Purification, characterization and evaluation of insecticidal activity of trypsin inhibitor from Albizia lebbeck seeds

Abstract

A Bowman-Birk inhibitor with activity against gut proteases of Helicoverpa armigera was extracted in 0.1 M sodium phosphate buffer from defatted seed flour of Albizia lebbeck. It was purified to 29.62 folds with 51.43% recovery using ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-100 column and ion exchange chromatography on DEAE-Sephadex A₅₀. The purified protein had a molecular weight of 12,303 daltons as determined by SDS-PAGE. It was found to be heat stable up to 60°C and had two pH optima of 7.5 and 9.0. The inhibitor exhibited non-competitive pattern of inhibition with a low K_i value of 0.2 μM. The inhibitor was found to be susceptible to varying concentrations of reducing agents like DTT and 2-mercaptoethanol, thereby indicating the role of disulphide bridges in maintaining its three dimensional structure and stability. The purified inhibitor caused mortality and suppressed larval growth of Pieris brassicae larvae. It was also found to be effective against gut trypsin extracted from Spodoptera littoralis. The sequence of the genes encoding for such inhibitors can be determined and the genes expressing protease inhibitors can be used in vegetable crops to confer resistance against insect pests and other plant pathogens.

Keywords

Albizia lebbeck / trypsin inhibitor / insecticidal activity purification / Pieris brassicae

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