A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune ( Prunus domestica ) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61   kDa by SDS-PAGE and 54   kDa by gel permeation chromatography. The enzyme has a p I of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55   °C. It is stable at temperatures up to 45   °C and in a broad pH range. Its activity was completely inhibited by 5   mM of Ag + and Hg 2+ . The enzyme hydrolyzed both p -nitrophenyl β- d -glucopyranoside with a K m of 3.09   mM and a V max of 122.1   μmol/min   mg and p -nitrophenyl β- d -fucopyranoside with a K m of 1.65   mM and a V max of 217.6   μmol/min   mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K i values of 0.033 and 468   mM, respectively.