High-performance biocatalysts for enhanced biodegradation of organophosphorus pesticides: Directed evolution and rational design.

摘要

The use of pesticides is necessary to increase food production and protect the public from diseases such as malaria. Organophosphates (OP), a class of highly toxic neurotoxins, account for about half of all pesticides used in the U.S. Due to the growing environmental concerns associated with these pesticides in food products and water supplies, there is a great need to develop a proper remediation method to deal with their wide spread contamination. Organophosphorus hydrolase (OPH) isolated from naturally occurring bacteria has been used for the degradation of a variety of organophosphates. Although this enzyme has a broad substrate specificity, the hydrolysis rate varies from diffusion-limited for paraoxon to poorly degraded for pesticides such as methyl parathion and chlorpyrifos and chemical warfare agents such as sarin.; Directed evolution was employed to improve the substrate specificity of OPH. Improved variants against methyl parathion were generated after only two rounds of DNA shuffling and screening. The best variant 22A11 hydrolyzed methyl parathion 30 times faster than the wild-type and also degraded paraoxon, parathion, and coumaphos 2–10 times faster. Several distal mutations were identified to be responsible for this global increase in hydrolysis and their importance in determining substrate specificity was investigated by site-directed mutagenesis and saturation mutagenesis. One particular mutation, K185R, was shown to enhance the overall hydrolysis rate when present in conjunction with other mutations.; Similar efforts to create improved variants for the degradation of chlorpyrifos were also performed. After two generations of DNA shuffling and screening, variants with ∼745-fold increased in k_cat/K _m value were obtained. The best variant B3561 also showed improved hydrolysis rates for paraoxon, methyl parathion, parathion, and coumaphos. The results suggest that the variant B3561 is one of the most efficient enzymes available to attack a wide spectrum of nerve agents.