Protein engineering of glutathione transferase for the development of optical biosensor to detect xenobiotics

摘要

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible enzymes that play essential role in detoxification and degradation of toxic compounds, including pesticides. The purpose of the present study is the development of an optical enzyme biosensor based on GSTs, for the detection and determination of pesticides in environmental samples. Protein engineering was used for the creation of a GST variant with higher selectivity towards pesticides. cDNA libraries were created from Phaseolus vulgaris and Glycine max stressed plants using degenerated primers and reverse transcription-PCR. Large diversity in GST genes was accomplished employing directed evolution through DNA shuffling of a mixture of GST genes from P. vulgaris and G. max stressed plants. The shuffled library of chimaeric GST genes was cloned in E. coli expression plasmid. Screening of the library led to the isolation of a novel GST enzyme that displays both glutathione transferase and glutathione peroxidase activities. The enzyme was purified by affinity chromatography and characterized by kinetic analysis towards 20 different substrates and 66 different pesticides. The results showed that the organoclorine insecticides and strobilurins (fungicides) are strong inhibitors of the enzyme. The specificity of the enzyme towards pesticides was further improved using site-saturation mutagenesis at position Phe117. The mutant Phe117Ile displays 5-fold higher catalytic efficiency and selectivity towards organochlorine insecticides. Therefore, the mutant GSTPhe117Ile was used for the development of an optical biosensor. The enzyme was immobilized in alkoxysilane (TEOS/PTMOS) sol-gel system in the presence of the pH indicators bromocresol purple (acidic) and phenol red (basic). The bioactive material exhibits linearity in the range of 0.625-30 μΜ α-endosulfan (pH=4-7) at 562 nm and was used for the development of an analytical method for the determination of α-endosulfan in environmental samples.