Partial Purification and Characterization of Phenoloxidase in Coconut Beetle Larvae (Oryctes rhinoceros L.)

Abstract

Coconut beetle larvae have a protein called phenoloxidase present in their hemolymph that is essential in arthropod immune response. Prior to characterization and isolation of an enzyme designing optimum assay conditions is crucial. This will stabilize the enzyme and ensure optimal activity. In this study, phenoloxidase activity was measured as a function of varying substrates, activators with their incubation periods and incubation temperatures, and buffer pH. The substrate 4-methylcatechol appeared as the best substrate for the enzyme. Among the activators tested, trypsin efficiently activated phenoloxidase within 20-60 minutes incubation periods at 30°C in sodium phosphate buffer with pH ranging from 6.0 to 8.0. The hemolymph was partially purified for pro-phenoloxidase through DEAE and SP ion-exchange chromatography. Fractions positive for enzyme activity were loaded on SOS-PAGE. Partially purified prophenoloxidase shows a molecular weight of 85-92 kDa while its active form (phenoloxidase) has a molecular weight of 62-67 kDa.