Glycoside Hydrolase Family 39 β-Xylosidases Exhibit β-1,2-Xylosidase Activity for Transformation of Notoginsenosides: A New EC Subsubclass

β-1,2-Xylosidase activity has not been recorded as an EC subsubclass. In this study, phylogenetic analysis and multiple sequence alignments revealed that characterized β-xylosidases of glycoside hydrolase family (GH) 39 were classified into the same subgroup with conserved amino acid residue positions participating in substrate recognition. Protein–ligand docking revealed that seven of these positions were probably essential to bind xylose–glucose, which is linked by a β-1,2-glycosidic bond. Amino acid residues in five of the seven positions are invariant, while those in two of the seven positions are variable with low frequency. Both the wild-type β-xylosidase rJB13GH39 and its mutants with mutation at the two positions exhibited β-1,2-xylosidase activity, as they hydrolyzed o-nitrophenyl-β-d-xylopyranoside and transformed notoginsenosides R1 and R2 to ginsenosides Rg1 and Rh1, respectively. The results suggest that all of these characterized GH 39 β-xylosidases probably show β-1,2-xylosidase activity, w...