Purification and biochemical properties of carboxylesterase from fasciola gigantica

ABSTRACT

Carboxylesterase was purified from Fasciola gigantica through ammonium sulfate precipitation, chromatography on DEAE-Sepharose and gel filtration on a sephacryl S300. Three enzymes [El, 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 SDS-polyacrylamide 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 beta- mercaptoethanol > PMSF > DTNB > PCMB > iodoacetate. While the order of inhibition on EIII was beta-mercaptoethanol > iodoacetate > DTNB> PCM > PMSF