Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination
In the present work, DNA recombination of three homologous tau class glutathione transferases (GSTUs) allowed the creation of a library of tau class <i>Gm</i>GSTUs. The library was activity screened for the identification of glutathione transferase (GST) variants with enhanced catalytic...
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MDPI AG
2021-03-01
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| Series: | Symmetry |
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| Online Access: | https://www.mdpi.com/2073-8994/13/3/461 |
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| author | Fereniki Perperopoulou Maria Fragoulaki Anastassios C. Papageorgiou Nikolaos E. Labrou |
| author_facet | Fereniki Perperopoulou Maria Fragoulaki Anastassios C. Papageorgiou Nikolaos E. Labrou |
| author_sort | Fereniki Perperopoulou |
| collection | DOAJ |
| description | In the present work, DNA recombination of three homologous tau class glutathione transferases (GSTUs) allowed the creation of a library of tau class <i>Gm</i>GSTUs. The library was activity screened for the identification of glutathione transferase (GST) variants with enhanced catalytic activity towards the herbicide alachlor (2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide). One enzyme variant (<i>Gm</i>GSTsf) with improved catalytic activity and binding affinity for alachlor was identified and explored for the development of an optical biosensor for alachlor determination. Kinetics analysis and molecular modeling studies revealed a key mutation (Ile69Val) at the subunit interface (helix α3) that appeared to be responsible for the altered catalytic properties. The enzyme was immobilized directly on polyvinylidenefluoride membrane by crosslinking with glutaraldehyde and was placed on the inner surface of a plastic cuvette. The rate of pH changes observed as a result of the enzyme reaction was followed optometrically using a pH indicator. A calibration curve indicated that the linear concentration range for alachlor was 30–300 μM. The approach used in the present study can provide tools for the generation of novel enzymes for eco-efficient and environment-friendly analytical technologies. In addition, the outcome of this study gives an example for harnessing protein symmetry for enzyme design. |
| first_indexed | 2024-03-10T13:18:26Z |
| format | Article |
| id | doaj.art-780b785b55fb4c6ea325a4e1bc6ee4c4 |
| institution | Directory Open Access Journal |
| issn | 2073-8994 |
| language | English |
| last_indexed | 2024-03-10T13:18:26Z |
| publishDate | 2021-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Symmetry |
| spelling | doaj.art-780b785b55fb4c6ea325a4e1bc6ee4c42023-11-21T10:12:40ZengMDPI AGSymmetry2073-89942021-03-0113346110.3390/sym13030461Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor DeterminationFereniki Perperopoulou0Maria Fragoulaki1Anastassios C. Papageorgiou2Nikolaos E. Labrou3Laboratory of Enzyme Technology, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, GR-11855 Athens, GreeceLaboratory of Enzyme Technology, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, GR-11855 Athens, GreeceTurku Bioscience Centre, University of Turku, 20521 Turku, FinlandLaboratory of Enzyme Technology, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, GR-11855 Athens, GreeceIn the present work, DNA recombination of three homologous tau class glutathione transferases (GSTUs) allowed the creation of a library of tau class <i>Gm</i>GSTUs. The library was activity screened for the identification of glutathione transferase (GST) variants with enhanced catalytic activity towards the herbicide alachlor (2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide). One enzyme variant (<i>Gm</i>GSTsf) with improved catalytic activity and binding affinity for alachlor was identified and explored for the development of an optical biosensor for alachlor determination. Kinetics analysis and molecular modeling studies revealed a key mutation (Ile69Val) at the subunit interface (helix α3) that appeared to be responsible for the altered catalytic properties. The enzyme was immobilized directly on polyvinylidenefluoride membrane by crosslinking with glutaraldehyde and was placed on the inner surface of a plastic cuvette. The rate of pH changes observed as a result of the enzyme reaction was followed optometrically using a pH indicator. A calibration curve indicated that the linear concentration range for alachlor was 30–300 μM. The approach used in the present study can provide tools for the generation of novel enzymes for eco-efficient and environment-friendly analytical technologies. In addition, the outcome of this study gives an example for harnessing protein symmetry for enzyme design.https://www.mdpi.com/2073-8994/13/3/461alachlorglutathione transferaseherbicidesprotein engineeringbiosensorpesticides |
| spellingShingle | Fereniki Perperopoulou Maria Fragoulaki Anastassios C. Papageorgiou Nikolaos E. Labrou Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination Symmetry alachlor glutathione transferase herbicides protein engineering biosensor pesticides |
| title | Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination |
| title_full | Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination |
| title_fullStr | Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination |
| title_full_unstemmed | Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination |
| title_short | Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination |
| title_sort | directed evolution of a glutathione transferase for the development of a biosensor for alachlor determination |
| topic | alachlor glutathione transferase herbicides protein engineering biosensor pesticides |
| url | https://www.mdpi.com/2073-8994/13/3/461 |
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