Purification and biochemical properties of carboxylesterase from fasciola gigantica

Carboxylesterase was purified from Fasciola gigantica through ammonium sulfate precipitation, chromatography on DEAE-Sepharose and gel filtration on a sephacryl S300. Three enzymes (EI, EII and EIII) were separated. EII and EIII were purified to homogeneity. The molecular weight of EII and EIII enzyme were 66 and 50 KDa, respectively as detected by gel filtration and SDSpolyacrylamide gel electrophoresis. EII and EIII had Km 1.3 and 1.7 mM of p-nitrophenyl acetate. Affinity of esterase EII and EIII decreased as increasing carbon atom number of the substrates. Esterase EII and EIII had optimum temperature at 40 °C. Esterase EII and EIII had pH optima at pH 7.5 in phosphate buffer and pH 8.0 in Tris–HCl buffer, respectively. Studying effect of metal ions on esterase EII and EIII indicated that Li+, Mn++, Ba++ and Mg++ had activation effect on each isoenzyme. An activation effects could be detected with N- ethylmalimaide on EII and EIII). The order of inhibition on EII was β- mercaptoethanol PMSF DTNB PCMB iodoacetate. While the order of inhibition on EIII was β-mercaptoethanol iodoacetate DTNB PCM PMSF