Biotransformation of Ginsenoside Rf to Rh₁ by Recombinant β-Glucosidase

Abstract

An Aspergillus niger strain was isolated from the soil around ginseng fruit. In vitro enzyme assays showed that this strain had the ability to transform total ginsenosides (TGS) into several new products. In a further biochemical study, a β-glucosidase gene isolated from this strain, bgl1, was expressed in Saccharomyces cerevisiae. His-tagged BGL1 protein (~170 kD) showed the ability to transform ginsenoside Rf into Rh₁.

Introduction

The root of Panax ginseng C.A. MEYER is frequently used in China as a traditional medicine [1]. Ginsenosides, as the major components of ginseng, have been reported to show various biological activities, eg. anti-tumor, anti-inflammatory, immune-modulatory and anti-aging effects [2,3,4,5]. Among the 30 previous reported ginsenosides, Rg₃, compound K (CK) and Rh₁ showed highly cytotoxicity against tumor cells [6,7,8,9]. CK was proven to be produced by intestinal microorganisms after oral administration of Rg₃, and then further esterified to sustain it longer in the body [10,11]. During our continued work on bioactive ginsenosides, an Aspergillus niger strain was isolated from the soil around ginseng fruit. In vitro enzyme assays showed that this strain had the ability to transform total ginsenosides (TGS) into several new products [12]. In the subsequent biochemical study, a β-glucosidase gene isolated from this strain, bgl1, showed the ability to transform ginsenoside Rf to Rh₁ (Figure 1).

Figure 1. Scheme of biotransformation from ginsenoside Rf to Rh₁ catalyzed by BGL1.

Figure 1. Scheme of biotransformation from ginsenoside Rf to Rh₁ catalyzed by BGL1.

Results and Discussion

Expression and purification of BGL1 in Saccharomyces cerevisiae

In the present study, the bgl1 gene isolated from an Aspergillus niger strain, which encodes a glucosidase, was cloned into the yeast shuttle vector pRS423 and introduced into Saccharomyces cerevisiae (MGY70). SDS-PAGE analysis showed strong expression of a ~170 kDa his-tagged BGL1 protein at 37 ºC (Figure 2, lane 2). The empty pRS423 vector was used as control (Figure 2, lane 3). Soluble recombinant protein purified from cultures grown at 37ºC by his-tagging yielded a single distinct band after SDS-PAGE (Figure 2, lane 4). After dialysis, the purified recombinant BGL1 was quantified at 0.9 µg µL^-1 (total of 5.1 mg from 6 g bacteria cell pellet).

Figure 2. SDS-PAGE gel of expression and purification of BGL1. Lane 1: Protein marker, 2: Over-expression of BGL1; 3: Empty control; 4: Purified protein.

Figure 2. SDS-PAGE gel of expression and purification of BGL1. Lane 1: Protein marker, 2: Over-expression of BGL1; 3: Empty control; 4: Purified protein.

In vitro biotransformation of ginsenoside Rf to Rh₁

Purified BGL1 protein was tested for biotransformation activities with ginsenoside Rf, DM₁, PM₁, SM₁ [11] and compound K. The recombinant protein didn’t show any glucosidase activities towards compound K, ginsenosides DM₁, PM₁ and SM₁ but did show the ability to transform ginsenoside Rf into new products, one of which has been identified as ginsenoside Rh₁ by comparison of the retention time with the authentic compound and further confirmed by LC-MS analysis (Figure 3A and Figure 3B).

Figure 3. HPLC trace of enzyme assay. A: Trace of substrate ginsenoside Rf; B: Trace of enzyme assay of ginsenoside Rf reacted with BGL1.

Figure 3. HPLC trace of enzyme assay. A: Trace of substrate ginsenoside Rf; B: Trace of enzyme assay of ginsenoside Rf reacted with BGL1.

Conclusions

Ginsenoside Rh₁ had been reported as a bioactive compound with various pharmacological effects [6,7,8,9], but the amount in the ginseng was relative minor. In the present study, a recombinant Aspergillus niger BGL1 protein showed the ability to transform ginsenoside Rf to Rh₁, increasing the availability of this compound and hence its potential as a drug.

Experimental

General

HPLC runs were carried out on a Zorbax C₁₈ column (150 x 25 mm, Phenomenex, Torrance, CA, USA) on an Agilent 1100 instrument and UV absorption data (λ₂₀₃) were analyzed with Agilent Chemstation Ver 8.01. All solvents used in this study were HPLC grade, purchased from the Chinese Chemical Group, Beijing, P.R. China. S. cerevisiae MGY70 was used as host strain and the yeast shuttle vector pRS423 was used for the construct.

Cloning and Expression of pRS-BGL1 in S. cerevisiae

Total RNA of overnight cultured Aspergillus niger was extracted using an RNAeasy mini kit, (Qiagen, USA). The full-length BGL1 cDNA was cloned using specific primers designed from the mRNA sequence deposited in GeneBank (Accession No. XM.001398779): sense primer 5'-GC CTCGAG ATGAGGTTCACTTCGATCGA-3' and antisense primer 5'- GC GAATTC TTAGTGAACAGTAGGCAGAG-3', with underlined nucleotides representing restriction sites included for XhoI and EcoRI. The PCR product was purified by a Mini-PCR purification kit (Invitrogen, USA), ligated into a pRS423 expression vector, sequenced and then introduced into Saccharomyces cerevisiae, selecting for growth on yeast nitrogen base (YNB) minimal medium (Difco) lacking histidine as appropriate.

Purification of recombinant BGL1 protein

All steps were carried out at 4 ºC. His-tagged BGL1 protein was purified from the soluble fraction using a His-Bind purification kit (Novagen) following the manufacture’s protocol. Briefly, cells were freeze-thawed 3 times in binding buffer (500 mM NaCl, 20 mM Tris-HCl and 20 mM imidazole, PH 7.9). The suspension was incubated with lysozyme on ice for 30 min, and sonicated, the supernatant was collected by centrifugation at 14,000 g for 20 min and applied to pre-equilibrated His-Bind resin. Bound resin was washed three times with wash buffer (500 mM NaCl, 20 mM Tris-HCl and 60 mM imidazole, PH 7.9), then his–tagged protein was elute twice with three bed volumes of elution buffer (500 mM NaCl, 20 mM Tris-HCl and 1 M imidazole, PH 7.9), dialyzed three times against 1×PBS (140 mM NaCl, 2.7 mM KCl, 1.4 mM KH₂PO₄ and 8 mM Na₂HPO₄, PH 7.4) to remove immidazole and examined by SDS-PAGE on 13% denaturing gel.

Enzyme Activity

To test the potential of BGL1 to catalyze the biotransformation of ginsenosides, in vitro enzyme assay conditions were altered to include incubation at 37 ºC for 12 h in 1 mL total volume containing 850 µL Tris-HCl buffer (100 mM, PH 7.0), 50µl purified BGl1 protein (0.9 µg µL^-1 ), 100 µL ginsenoside (1 µg µL^-1). Tested ginsenosides included ginsenosides Rf, DM₁, PM₁, SM₁ and compound K. The reaction mixture with ginsenosides was centrifuged and subjected to HPLC for analysis (20 µL injection), at ambient temperature, a linear gradient of 5% to 65% Acetonitrile (containing 0.05% formic acid), (v/v) (flow rate of 1.0 mL/min), and monitored by PDA at A₂₀₃.