Engineered glyphosate oxidase coupled to spore-based chemiluminescence system for glyphosate detection.

The extensive and intensive utilization of glyphosate (Glyp) caused public concerns on the potential risk of environment and health resulted from the chemical residues. Therefore, the development of a high-selective, low-cost and easy-operation Glyp detection methods is highly desired. Screening highly selective enzymes by directed evolution is important in practical applications. Herein, a glyphosate oxidase (GlypO) preferring substrate Glyp to produce H2O2 was obtained via directed evolution from glycine oxidase obtained from Bacillus cereus (BceGO). The catalytic efficiency, specificity constant, and affinity enhancement factor of GlypO toward Glyp were increased by 2.85 × 103-fold; 2.25 × 105-fold; and 9.64 × 104-fold, respectively, compared with those of BceGO. The catalytic efficiency toward glycine decreased by 78.60-fold. The spores of Bacillus subtilis (B. subtilis) effectively catalyzed luminol-H2O2 reaction to create excellent chemiluminescence (CL) signal because CotA-laccase exists on their surface. Based on these findings, a new CL biosensor via coupling to biological reaction system was presented for Glyp detection. The CL biosensor exhibited several advantages, such as eco-friendliness, low cost, high selectivity and sensitivity, and good practical application prospects for environmental pollution control.