KR101771577B1 - Alpha-N-arabinofuranosidase From Rhodanobacter ginsenosidimutans And Use Thereof - Google Patents

Abstract

The present invention relates to Rhodanobacter ginsenosidimutans) it relates to alpha -N- arabinose Pew pyrano let agents and their use in the origin, the protein is selectively hydrolyzed to the α-1,6 arabinose Pew pyrano room residue of ginsenoside PPD (protopanaxadiol) type Or protopanaxatriol (PPT) type ginsenoside into the body's absorbable, deglycosylated rare ginsenosides.

Description

N-arabinofuranosidase from Rhodonobacter ginsenoside mutans and its use {Alpha-N-arabinofuranosidase From Rhodanobacter ginsenosideimutans And Use Thereof}

The present invention relates to alpha-N-arabinofuranosidase from Rhodanobacter ginsenosidimutans and uses thereof.

Ginseng has been used as a medicinal product for promoting health and longevity in Korea, China and Japan including Asia, Japan, and also for therapeutic use at the plant level in the United States and in Oriental medicine treatment in Europe, especially Germany.

Saponin refers to a substance composed of a plurality of ring compounds in a part of glycosides which are widely present in the plant system, and triterpene saponin, which is a saponin component contained in ginseng or red ginseng as a major physiologically active ingredient, Since the chemical structure of saponin is different from that of saponin, it is called ginsenoside in order to distinguish saponin from other plant saponins.

To date, over 180 ginsenosides have been purified, characterized and classified (Christensen, L. P. 2008. Ginsenosides. Chemistry, biosynthesis, analysis, and potential health effects. Adv. Food Nutr. Res 55: 1-99). The basic structure of the ginsenoside is one to three residues attached to the main skeleton in a specific position, i.e., glycopyranosyl, arabinopyranosyl, arabinofuranosyl, xylopyranosyl, or rampopyranosyl groups A tetracyclic triterpene damermanane or a pentacyclic oleanane skeleton. Damaran-type ginsenosides are classified mainly by their own aglycone residues: protopanaxel (PPD), protopascan triol (PPT), and ocotillol.

The ginsenoside composition is diversified into various ginseng species. For example, Korean ginseng ( Panax ginseng ) contains 22 PPD type ginsenosides and 14 PPT type ginsenosides, while American ginseng ( Panax notoginseng ) contains 13 PPD type ginsenosides and 5 PPT types (Choi, KT 2008. Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginsengica Meyer. Acta Pharmacol. Sin. 29: 1109-1118). In certain species of Panax , the composition of ginsenosides also varies in different parts of ginseng bodies such as roots, leaves, and fruits (Wang, CZ, JA Wu, E. McEntee, and CS Yuan 2006. Saponins J. Agric. Food Chem. 54: 2261-2266).

In general, the efficacy of ginsenosides increases with the degree of deglycosylation, which enhances its hydrophobicity and cell wall permeability (Shibata, S. 2001. Chemistry and cancer prevention activities of ginseng saponins and some related triterpenoid compounds. Korean Med. Sci. 16 Suppl: S28-37). However, while deglycosylated ginsenosides are trace amounts in ginseng extract, Rb1 {3- O- [? - D-glycopyranosyl- (1-2) -? - D-glycopyranosyl] -20- O - [? - D-glycopyranosyl- (1-6) -? - D-glycopyranosyl] -20 ( S ) protopanaxanthooler system}, Rb2 {3- O- [ ( S ) -β-D-glycopyranosyl] -20- O- [α-L-arabinopyranosyl- (1-6) - Prototype wave incident yidayi olgye}, Rb3 {3- O - [ β-D- glycoside pyrazol nosil - (1-2) -β-D- glycoside pyrazol nosil] -20- O - [β-D- xylene to pyrazole O- [beta] -D-glycopyranosyl] - 20 ( S ) - protopanaxadiolide system), Rc {3- ? - D-glycopyranosyl] -20 ( S ) -protophenecarbocyanuric acid} -20- O- [? -L-arabinofuranosyl- (1-6) and Rd {3- O - [β- D- glycoside pyrazol nosil - (1-2) -β-D- glycoside pyrazol nosil] -20- O -β-D- glycoside pyrazol nosil -20 (S) - protocol This is my The larger ginsenoside of the same PPD family constitutes more than two thirds of the total ginsenoside extract.

Ginsenoside Rc is one of the major PPD type ginsenosides, accounting for 7-22% of total ginsenosides in white ginseng (Hu, C., and DD Kitts. 2001. Free radical scavenging capacity as related to antioxidant activity and Ginsenoside composition of Asian and North American Ginseng extracts. J. Am. Oil Chem. Soc. 78: 249-255). Alpha-L-arabinofuranosyl- (1-6) - beta -D-glucopyranose is bound to C20 (carbon at the 20 < th > position) , And? -D-glycopyranosyl- (1-2) -? - D-glucopyranose is bound to C3 (carbon at the third position) of the dermaran aglycone. Since ginsenoside Rc has a high molecular weight and low permeability in passing through a cell membrane, deglycosylation is required to exhibit high pharmacological activity. Until now, the genetic information and detailed biochemical characteristics of arabinofuranosidase that hydrolyzes arabinofuranosyl residues from ginsenoside Rc have not been disclosed. Α-L-arabinofuranosidase capable of producing Rd by hydrolyzing arabinofuranosyl residues of Rc has been isolated from intestinal bacteria, but the sequence information thereof has not been fully disclosed (Shin, HY, SY Park, JH Sung, and DH Kim 2003. Purification and characterization of aL-arabinopyranosidase and aL-arabinofuranosidase from Bifidobacterium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and R. Appl. Environ Microbiol., 69: 7116-7123).

In order to facilitate the medical application of rare ginsenosides and the study of biological properties at the molecular level, a method of producing deglycosylated ginsenosides is required. Steamed (heated) (Wang, CZ, B. Zhang, WX Song, A. Wang, M. Ni, X. Luo, HH Aung, JT Xie, R. Tong, ginseng berry: ginsenoside analyses and anticancer activities, J. Agric. Food Chem. 54: 9936-9942) and acid treatment (Bae, EA, MJ Han, EJ Kim, and DH Kim 2004. Transformation of ginseng saponins to ginsenoside Rh2 Physicochemical processes such as acid and human intestinal bacteria and their biological transformants, Arch Pharm Res. 27: 61-67, have been developed to convert the main constituents of ginsenosides into deglycosylated ginsenosides . However, the most preferable method is a bioconversion method in which an enzyme having known activity is sequentially reacted with a desired product for a desired product, or an enzyme having a known activity is used in combination of two or more, (Park, CS, MH Yoo, KH Noh, and DK Oh. 2010. Biotransformation of ginsenosides by hydrolyzing the sugar moieties of ginsenosides using microbial glycosidases. Appl. Microbiol Biotechnol. 87: 9-19). In recent years, some progress has been made in the identification of wild-type enzymes capable of converting large molecular weight ginsenosides from fungi and bacteria into low-molecular-weight ginsenosides and characterizing them for industrial applications there was. However, these studies faced two limitations: 1) there was little progress in the discovery of the biochemical properties of the ginsenoside biotransformation enzyme due to the lack of sequence information for the enzyme; and 2) It seems that the method of refining enzymes lacks economic feasibility.

Recently, sequence information of three enzymes exhibiting biosynthesis activity of ginsenosides from microorganisms has been identified and identified as an enzyme belonging to the family of Glygoside Hydrolase: (1) Rb1 or Rc as a compound (C ) -K [20- O - (β -D- glycoside pyrazol nosil) -20 (S) - Prototype wave incident yidayi olgye] Let a thermostable β- glucosidase to convert the (Noh, KH, JW Son, HJ Kim, and DK Oh. 2009. Ginsenoside compound K production from ginseng root extract by a thermostable β-glycosidase from Sulfolobus solfataricus . Biosci. Biotechnol. Biochem. 73: 316-321); (2) β-glucosidase from soil metagenomes converting Rb1 to Rd (Noh, KH, JW Son, HJ Kim, and DK Oh. 2009. Ginsenoside compound K production from ginseng root extract by a thermostable beta-glycosidase from Sulfolobus solfataricus . Biosci. Biotechnol. Biochem., 73: 316-321); And (3) reacting Rb1 with CK, Rb2 with CK, and Rc with C-Mc {20- O - [? - L-arabinofuranosyl- (1-6) -? - D-glycopyranosyl] (Noh, KH, and DK Oh. 2009. Production of the rare ginsenosides compound K, compound Y, and compound Mc from Sulfolobus acidocaldarius , which converts to -20 ( S ) by a thermostable beta-glycosidase from Sulfolobus acidocaldarius . Biol. Pharm. Bull. 32: 1830-1835). Gypenoside the Rb1 XVII {3- O -β-D- glycoside pyrazol nosil -20- O - [β-D- glycoside pyrazol nosil - (1-6) -β-D- glycoside pyrazol nosil] -20 (S) - a protocol wave incident yidayi olgye}, then Gypenoside LXXV {20- O - [β -D- glycoside pyrazol nosil - (1-6) -β-D- glycoside pyrazol nosil] -20 (S) - Prototype wave incident yidayi Recently, we have reported the enzymes of GH family 3, which catalyzes the successive conversion to CK (An, D.-S., C.-H. Cui, H.-G W. Chin, H.-K. Lee, W.-T. Im, and S.-T. Lee, F. Jin, H. Yu, Y.-W. Chin, G. Kim. 2010. Identification and characterization of a novel Terrabacter ginsenosidimutans sp. Nov. Beta -glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV Appl. Environ Microbiol. 76: 5827-5836).

However, β-galactosidase (Noh, KH, and DK Oh. 2009. Biol. Pharm. Bull. 32: 1830-1835) with activity to convert Rc to C-Mc, and Rc with C- The β-glucosidase from the Sulfolobus solfataricus , which subsequently converts to CK, mainly hydrolyzes the glucose residue on the C3-phase of the aglycone (Noh, KH, JW Son, HJ Kim, and DK Oh. And has different properties from arabinofuranosidase.

The present inventors have cloned and characterized the novel GH family 51 arabinofuranosidase from the soil microorganism Rhodanna bacteriocinosis mutans strain Gsoil 3054 isolated and identified in ginseng fields. As a result, it was found that the recombinant enzyme was overexpressed in Escherichia coli to show biotransformation activity against ginsenoside Rc and to convert Rc to Rd through hydrolysis of the arabinofuranoside residue at the 20th carbon position of the aglycon . In addition, it has been found that the recombinant enzyme has an exo-acting arabinofuranocidase activity acting on the terminal of the polysaccharide and acts only on the arabinofuranoside residue of the ginsenoside Rc or its analogous substance, Respectively.

A first aspect of the present invention provides a-N-arabinofuranosidase derived from Rhodanobacter ginsenosidimutans .

A second aspect of the present invention provides a nucleic acid encoding α- N -arabinofuranosidase according to the present invention and a recombinant vector comprising the same.

A third aspect of the present invention provides a transformant transformed with a nucleic acid or a recombinant vector according to the present invention.

A fourth aspect of the present invention is a method for producing α- N -arabinofuranosidase, comprising the steps of (a) culturing a transformant according to the present invention (b) producing α- N -arabinofuranocidase from the cultured transformant, and (c) N -arabinofuranosidase, comprising the step of recovering the produced α- N -arabinofuranosidase.

The fifth aspect of the present invention is α- N according to the present invention arabinose Pew pyrano let claim, α- N, prepared according to the invention arabinose Pew pyrano let claim, transformant or the transformant according to the present invention Lt; RTI ID = 0.0 > ginsenoside < / RTI > from the first ginsenoside by using a culture of the ginsenoside.

The sixth aspect of the present invention is α- N according to the present invention arabinose Pew pyrano let claim, α- N, prepared according to the invention arabinose Pew pyrano let claim, transformant or the transformant according to the present invention A kit for the conversion of a ginsenoside of PPD or PPT type to a deglycosylated rare ginsenoside containing a culture of a s body as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present inventors first purified and characterized? -N -arabinofuranosidase from Rhodobacter.

Specifically, the novel enzyme obtained from Rhodobacter geminosidymutans KCTC22231 ^T according to the present invention is a novel enzyme obtained from an oligosaccharide or polysaccharide containing an α-1,6 arabinofuranosyl residue of ginsenoside or arabinose, α N -arabinofuranosidase having selective hydrolysis ability to arabinofuranosyl residues, more preferably has the following characteristics:

a) has selective hydrolysis ability to the alpha-1,6 arabinofuranosyl residues located at the 20th carbon of protopanaxadiol-type ginsenoside.

b) through said selective hydrolysis ability, has a conversion activity which is able to convert the PPD type ginsenoside to the deglycosylated rare ginsenoside.

c) an exo-acting enzyme.

The term 'exogenous enzyme' refers to an enzyme that hydrolyzes glycosidic bonds from the ends of polysaccharides.

The α- N -arabinofuranosidase obtained from Rhodobacter geminosidymutans KCTC22231 ^T according to the present invention is registered as GenBank accession number HQ026482. The registered α- N -arabinofuranosidase of the present invention is composed of 518 amino acids, and the length of the nucleic acid encoding the protein is 1557 bp.

KCTC22231 ^T (= KACC 12822 ^T = DSM 21013 ^T = LMG 24457 ^T ) microorganisms were isolated from ginseng fields in the Pocheon area of Korea and deposited with the Korea Research Institute of Bioscience and Biotechnology on Jan. 25, 2008 Respectively.

The amino acid sequence constituting? -N -arabinofuranosidase derived from Rhodobacter ginsenoside mutans KCTC22231 ^T is the sequence described in SEQ ID NO: 1. The α- N -arabinofuranosidase according to the present invention has an amino acid sequence having preferably 70% or more homology with the above sequence, preferably 80% or more similarity, more preferably 90% or more, N-arabinofuranosidase as an amino acid sequence that exhibits at least 95% similarity, even more preferably at least 95% similarity, and most preferably at least 98% similarity. Further, as the sequence having this similarity, if it is an amino acid sequence having substantially the same or corresponding biological activity as that of alpha-N-arabinofuranosidase, a protein variant having an amino acid sequence in which a part of the sequence is deleted, Are also included within the scope of the present invention.

The nucleic acid encoding α- N -arabinofuranosidase according to the present invention may be a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 1, preferably a nucleic acid represented by SEQ ID NO: 2, A sequence which has at least 70% homology with the sequence, preferably at least 80% homology, more preferably at least 90% homology, even more preferably at least 95% homology, most preferably at least 98% And the nucleic acid-encoded protein comprises a nucleic acid having the activity of alpha-N-arabinofuranosidase.

According to one embodiment of the present invention, the nucleic acid of the present invention has been named by the present inventors as araf3054 (hereinafter, abbreviated as 'ARAF3054' and 'AbfA').

The term " similarity " of the present invention is intended to indicate the similarity with the amino acid sequence of the wild type protein or the base sequence encoding the amino acid sequence, and it is preferable that the amino acid sequence or base sequence of the present invention is identical to the above- The branch contains the sequence. Comparison of these similarities can be performed using a visual program or a comparison program that is easy to purchase. Commercially available computer programs can calculate the similarity between two or more sequences as a percentage, and the similarity (%) can be calculated for adjacent sequences.

Those skilled in the art will readily understand that such an artificial modification maintains a certain level of similarity and is an equivalent protein as long as it possesses the desired protein activity in the present invention. Thus, the protein of the present invention includes a wild-type amino acid sequence variant and a base sequence variant in which one or more amino acid residues or base sequences are deleted, inserted, non-conserved or conserved for a native amino acid sequence or a base sequence Quot; refers to a protein having a different sequence or a nucleic acid having a different sequence by virtue of an organic substitution or a combination thereof.

As used herein, the term "vector" refers to a nucleic acid construct comprising an essential control element operatively linked to the expression of a nucleic acid insert, as an expression vector capable of expressing a desired protein in a suitable host cell. The present invention can produce a recombinant vector comprising a nucleic acid encoding alpha-N-arabinofuranosidase.

In accordance with the present invention, a nucleic acid encoding α- N -arabinofuranosidase or a recombinant vector comprising the nucleic acid is transformed or transfected into a host cell, N -arabinofuranosidase can be obtained. In a specific embodiment of the present invention, an ORF (open reading frame) capable of coding alpha-N-arabinofuranosidase is screened through a fosmid library and inserted into a pCC1FOS expression vector To thereby prepare a recombinant vector pET-MBP-TEV-Araf3054 (FIG. 9).

The recombinant vector of the present invention can be obtained by linking (inserting) the nucleic acid of the present invention into an appropriate vector. The vector into which the nucleic acid of the present invention is to be inserted is not particularly limited as long as it can be replicated in the host. For example, plasmid DNA, phage DNA, etc. may be used. Specific examples of plasmid DNA include commercial plasmids such as pCDNA3.1 + (Invitrogen). Other examples of plasmids used in the present invention include plasmids derived from Escherichia coli (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis -derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50 ). Specific examples of phage DNA include lambda-phages (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11 and lambda ZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus may also be used. In a specific embodiment of the present invention, the recombinant vector pET-MBP-TEV-Araf3054 was prepared by insertion into pCC1FOS expression vector.

As a vector of the present invention, a fusion plasmid (for example, pJG4-5) to which a nucleic acid expression-activating protein (such as B42) is linked can be used. Such fusion plasmids include GST, GFP, His- tag, Myc-tag, etc. However, the fused plasmid of the present invention is not limited by the above examples. In a specific embodiment of the present invention, a maltose binding protein (MBP) tag was used to easily purify and recover the expressed alpha-N-arabinofuranosidase.

In order to insert the nucleic acid of the present invention as a vector, a method of inserting the purified DNA into a restriction site or a cloning site of a suitable vector DNA by cleaving the DNA with an appropriate restriction enzyme can be used. According to a specific embodiment of the present invention, the araf3054 gene was inserted between the cleaved BamHI and HindIII indicated by the arrows.

The nucleic acid of the present invention is preferably operably linked to a vector. The vector of the present invention may further comprise a promoter and a nucleic acid of the present invention in addition to a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker ), A ribosome binding sequence (SD sequence), and the like. Examples of selectable markers include, but are not limited to, chloramphenicol resistant nucleic acids, ampicillin resistant nucleic acids, dihydrofolate reductase, neomycin resistant nucleic acids, etc., but limiting the additional components operatively linked by the above examples no.

As used herein, the term "transformation" refers to the introduction of DNA as a host and DNA replication as a factor of a chromosome or by chromosomal integration, introducing an external DNA into a cell, It means a phenomenon that causes change.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. Generally, the transformation methods include the CaCl ₂ precipitation method, the Hanahan method which uses a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ method, the electroporation method, the calcium phosphate precipitation method, the protoplasm fusion method, Agrobacterium-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

The method for transforming the nucleic acid encoding the alpha-N-arabinofuranosidase of the present invention or a vector containing the same is not limited to the above examples, and a transformation or transfection method conventionally used in the art Can be used without limitation.

A transformant of the present invention can be obtained by introducing a nucleic acid encoding alpha-N-arabinofuranosidase, which is a target nucleic acid, or a recombinant vector containing the same into a host.

The host is not particularly limited as long as it is capable of expressing the nucleic acid of the present invention. Specific examples of hosts that can be used in the present invention include bacteria such as the bacterium Bacillus bacterium Pseudomonas putida , such as Escherichia genus Bacillus subtilis , such as E. coli, Yeast animal cells such as Saccharomyces cerevisiae , Pseudomonas sp., Schizosaccharomyces pombe , and insect cells.

Specific examples of the Escherichia coli strains usable in the present invention include CL41 (DE3), BL21 (DE3) and HB101, and specific examples of Bacillus subtilis strains include WB700 and LKS87. In a specific example of the present invention, BL21 (DE3) as a host cell to prepare a transformant transformed with a vector containing alpha-N-arabinofuranosidase.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention can autonomously replicate in a host, and is composed of a promoter, a ribosome binding sequence, a nucleic acid of the present invention, and a transcription termination sequence have.

The promoter of the present invention may be any promoter as long as the nucleic acid of the present invention is expressed in a host such as Escherichia coli. For example, E. coli infectious phage-derived promoters such as E. coli or phage-derived promoter T7 promoters such as trp promoter, lac promoter, PL promoter or PR promoter can be used. Artificially modified promoters such as the tac promoter may also be used.

In order to facilitate the purification of the target protein recovered in the present invention, the plasmid vector may further include other sequences as necessary. The additional sequence may be a tag sequence for protein purification, for example, glutathione S-transferase (Pharmacia, USA), MBP (Maltose Binding Protein, USA), FLAG (IBI, USA) and hexa histidine hexahistidine; Quiagen, USA), and most preferably MBP. However, the types of sequences necessary for purifying the target protein are not limited by the above examples. In a specific embodiment of the present invention, purification was facilitated using an MBP tag.

Further, in the case of a fusion protein expressed by a vector containing the fusion sequence, it can be purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of the enzyme, can be used. When MBP is used, desired protein can be easily recovered using an amylose column.

The host cell transformed by the above method for expressing the alpha-N-arabinofuranosidase of the present invention can be cultured by a conventional method used in the art. For example, the transformant expressing the above-mentioned alpha-N-arabinofuranosidase can be cultured in a variety of media, and can be fed-batch cultured and continuous cultured, By way of example, the method of culturing the transformant of the present invention is not limited. The carbon source that may be contained in the medium for the growth of the host cell may be appropriately selected according to the judgment of a person skilled in the art depending on the type of the transformant prepared and a suitable culture condition is adopted to control the culture time and amount .

When a suitable host cell is selected and the medium conditions are established, the transformant in which the target protein has been successfully transformed will produce alpha-N-arabinofuranosidase, which is produced according to the constitution of the vector and the characteristics of the host cell Alpha-N-arabinofuranosidase can be secreted into the cytoplasm, periplasmic space, or extracellular space of the host cell. The target protein may also be expressed in soluble or insoluble form.

Proteins expressed in or outside the host cell can be purified in a conventional manner. Examples of purification methods include salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (eg, protein fraction precipitation using acetone or ethanol), dialysis, gel filtration, ion exchange, reverse phase column chromatography Chromatography, ultrafiltration and the like can be used alone or in combination to purify the protein of the present invention.

In the case of alpha-N-arabinofuranosidase isolated by the method described above, the deglycosylated (alpha-N-arabinofuranosidase), which is readily absorbable in an in-vitro or in vivo system in which ginsenosides are present, It can produce ginsenosides.

At least one step in the production of the deglycosylated second ginsenoside from the first ginsenoside, the present invention relates to a process for producing the alpha- N -arabinofuranosidase of the invention, the transformant of the invention or said transformation A culture of sieve can be used.

The first ginenoside used as a starting material in the present invention may be PPD or PPT type ginsenoside and may be separated and purified form of ginsenoside or may be contained in powder or extract of ginseng or red ginseng You can also use the ginsenoside. That is, the method of the present invention may be carried out by directly using ginseng or red ginseng powder or extract containing ginsenoside as a starting material. As the ginseng to be used in the present invention, various known ginsengs can be used, and ginseng such as Panax ginseng , P. quiquefolius , P. notoginseng , P. japonicus , But are not limited to, P. trifolium , P. pseudoginseng , and P. vietnamensis .

Non-limiting examples of PPD (protopanaxadiol) type ginsenosides include the following compounds.

In addition, non-limiting examples of PPT (Protopanaxatriol) type ginsenosides include the following compounds.

The α- N -arabinofuranosidase of the present invention can be selectively hydrolyzed if it is a ginsenoside having a non-reduced α-1,6 arabinofuranosyl moiety, 1, 6 arabinofuranosyl residues of the PPD type or PPT type of ginsenosides, for example, Rc, C-Mc1, and Rc of the PPD type ginsenosides can be selectively hydrolyzed, And C-Mc have a non-reduced α-1,6 arabinofuranosyl moiety at C20 (meaning 20th carbon), and thus the non-reduced α-1 of Rc, C-Mc1 and C-Mc , 6 arabinofuranosyl residues can be selectively hydrolyzed by the? -N -arabinofuranosidase of the present invention.

The α- N -arabinofuranosidase of the present invention can convert PPD (protopanaxadiol) type or PPT type ginsenosides to relatively deglycosylated rare ginsenosides, which can be PPD type or PPT type ginsenosides 1, 6 arabinofuranosyl residues of the side-chain non-reduced α-1,6 arabinofuranosyl residues, more preferably, the non-reduced α-1,6 arabinofuranosyl residues located at the 20th carbon of the PPD type or PPT type ginsenosides Can be carried out by selective hydrolysis of the vinofuranosyl residue.

This conversion is accomplished by bioconversion and is achieved by selective hydrolysis of the non-reduced α-1,6 arabinofuranosyl residues of ginsenosides of PPD or PPT type either continuously or discontinuously .

Preferably, examples of biotransformation by the protein of the present invention include all of the methods of converting ginsenoside Rc to Rd, converting ginsenoside C-Mc1 to F2, and converting ginsenoside C-Mc to CK .

More specifically, in Example 4 of the present invention, when a composition comprising an α- N -arabinofuranocidase of the present invention is administered to ginsenoside Rc, it is deglycosylated with ginsenoside Rd. In addition, when α- N -arabinofuranocidase of the present invention was treated with ginsenoside C-Mc1 as the starting material, it was confirmed that the glycosenoside F2 was deglycosylated. In addition, when α- N -arabinofuranocidase of the present invention was treated with ginsenoside C-Mc as a starting material, it was confirmed that deglycosylation was caused by ginsenoside CK.

The deglycosylation of this PPD or PPT type ginsenoside is due to the selective hydrolysis ability of the α- N -arabinofuranosidase of the present invention to the non-reduced α-1,6 arabinofuranosyl residue, The deglycosylated rare ginsenoside produced through such hydrolysis is easily absorbed into the body. In addition, this bioconversion facilitates the conversion of PPD type or PPT type ginsenosides, such as ginsenoside Rc, to its most deglycosylated form, CK or Rh2.

The α- N -arabinofuranosidase of the present invention can be used as a non-reducing agent for protopanaxadiol type ginsenoside or PP (protopanaxatriol) type ginsenoside at various temperature and pH conditions as long as activity and stability can be maintained The selective hydrolysis of the α-1,6 arabinofuranosyl residues and the conversion of protopanaxadiol (PPD) type or PPT (protopanaxatriol) type ginsenosides into rare ginsenosides in a form that is easily absorbable into the body .

According to a specific embodiment of the present invention, the enzyme characteristics of the present invention were analyzed. As a result, araf3054 exhibited the highest stability and activity at pH 6-10 and highest activity at 37 ° C sodium phosphate buffer pH 7.5 4A). Also, it was stable at a temperature of 37 캜 or less, and lost 100% of its activity after incubation at 45 캜 for 40 minutes. The activity remained about 60% regardless of the temperature at the temperature of 4-35 ° C (FIG. 4B). Therefore, in order for the α- N -arabinofuranosidase of the present invention to function as an enzyme, the pH is preferably adjusted to 6 to 10 and the temperature is preferably adjusted to 4 to 37 ° C.

The α- N -arabinofuranosidase of the present invention can be used in combination with at least one metal selected from the group consisting of MgCl ₂ , EDTA, NaCl, KCl, DTT and β-mercaptoethanol, It does not. In addition, it is preferable to use pNP-alpha-L-arabinofuranoside as a substrate.

On the other hand, in the preparation of the deglycosylated second ginsenoside from the first ginsenoside, the? -N -arabinofuranosidase according to the present invention may be co-administered with other enzyme (s) Can be used. Non-limiting examples of other enzymes include? -Glucosidase,? -Galactosidase, glycosidase,? -L-arabinopyranosidase,? -L-arabinofuranosidase, Lysine, lysine, and alpha-L-lambsinidase. The second ginsenosides prepared by combination with? -N -arabinofuranosidase alone or in combination with other enzymes may be different, and such second ginsenosides may be one or more second ginsenosides Lt; / RTI >

The α- N -arabinofuranosidase according to the present invention may be provided in the same container as the other enzyme (s) or in a different container.

The alpha-N-arabinofuranosidase according to the present invention exhibits excellent activity of converting ginsenosides into deglycosylated rare ginsenosides. In addition, it can be mass-produced by culturing a nucleic acid encoding the above-mentioned alpha-N-arabinofuranosidase or a transformant transformed with a recombinant vector containing the same, thus being industrially versatile.

Figure 1 shows the results of purification of recombinant AbfA. Proteins were separated by 10% SDS-PAGE and visualized by Coomassie blue staining. Lane 1, a water-insoluble fraction lane 2 after induction with IPTG for 12 hours, a water soluble fraction lane 3 after induction for 12 hours, a protein lane 4 after amylose chromatography, a protein lane 5 after DEAE- Protein purified after DEAE-cellulose chromatography and TEV treatment. M: Molecular mass marker.
Figure 2 shows the TLC analysis results of AbfA hydrolysis products using ginsenoside substrates Rc (A), C-Mc1 (B) and C-Mc (C). STD, ginsenoside standard C, enzyme member P, AbfA. All reactions contained 0.5mgl / ml of substrate and were performed at 37 ° C for 24 hours.
Fig. 3 shows the result of analysis of the hydrolyzate of recombinant Araf3054 by HPLC. A. Standard B. Substrate Rc; C. Rc Metabolite (Rd); D. Substrate C-Mc1; E. C-Mc1 Metabolite (F2); F. Substrate C-Mc; G. C-Mc metabolite (CK). All hydrolysis was carried out for 12 hours at an enzyme concentration of 0.01 mg / ml.
Figure 4 shows the effect of pH (A) and temperature (B) on the stability and activity of recombinant Araf3054.
For the pH, an enzyme solution containing 2.0 mM pNP-alpha-L-arabinofuranoside (pNPAf) was incubated in buffers at various pHs between 2 and 10 at 4 ° C for 24 hours after the residual activity was determined. The following buffers (each 50 mM) were tested: KCl-HCl pH 2, glycine-HCl pH 3, sodium acetate pH 4 and 5, sodium phosphate pH 6 and 7, Tris- pH 8 and 9) and glycine-sodium hydroxide (pH 10). The influence of buffer composition on pH effects was also determined using McIlvaine buffer pH 6, 7, 7.5, 8 or 9.
The optimal temperature determination of the enzyme was analyzed in 50 mM sodium phosphate buffer at a temperature ranging from 4 to 90 < 0 > C. Thermal stability analysis was performed by incubating enzyme samples in 50 mM sodium phosphate buffer for 30 minutes at various temperatures. After cooling the sample on ice for 10 minutes, the residual activity was determined.
FIG. 5 is a graph showing the time course of conversion of ginsenoside Rc by recombinant β-glucosidase (BgpA) at an enzyme concentration of 0.1 mg / ml. Metabolites were analyzed by TLC. Line: Reference 0 to 24 h, operating time
6 shows a path (C) for biosynthesizing ginsenoside Rc by Rd (A), C-Mc1 by F2 (B) and C-Mc by CK by recombinant AbfA.
Figure 7 is a TLC analysis showing the substrate specificity of AbfA for various arabino oligosaccharides. 2, 4, 6, and 8 are the results of the reaction with the AbfA of the substance, respectively, and arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose before the reaction. The enzyme concentration was 0.01 mg / ml and reacted for 12 hours.
FIG. 8 is a double reciprocal graph showing the Km and Vmax of the AbfA protein for the substrates p-nitrophenyl-α-L-arabinofuranoside (A) and ginsenoside Rc (B) according to the present invention.
Figure 9 shows pET-MBP-TEV-Araf3054 as a recombinant vector.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1. Chemicals

The ginsenosides Rb1, Rb2, Rc, Rd, F2 and CK were purchased from Dalian Green Bio Ltd (Dalian, China). Ginsenosides C-Mc1 and C-Mc were prepared for this experiment. (X-Glc), pNP-alpha-L-arabinofuranoside (pNPAf) and L-arabinose were obtained from Sigma . Flour Arabi Noja Ilan, red pickled arabinan (RDA), arabinose bios, arabinote rios, arabinotetraose and arabinopentaose were purchased from Megazyme (Wicklow, Ireland). All other chemicals were at least analytical reagent grade and sources were specified in each method section respectively.

Example  2. Fosmid  Library screening and structuring

The strain Gsoil3054 was identified in soil in Pocheon area, Korea. In order to clone the arabinofuranosidase gene that hydrolyzes ginsenosides from Gsoil3054, it is possible to clone the phosphide library structure, the selection of a phosphide clone that exhibits enzymatic activity, the entire-sequence of the phosphide vector, Cloning of the target gene using the chain reaction (PCR) was performed sequentially as previously reported (An, D.-S., C.-H. Cui, H.-G. Lee, L. Wang , SC Kim, S.-T. Lee, F. Jin, H. Yu, Y.-W. Chin, H.-K. Lee, W.-T.Im, and S.-G. Kim. Identification and characterization of a novel Terrabacter ginsenosideimutans sp. nov. b-glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV Appl. Environ. Microbiol. 76: 5827-5836). The Gsoil3054 genomic DNA was isolated by phenol-chloroform extraction and the CopyControl Phosphamide Library Generation Kit (Epicenter, WI) was used to generate the phosphide library according to the manufacturer's protocol. The phosmid clone containing the putative β-glucosidase gene was visually screened through selection of blue colonies on LB plates containing 12.5 μg / ml chloramphenicol and 27 μg / ml X-Glc. Finally, one clone containing the [beta] -glucosidase gene and [alpha] -N-arabinofuranosidase was purified by thin film chromatography (TLC) analysis of the activity of hydrolyzing the ginsenoside Rc Lt; RTI ID = 0.0 > hydrolyzing < / RTI > side.

Example  3. Fosmid  order

The entire sequence of the selected phosmid clone was determined commercially by Macrogen (Seoul, Korea). Briefly, the phosphide DNA was digested with 2-5 kbp fragment, separated by agarose gel electrophoresis, and a Shotgun library was constructed by ligating the blunt end repair DNA with the plasmid vector pCB31. Was introduced into the vector in E. coli ampicillin (100μg / ml), 5- bromo-4-chloro-3-indolyl -β-D- galacto As going pyrano seed (80μg / ml) and isopropyl - β - White colonies on LB agar plates supplemented with D-thiogalactopyranoside (IPTG, 0.3 mM) were selected. A total of 398 reads with an average length of 700 bp was assembled into seven cones using the ABI3730 DNA sequencing system (Applied Biosystems). Six gaps were determined by pre-designed primer walking to obtain a total sequence of 34 kb to complete the sequence analysis.

Example  4. Recombination AbfA of Molecular Cloning , Expression and purification

The assembled DNA sequence was analyzed using the vector NTI (version 10.1) to identify the open reading frames (ORFs) of the putative. These ORFs were identified by BLASTP (http://blast.ncbi.nlm.nih.gov) to identify putative open reading frames for the? -N -arabinofuranosidase gene ( abf A) belonging to GH family 51 /Blast.cgi). In order to express soluble? -N -arabinofuranosidase in E. coli , maltose binding protein (MBP) was fused to the N-terminus of AbfA. Briefly, a DNA fragment coding for the MBP and Tobacco Etch Virus (TEV) protease cleavage site was cloned into the primer mbpF (5'-ATA CAT ATG AAA ATC GAA GAA GGT AAA CTG -3 ') and mbp-tevR (5'-CTC GAA TTC CGA CTG 045GAA GTA GAG ATT CTC TGA AAT CCT TCC CTC GAT CCC GAG GTT G-3' using a) E. coli V. Burley, M. Riley, J. Collado-Vides, JD Glasner, CK Rode, GF Mayhew, J. Gregor, NW (Genomic DNA of E. coli K-12, Escherichia coli K-12) was amplified by PCR from the genomic DNA of E. coli K., Davis, HA Kirkpatrick, MA Goeden, DJ Rose, B. Mau, and Y. Shao. Esposito, D., and DK Chatterjee, 2006. Enhancement of soluble protein expression through the use of fusion tags. Curr Opin. Biotechnol. 17: 353-358). The amplified fragments were digested with NdeI and EcoRI and inserted into the Nde I / EcoR I site of pET21a to generate pET21a-MBP (TEV). AbfA The gene was amplified by PCR using the following primers (italicized Eco RI and Hin dIII restriction sites): abfAF, 5'-CG G AAT TC C GTT GTA AAT TGA TTG CC-3 '; And abfAR, 5'-CCC G AA GCT T TC ACG ACC CCA CAG CCA GG-3 '. The amplified fragments were digested with EcoR I and Hin dIII and then inserted into pET21a-MBP (TEV) at the same position to generate pET21a-MBP - AbfA. pET21a-MBP - AbfA in E. coli Cells harboring the plasmid were cultured on LB medium containing 100 μg / ml ampicillin at 37 ° C and 50 μg / ml floramphenicol. The cells were harvested on a BL21-Codon Plus (DE3) -RIL (Agilent Technologies, Santa Clara, CA) The culture was allowed to grow until the OD reached 0.6 at 600 nm, at which point protein expression was induced by the addition of 0.1 mM IPTG. The culture was further incubated at 18 ° C for 8 hours and centrifuged at 5,000 × g for 20 minutes at 4 ° C. The cell pellet was resuspended in a solution consisting of 20 mM Tris-Cl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenyl methane esulfonyl fluoride and 1% Triton X-100 (pH 7.4) , And cells were ruptured through an ultrasonication (Vibra-cell, Sonics & Materials, CT). Unincorporated cells and debris were removed by centrifugation at 24,000 x g for 40 minutes at 4 ° C to obtain an unpurified cell extract. The MBP labeled fusion protein was purified by affinity chromatography on an amylose resin column (New England BioLabs) followed by DEAE-Cellulose DE-52 chromatography (Whatman). The MBP epitope was removed by incubation with TEV protease, and then the recombinant AbfA was purified by DEAE-cellulose DE-52 chromatography. Protein homology was evaluated by 10% SDS-PAGE and Coomassie blue staining. The purified protein was dialyzed against 50 mM sodium phosphate at pH 7.5 and concentrated to 1 mg / ml using an Amicon Ultra-15 filter (Millipore, CA). The protein was stored at -80 ° C until use. Enzyme analysis and kinetic analysis were performed using proteins purified from 50 mM sodium phosphate at pH 7.5. The molecular mass of the recombinant protein was determined by size exclusion chromatography using a Superose 6 10/300 GL column (GE Healthcare) followed by SDS-PAGE. Protein samples from Bio-Rad (catalog no. 151-1901) were used as reference samples.

Example  5. Ginenoside Enzymatic  Hydrolysis

Rb1, Rb2, Rc, Rd, C-Mc1 and C-Mc were used as substrates and the recombinant proteins were purified for the determination of the specificity and selectivity of AbfA. An enzyme solution at a concentration of 0.01 mg / ml in 50 mM sodium phosphate buffer (pH 7.5) was incubated at 37 ° C with an equal volume of Rc, C-Mc1 or C-Mc solution at a concentration of 0.1% (wt / vol) . The samples were removed regularly at regular intervals. An equal volume of water-saturated n -butanol was added to terminate the reaction and the reactants present in the n -butanol fraction were evaporated to dryness. The residue was dissolved in CH ₃ OH and analyzed using TLC and high performance liquid chromatography (HPLC).

Example  6. Enzyme characterization

The specific activity of purified AbfA was measured using pNPAf as an alternative substrate in 50 mM sodium phosphate buffer at pH 7.5, 37 ° C. The reaction was terminated by adding Na ₂ CO ₃ to a final concentration of 0.5 M and the release of p -nitrophenol was immediately measured using a microplate at 405 nm (Bio-Rad Model 680, Hercules, Calif.). One unit of activity was defined as the amount of protein required to produce 1 μmol of p - nitrophenol per minute. The specific activity was expressed as units per milligram of protein. Protein concentrations were determined using BCA protein assay (Pierce, Rockford, IL) with bovine serum albumin (Sigma) as the standard. All analyzes were performed three times.

6-1. pH  Measuring the Effect of Change

The effect of pH on the enzymatic activity was determined using 2.0 mM pNPAf as substrate in the following buffers (50 mM each): KCl-HCl (pH 2), glycine-HCl (pH 3), sodium acetate and 5), sodium phosphate (pH 6.0 and 7.0), Tris-HCl (pH 8.0, and 9.0) and glycine-sodium hydroxide (pH 10). To determine if the buffer composition affects the enzymatic activity, we also measured enzyme activity in McIlvaine buffer (100 mM citric acid, 200 mM disodium phosphate) at various pHs ranging from 6.0 to 8.0 (Margolles, A , and CG De los Reyes-Gavilan 2003. Purification and functional characterization of a novel α-L-arabinofuranosidase from Bifidobacterium longum B667. Appl. Environ Microbiol. 69: 5096-5103). The pH stability of recombinant AbfA was determined by measuring the enzymatic activity after incubation in each buffer (containing 2.0 mM pNPAf in 50 mM sodium buffer as substrate) for 24 hours at 4 ° C. Results are expressed as percent of activity obtained at optimal pH.

6-2. Measure the effect of temperature change

The effect of temperature on enzymatic activity was tested by incubating the enzyme at various temperatures ranging from 4 to 90 ° C at optimal pH for 5 minutes in 50 mM sodium phosphate buffer containing 2.0 mM pNPAf. The thermal stability of the enzyme was tested by incubating the enzyme in 50 mM sodium phosphate buffer for 30 minutes at various temperatures. After cooling the sample in ice for 10 minutes, the activity was determined using pNPAf as the substrate.

6-3. Measure the effectiveness of metals and chemicals

The effects of metals and other chemicals on AbfA activity were also determined. The AbfA activity was determined by incubation at 37 ° C for 30 minutes with 1 or 10 mM (final concentration) of MnCl ₂ , CaCl ₂ , CoCl ₂ , MgCl ₂ , EDTA, NaCl, KCl, CuCl ₂ , SDS, ZnCl _{2 ,} dithiothreitol ), And [beta] -mercaptoethanol. Residual activity was determined using pNPAf as substrate and expressed as a relative percentage with 100% activity obtained when the compound was absent.

6-4. Substrate specificity measurement

The substrate specificity of the enzyme was tested using the following chromogenic o -nitrophenyl (ONP) and p -nitrophenyl (PNP) -glycosides: PNP-β-D-glucopyranoside, PNP- Beta-D-glucosamides, PNP-beta-L-arabinopyranoside, and PNP-beta-D-galactopyranoside, PNP-beta-D-fucopyranoside, Alpha-L-arabinofuranoside, PNP-alpha-D-xylopyranoside, PNP- alpha -D-glucopyranoside, PNP- alpha -L- arabinofuranoside, Beta-D-glucopyranoside, ONP-alpha-D-xylopyranoside, and PNP- alpha -D-glucopyranoside, beta -D-galactopyranoside, ONP-beta-D-fucopyranoside and ONP- alpha -D-galactopyranoside (Sigma). Glycosidase activity was examined using a 2.0 mM chromogenic substrate defined as one active unit emitting 1 μmol of ONP or PNP per minute for 5 minutes at 37 ° C.

For other arabinoside-containing substrates, the reaction solution contained 0.1 ml of the AbfA solution (1 U) in 2% (wt / vol) flour arabinoselan, RDA, arabinose bios, arabinotriose, arabinotetraose Or arabinopentaose (0.5 ml). The reaction mixture was incubated at 37 ° C. Samples were taken at regular intervals and heated with boiling water for 5 minutes to stop the reaction. The products from flour arabinogaiiran, RDA, arabinose bios, arabinote rios, arabinotetraose and arabinopentaose were analyzed by TLC. Chromatography was carried out on silica gel 60 F ₂₅₄ TLC plates (Merck) as an ascending method. The solvent system consisted of ethyl acetate, acetic acid and water (2: 1: 1). The sugar on the plate was detected by heating at 120 ° C for 10 min and then developed with 5% (vol / vol) sulfuric acid in ethanol (Wong, DWS, VJ Chan, and SB Batt. 2008. Cloning and characterization of a novel exo-a-1,5-L-arabinanase gene and the enzyme Appl. Microbiol. Biotechnol. 79: 941-949).

Kinetic studies were performed with newly purified enzyme using pNPAf (0.05 mM to 5 mM) and ginsenoside Rc (0.1 mM to 2 mM) as substrates. For the former, the absorbance of p -nitrophenol was monitored at 405 nm for 20 min at 37 ° C. For the latter, the hydrolyzed product of ginsenoside Rc was determined as the HPLC analysis in the early stages of hydrolysis. The obtained data were analyzed using the Enzyme Kinetics Program reported by Cleland and the K _m and V _max (Cleland, WW 1979. Statistical analysis of the enzyme kinetic data. Methods Enzymol. 63: 103-138).

Example  7. Gin Senocide  C- Mc1  And C- Mc Preparation and identification

Ginsenosides C-Mc1 and C-Mc are Terrabcter sp. Was obtained by biotransformation of ginsenoside Rc using recombinant? -Glucosidase, BgpA from Gsoil 3082 (An, D.-S., C.-H. Cui, H.-G. Lee, L. et al. Wang, SC Kim, S.-T. Lee, F. Jin, H. Yu, Y.-W. Chin, H.-K. Lee, W.-T. Im, and S.-G. Kim. 2010. Identification and characterization of a novel Terrabacter ginsenosideimutans sp. nov. b-glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV Appl. Environ. Microbiol. 76: 5827-5836). Recombinant BgpA exhibits substrate specificity for the PPD type of ginsenoside having one or two glucose residues at C3 or C20 positions, and the outer glucose at the C3 position, then the inner glucose at the C3 position, and finally the outer glucose at the C20 position Is preferred. Since ginsenoside Rc has a terminal non-reducing α- L -arabinofuranosyl residue at the external site of C20, BgpA is an externally attached to the C3 position of Rc to produce C-Mc1 and C-Mc respectively And only internal residues are hydrolyzed (Figure 2). To prepare the reaction products of BgpA and Rc, a 500 ml reaction mixture containing 1 mg / ml of Rc and 0.1 mg / ml of enzyme was incubated at 37 ° C until all Rc had been converted to Metabolite 1, This was confirmed by TLC. 0.36 g of Metabolite 1 was obtained from this reaction product and 0.2 g of it was added to another round of catalysis (using 1.0 mg / ml of enzyme) to produce 0.15 g of Metabolite 2. This second reaction mixture is water-extracted twice with n- butanol in saturation and in evaporated in vacuo . To further purify the metabolites for nuclear magnetic resonance (NMR) analysis, 100 mg of each metabolite 1 and 2 was recycled with a UV / RI detector and a reversed phase column (ODS, 500 x 20 mm ID, 15 μm) Purification was carried out using a preparative HPLC system (LC-9201, Japan Analytical Instrument, Japan). A CH ₃ CN isocratic solvent system and deionized H ₂ O (7: 3) were used and the detection wavelength was set at 203 nm. While using this method, 42 mg of Metabolite 1 and 33 mg of Metabolite 2 were obtained and added for NMR and mass spectrometry (MS) analysis.

NMR spectral data were recorded on a Varian Unity 500 NMR spectrometer at pyridine. High resolution electrospray ionization mass spectra (HR-ESIMS) were measured on a Waters Q-Tof Premier mass spectrometer.

Example  8. Thin - Layer Chromatography  ( TLC ) Gin Senocide  analysis

TLC was carried out using 60 F ₂₅₄ silica gel plates (Merck, Germany) using CHCl ₃ -CH ₃ OH-H ₂ O (70: 30: 5, vol / vol, lower phase) as solvent. Spots on TLC plates were developed with 10% (vol / vol) H ₂ SO ₄ and then detected by heating at 110 ° C for 5 minutes.

Example  9. High - Performance Liquid Chromatography  ( HPLC ) analysis

HPLC analysis of ginsenoside was performed using an HPLC system (Younglin Co. Ltd, Korea) equipped with four solvent pumps, an automatic injector, and a single wavelength UV detector (model 730D) Peak identification and integration were performed using Younglin's AutoChro 3000 software.

The separation was carried out using a ZORBAX Eclipse XDB-C ₁₈ (with an Agilent XDB C ₁₈ , 5 μm, 12.5 mm x 4.6 mm inner diameter) Column (5 [mu] m, 250 x 4.6 mm inner diameter) (Agilent, USA). After starting the gradient elution with 25% acetonitrile and 75% solvent B, proceed as follows: A 25-32% (vol / vol), 0-10 min A 32 To 55% (vol / vol), 10-15 min A 55 to 60% (vol / vol), 15-20 min A 60 to 100% (vol / vol), 20-25 min A 100% 25-27 minutes A 100 to 25% (vol / vol), 27-30 minutes A 25% (vol / vol), 30-40 minutes. The flow rate was maintained at 1.0 ml / min and detection was performed by monitoring at an absorbance of 203 nm. The injection volume was 25 μl.

result

Example 10. abfA Cloning

Previously, strains of bacteria (named Gsoil3054) that hydrolyzes ginsenosides were identified from soil in ginseng fields in Pocheon, Korea. The strain Gsoil3054 can simultaneously convert the PPD type ginsenosides Rb1, Rb2 and Rc to Rd, which distinguishes Gsoil3054 from other identified ginsenoside metabolism bacteria. Gsoil3054 has been identified as Rhodanobacter ginsenosidimutans by polymorphism to clarify its location (An, DS, HG Lee, ST Lee, and WT Im. 2009. Rhodanobacter ginsenosideimutans sp. nov., isolated from the soil of a ginseng field in South Korea. Int. J. Syst. Evol. Microbiol. 59: 691-694).

Since the Gsoil3054 strain can hydrolyze Rb1, Rb2 and Rc, three types of glucosidase enzymatic activity,? -D-glucosidase,? -L-arabinopyranosidase and / or? Arabinofuranosidase activity. ≪ / RTI > In order to identify and clone each gene for an enzyme that hydrolyzes ginsenosides from Gsoil 3054, we generated a phosphide library and obtained the entire sequence of the phosphide vector. The average insertion size of the phosmid clone was about 40 kb, and the percentage of the clones containing the insert was about 91%. The phosmid clone producing β-D-glucosidase was screened by monitoring the chromogenic degradation of X-Glc, creating a blue region around the colonies growing on the agar medium. Following this initial screening protocol, the inventors reduced the number of selected clones according to the activity of? -L-arabinopyranosidase or? -L-arabinofuranosidase activity. Of the 1400 clones in the phosphamide library, 25 were associated with blue colonies on LB agar plates containing 12.5 μg / ml chloramphenicol and 27 μg / ml X-Glc. One of these fosmid clones was positive for? -D-glucosidase,? -L-arabinopyranosidase, and? -L-arabinofuranosidase activity, and the ginsenosides Rc, C-Mc1 And C-Mc to Rd. The above clones were selected for total sequencing.

Insertion of positive clones was a 34 kb genomic fragment. By analyzing the nucleotide sequence it has been found that it contains 34 putative ORFs. The recombinant enzyme of the first ORF can transform Rb1 and Rb2 into Rd. A second ORF was used for this study.

Example 11. Expression and purification of recombinant AbfA

Presumptive AbfA , which is an α-N-arabinofuranosidase gene, was amplified by PCR and then subjected to MBP gene fusion ( E. coli BL21-CodonPlus (DE3) -RIL) under the control of the IPTG-inducible T7 promoter MBP-AbfA) was subcloned into pET21a-MBP (TEV). In order to maximize the yield of the fusion protein, the present inventors have tested in various induction conditions. Induction with 0.1 mM IPTG at 18 ° C for 8 hours produced the highest level of soluble active fusion protein. The MBP-AbfA fusion protein was purified by amylose resin column followed by DEAE-cellulose DE-52 chromatography and digested with TEV protease to remove MBP residues. Recombinant AbfA was purified by continuous chromatography on a DEAE-cellulose column. This procedure resulted in 18.4-fold purity of AbfA and 57% recovery from crude extract (Table 1). The molecular mass of the native α-N-arabinofuranosidase was 247,000 Da as determined by size exclusion chromatography and 58,000 Da by SDS-PAGE (FIG. 1). The results of SDS-PAGE were consistent with the estimated size based on the translated polypeptide sequence (56,222 Da). These results suggest that α-N-arabinofuranosidase is physiologically active as a tetrameric protein (Canakci, S., AO Belduz, BC Saha, A. Yasar, FA Ayaz, and N. Yayli. 2007 Purification and characterization of a highly thermostable alpha-L-arabinofuranosidase from Geobacillus caldoxylolyticus TK4. Applied Microbiology and Biotechnology 75: 813-820).

Example 12. Enzymatic characterization

12-1. Effect by pH change

AbfA showed activity over a wide pH range (pH 5.0 to 10.0). The optimum pH was pH 7.5 in sodium phosphate buffer (Figure 4A). The enzyme retained its highest activity over 96% at pH 6.0 to 10.0. The enzyme showed residual activity at pH 5.0 and no activity at pH 4.0.

12-2. Effect by temperature change

The best temperature for AbfA activity was 37 ° C. The enzyme was stable at temperatures below 37 ° C, but lost almost 100% of its activity after 30 min incubation at 45 ° C (FIG. 4B).

12-3. Effects of metals and chemicals

The effects of metal ions, EDTA, β-mercaptoethanol and SDS on AbfA activity were also investigated (Table 2). The enzyme was not activated there is a need for Mg ^{+ 2} were significantly inhibited by 1mM of Mn ^{2 +,} Zn ^{2 +} or Cu ^{2 +} and 10 mM of Co ^{2 +} or Ca ^{2 +.} AbfA activity was not affected by DTT or? -Mercaptoethanol, a known thiol group inhibitor. These results suggest that the sulfhydryl group may not be involved in the catalytic center of the enzyme but would be essential for maintaining the three dimensional structure of the active protein. The chelating drug, EDTA, did not inhibit AbfA activity, indicating that divalent cations are not required for enzymatic activity . Enzyme activity was markedly inhibited in the presence of 10 mM SDS.

12-4. Substrate specificity

When the reaction constants of α-N-arabinofuranosidase were calculated from a double reciprocal graph, the K _m for the pNPAf hydrolysis And V _max are respectively 0.53 ± 0.07 mM, and ^{27.1 ± 1.7 μmolmin -1 mg of protein} - 1 and that, for each binary ginsenoside Rc 0.30 ± 0.07 mM, and ^{49.6 ± 4.1 μmolmin -1 mg of protein} - and ^1.

Example 13. Conversion of ginsenoside

AbfA did not react with Rb1 and Rb2 to generate any glucose or arabinopyranos. Of the reported ginsenosides, only Rc, C-Mc1 and C-Mc had terminal, non-reduced alpha -L-arabinofuranosyl residues at the C20 position. In contrast to ginsenoside Rc, C-Mc1 and C-Mc are either absent or present in minor amounts in raw ginseng and can be generated from Rc through hydrolysis of the glycopyranosyl moiety at the C3 position. Since C-Mc1 and C-Mc could not be obtained commercially, the inventors of the present invention have found that the recombinant BgpA, the β-galactosidase which hydrolyzes ginsenosides which specifically and continuously hydrolyze the terminal glycopyranosyl group from C3 of the aglycon, C-Mc1 and C-Mc were prepared using glucosidase (An, D.-S., C.-H. Cui, H.-G. Lee, L. Wang, Lee, W.-T. Im, and S.-G. Kim, 2010. Identification and characterization of a novel Terrabacter ginsenosidimutans glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV Appl. Environ. Microbiol. 76: 5827-5836). Since the terminal, unreduced α-L-arabinofuranosyl residue on Rc is not a substrate for BgpA, only the glucose residues inside and outside the C3 position of Rc are replaced by C-Mc1 (Metabolite 1) and C-Mc (metabolite 2) (Fig. 2), and their chemical structure was confirmed by NMR and MS data analysis. After confirming their chemical structure by NMR and MS analysis, C-Mc1 and C-Mc were used as substrates with Rc for recombinant AbfA. The hydrolysates of the ginsenosides Rc, C-Mc1 and C-Mc were identified at regular intervals by TLC and HPLC analysis. As shown in Figures 3 and 5, the enzyme hydrolyzes the arabinofuranosyl moiety from all three ginsenosides. The metabolites from Rc, C-Mc1 and C-Mc, based on a comparison of the Rs values (determined by TLC) of the standard ginsenosides with the metabolite retention times (determined by HPLC analysis) F2 and CK (An, D.-S., C.-H. Cui, H.-G. Lee, L. Wang, SC Kim, S.-T. Lee, F. Jin, H. Yu , Y.-W. Chin, H.-K. Lee, W.-T. Im, and S.-G. Kim. 2010. Identification and characterization of a novel Terrabacter ginsenosideimutans sp. nov. b-glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV Appl. Environ. Microbiol. 76: 5827-5836, Ko, SR, Y. Suzuki, K. Suzuki, KJ Choi, and BG Cho. 2007. Marked production of ginsenosides Rd, F2, Rg3, and compound K by enzymatic method. Chem. Pharm. Bull. 55: 1522-1527).

Example 14. Structural identification of C-Mc1 and C-Mc

The protonated molecular ion peak corresponding to metabolite 1 was observed with m / z 917 (C ₄₇ H ₈₁ O ₁₇ ) in the ESI MS spectrum of metabolite 1. The ¹ H NMR spectroscopic data of Metabolite 1 indicated two anomeric proton signals belonging to? -Glucose at 4.92 (1H, d, J = 7.8 Hz) and 5.12 And showed an anomeric proton signal belonging to α-arabinofuranos at 5.63 (1H, d, J = 1.6 Hz). The presence of anomeric protons indicated that one glucose unit was removed from the starting material, ginsenoside Rc. The ¹³ C NMR chemical changes of metabolite 1 were closely related to the signal for Mb in the literature (Bae, EA, MK Choo, EK Park, SY Park, HY Shin, and DH Kim, 2002. Metabolism of ginsenoside Rc by human intestinal bacteria and its related antiallergic activity. Biological and Pharmaceutical Bulletin 25: 743-747). This compound was therefore identified as C-Mc1. The ¹ H and ¹³ C NMR data of Metabolite 2 were very similar to the data for C-Mc1 except that there was no signal for the glucose unit and the ¹³ C chemical shift from 88.0 to 78.0 shifted. These differences suggest that glucose is hydrolyzed at the C3 position of C-Mc1 and free hydroxyl groups are present in the structure of Metabolite 2. This was confirmed by the detection of the protonated molecular ion peak at m / z 755 (C ₄₁ H ₇₁ O ₁₂ ) by ESI MS and comparison with the values reported in the literature. Thus, the structure of Metabolite 2 was identified as C-Mc.

Example 15. Substrate specificity of AbfA

Substrate specificity of AbfA was tested using 2.0 mM PNP- and ONP-glycosides with α and β configurations. Β-D-galactopyranoside, PNP-β-D-fucopyranoside, and PNP-N-acetyl-β-D-glucopyranoside were only active against pNPAf. Β-D-xylopyranoside, PNP-α-D-glucopyranoside, PNP-β-D-mannopyranoside, Alpha-D-xylopyranoside, PNP-alpha-L-arabinopyranoside, PNP- alpha -L-rhamphyranoside, Other substrates including -β-D-glucopyranoside, ONP-β-D-galactopyranoside, ONP-β-D-fucopyranoside and ONP- α-D-galactopyranoside It was not hydrolyzed. The decomposition pattern of the polymer containing arabinose revealed that AbfA is only active on the non-reducing terminal arabinofuranosyl residue. In order to identify the mode of action (external-versus-internal-acting) of AbfA, the hydrolysis of oligo- or polysaccharides containing arabinose was tested. The enzyme did not show any activity against RDA, indicating that it does not have endoarabinanase activity. AbfA showed activity with respect to the [alpha] -1, 5-linked arabinose bios, arabinotriose, arabinotetraose, and arabinopentaose. In all cases, only arabinose was detected as the final product and no other residues were detected by TLC (Fig. 7). No activity was detected for the flour arabinoselan containing α-1,2 and α-1,3 arabinofuranosyl residues. These results indicate that AbfA hydrolyzes the L-arabinofuranos residues at the nonreactive terminal α-1,5-linked, but the arabinofuranosyl residues at the nonreactive terminal α-1,2 or α- Lt; / RTI > enzyme that does not hydrolyze the residue. In addition, AbfA can also substitute the terminal non-afferent alpha -L-arabinofuranosyl residues to produce the gensenosides Rd, F2, and CK at the C20 position of ginsenosides Rc, C-Mc1 and C- It will be able to disassemble. In addition to the unique aglycone structure of Rc, C-Mc1 and C-Mc, the terminally non-reducing α-L-arabinofuranosyl moiety of such compounds is called α-1,6-linkage with internal glucose at the C20 position The fact suggests that Rc, C-Mc1 and C-Mc would be a good substrate for investigating the substrate specificity of alpha-arabinofuranosidase. In addition, the metabolites of these ginsenosides can be readily detected by TLC and HPLC. This means that the enzyme has very high specificity for the arabinofuranosyl group and, as opposed to the enzymes reported previously, acts exclusively as an exon-type enzyme. For example, the GH 51 arabinofuranosidase is an Arabinase gene expression in Aspergillus (Flipphi, MJA, J. Visser, P. Van der Veen, and LH De Graaff. niger : Indications for coordinated regulation. 1,2 < / RTI > and < RTI ID = 0.0 > a-1,3 < / RTI > arabinofuranosyl residues from < RTI ID = 0.0 > Glucanase activity (Eckert, K., and E. Schneider. 2003. A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases. Eur. J. Biochem. 270: 3593-3602).

In conclusion, the present inventors have found that the GH family 51, which removes the α-1,6 arabinofuranosyl residues from the ginsenosides Rc, C-Mc1 and C-Mc to produce minor amounts of ginsenosides Rd, F2 and CK, respectively α-N-arabinofuranosidase was cloned and characterized. The enzyme also hydrolyzes the alpha-1,5 arabinofuranosyl moiety from arabinose bios, arabinotriose, arabinotetraose, and arabinopentaose. This is the first report on the cloning of enzymes that convert ginsenosides C-Mc1 and C-Mc to F2 and C-K through the bioconversion of ginsenosides.

Korea Biotechnology Research Institute KCTC22231 20080125

<110> Korea Research Institute of Bioscience and Biotechnology          Korea Advanced Institute of Science and Technology <120> Alpha-N-arabinofuranosidase From Rhodanobacter ginsenosidimutans          And Use Thereof <130> PA110146KR <150> KR10-2010-0117515 <151> 2010-11-24 <150> KR10-2010-0123092 <151> 2010-12-03 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 518 <212> PRT <213> Artificial Sequence <220> <223> Alpha-N-arabinofuranosidase From Rhodanobcter ginsenosidimutans          KCTC22231T <400> 1 Arg Cys Lys Leu Ile Ala Glu Asp Thr Phe Val Thr Gln Pro Gln Pro   1 5 10 15 Ala Ala Ala Lys Ile Thr Ile Asp Pro Ser Phe Thr Val Gly Pro Val              20 25 30 Arg Arg Arg Thr Phe Gly Ala Phe Val Glu His Leu Gly Arg Cys Val          35 40 45 Tyr Thr Gly Ile Phe Glu Pro Gly His Pro Gly Ala Asp Gln Asp Gly      50 55 60 Phe Arg Lys Asp Val Leu Glu Leu Thr Arg Glu Leu Gly Val Thr Thr  65 70 75 80 Val Arg Tyr Pro Gly Gly Asn Phe Val Ser Gly Tyr Arg Trp Glu Asp                  85 90 95 Gly Val Gly Pro Val Asp Gln Arg Pro Val Arg Leu Asp Leu Ala Trp             100 105 110 His Ser Thr Glu Pro Asn Thr Val Gly Val Asp Glu Phe Ala Lys Trp         115 120 125 Ser Ala Lys Ala Gly Val Glu Leu Met Met Ala Val Asn Leu Gly Thr     130 135 140 Arg Gly Ile Gln Glu Ala Leu Asp Leu Leu Glu Tyr Cys Asn Ile Asp 145 150 155 160 Gly Gly Thr Thr Arg Ser Glu Gln Arg Arg Ala Asn Gly Ala Ala Asn                 165 170 175 Gly Tyr Gly Val Thr Met Trp Cys Leu Gly Asn Glu Met Asp Gly Pro             180 185 190 Trp Gln Ile Gly His Lys Asn Ala Leu Glu Tyr Gly Arg Leu Ala Ala         195 200 205 Asp Thr Ala Arg Gly Met Arg Met Ile Asp Pro Gly Leu Glu Leu Val     210 215 220 Ala Cys Gly Ser Ser Ser Ala Met Pro Thr Phe Gly Glu Trp Glu 225 230 235 240 Arg Val Val Leu Thr Glu Thr Tyr Asp Leu Val Asp Leu Ile Ser Ala                 245 250 255 His Gln Tyr Phe Glu Asp Thr Gly Asp Leu Gln Glu His Leu Ala Ala             260 265 270 Gly His Lys Met Glu Ala Phe Ile His Asp Leu Val Ser His Ile Asp         275 280 285 His Val Arg Ser Ala Arg Lys Ser Ser Arg Gln Val Asn Ile Ser Phe     290 295 300 Asp Glu Trp Asn Val Trp His Met Ser Ser Glu Ala Ser Arg Val Pro 305 310 315 320 Thr Gly Lys Asp Trp Pro Val Ala Pro Ala Leu Leu Glu Asp Ser Tyr                 325 330 335 Thr Val Ala Asp Ala Val Val Gly Asp Leu Leu Leu Thr Leu Leu             340 345 350 Arg Asn Thr Asp Arg Val His Ser Ala Ser Leu Ala Gln Leu Val Asn         355 360 365 Val Ile Ala Pro Ile Met Thr Glu Pro Gly Gly Arg Ala Trp Lys Gln     370 375 380 Thr Thr Phe His Pro Phe Ala Leu Thr Ser Arg His Ala Ser Gly Thr 385 390 395 400 Val Leu Gln Leu Ala Val Glu Ser Pro Pro Val Ser Gly Gly Thr Thr                 405 410 415 Ala Asp Phe Ala Ala Leu Ser Ala Val Ala Thr Phe Asp Arg Glu Lys             420 425 430 Gly Glu Ala Val Leu Phe Ala Val Asn Arg Ser Ala Arg Gln Ala Leu         435 440 445 Thr Leu Asp Ala Val Gly Ala Leu Gly Thr Met Val Val Leu Glu     450 455 460 Ala Val Thr Tyr Ala Asn Lys Asp Pro Tyr Trp Gln Ala Ser Ala Asp 465 470 475 480 Asp Ser Thr Ser Val Leu Pro Ser Gly Asn Ala Thr Val Lys Ala Asp                 485 490 495 Asp Gly Arg Leu Thr Ala Glu Leu Pro Ala Val Ser Trp Ser Met Ile             500 505 510 Arg Leu Ala Val Gly Ser         515 <210> 2 <211> 1557 <212> DNA <213> Artificial Sequence <220> <223> Alpha-N-arabinofuranosidase From Rhodanobcter ginsenosidimutans          KCTC22231T <400> 2 cgttgtaaat tgattgccga ggataccttc gtgacccagc cacagcccgc cgcagcaaaa 60 atcacaattg atccgtcgtt caccgtgggt cccgtccgtc gtcgtacgtt tggcgcgttc 120 gttgagcacc ttggccggtg cgtgtacacc gggatctttg aacccgggca cccgggggcc 180 gaccaggacg gcttccgcaa ggacgtcctg gaactgaccc gcgagctggg cgtcaccacc 240 gtccgctacc cgggcggcaa cttcgtctcg ggctaccgct gggaggacgg cgtgggtccg 300 gtggaccagc gtcccgtccg cctggacctg gcctggcatt cgactgaacc caacaccgtg 360 ggcgtggacg agttcgccaa atggtccgcc aaggcgggcg tggaactgat gatggccgtg 420 aacctgggca cgcgcggcat ccaggaggca ctggacctcc tggagtactg caacatcgac 480 ggcgggacaa cccgctccga acaacgccgc gccaacgggg ccgccaacgg ctacggcgtc 540 accatgtggt gcctgggcaa cgagatggac ggcccgtggc agatcggcca caagaacgcc 600 ctcgaatacg gcaggctggc cgcggacacc gcccgcggaa tgcgcatgat cgaccccggc 660 ctggagctcg tggcctgcgg cagctccagc cccgccatgc ccacctttgg cgagtgggag 720 cgtgtggtcc tcaccgaaac ctatgacctg gtggacctca tttccgcgca ccagtacttc 780 gaggacaccg gcgacctgca ggaacacctg gccgccgggc acaagatgga agccttcatc 840 cacgacctcg tgagtcacat cgaccatgtg agatcagcca ggaaatccag ccggcaggtg 900 aatatctcct tcgacgaatg gaacgtctgg cacatgagcc gcgaggcgtc cagggtcccc 960 accggcaagg actggccggt ggcacccgcc ctgctggagg acagctacac ggtggcggat 1020 gccgtggtgg tgggcgacct gctgctcacg ctgctccgga acaccgaccg cgtccactcg 1080 gccagcctgg cgcagctggt gaacgtgatc gcacccatca tgaccgaacc cggcggccgg 1140 gcctggaagc agaccacctt ccacccgttc gccctgacct cgcggcacgc atccggcacg 1200 gtgctgcagc tcgccgtcga atccccccct gtcagcggag gtacgacggc cgacttcgcc 1260 gccctgtccg ccgtcgccac gtttgaccgg gaaaagggcg aggccgtgct gttcgccgtg 1320 aaccgttcgg cgcgccaggc gctcaccctc gacgccgcag tgggtgccct aggtaccatg 1380 cgcgtgctgg aagcggtgac ctacgccaac aaggacccct actggcaggc cagcgccgat 1440 gactccactt ccgtcctgcc ttccggaaac gccacggtaa aggccgacga cgggcggctc 1500 accgctgagc tcccggcagt ctcctggtcc atgatccgcc tggctgtggg gtcctga 1557

Claims (21)

N-arabinofuranosidase having an amino acid sequence consisting of SEQ ID NO: 1. delete delete The method according to claim 1, wherein the α- N -arabinofuranosin having a selective hydrolysis ability to an α-1,6 arabinofuranosyl residue located at the 20th carbon of protopanaxadiol type ginsenoside Shidaje. A nucleic acid encoding the α- N -arabinofuranosidase of claim 1. 6. The nucleic acid according to claim 5, wherein the nucleic acid has the nucleotide sequence set forth in SEQ ID NO: 2. A recombinant vector comprising the nucleic acid of claim 5. 8. The recombinant vector according to claim 7, wherein said vector is pET-MBP-TEV-Araf3054 and has a cleavage map as shown in Fig. A transformant transformed with the nucleic acid of claim 5 or a recombinant vector comprising said nucleic acid. 10. The transformant according to claim 9, wherein the transformant is E. coli. (a) culturing the transformant of claim 9,
(b) producing? -N -arabinofuranosidase from the cultivated transformant, and
(c) recovering the produced? -N -arabinofuranosidase
N -arabinofuranosidase. &Lt; / RTI &gt; Paragraph (1) α- N - the arabinose Pew pyrano let claim, claim 9, or the transformant utilizing the culture of the transformant-arabino Pew pyrano let claim, the claim α- N produced by 11 A method for producing a deglycosylated second ginsenoside from a first ginsenoside, including 13. The method of claim 12, wherein the first ginsenoside is a protopanaxadiol (PPD) type or a protopanaxatriol (PPT) type ginsenoside. 13. The method according to claim 12, wherein the non-reduced alpha-1,6 arabinofurano at the 20th carbon of PPD (protopanaxadiol) type or PPT (protopanaxatriol) type ginsenoside by alpha -N -arabinofuranosidase Lt; RTI ID = 0.0 &gt; hydrolysis &lt; / RTI &gt; 13. The method according to claim 12, wherein the conversion of ginsenoside Rc to ginsenoside Rd, conversion of C-MC1 to ginsenoside F2, and conversion of ginsenoside C-MC to CKs by alpha -N -arabinofuranosidase, &Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; 13. The method according to claim 12, wherein the pH is adjusted to 6 to 10 and the temperature is adjusted to 4 to 37 DEG C so that? -N -arabinofuranosidase can act as an enzyme. The method according to claim 12, wherein the α- N -arabinofuranosidase is pNP-α-L-arabinofuranoside (pNP-α-L-arabinofuranoside). The method according to claim 12, characterized in that the enzymatic action of? -N -arabinofuranosidase is carried out with one or more substances selected from the group consisting of MgCl ₂ , EDTA, NaCl, KCl, DTT and beta-mercaptoethanol Lt; / RTI &gt; 13. The pharmaceutical composition according to claim 12, which is at least one selected from the group consisting of? -Glucosidase,? -Galactosidase, glycosidase,? -L-arabinopyranosidase,? -L-arabinofuranosidase, An enzyme, and an alpha-L-lambsosidase. With arabinose Pew pyrano let claim, claim claim 9, transformant or the culture of the transformant of the active ingredient - of claim 1 α- N - arabinose Pew pyrano let claim, the claim α- N produced by 11 A kit for the conversion of a first ginsenoside of a protopanaxadiol (PPD) type or a protopanaxatriol (PPT) type to a deglycosylated second ginsenoside, comprising: 21. The method of claim 20, wherein in the same or another container is added at least one compound selected from the group consisting of? -Glucosidase,? -Galactosidase, glycosidase,? -L-arabinopyranosidase,? -L-arabinofuranosidase, wherein the kit further comprises at least one selected enzyme selected from the group consisting of? -glucosidase,? -glucosidase, and? -L-lambosidase.

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