Enzymatic release of dipeptidyl peptidase-4(DPP-4) inhibitors from pigeon pea proteins: In silico and wet-lab assessments

Objective: Evidence supporting the accuracy of bioinformatics approach to predict bioactivity of peptides is limited. In this study, the potential of nutrient storage proteins in pigeon pea as precursors of DPP-4 inhibiting peptides were assessed with in silico simulations and validated with wet-lab experiments.
Methods Used: Pigeon pea proteins and sequences were obtained from UniProt Knowledgebase (UniProtKB). In silico physicochemical properties were determined with the ProtParam feature. Theoretical hydrolysis was conducted with ‘enzyme(s) action’ in BIOPEP-UWM. Activity of fragments potentially released by selected enzymes were calculated. In vitro, proteins were extracted from pigeon pea flour by alkaline solubilization and acid precipitation. Enzymatic hydrolysis was conducted, and free amino nitrogen content was determined using the o-phthalaldehyde (OPA) assay. Protein profile of proteins and their hydrolysates were analyzed with SDS-PAGE. Particle characteristics, surface hydrophobicity, and DPP-4 inhibition were also determined.

Results: In silico, 40 pigeon pea proteins evaluated had 46% of amino acids associated with DPP-4 inhibitory activity of peptides. After virtual hydrolysis, pepsin had the highest theoretical degree of hydrolysis, and bioactivity of released DPP-4 inhibiting peptides, compared to papain and thermolysin. In vitro, hydrolysates dose dependently inhibited DPP‐4 activity. Thermolysin however released the most active DPP-4 inhibitors with an IC₅₀ at 3.90 mg/ml. The hydrolysates contained similar amino acids but different particle sizes.

Conclusion: Using in silico and wet-lab analyses, this study demonstrated that proteins from pigeon pea, an underutilized legume, can be precursors of DPP-4 inhibitors for possible application in functional foods for managing Type 2 diabetes mellitus.