Purification, characterization and inhibition studies of α-amylase of Rhyzopertha dominica

Abstract

Rhyzopertha dominica causes extensive damage to stored wheat grains. α-Amylase, the major insect digestive enzyme, can be an attractive candidate to control the insect damage by inhibiting the enzyme through α-amylase inhibitors. R. dominica α-amylase (RDA) was purified to homogeneity by differential ammonium sulphate fractionation, Sephadex G-25 and Sephadex G-100 column chromatography. The homogenous α-amylase has a molecular weight of 52 kDa. The pH optima was 7.0 and temperature optima was 40 °C. Activation energy of RDA was 3.9 Kcal mol −1. The enzyme showed high activity with starch, amylose and amylopectin whereas dextrins were the poor substrates. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The enzyme showed Km of 0.98 mg ml −1 for starch as a substrate. Citric acid, oxalic acid, salicylic acid, HgCl 2, tannic acid and α-amylase inhibitors from wheat were inhibitors whereas; Ca 2+ and Mg 2+ were the activators of RDA. Ki values calculated from Dixon graphs with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitors were 6.9, 2.6–8.2, 3.2 mM and 0.013–0.018 μM, respectively. The Lineweaver–Burk plots with different inhibitors showed mixed type inhibition. Wheat α-amylase inhibitor showed mainly competitive inhibition with some non-competitive behaviour and other inhibitors showed mainly non-competitive inhibition with some un-competitive behaviour. Feeding trials with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitor showed significant effect of salicylic acid and oxalic acid along with wheat α-amylase inhibitor in controlling the multiplication of R. dominica.