Biotransformation of Ginsenoside Rf to Rh1 by Recombinant β-Glucosidase

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, Rg 3 , compound K (CK) and Rh 1 showed highly cytotoxicity against tumor cells [6][7][8][9]. CK was proven to be produced by intestinal microorganisms after oral administration of Rg 3 , 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 βglucosidase gene isolated from this strain, bgl1, showed the ability to transform ginsenoside Rf to Rh 1 ( Figure 1).

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).

In vitro biotransformation of ginsenoside Rf to Rh 1
Purified BGL1 protein was tested for biotransformation activities with ginsenoside Rf, DM 1 , PM 1 , SM 1 [11] and compound K. The recombinant protein didn't show any glucosidase activities towards compound K, ginsenosides DM 1 , PM 1 and SM 1 but did show the ability to transform ginsenoside Rf into new products, one of which has been identified as ginsenoside Rh 1 by comparison of the retention time with the authentic compound and further confirmed by LC-MS analysis (Figures 3A and B).

Conclusions
Ginsenoside Rh 1 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 Rh 1 , increasing the availability of this compound and hence its potential as a drug.

General
HPLC runs were carried out on a Zorbax C 18 column (150 x 25 mm, Phenomenex, Torrance, CA, USA) on an Agilent 1100 instrument and UV absorption data (λ 203 ) 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 KH 2 PO 4 and 8 mM Na 2 HPO 4 , PH 7.4) to remove immidazole and examined by SDS-PAGE on 13% denaturing gel.