Abstract

Thermotolerant yeasts, Candida easanensis strain JK-8 secreted a β-glucosidase when grown in liquid media containing various carbon sources. The greatest enzyme activity was found in medium containing 1% cellobiose as a sole carbon source. An extracellular β-glucosidase was purified to homogeneity by anion exchange chromatography and hydrophobic interaction chromatography. The molecular weight (Mw) of the β-glucosidase was estimated to be 48 kDa by SDS-PAGE. The optimal activity was at pH 4.5-5.5 and 60°C. The purified enzyme was stable in the pH range of 4.0-6.0 and had a 2 h half life at 50°C. The presence of metal ions (Ca2+, Mn2+, Zn2+ and Cu2+), mercaptoethanol and EDTA at 5 and 10 mM positively influenced its activity. It showed high specific activity for p-nitrophenyl-β-glucopyranoside (p-β-NPG) and laminarin. The similar Vmax of 10 μmol min-1mg protein-1 was found in both p-β-NPG (Km value of 5 mM) and laminarin (Km value of 5 mg mL-1). The enzyme displayed high tolerance to glucose with a Ki of 1.3 M and it was tolerant in the presence of methanol and ethanol at concentration up to 15%.

Keywords: β-glucosidase, thermotolerant yeasts, candida easanensis, glucose tolerant, laminarin.

Graphical Abstract