Abstract
A basic β-galactosidase with high specificity towards β-1,3- and β-1,6-galactosyl residues was cloned from radish plants by reverse transcriptase-PCR procedures. The gene, designated as RsBGAL1, contained an ORF consisting of 2,532 bp (851 amino acids), and is expressed in hypocotyls and young leaves. Recombinant RsBGAL1 was expressed in Pichia pastoris and purified to the homogeneity. RsBGAL1 appeared to share high similarity with β-galactosidases having exo-β-1,4-galactanase activity found in higher plants, and is classified into family 35 glycosyl hydrolase. The recombinant enzyme specifically hydrolyzed β-1,3- and β-1,6-galactooligosaccharides as did the native enzyme isolated from radish seeds, and split off more than 90% of the carbohydrate moieties of an AGP extracted from radish leaves under the concerted action with microbial α-L-arabinofuranosidase and β-glucuronidase. These results suggest that RsBGAL1 is a unique β-galactosidase showing characteristic substrate specificity different from that of β-galactosidases having exo-β-1,4-galactanase activity.
