Discovery to Engineering of a Bacterial Thermostable Secretory Phospholipase A ₂ Using Pretrained Computational Models

Secretory phospholipase A2 (sPLA2; EC 3.1.1.4) catalyzes the hydrolysis of the ester bond at the SN-2 position of phospholipids, generating lysophospholipids and free fatty acids. Therefore, it can be used for the enzymatic degumming of crude plant oils. This study focused on the discovery, characterization, and engineering of sPLA2s to obtain thermostable sPLA2s with high long-term bioconversion activity. Large-scale sPLA2 sequence mining and phylogenetic clustering led to the discovery of thermostable enzymes (e.g., sPLA2 from Marinactinospora thermotolerans (MtPLA2)). MtPLA2 was structurally stable at high temperatures with an apparent Tm of 79.8 °C, which is 14.2 °C higher than that of the previously reported thermostable sPLA2 from Sciscionella marina (SmPLA2). The application of a functional site prediction model allowed us to identify critical residues contributing to catalytic activity and structural stability. Ultimately, we constructed highly active and structurally stable variants of MtPLA2 (e.g., MtPLA2_Y53F). MtPLA2_Y53F converted phospholipids (e.g., soybean lecithin) to lysophospholipids and free fatty acids (e.g., linoleic acid and oleic acid) at a rate of 22.2 U/mg protein at 70 °C. Therefore, this study provides a deeper understanding of sPLA2s and will contribute to sustainable industrial practices (e.g., enzymatic oil degumming).