KR20220006893A - Ginsenoside G17 or CK production method using MT619 enzyme - Google Patents

Abstract

The present invention relates to a method for manufacturing ginsenoside G17 Or compound K (CK) using an MT619 enzyme. According to the present invention, β-glucosidase is highly concentrated and immobilized on amorphous cellulose, thereby exhibiting strong conversion activity for protopanaxadiol- and protopanaxatriol-type ginsenosides. In particular, β-glucosidase immobilized by using a gram-scale ginseng extract as a substrate produces a high concentration of minor ginsenoside CK within 24 hours and produces a minor ginsenoside, G_17 as an intermediate product. Such a safe, convenient, and efficient method for producing minor ginsenoside G_17 or CK can be effectively utilized to manufacture food processing recombinant enzymes in the pharmaceutical, functional food, and cosmetic industries.

Description

Ginsenoside G17 or CK production method using MT619 enzyme {Ginsenoside G17 or CK production method using MT619 enzyme}

The present invention relates to a _{method for producing ginsenoside G 17} or CK (Compound K) using MT619 enzyme.

Ginseng has been widely used to treat a variety of ailments in East Asia for over a thousand years, and is increasingly used in food and dietary supplements worldwide. Ginsenoside, a triterpene saponin found almost exclusively in ginseng, is considered to be the main active ingredient responsible for various pharmacological activities. Major ginsenosides known to constitute more than 90% of total ginsenosides in various parts of ginseng include Re, Rg ₁ , Rb ₁ , Rb ₂ , Rc and Rd. The main ginsenoside has a very low absorption rate in vivo due to its large size of around 1,000 Daltons. Therefore, in order to increase the medicinal efficacy of ginsenosides, it is necessary to convert the major ginsenosides into minor ginsenosides that are relatively well absorbed and have better medicinal efficacy. That is, major ginsenosides are required to be deglycosylated to remove glucose, arabinose, rhamnose, and xylose constituting sugars in order to effectively exhibit physiological activity in vivo.

On the other hand, minor ginsenosides with few sugar moieties attached to aglycones are mostly present less than major ginsenosides. Many minor ginsenosides are known to have higher chemical reactivity than major ginsenosides abundant in raw materials, absorb relatively well, and have superior medicinal effects. Minor ginsenosides include F ₂ , Rg ₃ , Rh ₁ , Rh ₂ , gypenoside (gypenoside, Gyp) XVII (ginsenoside G ₁₇ ), gypenoside LXXV (G75), CK (Compound K), Compound Mc and Compound Mc1, and the like.

In particular, among the minor ginsenosides, CK is a rare ginsenoside with low polarity, and less than 0.005% of raw ginseng or red ginseng contains it or does not contain it. Therefore, it is extremely difficult and expensive to purify large quantities of pure CK from raw plants, and it creates a bottleneck in food applications.

Accordingly, a recombinant enzyme conversion method has been studied to efficiently and specifically convert abundant major ginsenosides to CK. Recombinant glycosidases expressed from several E. coli produced minor ginsenosides on a gram scale from abundant major ginsenosides. Unfortunately, however, E. coli is not generally recognized as a food grade (Generally Recognized as Safe, GRAS) strain, and is not a suitable expression host for the production of food grade minor ginsenosides because of its potential to produce endotoxin.

Accordingly, recently, various attempts have been made to prepare minor ginsenosides from major ginsenosides using the expression system of the GRAS host strain (Li et al. (2016a) Production of ginsenoside F2 by Using Lactococcus lactis with enhanced expression). of β-glucosidase gene from Paenibacillus mucilaginosus. J Agric Food Chem 64:2506-2512.).

Under this background, the present inventors heterologous expression of β-glycosidase MT619 derived from Microbacterium testaceum in the GRAS strain Corynebacterium glutamicum In the process of converting the side, protopanaxadiol (PPD)-type ginsenoside mixture (PPDGM) to minor ginsenosides, the major ginsenosides are converted to minor ginsenosides CK in high yield Rather, the present invention was completed by confirming that _{G 17, which} is a minor ginsenoside, was produced as an intermediate product.

An object of the present invention is β-glucosidase comprising the amino acid sequence of SEQ ID NO: 3; a transformant into which a vector containing a nucleic acid encoding the protein has been introduced; And using one or more selected from the group consisting of the culture of the transformant, ginsenoside comprising the step of hydrolyzing the sugar specifically at the 6th carbon of ginsenoside to convert it into a deglycosylated form, ginseno To provide a method for manufacturing side CK (Compound K).

Another object of the present invention is β-glucosidase comprising the amino acid sequence of SEQ ID NO: 3; a transformant into which a vector containing a nucleic acid encoding the protein has been introduced; And converting to a deglycosylated form by hydrolyzing the 6th carbon-specific sugar of ginsenoside, including any one or more selected from the group consisting of a culture of the transformant as an active ingredient, ginseno To provide a composition for conversion to side CK.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object is β-glucosidase comprising the amino acid sequence of SEQ ID NO: 3; a transformant into which a vector containing a nucleic acid encoding the protein has been introduced; And using one or more selected from the group consisting of the culture of the transformant, ginsenoside comprising the step of hydrolyzing the sugar specifically at the 6th carbon of ginsenoside to convert it into a deglycosylated form, ginseno A method for manufacturing side CK (Compound K) is provided.

In the present invention, the term "ginsenoside" is a triterpene saponin derived from ginseng, and has a different chemical structure from saponins found in other plants. To do this, ginseng is called ginsenoside in the sense of glycoside. Ginsenosides are considered to be the main active ingredients responsible for various pharmacological activities.

Ginsenoside is protopanaxadiol (PPD)-type ginsenoside or protopanaxatriol (PPT)-type ginsenoside and It can be classified into three types of oleanolic acid (Oleanolic acid type) ginsenosides. These three groups are again classified according to the position and number of aglycones attached to carbon 3, carbon 6 and carbon 20 positions of the ring by glycosidic bonds in the compound structure. PPD-type ginsenosides include Rb ₁ , Rb ₂ , Rb ₃ , Rc, Rd, gypenoside (gypenoside, Gyp) XVII (ginsenoside G ₁₇ ), Compound O, Compound Mc1, F ₂ , Compound Y, Compound MC, Rg ₃ , Rh ₂ and Compound K(CK) and the like. PPT-type ginsenosides include Re, Rg ₁ , Rf, Rg ₂ , Rh ₁ and F ₁ .

In addition, ginsenosides are also classified into major ginsenosides or minor ginsenosides according to their content and molecular size in ginseng. Major ginsenosides with a high content include Re, Rg ₁ , Rb ₁ , Rb ₂ , Rc and Rd. The main ginsenoside has a very low absorption rate in vivo due to its large size of around 1,000 Daltons. Minor ginsenosides with low content include F ₂ , Rg ₃ , Rh ₁ , Rh ₂ , ginsenoside G ₁₇ , zipenoside LXXV(G75), CK, Compound Mc and Compound Mc1, etc. exist.

_{For the purpose of the present invention, the preparation method may be to produce ginsenoside G 17} as an intermediate product in the conversion process from the ginsenoside to the ginsenoside CK, but is not limited thereto.

As used herein, the term "β-glycosidase" is an enzyme that hydrolyzes glucoside molecular bonds, specifically hydrolyzing the sugar at carbon 6 of ginsenoside, or carbon 6 of ginsenoside. It refers to an enzyme that specifically trans-glycosylates. In the present invention, the β-glycosidase may be a β-glycosidase derived from a microorganism of the genus Microbacterium sp ., specifically, Microbacterium testaceum (Korean Patent Registration No. 10). -1785809), more specifically, may be a glycosidase represented by the amino acid sequence of SEQ ID NO: 3, but is not limited thereto. The β-glycosidase is used interchangeably with glycosidase or ginsenoside glycosidase in the present invention.

In the present invention, the β-glycosidase may be a β-glycosidase comprising the amino acid sequence of SEQ ID NO: 3.

The amino acid sequence of SEQ ID NO: 3 is 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% of the sequence and the gene including the amino acid sequence of SEQ ID NO: 3 Above, most specifically, as long as it is a protein or polypeptide substantially including the amino acid sequence of SEQ ID NO: 3 as an amino acid sequence showing 98% or more homology, it may be included in the present invention. In addition, as a sequence having such homology, if an amino acid sequence having the same or corresponding biological activity to the amino acid sequence of SEQ ID NO: 3 of the present invention, protein variants having an amino acid sequence in which some sequences are deleted, modified, substituted or added are also It is obvious that they are included within the scope of the present invention.

The β-glucosidase comprising the amino acid sequence of SEQ ID NO: 3 may be encoded by at least one of the nucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto.

For the purpose of the present invention, the gene encoding β-glycosidase comprising the amino acid sequence of SEQ ID NO: 3 may be a gene encoding β-glycosidase derived from Microbacterium testaceum and , The microbacterium testacium-derived β-glycosidase-derived gene encoding a GRAS strain, Corynebacterium glutamicum , may be a codon-optimized gene variant for heterologous expression, but limited thereto doesn't happen The gene encoding β-glycosidase derived from the Microbacterium testacium may include the nucleotide sequence of SEQ ID NO: 2, and the codon-optimized gene variant for heterologous expression of this in Corynebacterium glutamicum is It may include the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 1 was deposited with GenBank as accession number MK575514.

The nucleotide sequence of SEQ ID NO: 1 is not only the gene including the nucleotide sequence of SEQ ID NO: 1, but also 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% of the sequence Above, most specifically, any gene substantially including the nucleotide sequence of SEQ ID NO: 1 as a nucleotide sequence exhibiting 98% or more homology may be included in the present invention. In addition, as a sequence having such homology, if a nucleotide sequence encoding a protein having the same or corresponding biological activity as the nucleotide sequence of SEQ ID NO: 1 of the present invention, some sequences are deleted, modified, substituted or added nucleotide sequence It is apparent that variants of the gene having a gene are also included within the scope of the present invention.

The homology is intended to indicate a degree of similarity to the nucleotide sequence of a wild type gene or the amino acid sequence of a protein encoded therewith, and a sequence having the same percentage or more as the nucleotide sequence or amino acid sequence of the present invention as described above. includes Comparison of such homology can be performed visually or using an easily purchased comparison program.

In the present invention, the nucleotide sequence represented by SEQ ID NO: 1 is mixed with mt619, and β-glycosidase encoded by the nucleotide sequence represented by SEQ ID NO: 1 may be used interchangeably with MT619 in the present invention, but limited thereto doesn't happen

MT619 of the present invention can be used at various pH and temperature conditions as long as activity and stability can be maintained, and is composed of β-mercaptoethanol, Ca ²⁺ , Na ⁺ , K ⁺ and Mg ²⁺ One or more metal ions selected from the group and a chemical agent including the same may be used together, but the present invention is not limited thereto.

In an embodiment of the present invention, MT619 was relatively stable at pH 6.0 to 8.0, and had optimal activity at pH 7.0 (FIG. 2a).

In another embodiment of the present invention, the optimum temperature for the activity of MT619 was 45 ℃, and the activity was high at 30 ~ 55 ℃. Temperature stability exhibited stability in the range of 0 to 45 °C, specifically, excellent stability at 0 to 30 °C (Fig. 2b).

In another embodiment of the present invention, the activity of MT619 was not affected by ^{β-mercaptoethanol, Ca 2+} , Na ⁺ , K ⁺ and Mg ^{2+ .} However, activity was lost in the presence of Cu ^2+, Hg ^2+, and Zn ^2+, was significantly inhibited by Co ²⁺ and Mn ²⁺ (Fig. 3).

As a result of analyzing the substrate specificity of MT619 in another embodiment of the present invention, MT619 was activated only with respect to the glucose moiety of PNP-β-D-glucopyranoside and ONP-β-D-glucopyranoside, PNP Maximum activity against -β-D-glucopyranoside and 28.7% relative activity against ONP-β-D-glucopyranoside were shown.

In the present invention, the ginsenoside is not limited as long as it is a ginsenoside known in the art, but is specifically selected from the group consisting _{of Rb 1 and Rd, which are protopanaxadiol (PPD)-type ginsenosides.} It may be any one or more. However, any ginsenoside that can be converted to ginsenoside CK by MT619 may be included without limitation.

The ginsenoside may use the separated and purified form of ginsenoside, or ginsenosides contained in the powder or extract of ginseng or red ginseng may be used. That is, the method of the present invention can be carried out using not only the separated and purified form of ginsenoside, but also the powder or extract of ginseng or red ginseng containing ginsenoside as a direct starting material. Ginseng used in the present invention, it is possible to use the various well-known ginseng, goryeosam (Panax ginseng), hwagisam (P. quiquefolius), jeonchilsam (P. notoginseng), jukjeol three (P. japonicus), three yeopsam (P trifolium ), Himalayan ginseng ( P. pseudoginseng ) and Vietnamese ginseng ( P. vietnamensis ), but are not limited thereto.

In an embodiment of the _{present invention, it was confirmed that the ginsenoside Rb 1} and Rd is converted to the ginsenoside CK by MT619 of the present invention ( FIGS. 4 and 5 ).

The preparation of the ginsenoside CK may include any one or more conversion processes selected from the group consisting of a conversion process from _{Rb 1 to CK and a conversion process from Rd to CK, and the conversion process from Rb 1} to CK is the Rb ₁ to G ₁₇ conversion; and the _{conversion step of G 17} to CK; but is not limited thereto.

_{That is, the production method of the present invention may be to additionally produce another minor ginsenoside G 17} as an intermediate product in the conversion process from the ginsenoside to the minor ginsenoside CK, but is not limited thereto.

The method for preparing ginsenoside CK of the present invention includes: β-glucosidase comprising the amino acid sequence of SEQ ID NO: 3; a transformant into which a vector containing a nucleic acid encoding the protein has been introduced; Or it may include the step of reacting any one or more of the culture of the transformant and ginsenoside.

As used herein, the term "vector" refers to an expression vector capable of expressing a target protein in a suitable host cell, and refers to a nucleic acid preparation comprising essential regulatory elements operably linked to express a nucleic acid insert.

As used herein, the term "transformation" refers to a phenomenon in which DNA is introduced into a host to enable replication by DNA as a factor of chromosomes or by completion of chromosome integration, and artificially causes genetic changes by introducing external DNA into cells it means.

Any transformation method may be used for the transformation method of the present invention, and it can be easily performed according to a conventional method in the art.

The transformant transformed with the vector containing the nucleic acid encoding the β-glycosidase protein of the present invention by the above method can be converted into ginsenoside, particularly PPD-type ginsenoside, into ginsenoside CK. _{It refers to a transformant having the activity to convert Rb 1} and Rd, which are PPD-type ginsenosides, to ginsenoside CK, specifically, but is not limited thereto.

In the present invention, the host strain for the transformation is not particularly limited as long as it expresses the nucleic acid of the present invention. Hosts that can be used in the present invention include, but are not limited to, Corynebacterium glutamicum , such as Corynebacterium genus bacteria; Escherichia coli (Escherichia) bacteria belonging to the genus such as (Escherichia coli); Bacillus subtilis ( Bacillus subtilis ), such as Bacillus ( Bacillus ) genus bacteria; Pseudomonas putida ( Pseudomonas putida ), such as Pseudomonas ( Pseudomonas ) genus bacteria; Yeasts such as Saccharomyces cerevisiae , Schizosaccharomyces pombe; There are animal cells and insect cells, but is not limited thereto, and specifically, it may be a GRAS strain, Corynebacterium , genus bacteria, and more specifically, Corynebacterium glutamicum ). .

As used herein, the term "culture" may refer to a product obtained after culturing the transformant according to a known microbial culture method. The culture includes both culture supernatant and cell lysate, and may or may not contain cells. Specifically, the culture of the transformant into which the vector containing the gene encoding β-glycosidase, consisting of the nucleotide sequence shown in SEQ ID NO: 1, is introduced, is converted from PPD-type ginsenoside to ginsenoside CK activity, but is not limited thereto.

For the purposes of the present invention, the MT619 of the present invention may be fixed to cellulose and concentrated, but is not limited thereto. A cellulose-binding module was further bound to its N-terminus to immobilize MT619 to cellulose. The cellulose-binding module is a protein capable of increasing enzymatic activity by immobilizing MT619, but is not particularly limited , and may be specifically derived from Clostridium thermocellum , and more specifically, the amino acid sequence of SEQ ID NO: 4 may include.

The cellulose-binding module may be linked to MT619 through a linker, and the linker may be a peptide linker, but is not limited thereto.

In one embodiment of the present invention, MT619 to which the cellulose-binding module is bound was immobilized at a high density (maximum 984 mg/g cellulose) on amorphous cellulose, so that the enzyme concentration was increased 286 times ( FIG. 2 ). The concentrated and immobilized enzyme showed excellent conversion activity to ginsenoside CK, and the immobilized enzyme using a gram-scale ginseng extract as a substrate produced 7.59 g/L CK within 24 hours ( FIGS. 3 and 4 ). .

The present invention is meaningful in that a recombinant enzyme was first immobilized on cellulose for the production of minor ginsenosides, and through this, not only can minor ginsenoside CK be prepared at the highest yield among previously reported yields, but also _{As described above, it is significant that minor ginsenoside G 17} can be additionally produced as an intermediate product in the manufacturing process of minor ginsenoside CK.

Another aspect of the present invention is β-glucosidase comprising the amino acid sequence of SEQ ID NO: 3; a transformant into which a vector containing a nucleic acid encoding the protein has been introduced; And carbon 6-specific trans-glycosylation of ginsenoside, comprising at least one selected from the group consisting of a culture of the transformant as an active ingredient, provides a composition for conversion to ginsenoside CK .

The terms used herein are the same as described above.

_{The composition may be to produce ginsenoside G 17} as an intermediate product in the conversion process from the ginsenoside to the ginsenoside CK, but is not limited thereto.

The ginsenoside may be a PPD-type ginsenoside, and specifically may be _{any one or more selected from the group consisting of Rb 1} and Rd, but is not limited thereto, and this is the same as described above.

The β-glucosidase may be a cellulose binding module bound, fixed to cellulose and concentrated thereto, but is not limited thereto, and this is the same as described above.

The β-glucosidase according to the present invention is highly concentrated and immobilized on amorphous cellulose, thereby exhibiting strong conversion activity for protopanaxadiol- and protopanaxatriol-type ginsenosides. In particular, β-glucosidase immobilized using a gram-scale ginseng extract as a substrate produces a high concentration of minor ginsenoside CK within 24 hours, and a minor ginsenoside, G _17, is produced as an intermediate product. The safe, convenient, and efficient _{production method of minor ginsenoside G 17} or CK can be effectively used to manufacture food processing recombinant enzymes in the pharmaceutical, functional food and cosmetic industries.

1 is a diagram confirming the expression of glycosidase in Corynebacterium.
Figure 2 is a diagram confirming the (a) optimum pH and (b) temperature of glycosidase.
3 is a diagram confirming the effect of metal ions on glycosidase.
4 is a diagram confirming the conversion activity of glycosidase using _{ginsenosides Rb 1} , Re and Rg _{1 as substrates.}
5 is a schematic diagram showing the conversion activity of glycosidase to ginsenoside.
6 is an SDS-PAGE analysis result of ATCC13032 cell extract. lane M: protein marker; lane S: cell extract containing C3a-MT619; Lane P: sediment of cell extract; lane RAC-S: supernatant after cellulase fixation; Lane RAC: RAC adsorption C3a-MT619.
7 is a diagram showing the results of adsorption of various amounts of C3a-MT619 protein to a fixed amount of RAC at 25 °C.
8 is a diagram illustrating the effect of substrate concentration on the production of (a) CK and (b) F _{1 and} the production of scale-up (c) CK and (d) F _{1 using immobilized C3a-MT619.}
9 is an HPLC analysis result for transformation of ginsenoside using immobilized C3a-MT619. (a) Ginsenoside standards (G ₁₇ , G ₇₅ , Rh ₂ , PPD); (b) ginsenoside standards (Rb ₁ , Rd, F ₂ , CK); (c) PPDGM; (d) CK produced after 6 h reaction between immobilized C3a-MT619 and PPDGM; (e) CK produced after 24 h reaction; (f) ginsenoside standards (Rg ₂ , Rh ₁ , PPT); (g) ginsenoside standards (Re, Rg ₁ , F ₁ , PPT); (h) PPTGM; _{(i) F 1} produced after 6 hours of reaction between immobilized C3a-MT619 and PPTGM; (j) F ₁ produced after 24 hours.

Hereinafter, the present invention will be described in more detail by way of Examples. However, these Examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited by these Examples, and it will be apparent to those of ordinary skill in the art to which the present invention pertains.

Example 1. Construction of MT619 and C3a-MT619 expression vectors

The mt619 gene encoding MT619, a β-glycosidase derived from Microbacterium testaceum , is retranslated using a codon preferred by Corynebacterium glutamicum ATCC13032 and Mutagenex Co. (Suwanee, GA, USA) was synthesized and cloned into pGEX4T-1. The codon-optimized sequence of the mt619 gene (SEQ ID NO: 1) was deposited with GenBank under the accession number MK575514.

In addition, in order to high-density immobilize MT619 to cellulose using a cellulose-binding module (CBM), the CBM (C3a) of Clostridium thermocellum binds to the N-terminus of MT619. C3a-MT619 was constructed as a flexible linker (G4S)2 connecting the modules.

The mt619 and c3a-mt619 genes consist of 1878 and 2376 bps, respectively, and encode 625 and 791 amino acids, respectively, and were predicted to have molecular weights of 68.3 and 86.3 kDa.

c3a (using primer pair of P5 and P6), mt619-1 (using primer pair of P1 and P2), c3a-mt619 (using primer pair of P10 and P11) and mt619-2 (using primer pair of P2 and P3) DNA The fragment was amplified by PCR using Pfu polymerase (Elpis-Biotech, Korea), and PCR conditions were 95 °C for 3 min, 95 °C 30 sec, 56 °C 30 sec, 72 °C for 2 min, repeated 30 cycles, 72 °C was carried out for 3 minutes. The PCR product was purified using a gel extraction kit (Enzynomics Co. Ltd., Seoul), and plasmids pEX-mt619 (pEX backbone and mt619-1), pEX-c3a-mt619 (pEX backbone, c3a and mt619-2) and pH36-C3a-MT619 (pH 36 backbone and c3a-mt619) were constructed. The resulting plasmid was transformed with ATCC13032 according to a known electrical transformation method. MT619 is continuous in ATCC13032 by promoter H36 efficient for heterologous expression (Yim et al. (2014) High-level secretory production of recombinant single-chain variable fragment (scFv) in Corynebacterium glutamicum. Appl Microbiol Biotechnol 98:273-284.) was expressed as The primer sequences used herein are shown in Table 1 below.

SEQ ID NO: designation Sequence (5' to 3') 6 P1 caatttcacacaggaaacagtattcATGACCCACGCTCGCTTCC 7 P2 tcgagtcgacccgggaattcttagtgatggtgatggtgatgTCCGCGCTGTGCACCC 8 P5 caatttcacacaggaaacagtattcaTGAATTTGAAAGTGGAGTTTTAC 9 P6 gtcaggagcggtcaggaagcgagcgtgggtactaccacctccaccactaccacctccaccTGGCTCTTTACCCCAAACCAGGA 10 P10 ggttggtaggagtagcatgggatccATGAATTTGAAAGTGGAGTTTTACA 11 P11 tgccgccaggcagcggccgcTTAATGATGGTGATGGTGATGTCCGCG

After culturing the transformed ATCC13032 for 24 hours, it was confirmed that recombinant C3a-MT619 was contained in the ATCC13032 lysate at 1.41 ± 0.03% of the total protein.

Example 2. MT619 Purification and Enzyme Characterization Confirmation

2-1. MT619 Tablets

To obtain high cell density of MT619 via fed-batch culture, in a 10-L stirred-tank reactor (Fermentec Co., Korea) with a 6-L working volume using medium supplemented with kanamycin (30 mg/L). ATCC13032 with pH 36-C3a-MT619 was incubated at 400 rpm. As a seed culture, ATCC13032 with pH36-C3a-MT619 was inoculated into a 1-L baffle flask containing 200 mL of medium containing 20 g/L glucose, and incubated at 30°C at 200 rpm for 20 hours. did Per liter of medium, 10 g (NH ₄ ) ₂ SO ₄ , 3 g K ₂ HPO ₄ , 1 g KH ₂ PO ₄ , 2 g urea, 2 g MgSO ₄ , 200 μg biotin, 5 mg thiamine (thiamine), 10 mg of calcium pantothenate, 10 mg of FeSO ₄ , 1 mg of MnSO ₄ , 1 mg of ZnSO ₄ , 200 μg of CuSO ₄ , and 10 mg of CaCl ₂ with 25 mg/L composed of kanamycin.

After the cell density reached an OD600 of 100, the cells were harvested by centrifugation at 4000 g for 20 min. The pellet was suspended in 50 mM sodium phosphate buffer (pH 7.0), and the cells were disrupted by sonication (Branson Digital Sonifier, Mexico City, Mexico). Cell lysates were prepared by removing cell debris via centrifugation at 4000 g for 20 minutes. MT619 and C3a-MT619 proteins from the cell lysate were purified by Ni column and DEAE column chromatography.

As a result, the predicted molecular masses (68.3 and 86.3 kDa) of native β-glucosidase were verified by SDS-PAGE (Fig. 1).

In addition, as a result of the activity experiment using PNPGlc, recombinant C3a-MT619 expressed in ATCC13032 exhibited 61.9% enzymatic activity against MT619 heterologously expressed in E. coli.

2-2. Determine the optimum pH for MT619

The optimal pH for enzyme activity was 2.0 mM PNPGlc as a substrate, and the pH was adjusted using the following buffer (50 mM). Range of pH 2 to 10: KCl-HCl (pH 2), Glycine-HCl (pH 3), Sodium Acetate (pH 4 and pH 5), Sodium Phosphate (pH 6 and pH 7), Tris-HCl (pH 8 and 9) and glycine-sodium hydroxide (pH 10). After incubation in each buffer at 4° C. for 12 hours, enzyme stability according to pH fluctuations was measured by analyzing PNPGlc in 50 mM potassium buffer. Residue activity was analyzed by standard analytical methods to calculate the activity obtained at the optimum pH. The pH stability of recombinant C3a-MT619 was determined by measuring the enzyme activity after incubation in each buffer for 12 hours for 4 hours.

As a result, MT619 was relatively stable at pH 6.0 to 8.0 and had optimal activity at pH 7.0. In addition, the enzyme activity decreased sharply from pH 9.0, and the enzyme activity decreased to 54.0% at pH 5.0 (FIG. 2a).

2-3. Check the optimum temperature for MT619

The optimum temperature for enzyme activity was tested by incubating the enzyme with buffer and 50 mM potassium phosphate buffer containing 2.0 mM pNPGlc for 5 min.

As a result, recombinant MT619 was relatively stable at temperatures below 30°C. After incubation at 37°C and 45°C for 2 hours, enzyme activity of 76.3% and 54.9% was maintained, and no activity was detected above 55°C (Fig. 2b). That is, the optimum temperature for enzyme activity was 45° C., and the relative activities were 70.3% and 88.6% at 30°C and 37°C, respectively.

Based on the above results, in the following examples, the ginsenoside conversion reaction was performed at 37° C. to maximize the stable conversion activity.

2-4. Confirmation of the effect of metal ions on C3a-MT619

To analyze the effect of metals and other chemical reagents on the activity of MT619, MT619 was mixed with 10 mM β-mercaptoethanol, CaCl ₂ , CoCl ₂ , CuCl ₂ , dithio at 37°C for 30 minutes. After incubation with threitol (DTT), EDTA, HgCl ₂ , KCl, MgCl ₂ , MnCl ₂ , NaCl, SDS or ZnCl ₂ , the remaining activity was measured using pNPG as a substrate, and the activity was measured when the compound was not included. Expressed as a percentage of activity.

As a result, as shown in FIG. 3 , the enzyme activity was not affected by ^{β-mercaptoethanol, Ca 2+} , Na ⁺ , K ⁺ and Mg ^{2+ .} However, activity was lost in the presence of Cu ^2+, Hg ^2+, and Zn ^2+, was significantly inhibited by Co ²⁺ and Mn ^2+. EDTA did not affect enzyme activity. No significant positive effect on the activity of MT619 was found for the ions and agonists tested.

Example 3. Ginsenoside conversion activity of MT619

The glycoside specificity of MT619 is p-nitrophenyl (PNP) glycoside, o-nitrophenyl (ONP) glycoside, PNP-β-D-glucopyranoside (PNP-β-D) -glucopyranoside), PNP-β-D-galactopyranoside (PNP-β-D-galactopyranoside), PNP-β-D-fucopyranoside (PNP-β-D-fucopyranoside), PNPN-acetyl-β -D-glucosaminoid (PNPN-acetyl-β-D-glucosaminide), PNP-β-L-arabinopyranoside (PNP-β-L-arabinopyranoside), PNP-β-D-mannopyranoside ( PNP-β-D-mannopyranoside), PNP-β-D-xylopyranoside (PNP-β-D-xylopyranoside), PNP-α-D-glucopyranoside (PNP-α-D-glucopyranoside), PNP-α-L-arabinofuranoside (PNP-α-L-arabinofuranoside), PNP-α-L-arabinopyranoside (PNP-α-L-arabinopyranoside), PNP-α-L-rhamnophyll Ranoside (PNP-α-L-rhamnopyranoside), PNP-α-D-mannopyranoside (PNP-α-D-mannopyranoside), PNP-α-D-xylopyranoside (PNP-α-D- xylopyranoside), ONP-β-D-glucopyranoside (ONP-β-D-glucopyranoside), ONP-β-D-galactopyranoside (ONP-β-D-galactopyranoside), ONP-β-D- Fucopyranoside (ONP-β-D-fucopyranoside) and ONP-α-D-galactopyranoside (ONP-α-D-galactopyranoside) were used. Specifically, p-nitrophenyl (PNP) glycoside, o-nitrophenyl (ONP) glycoside, PNP-β-D-glucopyranoside (PNP-β) as a substrate at 37 ° C. -D-glucopyranoside), PNP-β-D-galactopyranoside (PNP-β-D-galactopyranoside), PNP-β-D-fucopyranoside (PNP-β-D-fucopyranoside), PNPN-acetyl -β-D-glucosaminoid (PNPN-acetyl-β-D-glucosaminide), PNP-β-L-arabinopyranoside (PNP-β-L-arabinopyranoside), PNP-β-D-mannopyrano Side (PNP-β-D-mannopyranoside), PNP-β-D-xylopyranoside (PNP-β-D-xylopyranoside), PNP-α-D-glucopyranoside (PNP-α-D-glucopyranoside) ), PNP-α-L-arabinofuranoside (PNP-α-L-arabinofuranoside), PNP-α-L-arabinopyranoside (PNP-α-L-arabinopyranoside), PNP-α-L- Rhamnopyranoside (PNP-α-L-rhamnopyranoside), PNP-α-D-mannopyranoside (PNP-α-D-mannopyranoside), PNP-α-D-xylopyranoside (PNP-α- D-xylopyranoside), ONP-β-D-glucopyranoside (ONP-β-D-glucopyranoside), ONP-β-D-galactopyranoside (ONP-β-D-galactopyranoside), ONP-β- Use 50 mM sodium phosphate buffer (pH 6.0) containing D-fucopyranoside (ONP-β-D-fucopyranoside) or ONP-α-D-galactopyranoside (ONP-α-D-galactopyranoside) to measure the enzyme activity. The reaction was terminated by treatment in 0.1 ml of 1M Na ₂ CO ₃ for 5 minutes, and the secretion of p-nitrophenol was immediately measured at 405 nm. One unit of activity was defined as the amount of secretion of 1 μmol of p-nitrophenol per minute. Specific activity was expressed in units per milligram of protein. Protein concentration was measured using Bradford reagent (Sigma).

As a result, MT619 was only activated against the glucose moieties of PNP-β-D-glucopyranoside and ONP-β-D-glucopyranoside, and maximal activity against PNP-β-D-glucopyranoside and ONP It showed 28.7% relative activity to -β-D-glucopyranoside.

Also, Sugar moieties attached to protopanaxatriol (PPD)-type ginsenoside (Re, Rg1) and protopanaxadiol (PPD)-type ginsenoside (Rb1, Rd) using MT619 For hydrolysis specificity was investigated. Specifically, purified MT619 was reacted with Re, Rg1, Rb1 or Rd (2.0 mg/mL, pH 7.0) in a shaking incubator at 37°C. Ginsenosides in the sample were extracted with butanol and confirmed by thin-layer chromatography (TLC). Briefly, TLC was performed using 60F254 silica gel plates (Merck, Germany) and CHCl ₃ -CH ₃ OH-H ₂ O (65:35:10, v/v/v) as developing solution. The result was visualized using _{10% H 2} SO ₄ heated at 110° C. for 5 minutes.

_{As a result, MT619 clearly converted the four major ginsenosides (Re, Rg 1} , Rb ₁ and Rd) as evidenced by the Rf values in TLC analysis (Fig. 4). The bioconversion pathway of PPD ginsenosides by MT619 is Rb ₁ → GypXVII (G ₁₇ ) → GypLXXV (G ₇₅ ) and F ₂ → CK → PPD; Rd → F ₂ → CK → PPD; Re → F ₁ → PPT; Rg ₁ → F ₁ → PPT (Fig. 5). This enzyme has low hydrolytic activity on glucose at the C20 position, which can induce the production of CK from highly polar ginsenosides.

Example 4. Adsorption of C3a-MT619 on regenerated amorphous cellulose (RAC)

To prepare RAC for adsorbing C3a-MT619 purified in Example 2-1, 10 g of microcrystalline cellulose (SigmaCell 20) and 30 mL of distilled water were mixed to form a suspension. 200 ml of ice-cold 86% H ₃ PO ₄ were carefully added to the mixture with stirring. After the cellulose solution became clear, it was placed on ice for 1 hour. Then, 800 mL of ice-cold water was added 200 mL at a time with vigorous stirring. The suspension mixture was centrifuged at 4000 g at 4° C. for 20 min and the supernatant removed. The cellulose pellets were washed 4 times with cold water to remove phosphoric acid. After neutralization with 2MNa ₂ CO ₃ , the pellet was washed twice with water. 0.2% sodium azide was added to the prepared RAC to prepare a 4 g RAC/L suspension and stored at 4°C.

Example 2-1 C3a-MT619 in cell lysate was absorbed into RAC with shaking at 25° C. for 15 min.

Then, the adsorption isotherm was measured to estimate the binding capacity of C3a-MT619 attached to RAC. Prepare a series of tubes containing 0.1-10 mL of C3a-MT619 cell lysate (pH 7.0) at a fixed concentration (0.5 mg/mL), add 0.4 mg of RAC to each tube and 200 for 10 min at 25 °C. The reaction was carried out while shaking at rpm. The enzyme immobilized RAC was centrifuged at 10,000 g for 5 min and the precipitate was washed twice with 1 mL of phosphate buffer (50 mM, pH 7.0). The obtained cellulose was analyzed for β-glucosidase activity adsorbed to cellulose. The maximum enzyme adsorption capacity (A _max ) of RAC was calculated by the Langmuir equation according to the previous method. W _max and K _p values were also calculated by a mathematical method, and the following formula was applied.

E _a is adsorbed C3a-MT619 (mg/g RAC),

W _max is the maximum adsorption of C3a-MT619 per liter (mg/g RAC),

E _f is free C3a-MT619 (mg/g RAC).

As a result, C3a-MT619 expressed in Example 2 was adsorbed to the surface of RAC, which had a much larger external surface area than that of microcrystalline cellulose. Specifically, the crude cell lysates of C3a-MT619 obtained by sonication of the cell pellet were mixed with RAC to adsorb C3a-MT619 to the surface of the cellulosic material, which was confirmed by SDS-PAGE analysis. became (FIG. 6).

In addition, the adsorption curve of the immobilized C3a-MT619 enzyme detected by glucosidase activity suggested monolayer adsorption according to the Langmuir isotherm (Fig. 7). The maximum adsorption capacity of C3a-MT619 was 984 mg/g RAC, and the maximum enzyme concentration concentrated by centrifugation at 1,000 g for 5 min was about 78.7 mg/mL RAC, which was 286 times higher than that of the cell lysate concentrate. .

Example 5. Optimization of substrate concentration for ginsenoside CK production

The enzymatic reaction was performed using immobilized C3a-MT619 (217 mg/g RAC), and PPDGM was used as a substrate because it was relatively abundant in crude ginseng extracts. Various substrate concentrations of PPDGM (10-100 mg/mL) were investigated to determine appropriate reaction conditions for reduced reactor volume and cost.

As a result, the immobilized enzyme converted 20 mg/mL PPDGM to CK at a product concentration of 8.25 mg/mL (Fig. 8a). PPDGM (10 mg/mL) reached the highest concentration after 18 h and decreased when CK (final product) was converted to PPD by C3a-MT619. Higher concentrations of PPDGM (>20 mg/mL) reduced productivity. A substrate concentration of 50 mg/mL induces similar CK concentrations, but 20 mg/mL was chosen for scale-up production.

Based on the above results, PPDGM at a concentration of 20 mg/mL, respectively, was used for the scale-up production of CK in the following examples.

Example 6. Ginsenoside CK production scale expansion

For scale-up of ginsenoside CK production, scale-up uptake was performed using 5000 mL of cell lysate and 50 mL of RAC. As a result, 66.0% of C3a-MT619 in the cell lysate was shaken for 10 minutes to fix it in RAC, and the concentration was increased by 61.8 times compared to the cell lysate. The fixed concentration of C3a-MT619 on RAC was 217 mg/g RAC (17.3 mg/mL RAC, concentrated by centrifugation at 10,000 g for 5 min), and SDS-PAGE showed that some non-specific proteins of ATCC13032 were simultaneously in cellulose. It was shown to be fixed (FIG. 6). The immobilized C3a-MT619 maintained more than 78.0% of its activity even after consecutive washing with 10 volumes of buffer repeated 10 times. Immobilization of C3a-MT619 attached fairly stably to RAC, but the reusability of the immobilized enzyme was limited by co-precipitation of the resulting CK.

Scale-up transformation of ginsenoside production was performed in a shaking incubator at 37°C and 200 rpm. 30 mL of C3a-MT619 immobilized on RAC and 3 g of PPDGM at 20 mg/mL were added and reacted for 24 hours.

The samples were then analyzed by HPLC to determine the concentration of CK produced. HPLC analysis was performed using an HPLC system (Agilent 1260 Infinity HPLC system, Agilent Co., USA). The separation of ginsenosides was performed on a YMC ODS C18 column (5 μm, 250 Х 4.6 mm; YMC, Japan) equipped with a guard column (Eclipse XDB C18, 5 μm, 12.5 Х 4.6 mm; Agilent Technologies, USA). The gradient elution system consisted of water (A) and acetonitrile (B) using the following gradient program: 0-8 min, 32% B; 8-12 min, 32-65% B; 12-15 min, 65-100% B; 15-15.1 min, 100% B; 15.1-25 min, 100-32% B; 25-26 min, 32% B. The flow rate was set to 1.0 mL/min, and the detection wavelength was set to 203 nm.

As a result, as shown in Fig. 8c, Rb ₁ and Rd in PPDGM were completely converted by C3a-MT619 immobilized for 6 h. CK was produced at a concentration of 7.59 g/L, 79.2 % molar yield for 24 hours. CK and F1 products extracted and analyzed by HPLC are shown in FIG. 9 .

The resulting CK was isolated from the reaction mixture using a macroporous resin. Finally, 1.38 g of CK product with a chromatographic purity of 51.6% was produced.

That is, immobilized C3a-MT619 produced 7.59 g/L CK within 24 hours when a gram-scale ginseng extract was used as a substrate.

From the results of the above examples, the β-glucosidase according to the present invention was immobilized at a high density on amorphous cellulose and concentrated, so that protopanaxadiol- and protopanaxatriol- type ginsenosides It exhibits strong conversion activity against As another minor ginsenoside, G _17, is additionally produced, such a safe, convenient and efficient minor ginsenoside G ₁₇ or The CK production method can be effectively utilized to manufacture food processing recombinant enzymes in the pharmaceutical, functional food and cosmetic industries.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

<110> Korea Advanced Institute of Science and Technology <120> Ginsenoside G17 or CK production method using MT619 enzyme <130> KPA191618-KR <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 1878 <212> DNA <213> Artificial Sequence <220> <223> Synthetic mt619 <400> 1 atgacccacg ctcgcttcct gaccgctcct gacggcacgc gcttccgtga tctcaacggt 60 aatggcgtca tggacccata cgagaaccca gccctctctg ttgaggagcg taccgacgac 120 ttggttagcc gcctctccct cgaagagaaa gtaggactta tgttccagac tgtaattgaa 180 gtcggtgacg aaggtgagct gttggaagca ccgggaaaaa tcagcaagtc cccgaccact 240 acagttgtcg tcggcaagca tatgaaccac ttcaacgtgc atgctatccg cactgctcgc 300 caggctgcca cctggaataa caacctccaa gccctggcgg aaacaacccc ccacggtatc 360 ccggtgacaa tctccactga tccacgacac gctttcgtgg agaatactgg cgtagctttc 420 tccgcaggcc cattctcgca gtggccagaa ggccttggcc tggcagcctt ggacgatgtg 480 gataccgtcc gtgaatttgc agaagtcgcg cgccgtgagt atgtcgcagt tggcattcgc 540 gccgctttgc atcctcagat tgatcttgca accgaacctc gctggggccg ccaggctcaa 600 acccttggtc aggatgcggc acgtgtgact gaattcaccg ctgcctactt gcagggcttc 660 caaggcgacg agcttggtcc agattcggta gcttgcacca ccaaacactt tcctggaggc 720 ggccctcaac tcgacggcga agatgcgcat ttcccctatg gccgcgagca agtgtaccca 780 ggtggtatgt ttgattatca tctcgagccg tttcgcgaag ctatccgttg cggtaccgct 840 ggcatgatgc catattacgg catgcctgtt ggtctcgagg ttgatggact ccccatcgaa 900 gaggtaggtt tcggatacaa tcgccaaatc attacgggac tgcttcgtga acaactcggc 960 tatgatggag ttgtcgtgac cgactgggaa ctcgtaaacg ataaccacgt cggcgatcag 1020 gttctcccgg cacgcgcgtg gggagtggaa cacctgtcag cagtagaacg tatggaaaag 1080 atcctggatg cgggctcaga tcagttcggc ggtgaggagt gcgtcgaaat gttggtggat 1140 ctggtccgtg caggtcgcgt aagcgaagag cgcattgacg agagcgttcg tcgcctcttg 1200 cgtgtaaagt ttcaactcgg tctgttcgac gatccctacg tcgatgtgga tgaggcggag 1260 cgaatcgtag gaaacgcaga gttccgtgcg ctcggcgagc gcgcccaggc acatccctc 1320 accgtgctgg ttaacgaagg cgacgtgttg cccctgcgcc ccgcaggcgc tgtctatatc 1380 gagggctttc gtcccgatga tgtggctgaa ctcggccgtg ttgttaccga ccctgccgag 1440 gcggacctcg caatcgtacg catcggcgca ccgttcgatc cccgcgatga tctgttcctg 1500 gaggcatggt tccatcaggg ttccttggaa tttgccccag gccttgtcta tcgcctgcaa 1560 cagatcgcgg cgtcctgccc attgatcctg gtcgtcaatc tcgaccgtcc ggctatcctg 1620 acgcctttcg ttggccacgc tgccgcaatc gtcgcagact acggctcttc gagcgctgcg 1680 gtcctggacg cattgaccgg tcgtgtgcct ccacgcggac gcctgcccat tgaaatccca 1740 cgttccatgg acgcggtgcg ctcctcgcgt gaagatgttc cctcagacac tggagatcct 1800 gttttccctg ttcaccacgg tgttgaattg aaagtttcga cgggtgcaca gcgcggacat 1860 caccatcacc atcattaa 1878 <210> 2 <211> 1860 <212> DNA <213> Unknown <220> <223> mt619 <400> 2 atgacccacg ctcgcttcct gaccgctcct gacggcacgc gcttccgtga tctcaacggt 60 aatggcgtca tggacccata cgagaaccca gccctctctg ttgaggagcg taccgacgac 120 ttggttagcc gcctctccct cgaagagaaa gtaggactta tgttccagac tgtaattgaa 180 gtcggtgacg aaggtgagct gttggaagca ccgggaaaaa tcagcaagtc cccgaccact 240 acagttgtcg tcggcaagca tatgaaccac ttcaacgtgc atgctatccg cactgctcgc 300 caggctgcca cctggaataa caacctccaa gccctggcgg aaacaacccc ccacggtatc 360 ccggtgacaa tctccactga tccacgacac gctttcgtgg agaatactgg cgtagctttc 420 tccgcaggcc cattctcgca gtggccagaa ggccttggcc tggcagcctt ggacgatgtg 480 gataccgtcc gtgaatttgc agaagtcgcg cgccgtgagt atgtcgcagt tggcattcgc 540 gccgctttgc atcctcagat tgatcttgca accgaacctc gctggggccg ccaggctcaa 600 acccttggtc aggatgcggc acgtgtgact gaattcaccg ctgcctactt gcagggcttc 660 caaggcgacg agcttggtcc agattcggta gcttgcacca ccaaacactt tcctggaggc 720 ggccctcaac tcgacggcga agatgcgcat ttcccctatg gccgcgagca agtgtaccca 780 ggtggtatgt ttgattatca tctcgagccg tttcgcgaag ctatccgttg cggtaccgct 840 ggcatgatgc catattacgg catgcctgtt ggtctcgagg ttgatggact ccccatcgaa 900 gaggtaggtt tcggatacaa tcgccaaatc attacgggac tgcttcgtga acaactcggc 960 tatgatggag ttgtcgtgac cgactgggaa ctcgtaaacg ataaccacgt cggcgatcag 1020 gttctcccgg cacgcgcgtg gggagtggaa cacctgtcag cagtagaacg tatggaaaag 1080 atcctggatg cgggctcaga tcagttcggc ggtgaggagt gcgtcgaaat gttggtggat 1140 ctggtccgtg caggtcgcgt aagcgaagag cgcattgacg agagcgttcg tcgcctcttg 1200 cgtgtaaagt ttcaactcgg tctgttcgac gatccctacg tcgatgtgga tgaggcggag 1260 cgaatcgtag gaaacgcaga gttccgtgcg ctcggcgagc gcgcccaggc acatccctc 1320 accgtgctgg ttaacgaagg cgacgtgttg cccctgcgcc ccgcaggcgc tgtctatatc 1380 gagggctttc gtcccgatga tgtggctgaa ctcggccgtg ttgttaccga ccctgccgag 1440 gcggacctcg caatcgtacg catcggcgca ccgttcgatc cccgcgatga tctgttcctg 1500 gaggcatggt tccatcaggg ttccttggaa tttgccccag gccttgtcta tcgcctgcaa 1560 cagatcgcgg cgtcctgccc attgatcctg gtcgtcaatc tcgaccgtcc ggctatcctg 1620 acgcctttcg ttggccacgc tgccgcaatc gtcgcagact acggctcttc gagcgctgcg 1680 gtcctggacg cattgaccgg tcgtgtgcct ccacgcggac gcctgcccat tgaaatccca 1740 cgttccatgg acgcggtgcg ctcctcgcgt gaagatgttc cctcagacac tggagatcct 1800 gttttccctg ttcaccacgg tgttgaattg aaagtttcga cgggtgcaca gcgcggataa 1860 1860 <210> 3 <211> 625 <212> PRT <213> Unknown <220> <223> MT619 <400> 3 Met Thr His Ala Arg Phe Leu Thr Ala Pro Asp Gly Thr Arg Phe Arg 1 5 10 15 Asp Leu Asn Gly Asn Gly Val Met Asp Pro Tyr Glu Asn Pro Ala Leu 20 25 30 Ser Val Glu Glu Arg Thr Asp Asp Leu Val Ser Arg Leu Ser Leu Glu 35 40 45 Glu Lys Val Gly Leu Met Phe Gln Thr Val Ile Glu Val Gly Asp Glu 50 55 60 Gly Glu Leu Leu Glu Ala Pro Gly Lys Ile Ser Lys Ser Pro Thr Thr 65 70 75 80 Thr Val Val Val Gly Lys His Met Asn His Phe Asn Val His Ala Ile 85 90 95 Arg Thr Ala Arg Gln Ala Ala Thr Trp Asn Asn Asn Leu Gln Ala Leu 100 105 110 Ala Glu Thr Thr Pro His Gly Ile Pro Val Thr Ile Ser Thr Asp Pro 115 120 125 Arg His Ala Phe Val Glu Asn Thr Gly Val Ala Phe Ser Ala Gly Pro 130 135 140 Phe Ser Gln Trp Pro Glu Gly Leu Gly Leu Ala Ala Leu Asp Asp Val 145 150 155 160 Asp Thr Val Arg Glu Phe Ala Glu Val Ala Arg Arg Glu Tyr Val Ala 165 170 175 Val Gly Ile Arg Ala Ala Leu His Pro Gln Ile Asp Leu Ala Thr Glu 180 185 190 Pro Arg Trp Gly Arg Gln Ala Gln Thr Leu Gly Gln Asp Ala Ala Arg 195 200 205 Val Thr Glu Phe Thr Ala Ala Tyr Leu Gin Gly Phe Gln Gly Asp Glu 210 215 220 Leu Gly Pro Asp Ser Val Ala Cys Thr Thr Lys His Phe Pro Gly Gly 225 230 235 240 Gly Pro Gln Leu Asp Gly Glu Asp Ala His Phe Pro Tyr Gly Arg Glu 245 250 255 Gln Val Tyr Pro Gly Gly Met Phe Asp Tyr His Leu Glu Pro Phe Arg 260 265 270 Glu Ala Ile Arg Cys Gly Thr Ala Gly Met Met Pro Tyr Tyr Gly Met 275 280 285 Pro Val Gly Leu Glu Val Asp Gly Leu Pro Ile Glu Glu Val Gly Phe 290 295 300 Gly Tyr Asn Arg Gln Ile Ile Thr Gly Leu Leu Arg Glu Gln Leu Gly 305 310 315 320 Tyr Asp Gly Val Val Val Thr Asp Trp Glu Leu Val Asn Asp Asn His 325 330 335 Val Gly Asp Gln Val Leu Pro Ala Arg Ala Trp Gly Val Glu His Leu 340 345 350 Ser Ala Val Glu Arg Met Glu Lys Ile Leu Asp Ala Gly Ser Asp Gln 355 360 365 Phe Gly Gly Glu Glu Cys Val Glu Met Leu Val Asp Leu Val Arg Ala 370 375 380 Gly Arg Val Ser Glu Glu Arg Ile Asp Glu Ser Val Arg Arg Leu Leu 385 390 395 400 Arg Val Lys Phe Gln Leu Gly Leu Phe Asp Asp Pro Tyr Val Asp Val 405 410 415 Asp Glu Ala Glu Arg Ile Val Gly Asn Ala Glu Phe Arg Ala Leu Gly 420 425 430 Glu Arg Ala Gln Ala His Ser Leu Thr Val Leu Val Asn Glu Gly Asp 435 440 445 Val Leu Pro Leu Arg Pro Ala Gly Ala Val Tyr Ile Glu Gly Phe Arg 450 455 460 Pro Asp Asp Val Ala Glu Leu Gly Arg Val Val Thr Asp Pro Ala Glu 465 470 475 480 Ala Asp Leu Ala Ile Val Arg Ile Gly Ala Pro Phe Asp Pro Arg Asp 485 490 495 Asp Leu Phe Leu Glu Ala Trp Phe His Gln Gly Ser Leu Glu Phe Ala 500 505 510 Pro Gly Leu Val Tyr Arg Leu Gln Gln Ile Ala Ala Ser Cys Pro Leu 515 520 525 Ile Leu Val Val Asn Leu Asp Arg Pro Ala Ile Leu Thr Pro Phe Val 530 535 540 Gly His Ala Ala Ala Ile Val Ala Asp Tyr Gly Ser Ser Ser Ala Ala 545 550 555 560 Val Leu Asp Ala Leu Thr Gly Arg Val Pro Pro Arg Gly Arg Leu Pro 565 570 575 Ile Glu Ile Pro Arg Ser Met Asp Ala Val Arg Ser Ser Arg Glu Asp 580 585 590 Val Pro Ser Asp Thr Gly Asp Pro Val Phe Pro Val His His Gly Val 595 600 605 Glu Leu Lys Val Ser Thr Gly Ala Gln Arg Gly His His His His His His 610 615 620 His 625 <210> 4 <211> 158 <212> PRT <213> Unknown <220> <223> C3a <400> 4 Met Val Ser Gly Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser 1 5 10 15 Asp Thr Thr Asn Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly 20 25 30 Ser Ser Ala Ile Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr 35 40 45 Val Asp Gly Gln Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile 50 55 60 Ile Gly Ser Asn Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly 65 70 75 80 Thr Phe Val Lys Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu 85 90 95 Glu Ile Ser Phe Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln 100 105 110 Ile Gln Gly Arg Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser 115 120 125 Asn Asp Tyr Ser Phe Lys Ser Ala Ser Gln Phe Val Glu Trp Asp Gln 130 135 140 Val Thr Ala Tyr Leu Asn Gly Val Leu Val Trp Gly Lys Glu 145 150 155 <210> 5 <211> 578 <212> DNA <213> Unknown <220> <223> C3a <400> 5 atgaagtgga gttttacaat tctaatccgt ctgatacaac gaattcgatc aacccccaat 60 ttaaagtaac taatactggt tcgagtgcaa ttgatctgtc aaaactgacc ctgcgttatt 120 attacacagt tgatggtcag aaggatcaga ctttttggtg tgaccacgcc gctattatcg 180 gtagcaatgg ttcatataat ggcattacat ctaatgttaa aggaacattt gtgaaaatga 240 gttcatctac caataacgcc gatacttatc ttgaaatatc cttcacagga ggtactttag 300 aaccaggggc acatgtacag attcagggtc gttttgctaa gaatgattgg agtaactata 360 cccagtcaaa tgactatagc ttcaaatctg catcccaatt cgtagaatgg gaccaagtga 420 ccgcgtacct gaatggtgtc ctggtttggg gtaaagagcc aggggggagc gtcgtgccgt 480 caactcagcc tgtcacgacc cctccggcca caacaaaacc tccggcgacg acaaagccgc 540 ctgcaaccac gatccctccg tccgacgatc cgaacgca 578 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> P1 <400> 6 caatttcaca caggaaacag tattcatgac ccacgctcgc ttcc 44 <210> 7 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> P2 <400> 7 tcgagtcgac ccgggaattc ttagtgatgg tgatggtgat gtccgcgctg tgcaccc 57 <210> 8 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> P5 <400> 8 caatttcaca caggaaacag tattcatgaa tttgaaagtg gagttttac 49 <210> 9 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> P6 <400> 9 gtcaggagcg gtcaggaagc gagcgtgggt actaccacct ccaccactac cacctccacc 60 tggctcttta ccccaaacca gga 83 <210> 10 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> P10 <400> 10 ggttggtagg agtagcatgg gatccatgaa tttgaaagtg gagttttaca 50 <210> 11 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> P11 <400> 11 tgccgccagg cagcggccgc ttaatgatgg tgatggtgat gtccgcg 47

Claims (17)

β-glucosidase comprising the amino acid sequence of SEQ ID NO: 3; a transformant into which a vector containing a nucleic acid encoding the protein has been introduced; And using one or more selected from the group consisting of the culture of the transformant, ginsenoside comprising the step of hydrolyzing the sugar specifically at the 6th carbon of ginsenoside to convert it into a deglycosylated form, ginseno A method of manufacturing side CK (Compound K).
The method according to claim 1, wherein the production method produces _{ginsenoside G 17} as an intermediate product in the conversion process from the ginsenoside to the ginsenoside CK.
The method of claim 1, wherein the β-glucosidase is encoded by at least one nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
The method according to claim 1, wherein the β-glucosidase is fixed and concentrated in cellulose.
The method according to claim 4, wherein the β-glucosidase is fixed to cellulose by binding a cellulose-binding module.
The method according to claim 5, wherein the cellulose binding module is derived from Clostridium thermocellum.
The method of claim 5, wherein the cellulose binding module comprises the amino acid sequence of SEQ ID NO: 4.
The method according to claim 1, wherein the ginsenoside is protopanaxadiol (PPD)-type ginsenoside.
The method according to claim 8, wherein the ginsenoside is at least one selected from the group consisting _{of Rb 1 and Rd.}
The method according to claim 1, wherein the preparation of the ginsenoside CK comprises any one or more conversion processes selected from the group consisting _{of Rb 1 to CK conversion and Rd to CK conversion.}
11. The method of claim 10, wherein _{the conversion of Rb 1} to CK comprises: the conversion of Rb ₁ to G ₁₇ ; And Containing the conversion step from the G ₁₇ to CK, the manufacturing method.
β-glucosidase comprising the amino acid sequence of SEQ ID NO: 3; a transformant into which a vector containing a nucleic acid encoding the protein has been introduced; And converting to a deglycosylated form by hydrolyzing the 6th carbon-specific sugar of ginsenoside, including any one or more selected from the group consisting of a culture of the transformant as an active ingredient, ginseno A composition for conversion to side CK.
The composition of claim 12, wherein the composition produces _{ginsenoside G 17} as an intermediate product in the conversion process from the ginsenoside to the ginsenoside CK.
13. The composition of claim 12, wherein the β-glucosidase is immobilized and concentrated in cellulose.
The composition according to claim 14, wherein the β-glucosidase is immobilized to cellulose by binding a cellulose-binding module.
The composition of claim 12, wherein the ginsenoside is protopanaxadiol (PPD)-type ginsenoside.
The composition of claim 16, wherein the ginsenoside is at least one selected from the group consisting _{of Rb 1 and Rd.}

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