

tttactttat actaaacatg tatcattggc cattacctct atggttacac gacccaataa
660

gagtaagaag aggagatttt actggaccaa gtggttggct aagtactaga acagtttacg
720

aattcgctag attctcagct tatatagctt ggaaattcga tgatctagtg gatgagtact
780

caacaatgaa tgaacctaac gttgttggag gtttaggata cgttggtgtt aagtccggtt
840

ttcccccagg atacctaagc tttgaacttt cccgtagggc aatgtataac atcattcaag
900

ctcacgcaag agcgtatgat gggataaaga gtgtttctaa aaaaccagtt ggaattattt
960

acgctaatag ctcattccag ccgttaacgg ataaagatat ggaagcggta gagatggctg
1020

aaaatgataa tagatggtgg ttctttgatg ctataataag aggtgagatc accagaggaa
1080

acgagaagat tgtaagagat gacctaaagg gtagattgga ttggattgga gttaattatt
1140

acactaggac tgttgtgaag aggactgaaa agggatacgt tagcttagga ggttacggtc
1200

acggatgtga gaggaattct gtaagtttag cgggattacc aaccagcgac ttcggctggg
1260

agttcttccc agaaggttta tatgacgttt tgacgaaata ctggaataga tatcatctct
1320

atatgtacgt tactgaaaat ggtattgcgg atgatgccga ttatcaaagg ccctattatt
1380

tagtatctca cgtttatcaa gttcatagag caataaatag tggtgcagat gttagagggt
1440

atttacattg gtctctagct gataattacg aatgggcttc aggattctct atgaggtttg
1500

gtctgttaaa ggtcgattac aacactaaga gactatactg gagaccctca gcactagtat
1560

atagggaaat cgccacaaat ggcgcaataa ctgatgaaat agagcactta aatagcgtac
1620

ctccagtaaa gccattaagg cactaaactt tctcaagtct cactatacca aatgagtttt
1680

cttttaatct tattctaatc tcattttcat tagattgcaa tactttcata ccttctatat
1740

tatttatttt gtaccttttg ggatc
1765

SEQ ID NO: 2

Met Tyr Ser Phe Pro Asn Ser Phe Arg Phe Gly Trp Ser Gln Ala Gly

Phe Gln Ser Glu Met Gly Thr Pro Gly Ser Glu Asp Pro Asn Thr Asp

Trp Tyr Lys Trp Val His Asp Pro Glu Asn Met Ala Ala Gly Leu Val

Ser Gly Asp Leu Pro Glu Asn Gly Pro Gly Tyr Trp Gly Asn Tyr Lys

Thr Phe His Asp Asn Ala Gln Lys Met Gly Leu Lys Ile Ala Arg Leu

Asn Val Glu Trp Ser Arg Ile Phe Pro Asn Pro Leu Pro Arg Pro Gln

Asn Phe Asp Glu Ser Lys Gln Asp Val Thr Glu Val Glu Ile Asn Glu

Asn Glu Leu Lys Arg Leu Asp Glu Tyr Ala Asn Lys Asp Ala Leu Asn

His Tyr Arg Glu Ile Phe Lys Asp Leu Lys Ser Arg Gly Leu Tyr Phe

Ile Leu Asn Met Tyr His Trp Pro Leu Pro Leu Trp Leu His Asp Pro

Ile Arg Val Arg Arg Gly Asp Phe Thr Gly Pro Ser Gly Trp Leu Ser

Thr Arg Thr Val Tyr Glu Phe Ala Arg Phe Ser Ala Tyr Ile Ala Trp

Lys Phe Asp Asp Leu Val Asp Glu Tyr Ser Thr Met Asn Glu Pro Asn

Val Val Gly Gly Leu Gly Tyr Val Gly Val Lys Ser Gly Phe Pro Pro

Gly Tyr Leu Ser Phe Glu Leu Ser Arg Arg Ala Met Tyr Asn Ile Ile

Gln Ala His Ala Arg Ala Tyr Asp Gly Ile Lys Ser Val Ser Lys Lys

Pro Val Gly Ile Ile Tyr Ala Asn Ser Ser Phe Gln Pro Leu Thr Asp

Lys Asp Met Glu Ala Val Glu Met Ala Glu Asn Asp Asn Arg Trp Trp

Phe Phe Asp Ala Ile Ile Arg Gly Glu Ile Thr Arg Gly Asn Glu Lys

Ile Val Arg Asp Asp Leu Lys Gly Arg Leu Asp Trp Ile Gly Val Asn

Tyr Tyr Thr Arg Thr Val Val Lys Arg Thr Glu Lys Gly Tyr Val Ser

Leu Gly Gly Tyr Gly His Gly Cys Glu Arg Asn Ser Val Ser Leu Ala

Gly Leu Pro Thr Ser Asp Phe Gly Trp Glu Phe Phe Pro Glu Gly Leu

Tyr Asp Val Leu Thr Lys Tyr Trp Asn Arg Tyr His Leu Tyr Met Tyr

Val Thr Glu Asn Gly Ile Ala Asp Asp Ala Asp Tyr Gln Arg Pro Tyr

Tyr Leu Val Ser His Val Tyr Gln Val His Arg Ala Ile Asn Ser Gly

Ala Asp Val Arg Gly Tyr Leu His Trp Ser Leu Ala Asp Asn Tyr Glu

Trp Ala Ser Gly Phe Ser Met Arg Phe Gly Leu Leu Lys Val Asp Tyr

Asn Thr Lys Arg Leu Tyr Trp Arg Pro Ser Ala Leu Val Tyr Arg Glu

Ile Ala Thr Asn Gly Ala Ile Thr Asp Glu Ile Glu His Leu Asn Ser

Val Pro Pro Val Lys Pro Leu Arg His

SEQ ID NO: 3

atgtcctaca ggatagtggt tgatccaaaa aaagttgtca agccgattag tagacacatc
60

tacggtcatt tcacggaaca tctgggaagg tgtatctacg gcggaattta tgaagaaggt
120

tctccgctct ccgatgaaag gggtttcaga aaggacgttc tggaggctgt aaagaggata
180

aaagttccga acttgagatg gcccggtgga aactttgtgt cgaactacca ctgggaagac
240

ggaataggtc ccaaagatca gaggcctgtc aggttcgatc tcgcctggca acaggaagag
300

acgaatagat ttggaacgga cgaattcatt gagtactgtc gtgagatagg agcagaacct
360

tacatcagta taaacatggg aactggaaca ctcgacgaag ctctccactg gcttgaatac
420

tgcaatggaa agggtaatac ctactacgct caactcagaa gaaagtacgg tcatccagaa
480

ccttacaacg taaagttctg gggaataggc aacgagatgt acggggaatg gcaggtaggc
540

cacatgacgg cggacgaata cgcaagagcc gccaaagaat acacgaaatg gatgaaggtt
600

ttcgatccta caattaaagc gatcgccgtg ggctgtgacg accctatatg gaatctcagg
660

gttcttcaag aagcaggtga tgtgattgac ttcatatcct accatttcta cacagggtcc
720

gaggattact acgaaacagt ttccacggtt taccttctca aagaaagact catcggagtg
780

aaaaagctca ttgatatggt ggatactgct agaaagagag gtgtcaaaat cgcccttgat
840

gaatggaacg tatggtacag agtgtccgat aacaagctcg aagaacctta cgatctcaaa
900

gatggtatct ttgcatgtgg agtgcttgta cttcttcaaa agatgagcga catagtccca
960

cttgccaatc tcgcacagct tgtaaacgcc cttggagcta tacacaccga gaaagacggt
1020

ctcattctca cacccgttta caaggctttt gaactcatcg tgaatcattc cggagaaaag
1080

cttgtcaaga cccatgttga atcggagact tacaacatag aaggagtcat gttcatcaac
1140

aaaatgcctt tctctgtcga gaacgcaccg ttccttgatg ccgccgcttc catctcagaa
1200

gatggcaaga aacttttcat cgctgttgta aactacagga aagaagacgc tttgaaggtt
1260

ccaatcagag tggaaggtct gggacagaaa aaagccaccg tttatacact cacaggtccg
1320

gacgtgaacg cgagaaacac catggaaaat ccgaacgtcg ttgatattac ctccgaaacc
1380

atcaccgttg acaccgaatt tgaacacacg tttaaaccat tctcttgcag tgtgattgag
1440

gtagaattgg agtaa
1455

SEQ ID NO: 4

Met Ser Tyr Arg Ile Val Val Asp Pro Lys Lys Val Val Lys Pro Ile

Ser Arg His Ile Tyr Gly His Phe Thr Glu His Leu Gly Arg Cys Ile

Tyr Gly Gly Ile Tyr Glu Glu Gly Ser Pro Leu Ser Asp Glu Arg Gly

Phe Arg Lys Asp Val Leu Glu Ala Val Lys Arg Ile Lys Val Pro Asn

Leu Arg Trp Pro Gly Gly Asn Phe Val Ser Asn Tyr His Trp Glu Asp

Gly Ile Gly Pro Lys Asp Gln Arg Pro Val Arg Phe Asp Leu Ala Trp

Gln Gln Glu Glu Thr Asn Arg Phe Gly Thr Asp Glu Phe Ile Glu Tyr

Cys Arg Glu Ile Gly Ala Glu Pro Tyr Ile Ser Ile Asn Met Gly Thr

Gly Thr Leu Asp Glu Ala Leu His Trp Leu Glu Tyr Cys Asn Gly Lys

Gly Asn Thr Tyr Tyr Ala Gln Leu Arg Arg Lys Tyr Gly His Pro Glu

Pro Tyr Asn Val Lys Phe Trp Gly Ile Gly Asn Glu Met Tyr Gly Glu

Trp Gln Val Gly His Met Thr Ala Asp Glu Tyr Ala Arg Ala Ala Lys

Glu Tyr Thr Lys Trp Met Lys Val Phe Asp Pro Thr Ile Lys Ala Ile

Ala Val Gly Cys Asp Asp Pro Ile Trp Asn Leu Arg Val Leu Gln Glu

Ala Gly Asp Val Ile Asp Phe Ile Ser Tyr His Phe Tyr Thr Gly Ser

Glu Asp Tyr Tyr Glu Thr Val Ser Thr Val Tyr Leu Leu Lys Glu Arg

Leu Ile Gly Val Lys Lys Leu Ile Asp Met Val Asp Thr Ala Arg Lys

Arg Gly Val Lys Ile Ala Leu Asp Glu Trp Asn Val Trp Tyr Arg Val

Ser Asp Asn Lys Leu Glu Glu Pro Tyr Asp Leu Lys Asp Gly Ile Phe

Ala Cys Gly Val Leu Val Leu Leu Gln Lys Met Ser Asp Ile Val Pro

Leu Ala Asn Leu Ala Gln Leu Val Asn Ala Leu Gly Ala Ile His Thr

Glu Lys Asp Gly Leu Ile Leu Thr Pro Val Tyr Lys Ala Phe Glu Leu

Ile Val Asn His Ser Gly Glu Lys Leu Val Lys Thr His Val Glu Ser

Glu Thr Tyr Asn Ile Glu Gly Val Met Phe Ile Asn Lys Met Pro Phe

Ser Val Glu Asn Ala Pro Phe Leu Asp Ala Ala Ala Ser Ile Ser Glu

Asp Gly Lys Lys Leu Phe Ile Ala Val Val Asn Tyr Arg Lys Glu Asp

Ala Leu Lys Val Pro Ile Arg Val Glu Gly Leu Gly Gln Lys Lys Ala

Thr Val Tyr Thr Leu Thr Gly Pro Asp Val Asn Ala Arg Asn Thr Met

Glu Asn Pro Asn Val Val Asp Ile Thr Ser Glu Thr Ile Thr Val Asp

Thr Glu Phe Glu His Thr Phe Lys Pro Phe Ser Cys Ser Val Ile Glu

Val Glu Leu Glu

SEQ ID NO: 5

catatgtact catttccaaa tagc
24

SEQ ID NO: 6

ctcgagttag tgccttaatg gctttac
27

SEQ ID NO: 7

catatgatgt cctacaggat agtggttgat c
31

SEQ ID NO: 8

ctcgagctcc aattctacct caatcac
27

SEQ ID NO: 9

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
180

cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata
240

tgtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc
300

acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc
360

cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
420

cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
480

cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
540

accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
600

gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
660

ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
720

cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
780

tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
840

cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
900

tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
960

ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
1020

gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
1080

tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
1140

ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
1200

atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
1260

cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
1320

aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
1380

tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
1440

tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
1500

gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
1560

tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
1620

gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
1680

tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
1740

ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
1800

aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
1860

agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
1920

cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
1980

attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta
2040

ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
2100

ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc
2160

ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
2220

ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
2280

acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
2340

tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
2400

acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
2460

atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
2520

gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
2580

aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
2640

cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
2700

gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc
2760

ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
2820

acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
2880

gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
2940

ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatata tgcggtgtga
3000

aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
3060

cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
3120

ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
3180

ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
3240

cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
3300

actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
3360

cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
3420

tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
3480

gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
3540

caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
3600

agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
3660

tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
3720

tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
3780

gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
3840

gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac
3900

tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
3960

cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
4020

ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg
4080

cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
4140

tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
4200

gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg
4260

tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
4320

gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
4380

tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
4440

agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
4500

caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
4560

ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
4620

ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
4680

aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
4740

agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa
4800

caaatagggg ttccgcgcac atttccccga aaagtgccac ctgaaattgt aaacgttaat
4860

attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
4920

gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
4980

ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
5040

accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
5100

tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
5160

cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
5220

agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
5280

gcgccgctac agggcgcgtc ccattcgcca
5310

SEQ ID NO: 10

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
540

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
600

gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
660

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
720

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
780

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
840

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
900

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
960

cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
1020

aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
1080

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
1140

tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
1200

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
1260

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
1320

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
1380

gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
1440

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
1500

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
1560

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
1620

aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
1680

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
1740

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
1800

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
1860

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
1920

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
1980

gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
2040

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
2100

cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
2160

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
2220

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
2280

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
2340

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
2400

gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg
2460

tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
2520

cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
2580

gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
2640

gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
2700

catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
2760

tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
2820

ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
2880

tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
2940

ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
3000

aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
3060

gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
3120

tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
3180

acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
3240

cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
3300

cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
3360

gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
3420

cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
3480

gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
3540

tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
3600

atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
3660

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
3720

gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
3780

tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
3840

cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
3900

tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
3960

atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
4020

atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
4080

tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
4140

cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
4200

aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
4260

ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
4320

tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
4380

tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
4440

gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
4500

gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
4560

gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
4620

ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
4680

taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
4740

ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
4800

atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
4860

tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
4920

gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
4980

gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
5040

aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
5100

ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
5160

cctctagaaa taattttgtt taactttaag aaggagatat acatatggct agcatgactg
5220

gtggacagca aatgggtcgc ggatccgaat tcgagctccg tcgacaagct tgcggccgca
5280

ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
5340

gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
5400

gtcttgaggg gttttttgct gaaaggagga actatatccg gat
5443

SEQ ID NO: 11

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagggatcaa tactaggagg
180

agtagcatat aattacgtta cacaatttta taacccaata tattcaatag accttatgct
240

tatcctatcc tctattctaa gattctcggt atctccccta ttcttgacca taaaagatac
300

tcgctcaaag cttaaataat attaatcata aataaagtca tgtactcatt tccaaatagc
360

tttaggtttg gttggtccca ggccggattt caatcagaaa tgggaacacc agggtcagaa
420

gatccaaata ctgactggta taaatgggtt catgatccag aaaacatggc agcgggatta
480

gtaagtggag atctaccaga aaatgggcca ggctactggg gaaactataa gacatttcac
540

gataatgcac aaaaaatggg attaaaaata gctagactaa atgtggaatg gtctaggata
600

tttcctaatc cattaccaag gccacaaaac tttgatgaat caaaacaaga tgtgacagag
660

gttgagataa acgaaaacga gttaaagaga cttgacgagt acgctaataa agacgcatta
720

aaccattaca gggaaatatt caaggatctt aaaagtagag gactttactt tatactaaac
780

atgtatcatt ggccattacc tctatggtta cacgacccaa taagagtaag aagaggagat
840

tttactggac caagtggttg gctaagtact agaacagttt acgaattcgc tagattctca
900

gcttatatag cttggaaatt cgatgatcta gtggatgagt actcaacaat gaatgaacct
960

aacgttgttg gaggtttagg atacgttggt gttaagtccg gttttccccc aggataccta
1020

agctttgaac tttcccgtag ggcaatgtat aacatcattc aagctcacgc aagagcgtat
1080

gatgggataa agagtgtttc taaaaaacca gttggaatta tttacgctaa tagctcattc
1140

cagccgttaa cggataaaga tatggaagcg gtagagatgg ctgaaaatga taatagatgg
1200

tggttctttg atgctataat aagaggtgag atcaccagag gaaacgagaa gattgtaaga
1260

gatgacctaa agggtagatt ggattggatt ggagttaatt attacactag gactgttgtg
1320

aagaggactg aaaagggata cgttagctta ggaggttacg gtcacggatg tgagaggaat
1380

tctgtaagtt tagcgggatt accaaccagc gacttcggct gggagttctt cccagaaggt
1440

ttatatgacg ttttgacgaa atactggaat agatatcatc tctatatgta cgttactgaa
1500

aatggtattg cggatgatgc cgattatcaa aggccctatt atttagtatc tcacgtttat
1560

caagttcata gagcaataaa tagtggtgca gatgttagag ggtatttaca ttggtctcta
1620

gctgataatt acgaatgggc ttcaggattc tctatgaggt ttggtctgtt aaaggtcgat
1680

tacaacacta agagactata ctggagaccc tcagcactag tatataggga aatcgccaca
1740

aatggcgcaa taactgatga aatagagcac ttaaatagcg tacctccagt aaagccatta
1800

aggcactaaa ctttctcaag tctcactata ccaaatgagt tttcttttaa tcttattcta
1860

atctcatttt cattagattg caatactttc ataccttcta tattatttat tttgtacctt
1920

ttgggatcca tatgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
1980

tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
2040

gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
2100

cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
2160

cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
2220

ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
2280

ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
2340

ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
2400

tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
2460

ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
2520

aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
2580

aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
2640

ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
2700

tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
2760

tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
2820

cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
2880

ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
2940

accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
3000

ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
3060

actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
3120

ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
3180

agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
3240

gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
3300

ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
3360

tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
3420

cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
3480

ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
3540

ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
3600

atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
3660

gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
3720

tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
3780

accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc
3840

atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
3900

cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
3960

acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
4020

aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
4080

tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
4140

tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
4200

tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
4260

cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
4320

cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
4380

ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
4440

taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
4500

ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
4560

gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
4620

ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
4680

tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
4740

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
4800

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
4860

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
4920

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
4980

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
5040

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
5100

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
5160

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
5220

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
5280

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
5340

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
5400

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
5460

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
5520

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
5580

gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
5640

aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg
5700

ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
5760

ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
5820

ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
5880

agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa
5940

cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
6000

cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
6060

gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
6120

attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
6180

ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
6240

tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
6300

gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
6360

aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
6420

tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt
6480

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt
6540

gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
6600

aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
6660

ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
6720

aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
6780

agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
6840

tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
6900

ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
6960

gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca
7002

SEQ ID NO: 12

ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
60

agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
120

tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
180

ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
240

cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
300

tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
360

tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
420

caaaaattta acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt
480

cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
540

ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
600

agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
660

tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
720

gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
780

gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
840

attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
900

gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
960

agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
1020

ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
1080

cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
1140

gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
1200

cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
1260

gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
1320

ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
1380

acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
1440

ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
1500

aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
1560

aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
1620

ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
1680

ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
1740

actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
1800

caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
1860

gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
1920

ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
1980

cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
2040

cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
2100

acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
2160

ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
2220

gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
2280

tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
2340

accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
2400

cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt
2460

gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
2520

gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
2580

acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
2640

catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
2700

atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
2760

gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
2820

tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
2880

gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
2940

gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
3000

aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
3060

ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
3120

ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
3180

cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
3240

agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
3300

ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
3360

accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
3420

gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
3480

cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
3540

gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
3600

tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
3660

ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
3720

cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
3780

gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
3840

ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
3900

cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
3960

tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
4020

tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
4080

cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
4140

gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
4200

atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
4260

tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
4320

ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
4380

gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
4440

acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
4500

acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
4560

ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
4620

tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
4680

aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
4740

tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
4800

tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
4860

agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
4920

cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
4980

agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
5040

accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
5100

tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
5160

ctctagaaat aattttgttt aactttaaga aggagatata catatgatgt cctacaggat
5220

agtggttgat ccaaaaaaag ttgtcaagcc gattagtaga cacatctacg gtcatttcac
5280

ggaacatctg ggaaggtgta tctacggcgg aatttatgaa gaaggttctc cgctctccga
5340

tgaaaggggt ttcagaaagg acgttctgga ggctgtaaag aggataaaag ttccgaactt
5400

gagatggccc ggtggaaact ttgtgtcgaa ctaccactgg gaagacggaa taggtcccaa
5460

agatcagagg cctgtcaggt tcgatctcgc ctggcaacag gaagagacga atagatttgg
5520

aacggacgaa ttcattgagt actgtcgtga gataggagca gaaccttaca tcagtataaa
5580

catgggaact ggaacactcg acgaagctct ccactggctt gaatactgca atggaaaggg
5640

taatacctac tacgctcaac tcagaagaaa gtacggtcat ccagaacctt acaacgtaaa
5700

gttctgggga ataggcaacg agatgtacgg ggaatggcag gtaggccaca tgacggcgga
5760

cgaatacgca agagccgcca aagaatacac gaaatggatg aaggttttcg atcctacaat
5820

taaagcgatc gccgtgggct gtgacgaccc tatatggaat ctcagggttc ttcaagaagc
5880

aggtgatgtg attgacttca tatcctacca tttctacaca gggtccgagg attactacga
5940

aacagtttcc acggtttacc ttctcaaaga aagactcatc ggagtgaaaa agctcattga
6000

tatggtggat actgctagaa agagaggtgt caaaatcgcc cttgatgaat ggaacgtatg
6060

gtacagagtg tccgataaca agctcgaaga accttacgat ctcaaagatg gtatctttgc
6120

atgtggagtg cttgtacttc ttcaaaagat gagcgacata gtcccacttg ccaatctcgc
6180

acagcttgta aacgcccttg gagctataca caccgagaaa gacggtctca ttctcacacc
6240

cgtttacaag gcttttgaac tcatcgtgaa tcattccgga gaaaagcttg tcaagaccca
6300

tgttgaatcg gagacttaca acatagaagg agtcatgttc atcaacaaaa tgcctttctc
6360

tgtcgagaac gcaccgttcc ttgatgccgc cgcttccatc tcagaagatg gcaagaaact
6420

tttcatcgct gttgtaaact acaggaaaga agacgctttg aaggttccaa tcagagtgga
6480

aggtctggga cagaaaaaag ccaccgttta tacactcaca ggtccggacg tgaacgcgag
6540

aaacaccatg gaaaatccga acgtcgttga tattacctcc gaaaccatca ccgttgacac
6600

cgaatttgaa cacacgttta aaccattctc ttgcagtgtg attgaggtag aattggagct
6660

cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
6720

gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
6780

cttgaggggt tttttgctga aaggaggaac tatatccgga t
6821

SEQ ID NO: 13

atggcagcta aagacgtaaa attcggtaac gacgctcgtg tgaaaatgct gcgcggcgta
60

aacgtactgg cagatgcagt gaaagttacc ctcggtccga aaggccgtaa cgtagttctg
120

gataaatctt tcggtgcacc gaccatcacc aaagatggtg tttccgttgc tcgtgaaatc
180

gaactggaag acaagttcga aaatatgggt gcgcagatgg tgaaagaagt tgcctccaaa
240

gcgaacgacg ctgcaggcga cggtaccacc actgcaaccg tactggctca ggctatcatc
300

actgaaggtc tgaaagctgt tgctgcgggc atgaacccga tggacctgaa acgtggtatc
360

gacaaagcgg ttaccgctgc agttgaagaa ctgaaagcgc tgtccgtacc gtgctctgat
420

tctaaagcga ttgctcaggt tggtaccatc tccgctaact ccgacgaaac cgtaggtaaa
480

ctgatcgcag aagcgatgga caaagtcggt aaagaaggcg ttatcaccgt tgaagacggt
540

accggtctgc aggacgaact ggacgtggtt gaaggtatgc agttcgaccg tggctacctg
600

tctccttact tcatcaacaa gccggaaact ggcgcagtag aactggaaag cccgttcatc
660

ctgctggctg acaagaaaat ctccaacatc cgcgaaatgc tgccggttct ggaagctgtt
720

gcaaaagcag gtaaaccgct gctgatcatc gctgaagatg tagaaggcga agcgctggca
780

actctggttg ttaacaccat gcgtggcatc gtgaaagtcg ctgcggttaa agcaccgggc
840

ttcggcgatc gtcgtaaagc tatgctgcag gatatcgcaa ccctgactgg cggtaccgtg
900

atctctgaag agatcggtat ggagctggaa aaagcaaccc tggaagacct gggtcaggct
960

aaacgtgttg tgatcaacaa agacaccacc actatcatcg atggcgtggg tgaagaagct
1020

gcaatccagg gccgtgttgc tcagatccgt cagcagattg aagaagcaac ttctgactac
1080

gaccgtgaaa aactgcagga acgcgtagcg aaactggcag gcggcgttgc agttatcaaa
1140

gtaggtgctg ctaccgaagt tgaaatgaaa gagaaaaaag cacgcgttga agatgccctg
1200

cacgcgaccc gtgcagcggt agaagagggc gtggttgctg gtggtggtgt tgcgctgatc
1260

cgcgtagcgt ctaaactggc tgacctgcgt ggtcagaacg aagaccagaa cgtgggtatc
1320

aaagttgcac tgcgtgcaat ggaagctccg ctgcgtcaga tcgtattgaa ctgcggcgaa
1380

gaaccgtctg ttgttgctaa caccgttaaa ggcggcgacg gcaactacgg ttacaacgca
1440

gcaaccgaag aatacggcaa catgatcgac atgggtatcc tggatccaac caaagtaact
1500

cgttctgctc tgcagtacgc agcttctgtg gctggcctga tgatcaccac cgagtgcatg
1560

gttaccgacc tgccgaaaaa cgatgcagct gacttaggcg ctgctggcgg tatgggcggc
1620

atgggtggca tgggcggcat gatgtaa
1647

SEQ ID NO: 14

atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa
60

tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg tggcgaagtg
120

ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa
180

gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat
240

gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gtaa
29

COMPOSITION FOR PRODUCTION OF GINSENOSIDE COMPOUND K COMPRISING HIGH TEMPERATURE alpha-L-ARABINOFURANOSIDASE, AND METHOD FOR PREPARING GINSENDOSIDE COMPOUND K

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