Purification and Characterization of an Extracellular High Molecular Mass Esterase from Bacillus pumilus

Abstract

Esterases (EC 3.1.1.x) represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds and are widely distributed in animals, plants and microorganisms. The esterase was purified from cell-free culture broth to homogeneity by size exclusion chromatography with 7.7 fold purification and 22.5% yield. The Mr of the purified esterase of B. pumilus was 17 kDa by SDS-PAGE. The esterase appeared to be a novel decameric protein as it possessed a single band of Mr 17 kDa in SDS PAGE and 170 kDa in Native PAGE. A Lineweaver-Burk plot was calibrated to determine K_m (3.94 mM), V_max (49.02 mM/mL/min), K_cat (138.89 sec^-1), K_spec (35.28) and K_si (26.58 mM) values of purified esterase of B. pumilus for its substrate p-nitrophenylacetate. Energy of activation (Ea) of purified esterase of Bacillus pumilus was 2.61 Jmol^-1as determined with the help of an Arrhenius plot. The DMSO drastically inhibited the activity of purified esterase while benzene and propan-2-ol were fairly tolerated by the esterase. The potential protease inhibitors such as PMSF, EDTA, SDS and DTT were found to decrease the activity of purified esterase. Thermostability of the purified esterase was checked at 45^oC. Purified esterase lost more than 50% of its initial activity at 45^oC after 6.5 h of incubation in a water-bath under shaking.