Purification and Characterization of Midgut α-Glucosidase from Larvae of the Rice Green Caterpillar, Naranga aenescens Moore

Abstract

Application of chemical pesticides has increased significantly worldwide and has raised serious concerns about environmental pollutions. One of the encouraging trends to minimize pesticide risk is production of resistant plants containing toxic proteins against insect pests. Considering the importance of purification and characterization of digestive enzymes in the production of resistant plants, in this study an α-glucosidase from the Naranga aenescens Moore's midgut was purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-sepharose, and concentrating through ultrafiltration. The apparent molecular mass of the enzyme was 48 kDa determined by SDS-PAGE. The optimum pH and temperature of the enzyme were 6.0 and 45°C, respectively. The irreversible thermoinactivation of the enzyme showed that it was highly stable at 35ºC but moderately stable at 40 and 45ºC. Zn2+, Hg2+, Co2+ at 10 and 20 mM, and Ba+2 only in 20 mM strongly inhibited the α-glucosidase activity. Ba2+ and Ca2+ only at 10 mM, EDTA and Hg2 2+ only at 20 mM and Mg2+ at 10 and 20 mM significantly increased the enzyme activity. The Km and Kcat values for the α-glucosidase were 0.54 mM and 3.62 min-1, respectively, when p-Nitrophenyl-α-D-glucopyranoside (pNαG) was used as a substrate. Keywords: Ion exchange chromatography, Enzyme activity, Kinetic parameters, Thermostability Kinetic parameters, Toxic proteins.