Enzymatic Biotransformation of Ginsenoside Rb2 into Rd by Recombinant α-L-Arabinopyranosidase from Blastococcus saxobsidens

In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenosideconverting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb2 into Rd. The gene, termed AbpBs, consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb2 under optimal conditions (pH 7.0 and 40°C). Kinetic parameters for α-Larabinopyranosidase showed apparent Km and Vmax values of 0.078 ± 0.0002 μM and 1.4 ± 0.1 μmol/min/mg of protein against p-nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 μg/ml), 0.1% of ginsenoside Rb2 was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb2.


Introduction
Ginseng has been used as a traditional herbal medicine to cure diseases and promote health in Asian countries for thousands of years, but has also gained recognition in the past decade in the West for its beneficial uses [1][2][3]. Many previous reports have shown that ginseng has extensive pharmacological and therapeutic effects on humans including anticancer [4], anti-inflammatory activity [5], neuro-protective effects [6], anti-amnestic [7], immunomodulatory [8], and radio protective properties [9]. Most medicinal effects of ginseng have been attributed to triterpene saponins, also referred to as ginsenosides. [10,11]. Hence, more than 180 kinds of naturally occurring saponins have been isolated for pharmaceutical usage [12].
Also among the major ginsenosides, ginsenoside Rb In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenosideconverting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb 2 into Rd. The gene, termed AbpBs, consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb comprises 1-22% of the total ginsenosides in ginseng root [18,19], and is thus required for conversion into Rd, which is an important divergent position in the biotransformation of ginsenoside: Rd → Rg 3 (S) → Rh 2 (S) pathway or Rd → F 2 → compound K pathway (Fig. 1A). One solution to this then is to obtain recombinant α-L-arabinopyranosidase to biotransform ginsenoside Rb 2 into Rd with high activity (Fig. 1). Up to now, several methods have been developed for producing minor ginsenosides such as heating, acid treatment and enzymatic methods [17,[20][21][22]. The enzymatic methods are considered as the most promising approach, the advantages being fewer byproducts, better environmental protection, and better stereo-specificity [23].
In this study, we report the cloning and characterization of a novel ginsenoside-transforming α-L-arabinopyranosidase (AbpBs) from Blastococcus saxobsidens, followed by expression in Escherichia coli and characterization of α-Larabinopyranosidase (AbpBs). AbpBs belongs to glycoside hydrolase family 2, and this recombinant enzyme could efficiently catalyze the conversion of ginsenoside Rb 2 to Rd by selectively hydrolyzing the outer arabinopyranoside moiety at the C20 position. In the same way, this enzyme could also hydrolyze ginsenoside compound O (C-O) and compound Y (C-Y) into ginsenoside F

Materials
Ginsenosides Rb , protopanaxadiol and C-K were purchased from AceEMzyme Co., Ltd. (Korea). Chromogenic substrates for an enzyme activity assay were obtained from Sigma. Blastococcus saxobsidens KACC 20608 T used for cloning α-L-arabinopyranosidase gene was cultivated on LB agar (BD, USA), under aerobic condition at 30°C. Escherichia coli BL21 (DE3) and pGEX-4T-1 plasmid (GE Healthcare, USA), for gene cloning and expression, were cultivated in a Luria-Bertani (LB) medium supplemented with ampicillin (70 mg/l). The other chemicals used in this study were at least of analytical reagent grade, and the sources are noted individually in the methods section below.

Analysis of AbpBs Sequence
Database homology search was performed with the BLAST program provided by NCBI. Furthermore, the multiple amino acid sequence alignment and the conserved patterns of discrete amino acid sequences of AbpBs and the known, most homologous α-L-arabinopyranosidase were performed by using the ClustalW program. (http://embnet.vital-it.ch/software/ClustalW.html).

Cloning, Expression, and Purification of Recombinant AbpBs
Genomic DNA of Blastococcus saxobsidens KACC 20608 T was extracted using a genomic DNA extraction kit (Macrogen, Korea). The gene, termed AbpBs, and encoding α-L-arabinopyranosidase, (GenBank Accession No. WP_104529000.1) was amplified by polymerase chain reaction (PCR) using Pfu DNA polymerase (BIOFACT, Korea) and the following primers containing BamHI and XhoI restriction sites (underlined): AbpBsF (5'-GGT TCC GCG TGG ATC CAT GCG ACG CAT CCC CTT C-3') and AbpBsR (5'-GAT GCG GCC GCT CGA GTC ATC GAG CCT CGA TTC C-3'). The amplified DNA fragments were purified and inserted into a pGEX 4T-1 glutathione S-transferase (GST) fusion vector using an EzCloning Kit (Enzynomics Co. Ltd., Korea) to generate a GST-AbpBs fusion gene. E. coli BL21 (DE3), transformed with recombinant pGEX-AbpBs, was grown in LB-ampicillin medium at 37°C until the culture reached an OD 6 0 0 of 0.5, at which point protein expression was induced by adding 0.1 mM isopropyl-β-Dthiogalactopyranoside (IPTG). Additionally, bacteria were incubated for 18 h at 20°C with shaking at 200 rpm and then harvested by centrifuging at 10,000 ×g for 20 min at 4°C. The cells were washed twice with 50 mM sodium phosphate buffer (pH 7.0, 5 mM EDTA, and 1% Triton X-100) and then suspended in 50 mM sodium phosphate buffer (pH 7.0). In order to obtain crude cells, the cells were disrupted by ultrasonication (Vibra-Cell, USA) on ice at 5 min and the intact cells and debris were removed by centrifugation at 13,000 ×g for 20 min at 4°C. The GST-tagged fusion protein was purified by GST-bind agarose resin (Elpis, Korea). The GST tag was removed from the resin after incubation with thrombin. The homogeneity of the protein was assessed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and EzStain AQUA (Atto, Japan).

Enzyme Characterization and Determination of Kinetic Parameters
To determine the optimum conditions for activity, pH, temperature, metal ions and chemical reagents were investigated as previously described [24]. The substrate specificity of AbpBs was tested using 2 mM p-nitrophenyl (pNP)-and o-nitrophenyl (oNP)-glycosides with α and β configurations described by [25]. The specific activity of purified AbpBs was determined using pnitrophenyl-α-L-arabinopyranoside (pNP-α-L-arabinopyranoside) as substrate in 50 mM sodium phosphate buffer (pH 7.0) at 40°C. p-Nitrophenol release was immediately measured using a microplate reader at 405 nm (Bio-Rad Model 680, USA). One unit of activity was defined as the amount of protein required to generate 1 μmol of p-nitrophenol per minute. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce, USA), with bovine serum albumin (Sigma) as the standard. All assays were performed in triplicate. Kinetic studies were performed using freshly purified enzyme (10 μg/ml), and pNP-α-L-arabinopyranoside and ginsenoside Rb 2 at concentrations ranging from 0.1 mM to 5 mM. All enzyme assays were performed in triplicates, and the parameters were determined as described by Cleland [26].

Enzymatic Hydrolysis of Ginsenosides
To investigate the biotransformation ability of recombinant AbpBs, three kinds of ginsenosides (Rb 2 , compound O, compound Y) having outer arabinopyranose moiety at C20 were evaluated as substrates. Initial biotransformation experiments using ginsenoside Rb 2 as substrate revealed that GST fused with AbpBs did not affect the activities of AbpBs. Each assay unit was composed of ginsenoside and fused protein solution (0.2 mg/ml in 50 mM sodium phosphate buffer, pH 7.0) in a 1:1 ratio (v/v) at 37°C. In addition, the hydrolyzing capacity of AbpBs (10 μg/ml) was determined using 2.0 mg/ml of Rb 2 , compound O and compound Y, respectively, as substrates in 50 mM sodium phosphate buffer (pH 7.0) at 40°C. Samples were withdrawn at regular intervals and an equal volume of water-saturated n-butanol was added to stop the reaction, and the reactant present in the n-butanol fraction was analyzed by TLC after pretreatment.
Analytical Methods TLC analysis. A reaction solution containing ginsenoside was extracted with an equal volume of water-saturated n-butanol; after centrifugation, the n-butanol fraction was examined by TLC using 60F , followed by heating at 110°C for 5 min to visualize ginsenoside spots, which were identified by comparing with a standard.
HPLC analysis. HPLC analysis of ginsenosides was performed using AutoChro 3000 software (Younglin, Korea) equipped with a quaternary pump, automatic injector, and single-wavelength UV detector (model 730D) for peak identification and integration. The HPLC column used was a Prodigy ODS (2) C18 column (4.6 × 150 mm, 5 μm) (Phenomenex, USA) combined with an Agilent safeguard column. Isocratic elution was performed, using acetonitrile (A) and water (B) at a ratio of 34:66 (v/v) as mobile phase, for 20 min, at a flow rate of 1.0 ml/min. Detection was performed by monitoring absorbance at 203 nm.

Analysis of AbpBs Sequence
The α-L-arabinopyranosidase gene (AbpBs) consists of 2,406 bp encoding 801 amino acids with a molecular mass of 86.4 kDa and a theoretical pI value of 5.00 (http:// web.expasy.org/compute_pi/). AbpBs has homology to the protein domain of glycoside hydrolase family 2 (GH2) of which enzyme activity showed β-galactosidase (E.C.

Cloning, Expression, and Purification of Recombinant AbpBs
GST-AbpBs was expressed in E. coli BL21 (DE3). To maximize the yield of the fusion protein in soluble form, we tested different induction conditions and found that induction with 0.1 mM IPTG at 20°C for 24 h produced a half-soluble active fusion enzyme. Supernatant from the cell lysates and purified protein samples were applied to SDS-PAGE. The calculated molecular mass of the AbpBs (86.4 kDa) was similar to the mass detected via SDS-PAGE (Fig. 2).

Enzyme Characterization
The optimal temperature for AbpBs activity was 40°C and the enzyme was stable at lower than 30°C. The enzyme lost 65% of its activity at 55°C (Fig. 3A). The enzyme activity retained more than 75% of its optimal activity from pH 6.5 to 9.0, while pH 10.0 enzyme activity decreased by more than 40% and at under pH 5.0 the enzyme activity decreased to 20% (Fig. 3B). Additionally, the effects of metal ions, EDTA, β-mercaptoethanol of AbpBs activity were investigated (Table 1). AbpBs activity was weakly affected by β-mercaptoethanol in accordance with concentration, which is well known of thiol group inhibitors. These results suggested that sulfhydryl groups may be involved in the catalytic center of the enzyme [27-29]. The enzyme did not require Mg   into Rd by GST-AbpBs, TLC analyses were carried out at regular intervals (Fig. 4). It is clear that GST-AbpBs could completely transform the ginsenosides Rb 2 and C-O into ginsenoside Rd and F 2 , respectively within 1 h. However, ginsenoside compound Y was partially converted to compound K regardless of long incubation time (Fig. 4). The HPLC chromatogram of a PPD mixture comprised of major ginsenosides (Rb1: 36.0%, Rc: 28.6%, Rb2: 17.0%, Rb3: 2.4%, Rd: 8.9%) was changed due to biotransformation following GST-AbpBs treatment. Ginsenoside Rb 2 was completely converted to Rd within 1 h. These HPLC results indicate that AbpBs selectively converts ginsenoside Rb     61.3 ± 1.5 μmol/min/mg/ of protein, respectively. These results show that AbpBs activity is faster for ginsenoside Rb 2 than pNP-α-L-arabinopyranoside.

Discussion
Until now, only one ginsenoside Rb 2 hydrolyzing α-Larabinopyranosidase (Bgp2) belonging to the glycoside hydrolase family 2 had been reported [32]. However, this Bgp2 has a side effect of cleaving the glucose moiety of C20, such that it produces ginsenoside Rg Compared to Bgp2, AbpBs has high stereo activity, making it unable to hydrolyze glucose moiety of any ginsenoside. Ginsenoside Rb 2 is one of the major PPD-type ginsenosides and accounts for 1-22% of the total ginsenosides in ginseng root or hair [18,19], so a sizable amount can be potentially exploited. Ginsenoside Rd, produced by AbpBs, is an important divergent position during the biotransformation of ginsenosides, Rd → Rg 3 (S) → Rh 2 (S) pathway or Rd → F 2 → compound K pathway (Fig. 1A), generated by the hydrolysis of the terminal or inner glucose moiety attached to the C3 or C20 carbon of ginsenoside Rd using βglucosidase [33-36]. Thus, biotransformation of ginsenoside In summary, a recombinant ginsenoside-hydrolyzing α-L-arabinopyranosidase (AbpBs) belonging to the glycoside hydrolase family 2 was cloned from Blastococcus saxobsidens and constructed for biotransformation of the major ginsenoside Rb 2 . The enzyme acts optimally at pH 7.0 and 40°C. AbpBs could hydrolyze Rb 2 , C-O, C-Y into Rd, F 2 , C-K, respectively, by selectively hydrolyzing the outer arabinopyranoside moiety at the C20 position. Biotransformed ginsenoside Rd and other minor ginsenosides derived from it are potentially useful in the cosmetic and pharmaceutical industry.