Purification and characterization of protease inhibitors from rice bean (Vigna umbellata) and their efficacy against Spodoptera litura (Fabricius) gut proteinases

A protease inhibitor from rice bean was isolated and purified to assess its potential against gut proteases of Spodoptera litura, a devastating pest of several economically important crops globally. The purified inhibitor showed a single band protein on SDS-PAGE corresponding to molecular weight of 24.0 kDa. The inhibitor was purified to near homogeneity with 181.55 fold purification and final yield of 29.18 per cent. The inhibitory potential of purified rice bean trypsin protease inhibitor on larvae of Spodoptera gut proteases was studied which showed maximum azocaseinolytic (1.93U), trypsin (0.66U) and chymotrypsin activity (0.37 U) in second larval instar which gradually decreased upto late fifth larval instar stage. Inhibitory assay revealed that rice bean trypsin inhibitors were strong inhibitor of Spodoptera litura and inhibited more than 80 per cent for trypsin and around 69 per cent for chymotrypsin activity. To further determine the efficacy of rice bean protease inhibitors, feeding assays were conducted by adding rice bean flour in larval artificial diet. High mortality rate was observed in Spodoptera litura larvae after 72 hrs when rice bean was given as sole diet (100% rice bean composition). The results obtained from present study provide important clues in designing strategies for sustainable use of rice bean protease inhibitors in developing insect-tolerant transgenic plants.