Detection of insecticides by Tetronarce californica acetylcholinesterase via expression and in silico analysis

The acetylcholinesterase (AChE) is involved in termination of synaptic transmission at cholinergic synapses and plays a vital role in the insecticide detection and inhibitor screening. Here, we report the heterologous expression of an AChE from Tetronarce californica ( Tc A) in Escherichia coli ( E. coli ) as a soluble active protein. Tc A was immobilized in calcium alginate beads; the morphology, biochemical properties, and insecticide detection performance of free and immobilized Tc A were characterized. Moreover, we used sequence, structure-based approaches, and molecular docking to investigate structural and functional characterization of Tc A. The results showed that Tc A exhibited a specific activity of 102 U/mg, with optimal activity at pH 8.0 and 30 °C. Immobilized Tc A demonstrated superior thermal stability, pH stability, and storage stability compared to the free enzyme. The highest sensitivity of free Tc A was observed with trichlorfon, whereas immobilized Tc A showed reduced IC 50 values towards tested insecticides by 3 to 180-fold. Molecular docking analysis revealed the interaction of trichlorfon, acephate, isoprocarb, λ-cyhalothrin, and fenpropathrin in the active site gorge of Tc A, particularly mediated through the formation of hydrogen bonds and π - π stacking. Therefore, Tc A expressed heterologously in E. coli is a promising candidate for applications in food safety and environmental analysis. Key points • T. californica AChE was expressed solubly in prokaryotic system. • The biochemical properties of free/immobilized enzyme were characterized. • The sensitivity of enzyme to insecticides was evaluated in vitro and in silico. Graphical Abstract