Two Key Amino Acids Variant of α-l-Arabinofuranosidase from <i>Bacillus subtilis</i> Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

• Main Authors: Ru Zhang, Shi Quan Tan, Bian Ling Zhang, Zi Yu Guo, Liang Yu Tian, Pei Weng, Zhi Yong Luo
• Format: Article
• Language: English
• Published: MDPI AG 2021-03-01
• Series: Molecules
• Subjects: <i>Bacilus subtilis</i>; α-l-arabinofuranosidase; ginsenoside Rc; biotransformation; ginsenoside Rd; site-directed mutagenesis
• Online Access: https://www.mdpi.com/1420-3049/26/6/1733

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, <i>BsAbfA,</i> was cloned from <i>Bacilus subtilis,</i> and its codons were optimized for efficient expression in <i>E. coli </i>BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other β-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters <i>K_m</i> of BsAbfA for <i>p</i>NP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the <i>K_cat/K_m</i>were 181.5 s⁻¹mM⁻¹ and 197.8 s⁻¹mM⁻¹, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C₂₀ position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.