Extraction of Amylases from Insects for Testing Inhibitory Compounds from Plants

α-Amylases (EC 3.2.1.1) are the hydrolytic enzymes that catalyze the breakdown of starch into maltose and glucose. In phytophagous insects, such as Helicoverpa armigera (cotton bollworm), α-amylases play a crucial role in carbohydrate digestion and, consequently, in larval growth and survival. Inhibition of these enzymes can impair nutrient assimilation, leading to reduced pest fitness. This chapter describes a clear step-by-step method to isolate α-amylases from H. armigera midguts, followed by the assessment of enzyme activity both quantitatively, using the classical Bernfeld dinitrosalicylic acid (DNS) assay, and qualitatively, using native-PAGE starch zymography. In-gel activity staining provides rapid visualization of distinct α-amylase isoforms and allows assessment of inhibitor effects directly on electrophoretic bands. This combined approach facilitates reproducible screening of α-amylases in diverse insect systems and provides a platform for evaluating plant-derived inhibitors for pest management applications.