RESUMO
Plant ß-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a ß-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-ß-glucosides, torvosides A and H. Furostanol glycoside 26-O-ß-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 ß-glucosidase, but could not hydrolyze ß-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum ß-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887bp ORF encoded 629 amino acids and showed high homology to other plant GH3 ß-glucosidases. Internal peptide sequences of purified native Sbgl3 determined by LC-MS/MS matched the deduced amino acid sequence of the Sbgl3 cDNA, suggesting that it encoded the natural enzyme. Recombinant SBgl3 with a polyhistidine tag (SBgl3His) was successfully expressed in Pichia pastoris. The purified SBgl3His showed the same substrate specificity as natural SBgl3, hydrolyzing torvoside A with much higher catalytic efficiency than other substrates. It also had similar biochemical properties and kinetic parameters to the natural enzyme, with slight differences, possibly attributable to post-translational glycosylation. Quantitative real-time PCR (qRT-PCR) showed that SBgl3 was highly expressed in leaves and germinated seeds, suggesting a role in leaf and seedling development. To our knowledge, a recombinant GH3 ß-glucosidase that hydrolyzes furostanol 26-O-ß-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.