Purification and characterization of a new protease inhibitor from the seeds of Hibiscus acetosella with potent insecticidal activity against the larvae of Spodoptera litura (Fabricius) Lepidoptera: Noctuidae

In this study we purified and characterized a Protease Inhibitor (PI) from the seeds of Hibiscus acetosella (HAPI). The inhibitor was purified using ammonium sulphate fractionation, Fast Protein Liquid Chromatography using anion exchanger UNOsphere Q column and trypsin affinity chromatography. The HAPI has a subunit molecular weight of 20 kDa and inhibited trypsin to the extent of 89.81 ± 0.61% and larval gut protease activity of Spodoptera litura to the extent of 84.61 ± 0.97%. The purified inhibitor showed 5690.00 U/mg specific inhibition activity with a fold purity of 158.10 and yield of 2.2 µg per gram of seed. The HAPI was quite stable in the range of pH 7–9 with a maximum activity around pH 8–9, the pH range in the gut of lepidopteran larvae. The inhibitor was found to be stable up to a temperature of 50 C and retained an inhibition of 73.54 ± 0.83% even at 70 °C but declined beyond 80 °C. Further, detergents, oxidizing agents and reducing agents altered the inhibition activity of HAPI. The kinetic analysis revealed that HAPI is a non-competitive inhibitor of trypsin with a Ki of 52 nM. Feeding experiments on S. litura larvae with 1.16 µg of the purified inhibitor twice daily resulted in only 5.5% of the larvae emerged as adults from the treated group as against 97% the larvae emerged as adults from control group indicating its potential in pest control.