Molecular cloning, expression, purification, and characterization of Bacillus subtilis hydrolyzed ginsenoside Rc of α-L-arabinofuranosidase in Escherichia coli.

The α-L-arabinofuranosidase enzyme plays a crucial role in the degradation of ginsenosides. In this study, we successfully cloned and expressed a novel α-L-arabinofuranosidase bsafs gene (1503 bp, 501 amino acids, 55 kDa, and pI = 5.4) belonging to glycosyl hydrolase (GH) family 51 from Bacillus subtilis genome in Escherichia coli BL21 cells. The recombinant protein Bsafs was purified using Ni2+ sepharose fastflow affinity chromatography and exhibited a specific activity of 2.91 U/mg. Bsafs effectively hydrolyzed the α-L-arabinofuranoside at C20 site of ginsenoside Rc to produce Rd as the product. The Km values for hydrolysis of pNP-α-L-arabinofuranoside (pNPαAraf) and ginsenoside Rc were determined as 0.74 and 4.59 mmol/L, respectively; while the Vmax values for these substrates were found to be 24 and 164 μmol/min/mg, respectively; furthermore, the Kcat values for these enzymes were calculated as 22.3 and 1.58 S-1 correspondingly.