The study figured out how to make a sea‑cucumber enzyme (PLA2) in bacteria so it stays soluble and active. They found the best conditions for producing it, purified it, and showed it works best at about 40 °C, pH 9, with calcium, breaking down a specific phospholipid and even killing some marine bacteria in lab tests.

Phospholipase A₂ (PLA₂) is a multifunctional enzyme involved in diverse physiological processes and industrial applications, including phospholipid modification, oil refining, and feed additive production. Consequently, the exogenous expression of PLA₂ from various species holds significant biotechnological potential. In this study, we aimed to heterologous express the PLA₂ gene from Apostichopus japonicus (AjPLA₂) in prokaryotic systems. Four plasmid vectors (pET-28a, pCold II, pMALc2x, and pET32a) were selected for constructing recombinant Aj-PLA₂-expressing strains. Among these constructs, only Rosetta gami (DE3)/pET32a-Aj-PLA₂ produced soluble and catalytically active recombinant TrxA-Aj-PLA₂ fusion protein upon induction. Optimal expression conditions were determined as follows: 0.1 mM IPTG induction for 2.5 h at 25°C, followed by 16 h of post-induction incubation. The active TrxA-Aj-PLA₂ and pure Aj-PLA₂ enzymes were subsequently purified via Ni²⁺-NTA affinity chromatography using a 300 mM imidazole elution buffer. Enzymatic characterization revealed that the TrxA-Aj-PLA₂ exhibited maximal activity at approximately 40°C and pH 9.0 in the presence of 5 mM Ca²⁺ ions. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed that TrxA-Aj-PLA₂ specifically hydrolyzed the sn-2 ester bond of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), releasing oleic acid (C18:0). Furthermore, turbidimetric assays demonstrated the antimicrobial potential of TrxA-Aj-PLA₂ against marine bacteria. Collectively, this study achieved the soluble expression of the TrxA-Aj-PLA₂ enzyme possessing dual functionalities: phospholipid hydrolysis and marine bacterial growth inhibition in vitro. These findings establish a critical foundation for future mechanistic investigations and biotechnological applications of Aj-PLA₂.