Biochemical and biophysical characterization of bio-active proteins in food and natural sources

Non-specific lipid transfer proteins (nsLTPs) are small, ubiquitous plant proteins associated with lipid transport. NsLTPs have a very high conserved structural fold and, despite the wide distribution, their precise biological role remains partially unknown. In this work, a multidisciplinary approach was applied combining biochemical, biophysical, and computational techniques to investigate the potential catalytic activity of purified nsLTP from hazelnut: Cor a 8. Hydrolytic activity was identified in both crude hazelnut extracts and purified protein fractions using the synthetic substrate p-nitrophenyl laurate (pNPL). Kinetic analyses revealed an important lipolytic activity, suggesting that Cor a 8 may play a role in lipid metabolism never identified so far. Molecular docking and molecular dynamics simulations were performed to obtain more specific insights into the observed activity. The analyses revealed stable lipid binding within the hydrophobic cavity of Cor a 8 and identified two key residues, Arg68 and Tyr103, as potential contributors to catalysis. The proposed mechanism suggests a noncanonical catalytic process, getting more information on the non-stereoselective hydrolysis products on triolein as substrate. Cor a 8 was discovered to have an unusually high solubility suggesting the presence of heterogeneous post-translational modifications. Moreover, chromatographic separation allowed to isolate two distinct protein populations, showing differences in enzymatic activity; this further supports the existence of functionally diverse isoforms. Overall, this work provides evidence that Cor a 8 possesses intrinsic catalytic capabilities, beyond its established role as lipid carriers. These findings contribute to re-think the pag. 9 biological role of this protein family and open new perspectives in plant biology and biotechnological applications, including lipid processing and raw material shelf-life assessment