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

Abstract

Phaseolus vulgaris α-amylase inhibitor (α-AI) is a protein that has recently gained commercial interest, as it inhibits mammalian α-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 α-amylase- and α-glucosidase-inhibiting activity, as well as for the absence of antinutritional compounds, such as phytohemagglutinin (PHA). All the cultivars presented α-glucosidase-inhibitor activity, while α-AI was missing in two of them. Only the Nieddone cultivar (ACC177) had no haemagglutination activity. In addition, the partial nucleotide sequence of the α-AI gene was identified with the degenerate hybrid oligonucleotide primer (CODEHOP) strategy to identify genetic variability, possibly linked to functional α-AI differences, expression of the α-AI gene, and phylogenetic relationships. Molecular studies showed that α-AI was expressed in all the cultivars, and a close similarity between the Pisu Grogu and Fasolu cultivars’ α-AI and α-AI-4 isoform emerged from the comparison of the partially reconstructed primary structures. Moreover, mechanistic models revealed the interaction network that connects α-AI with the α-amylase enzyme characterized by two interaction hotspots (Asp38 and Tyr186), providing some insights for the analysis of the α-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.