Evaluation of protease inhibitor on insect proteases and isolation of gene encoding protease inhibitor from rice bean (Vigna umbellata)

Abstract

In the present investigation, isolation and purification of protease inhibitor was carried out from rice bean genotype BRS-2. A rice bean trypsin protease inhibitor was isolated and purified to homogeneity by extraction with 0.1 M phosphate buffer (pH 7.5), 80 per cent ammonium sulfate precipitation, dialysis, ion exchange chromatography on DEAE-Sepharose, gel filtration through Superdex-75 and Sepharose-Trypsin affinity chromatography. The homogeneity of the purified inhibitor was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions showing a single band protein of molecular weight 24.0 kDa. The rice bean trypsin protease inhibitor was purified to 181.55 fold with a final yield of 29.18 per cent. Inhibition studies carried out between pH 2.2 to pH 9.0 showed two pH optima at pH 2.2 (85.65%) and other at pH4.0 (89.50%) suggesting the inhibitor to be more stable under acidic than alkaline solutions. The maximum trypsin inhibitory activity was achieved at 370C (65.50%) and lowest at 1000C (35.30%).To study the metabolic interaction of insect gut proteases with rice bean protease inhibitor, midguts of 1st to 5th instar larvae of Spodoptera litura were dissectedfor procuring gut protease. Maximum azocaseinolytic (1.93U), trypsin (0.66U) and chymotrypsin activity (0.37 U) was observed in second larval instar which gradually decreased upto late fifth larval instar stage.