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.
