Functional and Structural Characterization of a Thermostable Phospholipase A₂ from a Sparidae Fish (Diplodus...

Novel phospholipase (PLA₂) genes from the Sparidae family were cloned. The sequenced PLA₂ revealed an identity with pancreatic PLA₂ group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA₂ (DaPLA₂) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA₂ was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA₂ was constructed to gain insights into the functional properties of sparidae PLA₂. Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA₂. Using the monolayer technique, the purified DaPLA₂ was found to be active on various phospholipids ranging from 10 to 20 mN·m^-1, which explained the absence of the hemolytic activity for DaPLA₂.