Biochemical and Phylogenetic Analysis of Italian Phaseolus vulgaris Cultivars as Sources of a-Amylase and a-Glucosidase Inhibitors

Phaseolus vulgaris a-amylase inhibitor (a-AI) is a protein that has recently gained commercial interest, as it inhibits mammalian a-amylase activity, reducing the absorption of dietary carbohydrates. Numerous studies have reported the efficacy of preparations based on this protein on the control of glycaemic peaks in type-2 diabetes patients and in overweight subjects. A positive influence on microbiota regulation has also been described. In this work, ten insufficiently studied Italian P. vulgaris cultivars were screened for a-amylase- and a-glucosidase-inhibiting activity, as well as for the absence of antinutritional compounds, such as phytohemagglutinin (PHA). All the cultivars presented a-glucosidase-inhibitor activity, while a-AI was missing in two of them. Only the Nieddone cultivar (ACC177) had no haemagglutination activity. In addition, the partial nucleotide sequence of the a-AI gene was identified with the degenerate hybrid oligonucleotide primer (CODEHOP) strategy to identify genetic variability, possibly linked to functional a-AI differences, expression of the a-AI gene, and phylogenetic relationships. Molecular studies showed that a-AI was expressed in all the cultivars, and a close similarity between the Pisu Grogu and Fasolu cultivars' a-AI and a-AI-4 isoform emerged from the comparison of the partially reconstructed primary structures. Moreover, mechanistic models revealed the interaction network that connects a-AI with the a-amylase enzyme characterized by two interaction hotspots (Asp38 and Tyr186), providing some insights for the analysis of the a-AI primary structure from the different cultivars, particularly regarding the structure-activity relationship. This study can broaden the knowledge about this class of proteins, fuelling the valorisation of Italian agronomic biodiversity through the development of commercial preparations from legume cultivars.