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

Abstract

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, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli 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 Km of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the Kcat/Km were 181.5 s−1 mM−1 and 197.8 s−1 mM−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.

A Versatile β-Glycosidase from Petroclostridium xylanilyticum Prefers the Conversion of Ginsenoside Rb3 over Rb1, Rb2, and Rc to Rd by Its Specific Cleavage Activity toward 1,6-Glycosidic Linkages

“…The specificity of the ginsenoside structure and the style of the active pocket resulted in the binding of structures near ginsenoside C–20 to Pxbgl, while structures near C–3 were exposed to the solvent. This binding mode was consistent with that of ginsenoside and the other enzymes only generating ginsenoside Rd. , In general, the strong hydrogen bond is formed when the distance is less than 3.0 Å . Five hydrogen bonds were formed between ginsenoside Rb1 and five amino acid residues, including two strong hydrogen bonds between the glycosyl group at the C–20 position and Thr 450 and Glu 529 at distances of 2.6 and 2.5 Å, respectively (Figure b).…”

“…strain 22–3, and Bacillus subtilis Str. 168 showed more than 90% conversion of ginsenoside Rc. However, the conversion of ginsenoside Rc by Pxbgl was 100%.…”

Cerium-Promoted Ginsenosides Accumulation by Regulating Endogenous Methyl Jasmonate Biosynthesis in Hairy Roots of Panax ginseng

“…The total ginsenoside was filtered by a 0.22 µm membrane filter and analyzed by SHIMADZU LCMS-8050 at 203 nm with a ZORBAX SB-C18 column (3.5 μm, 2.1 mm × 150 mm). The mobile phase consisted of acetonitrile (A) and water (B), and the elute program was as follows: A:B (20:80) for 5 min; A:B (20:80) to (38:62) for 5–40 min; A:B (38:62) to (99:1) for 40–42 min; A:B (99:1) for 42–45 min; A:B (21:79) for 46–56 min [ 72 ]. The flow rate was 0.5 mL/min.…”

Biotransformation, Pharmacokinetics, and Pharmacological Activities of Ginsenoside Rd Against Multiple Diseases

“…The α-L-arabinosidase (AbpBs) from Caldicellulosiruptor saccharolyticus ( Shin et al, 2013 ), Thermotoga thermarum DSM 5069 ( Xie et al, 2016a ), Leuconostoc sp. 22-3 ( Liu et al, 2013 ), and Bacillus subtilis ( Zhang et al, 2021b ) converts ginsenoside Rc (Rc) into ginsenoside Rd by attacking the C-20 position of α-linked arabinoside, thereby releasing arabinose ( Liu et al, 2013 ; Zhang et al, 2021b ). AbpBs can promote the biotransformation of ginsenoside Rb2 (Rb2) to ginsenoside Rd by attacking C-20, thereby releasing arabinoside ( Kim et al, 2020 ).…”