Abstract
In the present work, DNA recombination of three homologous tau class glutathione transferases (GSTUs) allowed the creation of a library of tau class GmGSTUs. 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 (GmGSTsf) 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.
Highlights
The analysis showed that the GmGSTsf variant displayed about 17-fold lower Kinetic inhibition constant (Ki) and 4-fold lower IC50 values compared to the parent enzyme GmGSTU4-4, suggesting that the affinity of the variant has been improved significantly
The immobilized enzyme exhibited significantly higher stability and lost less than 30% of its initial activity after 15 days of incubation. These findings indicate that the immobilization of GmGSTsf on polyvinylidenefluoride membrane significantly improved the enzyme operational stability upon storage at 4 ◦ C, an important consideration that determines the practical viability of the biosensor
In the present study we report on a protein engineering approach for the creation of a mutant glutathione transferase with improved catalytic performance toward the alachlor/GSH conjugation reaction
Summary
2.5.1.18) are a widely spread family of enzymes found in both eukaryotes and prokaryotes [1,2] They catalyze the conjugation of glutathione (GSH) with a range of hydrophobic xenobiotic compounds such as drugs, environmental pollutants, and pesticides, including chloroacetanilide herbicides [3,4,5,6,7,8,9]. Their catalytic versatility and wide-substrate specificity stem from their structural flexibility and active-site plasticity [3,10,11,12,13].
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