earticle

영어

Synthetic organophosphates (OPs) have been used as nerve agents and pesticides due to their extreme toxicity and have caused serious environmental and human health problems. Therefore, effective methods for detoxification and decontamination of OPs are of great significance. We have working on the development of organophosphate-degrading enzymes through directed evolution and structure-based rational design. For directed evolution, we constructed and used a high throughput screening system that was based on phenolics-responsive transcription activator for directed evolution of organophosphate-degrading enzymes. In the screening system, phenolic compounds produced from substrates by OP-degrading enzymes bind a constitutively expressed transcription factor DmpR, initiating the expression of enhanced green fluorescent protein (EGFP) located at the downstream of the DmpR promoter. Fluorescence intensities of host cells are proportional to the levels of phenolic compounds, enabling the screening of OP-degrading enzymes with high catalytic activities by fluorescence-activated cell sorting (FACS). Methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate (pNDP) were used as a model enzyme and an analogue of G-type nerve agents, respectively. The utility of the screening system was demonstrated by generating a triple mutant with a 100-fold higher kcat/Km than the wild-type enzyme after three rounds of directed evolution. We also conducted structure-based rational design, and obtained enzymes with high catalytic activity. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. Our approach is expected to be widely used for developing organophosphate-degrading enzymes with greater potential.