Bowman–Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential

Abstract

Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3×10−10 and 1.5×10−10M, respectively, displaying a tight binding property. SDS–PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman–Birk inhibitor family. CFPI remained stable to progressive heating for 30min to each temperature range of 37 up to 100°C and CD analysis exhibited no changes in spectra at 207nm after heating at 90°C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2–10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.