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

Abstract

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 (TcA) in Escherichia coli (E. coli)as a soluble active protein. TcA was immobilized in calcium alginate beads; the morphology, biochemical properties, and insecticide detection performance of free and immobilized TcA were characterized. Moreover, we used sequence, structure-based approaches, and molecular docking to investigate structural and functional characterization of TcA. The results showed that TcA exhibited a specific activity of 102 U/mg, with optimal activity at pH 8.0 and 30 °C. Immobilized TcA demonstrated superior thermal stability, pH stability, and storage stability compared to the free enzyme. The highest sensitivity of free TcA was observed with trichlorfon, whereas immobilized TcA showed reduced IC50 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 TcA, particularly mediated through the formation of hydrogen bonds and π-π stacking. Therefore, TcA 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.