Strategies to Express Soluble Recombinant Apostichopus japonicus Phospholipase A 2 in Escherichia coli and Its Activities
Year: 2025
Type: article
Abstract: ABSTRACT Phospholipase A 2 (PLA 2 ) 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 PLA 2 from various species holds significant biotechnological potential. In this study, we aimed to heterologous express the PLA 2 gene from Apostichopus japonicus (AjPLA 2 ) in prokaryotic systems. Four plasmid vectors (pET‐28a, pCold II, pMALc2x, and pET32a) were selected for constructing recombinant Aj‐PLA 2 ‐expressing strains. Among these constructs, only Rosetta gami (DE3)/pET32a‐Aj‐PLA 2 produced soluble and catalytically active recombinant TrxA‐Aj‐PLA 2 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‐PLA 2 and pure Aj‐PLA 2 enzymes were subsequently purified via Ni 2+ ‐NTA affinity chromatography using a 300 mM imidazole elution buffer. Enzymatic characterization revealed that the TrxA‐Aj‐PLA 2 exhibited maximal activity at approximately 40°C and pH 9.0 in the presence of 5 mM Ca 2+ ions. Gas chromatography–mass spectrometry (GC–MS) analysis confirmed that TrxA‐Aj‐PLA 2 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‐PLA 2 against marine bacteria. Collectively, this study achieved the soluble expression of the TrxA‐Aj‐PLA 2 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‐PLA 2 .
DOI: 10.1002/bab.70080
Authors: Jia Li, Xiaoqing Li, Ying Liu, Cheng Li, Benkang Liu
Institutions: Dalian Polytechnic University, Dalian Minzu University
Language: English
Subfield: Biotechnology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4416825066