Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates

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• 作者：Xiangjiang Tang ; Bo Liang ; Tuyong Yi ; Giuseppe Manco ; IlariaPalchetti ; Aihua Liu
• 关键词：Bacterial surface display ; Ice nucleation protein ; Organophosphorus hydrolase ; p-Nitrophenol ; Spectrophotometric detection of organophosphates
• 刊名：Enzyme and Microbial Technology
• 出版年：5 February, 2014
• 年：2014
• 卷：55
• 期：Complete
• 页码：107-112
• 全文大小：1103 K

文摘

Organophosphates (OPs) widely exist in ecosystem as toxic substances, for which sensitive and rapid analytical methods are highly requested. In the present work, by using N-terminal of ice nucleation protein (INP) as anchoring motif, a genetically engineered Escherichia coli (E. coli) strain surface displayed mutant organophosphorus hydrolase (OPH) (S5) with improved enzyme activity was successfully constructed. The surface location of INP-OPH fusion was confirmed by SDS-PAGE analysis and enzyme activity assays. The OPH-displayed bacteria facilitate the hydrolysis of p-nitrophenol (PNP) substituted organophosphates to generate PNP, which can be detected spectrometrically at 410 nm. Over 90% of the recombinant protein present on the surface of microbes demonstrated enhanced enzyme activity and long-term stability. The OPH activity of whole cells was 2.16 U/OD₆₀₀ using paraoxon as its substrate, which is the highest value reported so far_. The optimal temperature for OPH activity was around 55 掳C, and suspended cultures retained almost 100% of its activity over a period of one month at room temperature, exhibiting the better stability than free OPH. The recombinant E. coli strain could be employed as a whole-cell biocatalyst for detecting PNP substituted OPs at wider ranges and lower detection limits. Specifically, the linear ranges of the calibration curves were 0.5-150 渭M paraoxon, 1-200 渭M parathion and 2.5-200 渭M methyl parathion, and limits of detection were 0.2 渭M, 0.4 渭M and 1 渭M for paraoxon, parathion and methyl parathion, respectively (S/N = 3). These results indicate that the engineered OPH strain is a promising multifunctional bacterium that could be used for further large-scale industrial and environmental applications.