研究目的
To develop an optimized glutathione transferase mutant for creating an optical biosensor for α-endosulfan determination in environmental samples.
研究成果
The structure-based engineering of GST successfully produced a mutant (Phe117Ile) with enhanced sensitivity to α-endosulfan, enabling the development of a reliable optical biosensor. The biosensor demonstrated linear response, good reproducibility, and applicability to real water samples, offering a low-cost alternative to chromatographic methods. Future work could extend this approach to other pesticides and incorporate data processing techniques for improved selectivity.
研究不足
The biosensor may suffer from poor specificity as GSTs have wide inhibition capabilities, making it difficult to discriminate between different pesticides without additional methods like artificial neural networks. Leaching and diffusion barriers in sol-gel entrapment are potential issues, though not detected in this study.
1：Experimental Design and Method Selection:
A structure-based design approach combined with site-saturation mutagenesis was used to engineer a GST variant. Molecular docking and dynamics simulations guided the mutagenesis. Enzyme activity assays and inhibition studies were performed to screen mutants. An optical biosensor was developed using sol-gel entrapment of the enzyme and pH indicators.
2：Sample Selection and Data Sources:
The synthetic GST enzyme PvGmGSTUG was used, derived from Phaseolus vulgaris and Glycine max genes. Pesticides were sourced from Riedel de Haen. Water samples included drinking water from Athens and mineral water (Korpi, NESTLE Hellas).
3：List of Experimental Equipment and Materials:
Equipment includes ultrasonic bath, spectrophotometer for absorbance measurements, PCR thermocycler, chromatography setup for purification. Materials include TEOS, PTMOS, PEG, CTAB, pH indicators (bromocresol purple and phenol red), pesticides, enzymes, buffers, and reagents from Sigma-Aldrich and other suppliers.
4：Experimental Procedures and Operational Workflow:
Site-saturation mutagenesis at Phe117 position using PCR with degenerate primers, expression in E. coli, purification by affinity chromatography. Kinetic and inhibition assays with CDNB/GSH system. Sol-gel entrapment of enzyme and indicators, aging process, and spectroscopic measurements for biosensor assessment. Application to spiked water samples for recovery experiments.
5：Data Analysis Methods:
Kinetic parameters determined using GraphPad Prism software. Inhibition data fitted to equation for IC50 calculation. Absorbance measurements at 562 nm for calibration curves. Statistical analysis for reproducibility (RSD).
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