In this study, we have produced for the first time a fish phospholipase (PLA2) in heterologous system (E. coli). The Diplodus annularis PLA2 (DaPLA2) was then refolded from inclusion bodies and purified by Ni-affinity chromatography. We used the pH-stat method (with emulsified phosphatidylcholine as substrate) and the monomolecular film technique (using various glycerophospholipids substrates spread in the form of monomolecular films at the air-water interface) to access the biochemical and kinetic properties of the recombinant DaPLA2. The DaPLA2 was found to be active and stable at higher temperatures (37-50 °C) than expected. Interestingly, DaPLA2 hydrolyzes efficiently both purified phosphatidylglycerol and phosphatidylethanolamine at 20 mN/m. These analytical results corroborate with the fact that the catalytic activity of DaPLA2, measured with the pH-stat using egg yolk as substrate, is mainly due to the hydrolysis of the PE fraction present in egg yolk, whereas the phosphatidylglycerol is a hallmark substrate for the most secreted PLA2-IB.
Keywords: Biochemical characterization; Cloning; Diplodus annularis PLA2 (DaPLA2); Heterologous expression and purification; Sparidae.