Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Library Construction
Expression, Purification, and Screening of the Wild-Type and Mutant Enzymes
2.2.2. Assay of Enzyme Activity and Kinetic Analysis
2.2.3. Molecular Modeling
2.2.4. Immobilization of Enzyme and Assessment of Enzyme Activity
2.2.5. Stability Analysis of the Free and Immobilized Enzyme
2.2.6. Colorimetric Assays for the GST-Catalyzed Alachlor Conjugation Reaction
2.2.7. Determination of Alachlor in Natural Water Samples
3. Results and Discussion
3.1. Screening of GmGSTUs Library for Identifying a Shuffled Enzyme for the Development of a Biosensor for Alachlor Determination
3.2. Molecular Modeling
3.3. Enzyme Immobilization
3.4. Thermal Stability of the Immobilized Enzyme
3.5. The Basis of an Optometric Biosensor for Alachlor Determination
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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GmGSTsf (Amino Acid Replacement in GmGSTU4-4 Sequence) | Source of Mutations |
---|---|
Ser2Gln | Spontaneous mutation |
Glu4Gln | Spontaneous mutation |
Tyr32Ser | GmGSTU2-2 |
Ile69Val | GmGSTU2-2 |
Asn149Asp | GmGSTU2-2 |
Val158Ile | GmGSTU2-2 |
Tyr161Asp | GmGSTU2-2 |
Deletion (166AlaTyrGlu168) | GmGSTU2-2 |
Thr172Ser | GmGSTU2-2 or GmGSTU10-10 |
Ile183Val | GmGSTU2-2 or GmGSTU10-10 |
Met210Val | GmGSTU10-10 |
Enzyme | kcat (min−1) | Κm (μΜ)(GSH) | Κm (μΜ)(CDNB) | kcat/Κm(GSH)(min−1μM−1) | kcat/Κm(CDNB)(min−1μM−1) |
---|---|---|---|---|---|
GSH/CDNB | |||||
GmGSTU4-4 1 | 149 ± 18.6 | 159 ± 18.9 | 158 ± 31.6 | 0.9 | 0.9 |
GmGSTsf | 472 ± 42.6 | 223 ± 23.3 | 351 ± 48.1 | 2.1 | 1.3 |
Enzyme | kcat (min−1) | Κm (μΜ)(GSH) | Κm (μΜ)(Alachlor) | kcat/Κm(GSH)(min−1μM−1) | kcat/Κm(CDNB)(min−1μM−1) |
GSH/Alachlor | |||||
GmGSTU4-4 | 4.5 | 210 | 225 | 0.021 | 0.020 |
GmGSTsf | 15.2 | 319 | 352 | 0.047 | 0.043 |
Enzyme | Ki (μΜ) | IC50 (μΜ) |
---|---|---|
GmGSTU4-4 | 127.3 ± 12 | 65.8 ± 11.8 |
GmGSTsf | 7.5 ± 0.9 | 17.5 ± 5.2 |
Type of Interaction | GmGSTU4-4 | GmGSTsf |
---|---|---|
Polar contacts | 4 | 3 |
Weak polar contacts | 3 | 2 |
Hydrogen bonds | 2 | 2 |
Weak hydrogen bonds | 4 | 2 |
Hydrophobic contacts | 11 | 6 |
Added Alachlor Concentrations (mM) | Found Alachlor Concentrations (mM) | % |
---|---|---|
0 0.02 | 0 0.019 | 100 95.0 |
0.04 | 0.037 | 92.5 |
0.08 | 0.084 | 105 |
0.10 | 0.09 | 90 |
0.17 | 0.19 | 111.8 |
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Perperopoulou, F.; Fragoulaki, M.; Papageorgiou, A.C.; Labrou, N.E. Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination. Symmetry 2021, 13, 461. https://doi.org/10.3390/sym13030461
Perperopoulou F, Fragoulaki M, Papageorgiou AC, Labrou NE. Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination. Symmetry. 2021; 13(3):461. https://doi.org/10.3390/sym13030461
Chicago/Turabian StylePerperopoulou, Fereniki, Maria Fragoulaki, Anastassios C. Papageorgiou, and Nikolaos E. Labrou. 2021. "Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination" Symmetry 13, no. 3: 461. https://doi.org/10.3390/sym13030461
APA StylePerperopoulou, F., Fragoulaki, M., Papageorgiou, A. C., & Labrou, N. E. (2021). Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination. Symmetry, 13(3), 461. https://doi.org/10.3390/sym13030461