Structure-based design and application of an engineered glutathione transferase for the development of an optical biosensor for pesticides determination
| Autor: | Farid S. Ataya, Evangelia G. Chronopoulou, Dimitrios Vlachakis, Nikolaos E. Labrou, Anastassios C. Papageorgiou |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Biophysics
Biosensing Techniques Protein Engineering 01 natural sciences Biochemistry Catalysis Structure-Activity Relationship 03 medical and health sciences chemistry.chemical_compound Catalytic Domain Sample preparation Pesticides ta414 Molecular Biology Glutathione Transferase 030304 developmental biology Phaseolus chemistry.chemical_classification Phenol red 0303 health sciences Binding Sites Chromatography 010401 analytical chemistry Reproducibility of Results Protein engineering Enzymes Immobilized Glutathione 0104 chemical sciences Enzyme chemistry Mutagenesis Site-Directed Environmental Pollutants Mutant Proteins Soybeans Directed Molecular Evolution Xenobiotic Bromocresol purple Biosensor Endosulfan Environmental Monitoring |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - General Subjects. 1863:565-576 |
| ISSN: | 0304-4165 |
| DOI: | 10.1016/j.bbagen.2018.12.004 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect