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

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 ۴۸ kDa determined by SDS-PAGE. The optimum pH and temperature of the enzyme were ۶.۰ and ۴۵°C, respectively. The irreversible thermoinactivation of the enzyme showed that it was highly stable at ۳۵ºC but moderately stable at ۴۰ and ۴۵ºC. Zn۲+, Hg۲+, Co۲+ at ۱۰ and ۲۰ mM, and Ba+۲only in ۲۰ mM strongly inhibited the α-glucosidase activity. Ba۲+ and Ca۲+ only at ۱۰ mM, EDTA and Hg۲۲+ only at ۲۰ mM and Mg۲+ at ۱۰ and ۲۰ mM significantly increased the enzyme activity. The Km and Kcat values for the α-glucosidase were ۰.۵۴ mM and ۳.۶۲ min-۱, respectively, when p-Nitrophenyl-α-D-glucopyranoside (pNαG) was used as a substrate.

کلیدواژه ها:

Ion exchange chromatography ، Enzyme activity ، Kinetic parameters ، Thermostability Kinetic parameters ، Toxic proteins