Salivary amylase from Chilo partellus (Swinhoe) — Characterization and mode of inhibition through analysis of double reciprocal, fractional velocity and combination plots

Abstract

The stem borer <em>Chilo partellus</em> (Swinhoe) is the most devastating pest that causes huge losses to agricultural productivity. Being a herbivorous insect, the starch degrading enzyme, salivary amylase, plays a crucial role in its digestive system. The characterization of salivary amylase and targeting it with potent inhibitors could help in managing the pest by hindering its normal digestive process. Therefore, we have made an attempt to characterize the enzyme and analyze its nature of interaction with organic acids and inorganic salts. The salivary amylase was purified by G-100 column chromatography to 16.02 folds and biochemically characterized. The purified fraction consisted of &alpha;-amylase activity with a single isoform of 59.26 kDa. It showed an optimum pH between 6.0 and 7.0. Its optimum temperature was 40 &deg;C and was thermally stable up to 70 &deg;C. Starch was the preferred substrate of salivary amylase. By critical analysis of Lineweaver-Burk, Eadie- Hofstee and Hanes plots, the Km and Vmax values of salivary amylase for starch were confirmed to be 0.49 mg/mL and 1.67 nmoles of reducing sugars formed/min/mL, respectively. Zinc chloride, calcium nitrate, salicylic acid, citric acid and oxalic acid were found to be potent inhibitors of salivary &alpha;-amylase activity. From Lineweaver-Burk, fractional velocity and combination plots, it was inferred that citric acid was a pure non-competitive inhibitor of salivary &alpha;-amylase while salicylic acid, oxalic acid and zinc chloride caused complete mixed non-competitive- competitive inhibitions. The inhibition of salivary &alpha;-amylase with calcium nitrate was of complete mixed non-competitive- uncompetitive type.