State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China; College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
Anal Chim Acta. 2020 Oct 9;1133:39-47. doi: 10.1016/j.aca.2020.07.077. Epub 2020 Aug 12.
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 HO 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 × 10-fold; 2.25 × 10-fold; and 9.64 × 10-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-HO 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.
草甘膦（Glyp）的广泛和密集利用引起了公众对化学残留可能对环境和健康造成潜在风险的关注。因此，人们非常希望开发出一种高选择性、低成本和易于操作的 Glyp 检测方法。通过定向进化筛选高选择性酶在实际应用中非常重要。本文通过定向进化从蜡状芽孢杆菌（Bacillus cereus）获得的甘氨酸氧化酶（BceGO）中获得了一种对草甘膦有偏好的草甘膦氧化酶（GlypO），用于产生 HO。与 BceGO 相比，GlypO 对 Glyp 的催化效率、比活性常数和亲和力增强因子分别提高了 2.85×10 倍、2.25×10 倍和 9.64×10 倍。对甘氨酸的催化效率降低了 78.60 倍。枯草芽孢杆菌（Bacillus subtilis）的孢子由于表面存在 CotA-漆酶，能够有效地催化发光氨-HO 反应，产生极好的化学发光（CL）信号。基于这些发现，通过与生物反应系统偶联，提出了一种用于 Glyp 检测的新型 CL 生物传感器。该 CL 生物传感器具有环保、低成本、高选择性和灵敏度以及良好的实际应用前景等优点，可用于环境污染控制。
