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

Abstract

This study reports the purification and characterization of ß-glucosidase from a newly isolated thermophilic fungus, Melanocarpus sp. Microbial Type Culture Collection (MTCC) 3922. The molecular weight of ß-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 ß-glucosidase but the activity was inhibited in the presence of CuSO4. ß-Glucosidase recognized pNP- ß-glucopyranoside (pNPG) as the preferred substrate, and showed very low affinity for pNP- ß-D-cellobioside. Km and Vmax for the hydrolysis of pNPG by ß-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. ß-Glucosidase activity was enhanced appreciably in the presence of alcohols (methanol and ethanol) moreover, purified ß-glucosidase showed putative transglycosylation activity that was positively catalyzed in presence of methanol as an acceptor molecule