Isolation and partial characterization of an α-amylase inhibitor present in Phaseolus lunatus seeds and its relationship with the induction of α-amylases in larval Zabrotes subfasciatus

The Mexican bean weevil, Zabrotes subfasciatus, causes significant economic losses by infesting the seeds of three major legume species cultivated in Brazil, namely Phaseolus lunatus, Phaseolus vulgaris, and Vigna unguiculata. α-Amylase inhibitors found in P. vulgaris seeds, known as α-AI-1, are efficient against Old World bruchids but are ineffective against Z. subfasciatus. Exposure to α-AI-1 induces the expression and secretion of two α-amylase isoforms, both insensitive to this inhibitor when compared to the constitutive isoform. Interestingly, this induction was also observed when these insects were cultivated on P. lunatus seeds. In this study, we demonstrated the induction of digestive amylases in Z. subfasciatus on P. lunatus seeds through feeding trials. Separation of P. lunatus seed extracts via ion-exchange chromatography led to the identification of fractions containing inhibitory activity against porcine pancreatic amylase. Enzymatic kinetic parameters revealed the presence of a mixed non-competitive inhibitor in P. lunatus fractions, akin to the pattern observed in P. vulgaris. Furthermore, the protein profiles of both types of seeds were similar, with the enrichment of a 30–32 kD protein, possibly representing a precursor form of the α-amylase inhibitor. Thus, our findings show the presence of an α-AI-1-like inhibitor in P. lunatus seeds, which may play a pivotal role in modulating digestive amylases in Z. subfasciatus larvae.