Structure-based design and application of an engineered glutathione transferase for the development of an optical biosensor for pesticides determination

Biochim Biophys Acta Gen Subj. 2019 Mar;1863(3):565-576. doi: 10.1016/j.bbagen.2018.12.004. Epub 2018 Dec 24.

Abstract

In the present work, a structure-based design approach was used for the generation of a novel variant of synthetic glutathione transferase (PvGmGSTU) with higher sensitivity towards pesticides. Molecular modelling studies revealed Phe117 as a key residue that contributes to the formation of the hydrophobic binding site (H-site) and modulates the affinity of the enzyme towards xenobiotic compounds. Site-saturation mutagenesis of position Phe117 created a library of PvGmGSTU variants with altered kinetic and binding properties. Screening of the library against twenty-five different pesticides, showed that the mutant enzyme Phe117Ile displays 3-fold higher catalytic efficiency and exhibits increased affinity towards α-endosulfan, compared to the wild-type enzyme. Based on these catalytic features the mutant enzyme Phe117Ile was explored for the development of an optical biosensor for α-endosulfan. The enzyme was entrapped in alkosixylane sol-gel system in the presence of two pH indicators (bromocresol purple and phenol red). The sensing signal was based on the inhibition of the sol-gel entrapped GST, with subsequent decrease of released [H+] by the catalytic reaction, measured by sol-gel entrapped indicators. The assay response at 562 nm was linear in the range pH = 4-7. Linear calibration curves were obtained for α-endosulfan in the range of 0-30 μΜ. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of α-endosulfan in real water samples without sample preparation steps.

Keywords: Biosensor; Glutathione transferase; Pesticides determination; Protein engineering; α-Endosulfan.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites / genetics
  • Biosensing Techniques / methods*
  • Catalysis
  • Catalytic Domain / genetics
  • Directed Molecular Evolution / methods
  • Endosulfan / analysis
  • Endosulfan / isolation & purification
  • Environmental Monitoring / methods*
  • Environmental Pollutants / analysis
  • Environmental Pollutants / isolation & purification
  • Enzymes, Immobilized / genetics
  • Enzymes, Immobilized / metabolism
  • Glutathione / metabolism
  • Glutathione Transferase / chemistry*
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism*
  • Mutagenesis, Site-Directed
  • Mutant Proteins / metabolism
  • Pesticides / analysis*
  • Pesticides / isolation & purification
  • Phaseolus / enzymology
  • Phaseolus / genetics
  • Protein Engineering / methods*
  • Reproducibility of Results
  • Soybeans / enzymology
  • Soybeans / genetics
  • Structure-Activity Relationship

Substances

  • Environmental Pollutants
  • Enzymes, Immobilized
  • Mutant Proteins
  • Pesticides
  • Glutathione Transferase
  • Glutathione
  • Endosulfan