Here we report the cDNA cloning of a phospholipase A₂ (PLA₂) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA₂. In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA₂ are closer to avian PLA₂ group IB than to mammals’ ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA₂ (AsPLA₂). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA₂ with a molecular mass of 15 kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400 U.mg⁻¹ measured on phosphatidylcholine at pH 8.5 and 50 °C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA₂. By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA₂ at high temperatures was explained. Using the monomolecular film technique, AsPLA₂ was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25 dyn cm⁻¹. Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA₂, provides new insights on structure-activity relationships of fish PLA₂.

在这里，我们报告了五个Spa科的磷脂酶A ₂（PLA ₂）的cDNA克隆。推导的氨基酸序列显示出与胰腺PLA _2的高度相似性。此外，从各种可用序列的比对得出的系统发育树显示，Sparidae PLA ₂比鸟类PLA ₂组更接近禽类PLA ₂组IB。为了了解这些酶的结构-功能关系，我们在此报告了在大肠杆菌中的重组表达，带有His标签的环鲷PLA ₂（AsPLA _2）的重折叠和表征。）。通过凝胶电泳和MALDI-TOF质谱数据证明，使用单个Ni亲和层析步骤即可获得分子量为15 kDa的高度纯化的重组AsPLA ₂。该酶在pH 8.5和50°C下在磷脂酰胆碱上测得的比活为400 U.mg ^-1。该酶的高热活性和热稳定性使其成为各种生物学应用中的潜在候选者。将这些酶的3D结构模型与系统发育相关的胰腺PLA _2的结构进行了比较。通过遵循这些模型并利用分子动力学模拟，解释了AsPLA ₂在高温下的电阻。使用单分子膜技术AsPLA ₂发现在表面压力在12至25达因厘米^-1之间的空气/水界面处分布的各种磷脂上具有活性。有趣的是，该酶在二月桂酰磷脂酰甘油单层上显示出主要活性，这种行为已通过分子对接和动力学模拟分析得到证实。Sparidae PLA ₂的热活性新成员的发现，为鱼类PLA _2的构效关系提供了新的见解。