Biochemical characterization of the α-Amylase inhibitor in mungbeans and its application in inhibiting the growth of callosobruchus maculatus

Abstract

The insect Callosobruchus maculatus causes considerable damage to harvested mungbean seeds every year, which leads to commercial losses. However, recent studies have revealed that mungbean seeds contain α-amylase inhibitors that can inhibit the protein C. maculatus, preventing growth and development of the insect larvae in the seed, thus preventing further damage. For this reason, the use of α-amylase inhibitors to interfere with the pest's digestion process has become an interesting alternative biocontrolling agent. In this study, we have isolated and purified the a-amylase inhibitor from mungbean seeds (KPSl) using ammonium sulfate precipitation, gel filtration chromatography and reversed phase HPLC. We found that the αa-amylase inhibitor, isolated as a monomer, had a molecular weight of 27 kDa. The α-amylase inhibitor was purified 750-fold with a final yield of 0.4 mg of protein per 30 g of mungbean seeds. Its specific activity was determined at 14.5 U (mg of protein) -1 . Interestingly, we found that the isolated α-amylase inhibitor inhibits C. maculatus a-amylase but not human salivary α-amylase. After preincubation of the enzyme with the inhibitor, the mungbean α-amylase inhibitor inhibited C. maculatus α-amylase activity by decreasing Vmaxwhile increasing the Km constant, indicating that the mungbean α-amylase is a mix noncompetitive inhibitor. The in vivo effect of α-amylase inhibitor on the mortality of C. maculatus shows that the α-amylase inhibitor acts on C. maculatus during the development stage, by reducing carbohydrate digestion necessary for growth and development, rather than during the end laying/ hatching stage. Our results suggest that mungbean α-amylase inhibitor could be a useful future biocontrolling agent. © 2010 American Chemical Society.