Purification and Properties of β-Glucosidase from Candida pelliculosa var. acetaetherius

Abstract

Candida pelliculosa var. acetaetherius was found to produce a β-glucosidase intracellularly. The enzyme was purified 200-fold by fractionation with ammonium sulfate and chromatography on Sephadex G-100 and DEAE Sepharose CL-6B. After polyacrylamide gel electrophoresis of the final fraction, one protein band corresponding to β-glucosidase was detected. The molecular weights determined by SDS-PAGE and by Sephacryl S-300 chromatography were 90,000 and 360,000, respectively, suggesting that the enzyme was a tetramer. The enzyme was a glycoprotein and its isoelectric point was at pH 4.9. It’s optimum pH and temperature were 6.5 and 50°C, respectively. The enzyme activity was inhibited by Zn^{2 +}, Hg^{2 +}, Cu^{2 +}, Co^{2 +}, p-chloromercuribenzoate, and glucose. Inhibition by glucose was competitive with a Ki value of 6.5 mm. Specificity studies for substrates indicated that the enzyme was specific for the p-configuration of sugars. Km values measured at 50°C were 0.5 mm for p-nitrophenyl-β-glucoside and 37 mm for cellobiose.