Development of a rapid process for purification of Bowman-Birk and Kunitz inhibitors from legume seeds, and evaluation of their biophysical, insecticidal, and antimicrobial properties

Abstract

Bowman-Birk inhibitor (BBI ~10 kDa) and Kunitz inhibitor (KI ~20 kDa) are serine protease/proteinase inhibitor(s) [PI(s)] ubiquitously found in several Leguminous plant species with insecticidal and therapeutic properties. Due to narrow molecular mass differences, the separation of these inhibitors from a single seed variety is tedious. The present study is aimed to develop a rapid protocol (<24 h) for purifying BBI and KI from legume seeds using mild trichloroacetic acid (TCA) extraction followed by trypsin-affinity chromatography. The mature seeds of Vigna radiata and Cajanus platycarpus are used as a model to purify BBI and KI using this protocol. The BBI and KI purified from the seeds of V. radiata are labeled as VrBBI & VrKI, and C. platycarpus are labeled as CpBBI & CpKI, respectively. These PIs are confirmed by immunodetection and MALDI-TOF studies and further characterized for their structural (CD & fluorescence spectroscopy) and functional properties (temperature & DTT stability). BBI(s) purified using the above process are effective in the management of castor semi-looper 'Achaea janata', while KI(s) are effective in the management of pod borer 'Helicoverpa armigera'. Besides, both BBI(s) and KI(s) have significant potential in controlling the growth of methicillin-sensitive 'Staphylococcus aureus', a gram-positive pathogenic bacterium.

Keywords: Bowman-Birk inhibitor and Kunitz inhibitor; Insecticidal and antibacterial activity; Trichloroacetic acid extraction; Trypsin-affinity chromatography.