Purification and characterization of β-glucosidase from Melanocarpus sp. MTCC 3922

Abstract

This study reports the purification and characterization of β-glucosidase from a newly isolated thermophilic fungus, Melanocarpus sp. Microbial Type Culture Collection (MTCC) 3922. The molecular weight of β-glucosidase was determined to be ~ 92 and 102 kDa with SDS PAGE and gel filtration, respectively, and pI of ~ 4.1. It was optimally active at 60ºC and pH 6.0, though was stable at 50ºC and pH 5.0 - 6.0. The presence of DTT, mercaptoethanol and metal ions such as Na⁺, K⁺, Ca²⁺, Mg²⁺and Zn²⁺ positively influenced the activity of β-glucosidase but the activity was inhibited in the presence of CuSO₄. β-glucosidase recognized pNP- β-glucopyranoside (pNPG) as the preferred substrate, and showed very low affinity for pNP- β-D-cellobioside. K_m and V_max for the hydrolysis of pNPG by β-glucosidase was calculated as 3.3 mM and 43.68 µmolmin^-1mg protein^-1, respectively and k_cat was quantified as 4 x 10³ min^-1. β-glucosidase activity was enhanced appreciably in the presence of alcohols (methanol and ethanol) moreover, purified β-glucosidase showed putative transglycosylation activity that was positively catalyzed in presence of methanol as an acceptor molecule.