Strategies to Express Soluble Recombinant Apostichopus japonicus Phospholipase A2 in Escherichia coli and Its Activities

Abstract

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.