Multiple mutations in the aglycone binding pocket to convert the substrate specificity of dalcochinase to linamarase.

Dalcochinase from Dalbergia cochinchinensis Pierre and linamarase from Manihot esculenta Crantz are β-glucosidases which share 47% sequence identity, but show distinct substrate specificities in hydrolysis and transglucosylation. Previously, three amino acid residues of dalcochinase, namely I185, N189 and V255, were identified as being important for determining substrate specificity. In this study, kinetic analysis of the ensuing double and triple mutants of dalcochinase showed that only those containing the 185A mutation could appreciably hydrolyze linamarin as well as transfer glucose to 2-methyl-2-propanol.

Mutational analysis in the glycone binding pocket of Dalbergia cochinchinensis β-glucosidase to increase catalytic efficiency toward mannosides.

Dalcochinase and Abg are glycoside hydrolase family 1 β-glucosidases from Dalbergia cochinchinensis Pierre and Agrobacterium sp., respectively, with 35% sequence identity. However, Abg shows much higher catalytic efficiencies toward a broad range of glycone substrates than dalcochinase does, possibly due to the difference in amino acid residues around their glycone binding pockets.

Expression and purification of dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, in yeast and bacterial hosts.

The coding sequence of the mature dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, was cloned and expressed in various systems. Expression in Escherichia coli resulted in an insoluble protein, which could be made soluble by co-expression with bacterial chaperonin GroESL. However, the enzyme had no activity.