Enzymatic biotransformation of ginsenosides $Rb_1$, $Rb_2$, Rc and Rd into $F_2$ by novel recombinant ginsenoside hydrolyzing glycosidase from Novosphingobium aromaticivorans

Abstract

This study focuses on the cloning, expression, and characterization of a recombinant ginsenoside hy-drolyzing glycosidase from Novosphingobium aromaticivorans KCTC $2888^{T}$ in order to efficiently biotransform protopanaxadiol type major ginsenosides ($Rb_1$, $Rb_2$, Rc, Rd) into ginsenoside $F_2$. The gene (BglNar) consists of 1,320 bp (439 amino acid residues) and revealed significant homology to that of glycoside hydrolase family 1. The gene was cloned and the recombinant enzyme overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system was characterized at conditions of pH 6.0 and $37^\circ C$. BglNar could convert gin-senosides $Rb_1$, $Rb_2, Rc$ into $F_2$ via Rd or gypenoside XVII, Rd, $C-Mc_1$, respectively, through selective hydrolysis of the glucose or arabinosides moieties. $K_m$ value of p-nitrophenyl- $\beta$ -D-glucopyranoside was 6.73 $\pm$ 0.27 and the $V_{max}$ value was 21.2 $\pm$ 0.36 $\mu mol min^{-1} mg^{-1}$ of protein, respectively. A crude protopanaxadiol-type ginsenoside mixture (PPDGM) was treated with BglNar followed by a column purification packed with HP20 resin, to pro-duce $F_2$ at chromatographic purities of 82.5 $\pm$ 1.3%. This is the first report of gram-scale production of $F_2$ from PPDGM using a novel ginsenoside-transforming $\beta$ -glucosidase of glycoside hydrolase family 1.