Biochemical Characterization of the Larval Alpha-amylase of Leucinodes orbonalis Guenee (Lepidoptera: Pyralidae) from the Field Collected Population of Uttar Dinajpur, West Bengal, India

Background: Brinjal shoot and fruit borer (Leucinodes orbonalis Guenee) significantly harms brinjal crops throughout South Asia. The internal feeding habit of larvae renders pesticides ineffective and contributes to resistance against broad-spectrum insecticides, posing severe management challenges. Targeting insect α-amylase to design enzyme inhibitors presents a promising approach for pest suppression. Therefore, biochemical characterization of larval α-amylase of L. orbonalis was carried out. Methods: Spectrophotometric assays were performed to determine the pH optima and optimal temperature range for α-amylase activity. Effects of various activators and inhibitors on the enzyme were also calculated. Enzyme kinetics study was done to decipher the Km and Vmax of the enzyme-substrate reaction. Enzyme activity was also noted in by zymographic study. Result: The α-amylase enzyme exhibited a specific activity of 0.294 U/mg of insect protein. It operates optimally at pH 9 and within a temperature range of 45-50oC. Activity of α-amylase was moderately enhanced by Na+ and K+ ions, while Mg²+ and Ca²+ ions significantly increased enzymatic activity. Inhibitory effects were observed with ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS) and gallic acid (GA) at 5 mM concentrations, reducing activity by 54.62%, 29.07% and 74.04%, respectively. Kinetic analysis of α-amylase revealed that its Km was 0.315% and Vmax was 0.099 mM maltose/min. Two clear zones observed through Native PAGE zymography confirmed the identification of two distinct α-amylase isoforms in the gut.