WO2012070724A1 - Alpha-n-arabinofuranosidase derived from rhodanobacter ginsenosidimutans and uses thereof - Google Patents

Abstract

The present invention relates to an alpha-N-arabinofuranosidase derived from Rhodanobacter ginsenosidimutans and uses thereof, wherein the protein has an activity of converting PPD(protopanaxadiol)- or PPT(protopanaxatriol)-type ginsenosides into deglycosylated rare ginsenosides capable of in vivo absorption by selectively hydrolyzing the á-1,6 arabinofuranosyl residues of ginsenosides.

Description

Alpha-N-Arabinofuranosidase from Rhodanobacter ginsenocidimutans and uses thereof

The present invention relates to alpha-N-arabinofuranosidase and its use derived from Rhodanobacter ginsenosidimutans .

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

Saponin means a substance consisting of several cyclic compounds in the glycosides widely present in the plant system, and triterpene saponin, a saponin component included as a main bioactive component in ginseng or red ginseng, is used in other plants. Since ginseng saponins are different from other saponins, they are called ginsenosides in the sense of Ginseng glycosides to distinguish them from other plant-based 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 ginsenosides comprises one to three residues, ie, glycofyranosyl, arabinofyranosyl, arabinofuranosyl, xylopyranosyl, or rhamnopyranosyl groups, attached at specific positions to the main backbone. It consists of a tetracyclic triterpene dammarane or pentacyclic oleanane skeleton. Damaren type ginsenosides are mainly classified according to their intrinsic aglyconic residues: protopanaxanadiol (PPD), protopanasatriol (PPT), and ocotillol.

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 US ginseng ( Panax notoginseng ) contains 13 PPD type ginsenosides and 5 PPT types. Ginsenosides (Choi, KT 2008. Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng CA Meyer. Acta Pharmacol. Sin. 29: 1109-1118). In certain species of Panax , the composition of ginsenosides also varies in other parts of the ginseng body such as roots, leaves, and berries (Wang, CZ, JA Wu, E. McEntee, and CS Yuan. 2006. Saponins composition in American ginseng leaf and berry assayed by high-performance liquid chromatography.J. Agric.Food Chem. 54: 2261-2266).

In general, the effectiveness of ginsenosides increases with the degree of deglycosylation, enhancing their hydrophobicity and cell wall permeability (Shibata, S. 2001. Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds. Korean Med.Sci. 16 Suppl: S28-37). However, deglycosylated ginsenosides are traces in ginseng extracts, whereas Rb1 {3- O- [β-D-glycopyrronosyl- (1-2) -β-D-glycopyranosyl] -20- O -[β-D-glycopyranosyl- (1-6) -β-D-glycopyranosyl] -20 ( S ) -protopanaxadianiole}, Rb2 {3- O- [β-D-glycopyra Nosyl- (1-2) -β-D-glycopyranosyl] -20- O- [α-L-arabinopyranosyl- (1-6) -β-D-glycopyranosyl] -20 ( S ) -Protopanaxadiol-based}, Rb3 {3- O- [β-D-glycopyranosyl- (1-2) -β-D-glycopyranosyl] -20- O- [β-D-xylpyra Nosyl- (1-6) -β-D-glycopyranosyl] -20 ( S ) -protopanaxadiandiol}, Rc {3- O- [β-D-glycopyranosyl- (1-2)- β-D-glycopyranosyl] -20- O- [α-L-arabinofuranosyl- (1-6) -β-D-glycopyranosyl] -20 ( S ) -protopanaxadiol type} , And Rd {3- O- [β-D-glycopyranosyl- (1-2) -β-D-glycopyranosyl] -20- O- β-D-glycopyranosyl-20 ( S ) -proto And panaxanaid system Larger ginsenosides of the same PPD class constitute more than two thirds of the ginsenosides in the ginseng extract.

Ginsenoside Rc is one of the major PPD-type ginsenosides and accounts for 7-22% of all 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). Four sugar residues are attached to the Dmarene aglycone, the main skeleton of Rc. Α-L-arabinofuranosyl- (1-6) -β-D-glucopyranose is C20 (carbon at position 20) Β-D-glycopyranosyl- (1-2) -β-D-glucopyranose is bonded to C3 (carbon at third position) of the damarene aglycone. Ginsenoside Rc has a high molecular weight and low permeability in passing through cell membranes, so deglycosylation is required to exhibit high pharmacological activity. To date, no genetic information and detailed biochemical properties have been disclosed for arabinofuranosidase that hydrolyzes arabinofuranosyl residues from ginsenoside Rc. Although α-L-arabinofuranosidase capable of hydrolyzing the arabinofuranose residues of Rc to produce Rd has been reported from intestinal bacteria, its sequence information is not fully understood (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 Rc.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 needed. Steaming (heating) (Wang, CZ, B. Zhang, WX Song, A. Wang, M. Ni, X. Luo, HH Aung, JT Xie, R. Tong, TC He, and CS Yuan. 2006. Steamed American 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 by Physicochemical processes such as acids and human intestinal bacteria and biological activities of their transformants.Arch. Pharm.Res. 27: 61-67) were developed to convert the major components of ginsenosides into deglycosylated ginsenosides. It became. However, the most preferred method is a bioconversion method in which the enzymes with known activities are sequentially reacted with a desired plan or a combination of two or more enzymes with known activities, according to a preliminary plan. Avoiding such problems and producing known, defined products (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). Recently, some progress has been made to identify wild-type enzymes that can convert large molecular weight ginsenosides into rare low molecular weight ginsenosides from molds and bacteria, and to characterize them for industrial use. there was. However, these studies faced two limitations: 1) little progress was made regarding the discovery of the biochemical properties of ginsenoside bioconverting enzymes due to the lack of sequence information on enzymes, and 2) culturing wild-type microorganisms and wild-type. The method of purifying enzymes seems to lack economic feasibility.

Recently, sequence information of three enzymes showing ginsenoside bioconversion activity from microorganisms has been revealed and identified as enzymes belonging to the glycoside hydrolase family: (1) Rb1 or Rc is represented by Compound (C Thermostable β-glucosidase (Noh, KH, JW Son, HJ Kim, conversion to) -K [20- O- (β-D-glycopyranosyl) -20 ( S ) -protopanaxadiol system] 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 metagenome 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) Rb1 to CK, Rb2 to CK, and Rc to C-Mc {20- O- [α-L-arabinofuranosyl- (1-6) -β-D-glycopyranosyl] -20 (S) -. Prototype wave incident β- galactosidase yidayi let go from the Sulfolobus acidocaldarius switching to claim olgye} (Noh, KH, and DK Oh 2009. Production of the rare ginsenosides compound K, compound Y, and compound Mc by a thermostable β-glycosidase from Sulfolobus acidocaldarius.Biol . Pharm. Bull. 32: 1830-1835). Rb1 is Gypenoside XVII {3- O- β-D-glycopyranosyl-20- O- [β-D-glycopyranosyl- (1-6) -β-D-glycopyranosyl] -20 ( S )- Protopanaxidaiol system}, followed by Gypenoside LXXV {20- O- [β-D-glycopyranosyl- (1-6) -β-D-glycopyranosyl] -20 ( S ) -protopanaxadie Enzymes of GH family 3 that catalyze the continuous conversion to all systems and finally to CK have recently been reported by the inventors (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 ginsenosidimutans sp.nov.β -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), having the activity of converting Rc to C-Mc, and Rc to C-Mc, Β-glucosidase from Sulfolobus solfataricus (Noh, KH, JW Son, HJ Kim, and DK Oh. 2009. 73: 316-321), which then converts to CK, mainly hydrolyzes the glucose residues on C3 of aglycone. It has been shown to have different properties from arabinofuranosidase.

We cloned and characterized a novel GH family 51 arabinofuranosidase from the soil bacterium Rhodanobacter ginsenocidimutans strain Gsoil3054, isolated and identified in ginseng fields. As a result, the recombinant enzyme was overexpressed in Escherichia coli and showed bioconversion activity against ginsenoside Rc, and was found to convert Rc to Rd through hydrolysis of the arabinofuranoside residue at the 20th carbon position of aglycone. . In addition, the recombinant enzyme has an exo-acting arabinofuranosidase activity that acts on the end of the polysaccharide, and found that it acts only on the arabinofuranoside residues of ginsenoside Rc or the like, and completed the present invention. It was.

A first aspect of the present invention provides α-N-arabinofuranosidase 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.

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

A fourth aspect of the invention comprises the steps of (a) culturing a transformant according to the invention (b) producing α- N -arabinofuranosidase from the cultured transformant and (c) the It provides arabinose Pew pyrano let the production process of-the production of α- N - arabinose Pew pyrano Let α- N comprises the number of times a.

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 A method of preparing a deglycosylated second ginsenoside from a first ginsenoside is provided, including using a culture of a sieve.

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 Provided is a kit for converting a ginsenoside of PPD or PPT type to deglycosylated rare ginsenoside, comprising a culture of a sieve as an active ingredient.

Alpha-N-arabinofuranosidase according to the present invention exhibits excellent activity of converting ginsenosides to deglycosylated rare ginsenosides. In addition, by culturing a transformant transformed with a nucleic acid encoding the alpha-N-arabinofuranosidase or a recombinant vector comprising the same, it can be mass-produced and variously used industrially.

1 shows the results of purification of recombinant AbfA. Proteins were separated using 10% SDS-PAGE and visualized through Coomassie blue staining. Lane 1, insoluble fraction after induction for 12 hours with IPTG lane 2, water soluble fraction after induction for 12 hours lane 3, purified protein lane 4 after amylose chromatography, purified protein lane 5 after DEAE-cellulose chromatography, Protein purified after DEAE-cellulose chromatography and TEV treatment. M: molecular mass marker.

2 shows the results of TLC analysis of AbfA hydrolysis products using ginsenoside substrates Rc (A), C-Mc1 (B) and C-Mc (C). Reacts with STD, Ginsenoside Standard C, Enzyme-free P, AbfA. All reactions included 0.5 mgl / ml of substrate and were performed at 37 ° C. for 24 hours.

3 shows the results of HPLC analysis of the hydrolyzate of recombinant Araf3054. A. Standard B. Substrate Rc; C. Rc metabolites (Rd); D. Substrate C-Mc1; E. C-Mc1 metabolite (F2); F. Substrates C-Mc; G. C-Mc Metabolites (C-K). All hydrolysis was performed for 12 hours at enzyme concentration of 0.01 mg / ml.

4 shows the effect of pH (A) and temperature (B) on the stability and activity of recombinant Araf3054.

For pH, enzyme solutions comprising 2.0 mM pNP-α-L-arabinofuranoside (pNPAf) were incubated in buffers at various pHs of pH 2 to 10 for 24 hours at 4 ° C. after residual activity was determined. The following buffers (50 mM each) were tested: KCl-HCl (pH 2), glycine-HCl (pH 3), Nat acetate (pH 4 and 5), Sodium Phosphate (pH 6 and 7), Tris-HCl ( pH 8 and 9) and glycine-sodium hydroxide (pH 10). The influence of buffer composition on pH influence was also determined using McIvavaine buffer pH 6, 7, 7.5, 8 or 9.

Determination of the optimal temperature of the enzyme was analyzed in 50 mM sodium phosphate buffer at temperatures ranging from 4 to 90 ° C. Thermostability analysis was performed by incubating enzyme samples in 50 mM sodium phosphate buffer at various temperatures for 30 minutes. After cooling the sample on ice for 10 minutes, the remaining activity was determined.

5 shows the bioconversion of ginsenoside Rc by BgpA (recombinant β-glucosidase) at an enzyme concentration of 0.1 mg / ml over time. Metabolites were analyzed by TLC. Line: reference 0 ~ 24 h, operating time

FIG. 6 shows the pathway (C) for bioconversion of ginsenoside Rc to Rd (A), C-Mc1 to F2 (B) and C-Mc to C-K by recombinant AbfA.

7 is a TLC analysis showing the substrate specificity for various arabino oligosaccharides of AbfA. 1, 10 are standard arabinose, 2, 4, 6, 8 are arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose before the reaction, and 3, 5, 7, 9 are the results after the reaction with AbfA of the substance, respectively. The enzyme concentration was 0.01 mg / ml and reacted for 12 hours.

8 is a double reciprocal graph showing the Km and Vmax of AbfA proteins for the substrates p-nitrophenyl-α-L-arabinofuranoside (A) and ginsenoside Rc (B) according to the present invention.

9 shows the recombinant vector pET-MBP-TEV-Araf3054.

Hereinafter, the present invention will be described in detail.

We first purified and characterized α- N -arabinofuranosidase from Rhodanobacter.

Specifically, the novel enzymes obtained from Rhodanobacter ginsenosididimutans KCTC22231 ^T according to the present invention are α-1,6 arabinofuranosyl residues of ginsenosides or α of oligosaccharides or polysaccharides containing arabinose. Α- N -arabinofuranosidase having selective hydrolytic ability to -1,5 arabinofuranosyl residues, more preferably having the following properties:

a) has a selective hydrolysis capacity for α-1,6 arabinofuranosyl residues located at the 20th carbon of the protopanaxadiol (PPD) type ginsenoside.

b) through the selective hydrolysis, it has a conversion activity capable of converting ginsenosides of the PPD type to deglycosylated rare ginsenosides.

c) exo-acting enzymes.

The 'externally-acting enzyme' refers to one having the activity of hydrolyzing glycoside bonds from the ends of the polysaccharides.

Α- N -arabinofuranosidase obtained from Rhodanobacter ginsenosididimutans KCTC22231 ^T according to the present invention is registered under GenBank accession number HQ026482. The registered α- N -arabinofuranosidase of the present invention consists of 518 amino acids, and the nucleic acid encoding the protein is 1557 bp in length.

Rodanobacter ginsenocidi mutans KCTC22231 ^T (= KACC 12822 ^T = DSM 21013 ^T = LMG 24457 ^T ) Microorganisms are isolated from ginseng fields in Pocheon, Korea and deposited on January 25, 2008 with the Korea Research Institute of Bioscience and Biotechnology. It was.

The amino acid sequence which comprises (alpha) -N -arabinofuranosidase derived from Rodanobacter ginsenocidimutans KCTC22231 ^T is the sequence shown by SEQ ID NO: 1. Α- N -arabinofuranosidase according to the present invention is an amino acid sequence having at least 70% similarity to the sequence described in SEQ ID NO: 1 as well as the sequence, preferably at least 80% similarity, more preferably 90% Amino acid sequences exhibiting the above similarities, even more preferably at least 95% similarity, most preferably at least 98% similarity, include proteins having substantially the activity of alpha-N-arabinofuranosidase. Further, if the sequence having such similarity is an amino acid sequence having a biological activity substantially the same as or corresponding to that of alpha-N-arabinofuranosidase, a protein variant having an amino acid sequence in which some sequences are deleted, modified, substituted or added It is obvious that it is included in 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 set forth in SEQ ID NO: 1, preferably the nucleic acid set forth in SEQ ID NO: 2, as well as the sequence A sequence having at least 70% similarity with the sequence, preferably at least 80% similarity, more preferably at least 90% similarity, even more preferably at least 95% similarity, most preferably at least 98% similarity The protein encoded by the nucleic acid includes 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 inventors araf3054 (hereinafter, interchangeably with 'ARAF3054' and 'AbfA').

The term 'similarity' of the present invention is intended to represent a degree of similarity with the amino acid sequence of the wild type protein or the nucleotide sequence encoding the same, and the same sequence as the amino acid sequence or nucleotide sequence of the present invention or more Branches include sequences. This similarity can be compared with the naked eye or using 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 artificial modifications are equivalent proteins so long as they retain a certain level of similarity and retain the activity of the protein of interest in the present invention. Thus, the proteins of the present invention include wild-type amino acid sequence variants and nucleotide sequence variants, wherein the 'variant' refers to the deletion or insertion, non-conservation or preservation of one or more amino acid residues or nucleotide sequences relative to a native amino acid sequence or nucleotide sequence. By protein substitution or combination thereof, it is meant a protein having a different sequence or a nucleic acid having a different sequence.

As used herein, the term 'vector' refers to a nucleic acid construct that is an expression vector capable of expressing a protein of interest in a suitable host cell and that includes essential regulatory elements operably linked to express the nucleic acid insert. The present invention can produce a recombinant vector comprising a nucleic acid encoding alpha-N-arabinofuranosidase.

Meanwhile, according to 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, thereby producing α- of the present invention. N -arabinofuranosidase can be obtained. In a specific embodiment of the present invention, while screening through a fosmid library, an ORF (open reading frame) capable of encoding alpha-N-arabinofuranosidase is identified and inserted into the pCC1FOS expression vector. By doing so, the recombinant vector pET-MBP-TEV-Araf3054 was prepared (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 and the like can 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 Escherichia coli derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis -derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50). There is). Specific examples of phage DNA include λ-phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP). In addition, animal viruses such as retroviruses, adenoviruses or vaccinia viruses, insect viruses such as baculoviruses can also be used. In a specific embodiment of the present invention, a recombinant vector pET-MBP-TEV-Araf3054 was prepared by inserting into a pCC1FOS expression vector.

In addition, as a vector of the present invention, a fusion plasmid (eg, pJG4-5) to which a nucleic acid expression activating protein (eg, B42) is linked may be used, and the fusion plasmid may be GST, GFP, His- tag, Myc-tag and the like, but the fusion 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 into a vector, a method of cutting the purified DNA with a suitable restriction enzyme and inserting it into a restriction site or a cloning site of the appropriate vector DNA 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 arrows.

The nucleic acid of the present invention is preferably operably linked to a vector. In addition to the promoter and the nucleic acid of the present invention, the vector of the present invention includes a cis element such as an enhancer, a splicing signal, a poly A addition signal, and a selection marker. ), A ribosome binding sequence (ribosome binding sequence, SD sequence) and the like can be further included. As an example of a selection marker, chloramphenicol resistance nucleic acid, ampicillin resistance nucleic acid, dihydrofolate reductase, neomycin resistance nucleic acid, and the like may be used, but the additional components that are operably linked by the above examples are limited. no.

As used herein, the term 'transformation' means that DNA is introduced into a host so that the DNA can be reproduced as a factor of a chromosome or by completion of chromosome integration. It means a phenomenon causing change.

As the transformation method of the present invention, any transformation method may be used, and may be easily performed according to conventional methods in the art. In general, the Hanahan method, the electroporation method, the calcium phosphate precipitation method, the plasma fusion method, the silicon carbide, which have improved efficiency by using a CaCl ₂ precipitation method and a reducing material called DMSO (dimethyl sulfoxide) in the CaCl ₂ method Agitation with fibers, agrobacterial mediated transformation, transformation with PEG, dextran sulfate, lipofectamine and dry / inhibition mediated transformation.

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

The 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 comprising the same into a host.

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

Specific examples of E. coli strains that can be used in the present invention are CL41 (DE3), BL21 (DE3) and HB101, specific examples of Bacillus subtilis strains are WB700 and LKS87, in a specific embodiment of the present invention E. coli cells A transformant transformed with a vector containing alpha-N-arabinofuranosidase was prepared using BL21 (DE3) as a host cell.

When a bacterium such as E. coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the host and consists of a promoter, a ribosomal binding sequence, a nucleic acid of the present invention, and a transcription termination sequence. have.

The promoter of the present invention can be used as long as it allows expression of the nucleic acid of the present invention in a host such as E. coli. For example, E. coli or phage-derived promoters such as the trp promoter, lac promoter, PL promoter or PR promoter or E. coli infection phage-derived promoters such as the T7 promoter can be used. Artificially modified promoters can also be used, such as the tac promoter.

In order to facilitate purification of the protein of interest recovered in the present invention, the plasmid vector may further comprise other sequences as necessary. The sequence which may be further included may be a tag sequence for protein purification, such as glutathione S-transferase (Pharmacia, USA), MBP (maltose binding protein, USA), FLAG (IBI, USA) and hexahistidine ( hexahistidine; Quiagen, USA) and the like, and most preferably MBP, but the examples do not limit the type of sequence required for purification of the target protein. In a specific embodiment of the present invention, purification is facilitated by using an MBP tag.

In addition, 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, and when MBP is used, a desired target protein can be easily recovered by using an amylose column.

Host cells transformed by the above method to express the alpha-N-arabinofuranosidase of the present invention can be cultured by conventional methods used in the art. For example, the transformant expressing the alpha-N-arabinofuranosidase may be cultured in various media, and may be fed-batch culture and continuous culture. By way of example, the method for culturing the transformant of the present invention is not limited. In addition, the carbon source that may be included in the medium for the growth of the host cell may be appropriately selected according to the judgment of those skilled in the art according to the type of transformant produced, and appropriate culture conditions may be adopted to control the timing and amount of culture. Can be.

By selecting the appropriate host cell and formulating the medium conditions, the transformant which is successfully transformed with the target protein produces alpha-N-arabinofuranosidase, which is produced according to the composition of the vector and the characteristics of the host cell. The alpha-N-arabinofuranosidase may be secreted into the cytoplasm of the host cell, into the periplasmic space or extracellularly. The protein of interest 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 with acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, reverse phase column chromatography. Techniques such as chromatography and ultrafiltration may be applied alone or in combination to purify the proteins of the invention.

Deglycosylated, easily absorbed in an in-vitro or in vivo system with ginsenosides using alpha-N-arabinofuranosidase isolated by the above method Ginsenosides can be produced.

In the preparation of a deglycosylated second ginsenoside from the first ginsenoside, in at least one step the present invention is characterized in that the α- N -arabinofuranosidase of the invention, the transformant of the invention or the transformation Sieve culture can be used.

The first ginsenoid used as a starting material in the present invention may be a ginsenoside of PPD type or PPT type, may use a separated and purified form of ginsenoside, or may be included in powder or extract of ginseng or red ginseng. You can also use ginsenosides. That is, the method of the present invention may be carried out using a powder or extract of ginseng or red ginseng including ginsenosides as a starting material. As the ginseng used in the present invention, various known ginsengs can be used, and Korean ginseng ( Panax ginseng ), Gyegi ( P. quiquefolius ), Jeonchisam ( P. notoginseng ), Bamboo ginseng ( P. japonicus ), and three leaf ginseng ( P. trifolium ), Himalayan ginseng ( P. pseudoginseng ) and Vietnamese ginseng ( P. vietnamensis) , but are not limited thereto.

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

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

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[Revision under Rule 26 03.05.2011]

The α- N -arabinofuranosidase of the present invention may selectively hydrolyze all ginsenosides having non-reduced α-1,6 arabinofuranosyl residues, and preferably all known in the art Non-reduced α-1,6 arabinofuranosyl residues of ginsenosides of PPD type or PPT type can be selectively hydrolyzed, for example, Rc, C-Mc1, And non-reduced α-1 of Rc, C-Mc1, and C-Mc because C-Mc has a non-reduced α-1,6 arabinofuranosyl residue at C20 (meaning the 20th carbon) The, 6 arabinofuranosyl moiety can be selectively hydrolyzed by the α- N -arabinofuranosidase of the present invention.

Α- N -arabinofuranosidase of the present invention can convert protopanaxadiol (PPD) type or PPT type ginsenosides into relatively deglycosylated rare ginsenosides, which are PPD type or PPT type ginsenosides. Selectively hydrolyzes the non-reduced α-1,6 arabinofuranosyl residues of the side, more preferably the non-reduced α-1,6 ara located at the 20th carbon of the PPD type or PPT type ginsenoside It can be carried out by selectively hydrolyzing nonfuranosyl residues.

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

Preferably, examples of bioconversion by the protein of the present invention include all 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 the composition comprising α- N -arabinofuranosidase of the present invention was administered to ginsenoside Rc, it was deglycosylated with ginsenoside Rd. In addition, when the α- N -arabinofuranosidase of the present invention was treated with ginsenoside C-Mc1 as a starting material, it was confirmed that deglycosylation was performed with ginsenoside F2. In addition, when the α- N -arabinofuranosidase of the present invention was treated with ginsenoside C-Mc as a starting material, it was confirmed that deglycosylation was performed with ginsenoside CK.

Deglycosylation of such PPD or PPT type ginsenosides is due to the selective hydrolysis capacity of the non-reduced α-1,6 arabinofuranosyl residues of the α- N -arabinofuranosidase of the present invention, Deglycosylated rare ginsenosides produced through such hydrolysis are easily absorbed by the body. In addition, this bioconversion facilitates the conversion of PPD or PPT type ginsenosides, such as ginsenoside Rc, to CK or Rh2, which is its most deglycosylated form.

Α- N -arabinofuranosidase of the present invention is non-reducing of protopanaxadiol (PPD) type ginsenosides or PPT (Protopanaxatriol) type ginsenosides at various temperature and pH conditions as long as activity and stability can be maintained. Selective hydrolysis of α-1,6 arabinofuranosyl residues and conversion of protopanaxadiol (PPD) type or protopanaxatriol (PPT) type ginsenosides into rare ginsenosides in an easily absorbable form .

According to a specific embodiment of the present invention, the enzyme properties of the present invention was analyzed araf3054 was excellent in the stability and activity of the enzyme at pH 6-10, the highest activity in sodium phosphate buffer pH 7.5 of 37 ° C (FIG. 4A). It was also stable at temperatures below 37 ° C. and lost 100% of activity after incubation at 45 ° C. for 40 minutes. The activity was maintained at about 60% at a temperature of 4-35 ° C. regardless of the elevation of the temperature (FIG. 4B). Therefore, in order for the α- N -arabinofuranosidase according to the present invention to enzymatically, it is preferable to adjust the pH to 6 to 10 and the temperature to 4 to 37 ° C.

In addition, the α- N -arabinofuranosidase of the present invention may be used with one or more metals and chemical agents selected from the group consisting of MgCl ₂ , EDTA, NaCl, KCl, DTT and beta-mercaptoethanol, but is not limited thereto. It doesn't work. In addition, the substrate is preferably used pNP-α-L-arabinofuranoside (pNP-α-L-arabinofuranoside).

On the other hand, in preparing a deglycosylated second ginsenoside from the first ginsenoside, the α- N -arabinofuranosidase according to the present invention, together with other enzyme (s) at the same time or in a certain order, Can be used. Non-limiting examples of other enzymes include β-glucosidase, β-galactosidase, glycosidase, α-L-arabinofyranosidase, α-L-arabinofuranosidase, β-xyl Rosidase, α-L-rhamnosidase and the like. The second ginsenosides prepared by α- N -arabinofuranosidase alone or in combination with other enzymes may be different, and such second ginsenosides may be one or two or more second ginsenosides. Can be.

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

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for carrying out the present invention by way of example, but the scope of the present invention is not limited to these examples.

Example 1 Chemicals

Ginsenosides Rb1, Rb2, Rc, Rd, F2 and C-K were purchased from Dalian Green Bio Ltd (Dalian, China). Ginsenosides C-Mc1 and C-Mc were prepared for this experiment. 5-Bromo-4-chloro-3-indolyl β-D-glucopyranoside (X-Glc), pNP-α-L-arabinofuranoside (pNPAf) and L-arabinose were obtained from Sigma. . Flour arabinoxylan, red-branched arabinane (RDA), arabinobiose, arabintriose, arabinotetraose and arabinofentaose were purchased from Megazyme (Wicklow, Ireland). All other chemicals were at least analytical reagent grades and their respective sources are specified in the respective method sections.

Example 2. Phosmid Library Screening and Structure

The strain Gsoil3054 was identified in the soil of Pocheon, Korea. To clone the arabinofuranosidase gene that hydrolyzes ginsenosides from Gsoil3054, the phosphide library structure, the selection of phosphide clones showing enzymatic activity, the full-sequence of the phosphide vector, and finally the polymerase Cloning of the target gene using 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. 2010. Identification and characterization of a novel Terrabacter ginsenosidimutans sp.nov.b -glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV Appl.Environ.Microbiol. 76: 5827-5836). Gsoil3054 genomic DNA was isolated by phenol-chloroform extraction and CopyControl phosphide library generation kit (Epicentre, WI) was used to generate phosphide libraries according to the manufacturer's protocol. Phosmid clones with putative β-glucosidase gene were 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 comprising the β-glucosidase gene and α-N-arabinofuranosidase was analyzed by thin membrane chromatography (TLC) analysis of active hydrolysis of ginsenoside Rc. Selection was made after the activity of hydrolyzing the side was demonstrated.

Example 3. Phosmid Sequences

The total sequence of selected phosphide clones was determined commercially by Macrogen (Seoul, Korea). In summary, after crushing the phosmid DNA into 2-5kbp fragments, it was isolated by agarose gel electrophoresis, and the shotgun library was constructed by ligation of blunt end repair DNA with plasmid vector pCB31. The vector was introduced into E. coli and was ampicillin (100 μg / ml), 5-bromo-4-chloro-3-indolyl-β-D-galactosypyranoside (80 μg / 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 were aggregated into 7 contigs using the ABI3730 DNA Sequencing System (Applied Biosystems). Six gaps were determined by pre-designed primer walking to obtain 34 kb total sequence to complete sequencing.

Example 4. Molecular Cloning, Expression and Purification of Recombinant AbfA

Collected DNA sequences were analyzed using vector NTI (version 10.1) to identify putative open reading frames (ORFs). These ORFs were identified by BLASTP (http://blast.ncbi.nlm.nih.gov) to identify an open reading frame of the putative for the α- N -arabinofuranosidase gene ( abf A) belonging to GH family 51. /Blast.cgi) to investigate similarity. To express soluble α- N -arabinofuranosidase in E. coli , maltose binding protein (MBP) was fused to the N terminus of AbfA. In summary, DNA fragments encoding MBP and Tobacco Etch Virus (TEV) protease cleavage sites were identified by primers mbpF (5'-ATA CAT ATG AAA ATC GAA GAA GGT AAA CTG-3 ') and mbp-tevR (5'- E. coli K-12 strain MG1655 (Blattner, FR, G. Plunkett, 3rd, CA Bloch, NT) using CTC GAA TTC CGA CTG 045GAA GTA GAG ATT CTC TGA AAT CCT TCC CTC GAT CCC GAG GTT G-3 ' Perna, V. Burland, M. Riley, J. Collado-Vides, JD Glasner, CK Rode, GF Mayhew, J. Gregor, NW Davis, HA Kirkpatrick, MA Goeden, DJ Rose, B. Mau, and Y. Shao. 1997.The complete genome sequence of Escherichia coli K-12.Science 277: 1453-62 was amplified by PCR (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 fragment was digested with Nde I and EcoR I and then inserted into the Nde I / EcoR I position of pET21a to produce pET21a-MBP (TEV). The AbfA gene was amplified by PCR using the following primers (italic in Eco RI and Hin dIII restriction positions): 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 fragment was 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 was introduced into E. coli BL21-Codon Plus (DE3) -RIL (Agilent technologies, Santa Clara, Calif.) and cells containing plasmids were treated with 100 μg / ml ampicillin at 37 ° C and 50 μg / ml Cultures were grown on LB medium containing fluoramphenicol at 600 nm until the OD reached 0.6, at which point protein expression was induced by addition of 0.1 mM IPTG. Cultures were further incubated at 18 ° C. for 8 hours and centrifuged at 5,000 × g for 20 minutes at 4 ° C. The cell pallet was resuspended in a solution consisting of 20 mM Tris-Cl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride and 1% Triton X-100 (pH 7.4). Cells were ruptured through ultrasonography (Vibra-cell, Sonics & Materials, CT). Intact cells and debris were removed by centrifugation at 24,000 × g for 40 minutes at 4 ° C. to obtain a crude cell extract. MBP labeled fusion proteins were purified by affinity chromatography followed by DEAE-cellulose DE-52 chromatography (Whatman) on amylose resin columns (New England BioLabs). MBP epitopes were removed by incubation with TEV protease, and then recombinant AbfA was purified by DEAE-cellulose DE-52 chromatography. Homogeneity of the protein was assessed by 10% SDS-PAGE and Coomassie blue staining. Purified protein was dialyzed with 50 mM sodium phosphate at pH 7.5 and concentrated to 1 mg / ml using Amicon Ultra-15 filter (Millipore, Calif.). Proteins were stored at -80 ° C until use. Enzyme and kinetic analyzes were performed using protein purified from 50 mM sodium phosphate at pH 7.5. The molecular mass of the recombinant protein was determined by size exclusion chromatography using 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 Enzymatic Hydrolysis of Ginsenosides

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

Example 6 Enzyme Characterization

Specific activity of the purified AbfA was measured using pNPAf as a replacement substrate in 50 mM sodium phosphate buffer at pH 7.5, 37 ° C. The reaction was terminated by addition of 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 needed to produce 1 μmol of p -nitrophenol per minute. Specific activity was expressed as units per milligram of protein. Protein concentration was measured using BCA protein assay (Pierce, Rockford, IL), based on bovine serum albumin (Sigma). All analyzes were performed three times.

6-1. Measure the effect of pH changes

The effect of pH on 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 (pH 4). 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 whether the buffer composition affects enzymatic activity, we also measured enzyme activity in McIlvaine buffer (100 mM citric acid, 200 mM disodium phosphate) at various pHs in the range 6.0-8.0 (Margolles, A). ., and CG De los Reyes-Gavila'n. 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. The results are expressed as percent of activity obtained at optimal pH.

6-2. Measure the effect of temperature changes

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

6-3. Measure the effects of gold and chemicals

The influence of metals and other chemicals on AbfA activity was also determined. AbfA activity is 1 or 10 mM (final concentration) MnCl ₂ , CaCl ₂ , CoCl ₂ , MgCl ₂ , EDTA, NaCl, KCl, CuCl ₂ , SDS, ZnCl _2, Dithiothreitol (DTT) for 30 minutes at 37 ° C. ), And β-mercaptoethanol. Residual activity was determined using pNPAf as the substrate and expressed as relative percentage with 100% of activity obtained when the compound was absent.

6-4. Group 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-β-D Galactopyranoside, PNP-β-D-fucopyranoside, PNP-N-acetyl-β-D-glucoamide, PNP-β-L-arabinofyranoside, PNP-β-D-only Nopyranoside, PNP-β-D-xylpyranoside, PNP-α-D-glucopyranoside, PNP-α-L-arabinofuranoside, PNP-α-L-arabinofyranoside , PNP-α-L-rhamnopyranoside, PNP-α-D-mannopyranoside, and PNP-α-D-xylpyranoside, and ONP-β-D-glucopyranoside, ONP- β-D-galactopyranoside, ONP-β-D-fucopyranoside and ONP-α-D-galactopyranoside (Sigma). Glycosidase activity was tested using a 2.0 mM chromogenic substrate, defined as one active unit releasing 1 μmol of ONP or PNP per minute at 37 ° C. for 5 minutes.

For other arabinoside-containing substrates, the reaction solution contains 0.1 ml of AbfA solution (1 U) in 2% (wt / vol) wheat flour arabinoxylan, RDA, arabinobiose, arabintriose, arabinotetraose. Or by adding 0.5 ml of arabinopentose. The reaction mixture was incubated at 37 ° C. Samples were taken out at regular times and heated with boiling water for 5 minutes to stop the reaction. Products from wheat flour arabinoxylan, RDA, arabinobiose, arabintriose, arabinotetraose and arabinopentaos were analyzed by TLC. Chromatography was performed as a synergistic method on silica gel 60 F ₂₅₄ TLC plates (Merck). 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 minutes 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-α-1,5-L-arabinanase gene and the enzyme.Appl.Microbiol.Biotechnol. 79: 941-949).

Kinetic studies were performed with freshly 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 minutes at 37 ° C. For the latter, the hydrolyzed product of ginsenoside Rc was determined as HPLC analysis at the initial stage of hydrolysis. The data obtained were used to determine K _m and V _max using the Enzyme Kinetics Program reported by Cleland (Cleland, WW 1979. Statistical analysis of enzyme kinetic data. Methods Enzymol. 63: 103-138).

Example 7 Preparation and Identification of Ginsenosides C-Mc1 and C-Mc

Ginsenosides C-Mc1 and C-McTerrabctersp. Obtained by bioconversion of ginsenoside Rc using recombinant β-glucosidase from Gsoil3082, BgpA (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 novelTerrabacter ginsenosidimutans 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 ginsenosides having one or two glucose residues at the C3 or C20 position, followed by external glucose at C3, then internal glucose at C3, and finally external glucose at C20. Prefer hydrolysis of. Ginsenoside Rc is terminally non-reducing α- at the outer position of C20LSince having arabinofuranosyl residues, BgpA hydrolyzes only external and internal residues attached to the C3 position of Rc to produce C-Mc1 and C-Mc, respectively (FIG. 2). To prepare reaction products of BgpA and Rc, 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 were converted to metabolite 1, This was confirmed by TLC. From this reaction product, 0.36 g of metabolite 1 was obtained, 0.2 g of which was subjected 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 extracted twice with water-saturated n-butanol andin vacuoEvaporated at For further purification of metabolites for nuclear magnetic resonance (NMR) analysis, 100 mg of each metabolite 1 and 2 were recycled with a UV / RI detector and a reversed phase column (ODS, 500 × 20 mm ID, 15 μm). Purification was performed using a preparative HPLC system (LC-9201, Japan Analytical Instrument, Japan). CH₃CN's isocratic solvent system and deionized H₂O (7: 3) was used and the detection wavelength was set to 203 nm. While using this method, 42 mg of metabolite 1 and 33 mg of metabolite 2 were obtained and placed for NMR and mass spectrometry (MS) analysis.

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

Example 8 Ginsenoside Analysis Using Thin-Layer Chromatography (TLC)

TLC was performed using a 60F ₂₅₄ silica gel plate (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 heated at 110 ° C. for 5 minutes.

Example 9 High-Performance Liquid Chromatography (HPLC) Analysis

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

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

result

Example 1. abfA Cloning of

Previously, strains of bacteria (named Gsoil3054) that hydrolyze ginsenosides have been identified from soils of ginseng fields in Pocheon, Korea. Strain Gsoil3054 can simultaneously convert PPD type ginsenosides Rb1, Rb2 and Rc to Rd, which characterizes Gsoil3054 from other identified ginsenoside metabolizing bacteria. Gsoil3054 has been identified as Rhodanobacter ginsenosidimutans through a polymorphic characterization process to clarify its classification site (An, DS, HG Lee, ST Lee, and WT Im. 2009. Rhodanobacter ginsenosidimutans sp. Nov. , Isolated from 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 glucoside-hydrolase activity, β-D-glucosidase, α-L-arabinofyranosidase and / or α- It may have L-arabinofuranosidase activity. In order to identify and clone individual genes for enzymes that hydrolyze ginsenosides from Gsoil3054, we generated a phosmid library and obtained the full sequence of the phosphide vector. The average insertion size of the phosphide clones was about 40 kb and the proportion of clones containing the inserts was about 91%. Phosmid clones producing β-D-glucosidase were screened by monitoring the gradation of X-Glc, creating a green area around colonies growing on agar media. Following this initial screening protocol, we have reduced the range of number of clones selected according to α-L-arabinofyranosidase or α-L-arabinofuranosidase activity. 25 of the 1400 clones in the phosmid library were associated with blue colonies on LB agar plates containing 12.5 μg / ml chloramphenicol and 27 μg / ml X-Glc. One of these phosphide clones was positive for β-D-glucosidase, α-L-arabinopyranosidase and α-L-arabinofuranosidase activity, and ginsenoside Rc, C-Mc1 And C-Mc could be converted to Rd. The clones above were selected for overall sequencing.

The insertion of positive clones was a 34 kb genomic fragment. Analysis of the nucleotide sequence revealed that it contained 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 2. Expression and Purification of Recombinant AbfA

Presumption α-N-arabinofuranosidase geneabfAAmplified by PCR, and under the control of the IPTG-induced T7 promoterE. coli To produce MBP gene fusion (MBP-AbfA) that can be expressed in BL21-CodonPlus (DE3) -RIL, it was subcloned into pET21a-MBP (TEV). In order to maximize the yield of fusion proteins, we tested in various induction states. Induction with 0.1 mM IPTG for 8 hours at 18 ° C. produced the highest levels of soluble active fusion protein. MBP-AbfA fusion protein was purified by amylose resin column followed by DEAE-cellulose DE-52 chromatography and digested with TEV proteolytic enzymes to remove MBP residues. Recombinant AbfA was purified by continuous chromatography on DEAE-cellulose column. This procedure resulted in 18.4 times purity of AbfA and 57% recovery from the crude extract (Table 1). The molecular mass of 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 converted 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 α-L-Arabinofuranosidase from Geobacillus caldoxylolyticus TK4.Applied Microbiology and Biotechnology 75: 813-820).

Table 1

Example 3. Enzyme Characterization

3-1. Effect of pH Change

AbfA showed activity over a wide pH range (pH 5.0-10.0). The optimal pH was pH 7.5 in sodium phosphate buffer (FIG. 4A). The enzyme maintained at least 96% of its best activity at pH 6.0-10.0. The enzyme showed residual activity at pH 5.0 and no activity at pH 4.0.

3-2. Effect of temperature change

The best temperature for AbfA activity was 37 ° C. The enzyme was stable at temperatures lower than 37 ° C., but after culturing for 30 minutes at 45 ° C., nearly 100% of its activity was lost (FIG. 4B).

3-3. Effect of metals and chemicals

The effects of metal ions EDTA, β-mercaptoethanol and SDS on AbfA activity were also investigated (Table 2). The enzyme did not require Mg ²⁺ to be activated and was significantly inhibited by 1 mM Mn ²⁺ , Zn ²⁺ or Cu ²⁺ and 10 mM Co ²⁺ or Ca ²⁺ . AbfA activity was not affected by DTT or β-mercaptoethanol, which are known thiol group inhibitors. These results suggest that sulfhydryl groups may not be involved in the catalytic center of the enzyme, but will be quite 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 . Enzymatic activity was markedly inhibited in the presence of 10 mM SDS.

TABLE 2

3-4. Substrate specificity

When the reaction constant of α-N-arabinofuranosidase was calculated from a double reciprocal graph, for pNPAf hydrolysis, K _m and V _max were 0.53 ± 0.07 mM and 27.1 ± 1.7 μmol min ^-1 mg, respectively. of protein ^-1 and 0.30 ± 0.07 mM and 49.6 ± 4.1 μmol min ^-1 mg of protein ^-1 for ginsenoside Rc, respectively.

Example 4 Bioconversion of Ginsenosides

AbfA did not react with Rb1 and Rb2 to produce any glucose or arabinopyranose. Of the reported ginsenosides, only Rc, C-Mc1 and C-Mc had terminally non-reducing α-L-arabinofuranosyl residues at the C20 position. In contrast to ginsenosides Rc, C-Mc1 and C-Mc are either absent or present in trace amounts in raw ginseng and can be generated from Rc via hydrolysis of the glycofyranosyl residue at the C3 position. Since the inventors were unable to obtain C-Mc1 and C-Mc commercially, β- which hydrolyzes ginsenosides that specifically and continuously hydrolyze the terminal glycofyranosyl groups from recombinant BgpA, C3 of aglycone. Glucosidase was used to prepare C-Mc1 and C-Mc (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 novelTerrabacter ginsenosidimutanssp. nov. b-glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV Appl. Environ. Microbiol. 76: 5827-5836). Since the terminal, non-reducing α-L-arabinofuranosyl residue on Rc is not a substrate of BgpA, the enzyme only contains glucose residues outside and inside the C3 position of Rc, respectively, C-Mc1 (metabolite 1) and Hydrolysis to C-Mc (metabolic 2) was possible (FIG. 2), and their chemical structures were confirmed by NMR and MS data analysis. After confirming their chemical structures by NMR and MS analysis, C-Mc1 and C-Mc were used as substrates with Rc for recombinant AbfA. Hydrolysis products of ginsenosides Rc, C-Mc1 and C-Mc were identified at regular intervals by TLC and HPLC analysis. As shown in FIGS. 3 and 5, the enzyme hydrolyzed the arabinofuranosyl residues from all three ginsenosides. Based on the comparison of the Rf values (determined by TLC) and metabolite retention time (determined by HPLC analysis) of the standard ginsenosides, the metabolites from Rc, C-Mc1 and C-Mc are Rd, 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. 2010. Identification and characterization of a novelTerrabacter ginsenosidimutans sp. nov. b-glucosidase that transforms ginsenoside Rb1 into the rare gypenoside XVII and gypenoside LXXV Appl. Environ. Microbiol. 76: 5827-5836, Ko, S. R., Y. Suzuki, K. Suzuki, K. J. Choi, and B. G. Cho. 2007. Marked production of ginsenosides Rd, F2, Rg3, and compound K by enzymatic method. Chem. Pharm. Bull. 55: 1522-1527).

Example 5 Structural Identification of C-Mc1 and C-Mc

Protonated molecular ion peaks corresponding to metabolite 1 were observed at m / z 917 (C ₄₇ H ₈₁ O ₁₇ ) in the ESI MS spectrum of metabolite 1. ¹ H NMR spectral data of Metabolite 1 (Table 3) show two anomeric proton signals, belonging to β-glucose at 4.92 (1H, d, J = 7.8 Hz) and 5.12 (1H, d, J = 7.8 Hz) It showed one 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. ¹³ C NMR chemical changes of metabolite 1 have been shown to be closely associated with signals 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). Thus this compound was identified as C-Mc1. The ¹ H and ¹³ C NMR data of Metabolite 2 were very similar to the data of C-Mc1 except for the absence of a signal for glucose units and the shift of ¹³ C chemical changes from 88.0 to 78.0. These differences suggest that glucose is hydrolyzed at the C3 position of C-Mc1 and that free hydroxyl groups are present in the structure of metabolite 2. This was confirmed by detection of protonated molecular ion peaks 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.

TABLE 3

Example 6. Substrate Specificity of AbfA

Substrate specificity of AbfA was tested using 2.0 mM PNP- and ONP-glycosides with α and β configurations. AbfA was only active against pNPAf PNP-β-D-glucopyranoside, PNP-β-D-galactopyranoside, PNP-β-D-fucopyranoside, PNP-N-acetyl-β- D-glucoseamide, PNP-β-L-arabinopyranoside, PNP-β-D-mannopyranoside, PNP-β-D-xylopyranoside, PNP-α-D-glucopyranoside , PNP-α-L-arabinopyranoside, PNP-α-L-rhamnopyranoside, PNP-α-D-mannopyranoside, and PNP-α-D-xylpyranoside, and ONP Other substrates including -β-D-glucopyranoside, ONP-β-D-galactopyranoside, ONP-β-D-fucopyranoside and ONP-α-D-galactopyranoside Not hydrolyzed. Degradation aspects of the polymer comprising arabino revealed that AbfA was only active on the non-reducing terminal arabinofuranosyl moiety. To identify the mode of action (outer-to-inner) of AbfA, hydrolysis of oligos or polysaccharides comprising arabinose was tested. The enzyme did not show any activity against RDA, indicating that it did not have internal arabinose hydrolase (endoarabinanase) activity. AbfA has been shown to be active against α-1,5-linked arabinobiose, arabintriose, arabinotetraose, and arabinofentaose. In all cases, only arabinose was detected as the final product and other residues were not detected by TLC (FIG. 7). No activity was detected for wheat flour arabinoxylan comprising α-1,2 and α-1,3 arabinofuranosyl residues. These results indicate that AbfA hydrolyzes non-reducing terminal α-1,5-linked L-arabinofuranos residues, while non-reducing terminal α-1,2 or α-1,3-linked arabinofuranosyl It indicates that the residue is an external-acting enzyme that does not hydrolyze the residue. AbfA also hydrolyzes terminally non-reducing α-L-arabinofuranosyl residues to produce ginsenosides Rd, F2, and CK at the C20 positions of ginsenosides Rc, C-Mc1 and C-Mc, respectively. It can be disassembled. In addition to the unique aglycone structures of Rc, C-Mc1 and C-Mc, the terminally non-reducing α-L-arabinofuranosyl residues of these compounds are termed α-1,6-linkages with internal glucose at the C20 position. The fact suggests that Rc, C-Mc1 and C-Mc would be excellent substrates to investigate the substrate specificity of α-arabinofuranosidase. In addition, metabolites of these ginsenosides can be easily detected by TLC and HPLC. This means that the enzyme has very high specificity for the arabinofuranosyl group and, unlike previously reported enzymes, acts only as an external-type enzyme. For example, GH 51 arabinofuranosidase (Flipphi, M. J. A., J. Visser, P. Van der Veen, and L. H. De Graaff. 1994. Arabinase gene expression inAspergillus nigerIndications for coordinated regulation. Microbiology 140: 2673-2682) removes both α-1,2 and α-1,3 arabinofuranosyl residues, and some enzymes in this family are β-1,4-internal to carboxyl cellulose and xylan Exhibits glucanase activity (Eckert, K., and E. Schneider. 2003. A thermoacidophilic endoglucanase (CelB) fromAlicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases. Eur. J. Biochem. 270: 3593-3602).

In conclusion, we remove the α-1,6 arabinofuranosyl residues from ginsenosides Rc, C-Mc1 and C-Mc to produce trace amounts of ginsenosides Rd, F2 and CK, respectively. α-N-arabinofuranosidase was cloned and characterized. The enzyme also hydrolyzes α-1,5 arabinofuranosyl residues from arabinobiose, arabintriose, arabinotetraose, and arabinofentaose. This is the first report on cloning enzymes that convert ginsenosides C-Mc1 and C-Mc to F2 and C-K through the bioconversion of ginsenosides.

[Revision under Rule 26 03.05.2011]

Claims (21)

1. Α-N-arabinofuranosidase derived from Rhodanobacter ginsenosidimutans .
2. The α-N-arabinofuranosidase of claim 1, wherein Rhodanobacter ginsenosidimutans is KCTC22231T.
3. The α-N-arabinofuranosidase according to claim 1, which has an amino acid sequence consisting of SEQ ID NO: 1.
4. The α- N -arabinofurano according to claim 1, wherein the α- N -arabinofurano has a selective hydrolysis of α-1,6 arabinofuranosyl residues located at the 20th carbon of the protopanaxadiol (PPD) type ginsenoside. Sidaze.
5. A nucleic acid encoding the α- N -arabinofuranosidase of any one of claims 1 to 4.
6. The nucleic acid according to claim 5, wherein the nucleic acid has the nucleotide sequence set forth in SEQ ID NO: 2. 7.
7. A recombinant vector comprising the nucleic acid of claim 5.
8. 8. The recombinant vector of claim 7, wherein said vector has a cleavage map as described in FIG. 9 as pET-MBP-TEV-Araf3054.
9. A transformant transformed with the nucleic acid of claim 5 or a recombinant vector comprising the nucleic acid.
10. The transformant of claim 9, wherein the transformant is E. coli.
11. (a) culturing the transformant of claim 9

    (b) producing α- N -arabinofuranosidase from the cultured transformant, and

    (c) recovering the produced α- N -arabinofuranosidase

    Method for producing α- N -arabinofuranosidase comprising a.
12. The α- N one claim any of items 1 to 4, wherein - arabinose Pew pyrano let claim, the claim α- N produced by the 11-arabino Pew pyrano let claim, claim 9 transformants or the transformants A method for preparing a deglycosylated second ginsenoside from a first ginsenoside, comprising using a culture of a sieve.
13. The method of claim 12, wherein the first ginsenoside is protopanaxadiol (PPD) type or protopanaxatriol (PPT) type ginsenoside.
14. 13. The non-reduced α-1,6 arabinofurano according to claim 12, wherein the non-reduced α-1,6 arabinofurano is located at the 20th carbon of protopanaxadiol (PPD) type or protopanaxatriol (PPT) type ginsenoside by α- N -arabinofuranosidase. And wherein the real residues are hydrolyzed.
15. 13. The process of claim 12, wherein the conversion of ginsenoside Rc to ginsenoside Rd, conversion of C-MC1 to ginsenoside F2, and ginsenoside C-MC to CK by α- N -arabinofuranosidase And at least one conversion step selected from the group consisting of conversion to.
16. The method of claim 12, wherein the pH is adjusted to 6 to 10 and the temperature is adjusted to 4 to 37 DEG C to allow the α- N -arabinofuranosidase to be enzymatically active.
17. 13. The method of claim 12, α- N - arabinose Pew pyrano let the method of the substrate, wherein the ginsenoside is pNP-α-L- arabino Pew pyrano a side (pNP-α-L-arabinofuranoside ).
18. The method according to claim 12, wherein the enzymatic action of α- N -arabinofuranosidase is performed with at least one substance selected from the group consisting of MgCl ₂ , EDTA, NaCl, KCl, DTT and beta-mercaptoethanol. How to.
19. 13. β-glucosidase, β-galactosidase, glycosidase, α-L-arabinofyranosidase, α-L-arabinofuranosidase, β-xylosida And at least one enzyme selected from the group consisting of an azeta and an α-L-lamnosidase.
20. The α- N one claim any of items 1 to 4, wherein - arabinose Pew pyrano let claim, the claim α- N produced by the 11-arabino Pew pyrano let claim, claim 9 transformants or the transformants Kit for conversion from protopanaxadiol (PPD) type or protopanaxatriol (PPT) type ginsenoside to deglycosylated rare ginsenoside, comprising a culture of a sieve as an active ingredient.
21. The method of claim 20, wherein the same or different container, β-glucosidase, β-galactosidase, glycosidase, α-L-arabinofyranosidase, α-L-arabinofuranosidase, The kit further comprises at least one enzyme selected from the group consisting of β-xylosidase, and α-L-rhamnosidase.

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