Identification, purification and characterization of β-glucosidase from apple seed as a novel catalyst for synthesis of O-glucosides

Among several fruit seeds, apple seed was identified as a new promising β-glucosidase source for alkyl O-glucoside synthesis by reverse hydrolysis, since it showed high hydrolytic activities on a broad spectrum of β-glucosides. From the crude extract of apple seed meal, a major glucosidase isoenzyme was purified to homogeneity, with a purification factor of 47-fold and an overall yield of 12.8%, by ammonium sulfate fractionation and chromatographic separation through DEAE-Cellulose, Butyl-Toyopearl and Sephadex G150 columns. The purified enzyme is a homodimer; each subunit has a molecular mass of about 60 kDa as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the pI of this β-glucosidase is about 5.7. These properties indicate that the apple seed β-glucosidase is totally different from any of the almond β-glucosidase isozymes reported so far. Furthermore, the purified enzyme of apple seed displays higher thermal stability than the commercially supplied β-glucosidase from almond, with half-lives of 42.9 h and 14.2 h, respectively, as preserved at 50 °C in an aqueous environment. The optimum pH of the apple seed β-glucosidase was 6.0 and the stable pH range was 5.0–9.0, both similar to those of the commercial almond β-glucosidase.