title: Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates.
abstract: 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 °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 μM paraoxon, 1-200 μM parathion and 2.5-200 μM methyl parathion, and limits of detection were 0.2 μM, 0.4 μM and 1 μM 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.
authors.affiliation: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China; University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China.
authors.forename: Xiangjiang
authors.initials: X
authors.lastname: Tang
authors.affiliation: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Liang
authors.affiliation: Hunan Provincial Key Laboratory for Biology and Control of Plant Pests, College of Plant Protection, Hunan Agricultural University, 1 Nongda Road, Furong District, Changsha 410128, China. Electronic address: yituyong@hunau.net.
authors.forename: Tuyong
authors.initials: T
authors.lastname: Yi
authors.affiliation: Institute of Protein Biochemistry, Italian National Research Council, Via Pietro Castellino 111, Naples 80131, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Manco
authors.forename: Ilaria
authors.initials: I
authors.lastname: Palchetti
authors.affiliation: Dipartimento di Chimica, Universita degli Studi di Firenze, Via della Lastruccia 3, Sesto Fiorentino 50019, Italy.
authors.lastname: Ilariapalchetti
authors.affiliation: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China; University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China. Electronic address: liuah@qibebt.ac.cn.
authors.forename: Aihua
authors.initials: A
authors.lastname: Liu
mesh_terms: D043303:Aryldialkylphosphatase / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002138:Calibration; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry*; D004785:Environmental Pollutants / Q000032:analysis; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005278:Fenitrothion / Q000032:analysis / Q000378:metabolism; D005813:Genes, Synthetic; D017261:Glycosylphosphatidylinositols / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008743:Methyl Parathion / Q000032:analysis / Q000378:metabolism; D015394:Molecular Structure; D009596:Nitrophenols / Q000032:analysis*; D010755:Organophosphates / Q000032:analysis*; D010261:Paraoxon / Q000032:analysis / Q000378:metabolism; D010278:Parathion / Q000032:analysis / Q000378:metabolism; D010573:Pesticide Residues / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*; D013696:Temperature
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D004785:Environmental Pollutants; D029968:Escherichia coli Proteins; D017261:Glycosylphosphatidylinositols; D009596:Nitrophenols; D010755:Organophosphates; D010573:Pesticide Residues; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein; D008743:Methyl Parathion; D010278:Parathion; D043303:Aryldialkylphosphatase; D010261:Paraoxon; D005278:Fenitrothion; C024836:4-nitrophenol
keywords: Bacterial surface display; Ice nucleation protein; Organophosphorus hydrolase; Spectrophotometric detection of organophosphates; p-Nitrophenol