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Title 

High-throughput screening system based on phenolics-responsive transcription activator for directed evolution of organophosphate-degrading enzymes

 

MPH 효소 진화를 위한 고속탐색 기술

Authors 

Y S JeongSu-Lim ChoiH H KyeongJ H KimE J KimJae Gu PanEugene RhaSeung Goo LeeH S Kim

Publisher 

Oxford University Press

Issue Date 

2012

Citation 

Protein Engineering, Design and Selection, vol. 25, no. 11, pp. 725-731

Keywords 

directed evolutionhigh-throughput screeningorganophosphate-degrading enzymestranscription activator

Abstract 

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. Hence, effective methods for detoxification and decontamination of OPs are of great significance. Here we constructed and used a high-throughput screening (HTS) system that was based on phenolics-responsive transcription activator for directed evolution of OP-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 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. Methyl parathion hydrolase from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate 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 k cat/K m than the wild-type enzyme after three rounds of directed evolution. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. The DmpR-based screening system is expected to be widely used for developing OP-degrading enzymes with greater potential.

Citation 

Protein Engineering, Design and Selection , 25(11): 725-731

ISSN 

1741-0126

Link 

http://dx.doi.org/10.1093/protein/gzs071

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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