Abstract
A putative glycoside hydrolase family 78 α-l-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron VPI-5482 was heterologously over-expressed in Escherichia coli. Enzymatic properties of recombinant BtRha78A were characterized in detail. Recombinant BtRha78A might efficiently hydrolyze p-nitrophenyl α-l-rhamnopyranoside. BtRha78A displayed the highest activity at 60 °C in pH 6.5. BtRha78A exhibited a good pH stability and relatively high thermostability. BtRha78A could be tolerant of a low concentration of alcohols. These attractive advantages made it a promising alternative biocatalyst for industrial applications. The catalytic general acid Asp335 and general base Glu595 of BtRha78A were confirmed by site-directed mutagenesis. Alanine scanning mutagenesis based on sequence alignment and structural analysis revealed that the conserved residues Asp330, Arg334, Trp339, Asp342, Tyr383, Trp440, and His620 were crucial for enzyme catalysis. Most functional residues located at the conserved general acid motif (Asp330-Asp342) and were completely conserved in the subfamily I Rha78s.