Abstract
Glyphosate, the most widely used pesticide worldwide, is under debate due to its potentially cancerogenic effects and harmful influence on biodiversity and environment. Therefore, the detection of glyphosate in water, food or environmental probes is of high interest. Currently detection of glyphosate usually requires specialized, costly instruments, is labor intensive and time consuming. Here we present a fast and simple method to detect glyphosate in the nanomolar range based on the surface immobilization of glyphosate’s target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) via fusion to the hydrophobin Ccg2 and determination of enzyme activity with a malachite green assay, which is a common photometric technique to measure inorganic phosphate (Pi). The assay demonstrates a new approach for a fast and simple detection of pesticides.
Highlights
Glyphosate is a potent post-emergent total herbicide.It belongs to the group of organophosphonate-pesticides and is one of the most widely used herbicides worldwide
To prepare a functionalized surface presenting the E. coli enzyme 5-enolpyruvyl-shikimate-3phosphate synthase (EcEPSPS) to detect glyphosate, we investigated two different variants of fusion proteins between the hydrophobin Ccg2 and the enzyme EcEPSPS, both separated by a flexible glycine-serine-linker (G4 S)3
The results suggest that the fusion protein Ccg2_GS_EcEPSPS exhibits an enzymatic activity that is inhibited by glyphosate, which was a prerequisite for the aimed assay
Summary
Glyphosate is a potent post-emergent total herbicide.It belongs to the group of organophosphonate-pesticides and is one of the most widely used herbicides worldwide. Because of its physicochemical properties, i.e., its small size, its polarity and the high water solubility, the detection of glyphosate is difficult. It is non-volatile and zwitterionic [17,18]. Most available methods for detection of glyphosate are costly in terms of sample preparation, technical equipment and time consumption. They require qualified personnel as detection mostly relies on ELISA techniques or chromatography methods coupled with mass spectrometry [10,19,20,21]
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