ORIGINAL ABSTRACT

Glyphosate persistence in the environment may threaten human health and ecosystems. The development of a cost-effective, portable and sensitive method for glyphosate detection is crucial. In this study, we conducted directed evolution of the highly active glyphosate oxidase B5R18 in our laboratory and obtained a variant with increased enzyme activity and thermostability. Subsequently, via surface display technology for the S-layer protein from the Bacillus thuringiensis (Bt) strain CTC, the glyphosate oxidase variant was actively anchored onto the surface of the Bt strain. Compared with the purified enzyme, the surface-displayed glyphosate oxidase exhibited better storage stability, with more than 95 % activity remaining after 35 days at 28 °C, whereas the free enzyme activity was only 20 %. Under optimal reaction conditions, surface-displayed glyphosate oxidase can catalyze glyphosate to generate H2O2. Using a horseradish peroxidase coupling assay, a visible orange-red substance, which can also be detected spectrophotometrically at 450 nm, was produced. The increase in absorbance was linearly correlated with the concentration of glyphosate (5-400 μM). The detection limit was 0.82 μM, and the recovery rates of real samples reached 97.5-109.1 %. Additional orange-red product is deposited on the surface of the whole-cell catalyst, which can be completely intercepted by a 0.22 μm filter membrane. A portable biosensor and color comparison card for detecting glyphosate were subsequently prepared, which allowed qualitative and semiquantitative analysis of glyphosate levels (0.05-1 mM, LOD of 0.013 mM). This biosensing device provides an easy-to-operate and effective strategy for onsite glyphosate detection.