Preparation of a Bombyx mori acetylcholinesterase enzyme reagent through chaperone protein disulfide isomerase co-expression strategy in Pichia pastoris for detection of pesticides.

Abstract

The cholinesterase-based spectrophotometric methods for detection of organophosphate pesticides (OPs) and carbamate pesticides (CPs) have been proposed as a good choice for their high efficiency, simplicity and low cost. The enzyme, as a core reagent, is of great importance for the developed method. In this study, a protein disulfide isomerase (PDI) co-expression strategy in Pichia pastoris was employed to enhance the yield of recombinant Bombyx mori acetylcholinesterase 2 (rBmAChE2). Subsequently, the prepared enzyme reagent was used to detect the pesticides in real samples. The results showed that the co-expression of rBmAChE2 with PDI increased the enzyme activity of the supernatant and the yield of purified rBmAChE2 up to 60 U/mL and 6 mg/L respectively, both almost 5-fold higher than those of original recombinant strain. In addition, 5 g/L gelatin reagent could help to preserve nearly 90% of the rBmAChE2 activity for 90 days in 4°C and the limits of detections (LODs) of the rBmAChE2-based assay for 20 kinds of OPs or CPs ranged from 0.010 to 2.725 mg/kg, which were lower than most of indexes present in current Chinese National Standard (GB/T 5009.199-2003) or the maximum residue limits (GB 2763-2019). Furthermore, the detection results of 23 vegetable samples were verified by the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, which indicated that the rBmAChE2-based assay in this work is suitable for pesticide residues rapid detection.