Proteinaceous inhibitors of α-amylase and α-glucosidase from common bean (Phaseolus vulgaris L.): biochemical characterization and phylogenetic analysis of Sardinian cultivars

The common bean Phaseolus vulgaris is a good source of protein fiber and bioactive compounds. Among these proteinaceous α-amylase inhibitors (α-AIs) and α-glucosidase inhibitor could play an important role in weight loss and control of glycemic index above all in overweight and diabetic subjects. In this study 10 poorly studied Italian P. vulgaris cultivars were screened about their content in α-amylase/glucosidase inhibitors, antinutritional factor phytohemagglutinin (PHA), and about the expression of α-AI gene and their phylogenetic relationship. All the cultivars presented α-glucosidase inhibitor activity, while α-AI was missing in two of them. Only Nieddone cultivar (ACC177) had no hemagglutination activity and its α-AI was extracted, purified and characterized. The purified inhibitor has a tetrameric structure with a molecular weight of about 42 kDa, a temperature optimum of about 40°C and two pH optima (5 and 6.5). Thermal stability is remarkably important, since the inhibitory activity was maintained at 25% after 5 hours of incubation at 100°C. The purified inhibitor had the highest affinity toward insect α-amylase about 3-fold more susceptible than human salivary amylase and porcine pancreatic α-amylase. The kinetic characterization showed a mixed-type inhibition, suggesting a multiple site protein/protein interaction between enzyme (α-amylase) and inhibitor. Molecular studies showed that α-AI is expressed in all cultivars and a close similarity between the two cultivars Pisu Grogu and Fasolu α-AI and isoform α-AI-4 emerged from the comparison of the partially reconstructed primary structures. Moreover, from mechanistic models emerge the interaction network that connects the αAI with the α-amylase enzymecharacterized from two interaction hotspots (Asp38, and Tyr186), giving some insights for the analysis of α-AI primary structure from the different cultivars, particularly about structure-activity relationship. This study can be lay the basis for increase the knowledge about this class of proteins and developing commercial preparations from Italian common beans cultivars.