title: Surface display of MPH on Pseudomonas putida JS444 using ice nucleation protein and its application in detoxification of organophosphates.
abstract: Methyl parathion hydrolase (MPH) has been displayed on the surface of microorganisms for the first time using only N- and C-terminal domains of the ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. A shuttle vector pINCM coding for INPNC-MPH was constructed and used to target MPH onto the surface of a natural p-nitrophenol (PNP) degrader, Pseudomonas putida JS444, overcoming the potential substrate uptake limitation. Over 90% of the MPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of the INPNC-MPH fusion was further verified by Western blot analysis and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without growth inhibition. Compared to organophosphorus hydrolase-displaying systems reported, changes in substrate specificity highlight an important potential use of the engineered strain for the clean-up of specific organophosphate nerve agents.
authors.affiliation: State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Yang
authors.forename: Ning
authors.initials: N
authors.lastname: Cai
authors.forename: Ming
authors.initials: M
authors.lastname: Dong
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.forename: Jinming
authors.initials: J
authors.lastname: Li
authors.forename: Chuanling
authors.initials: C
authors.lastname: Qiao
authors.forename: Ashok
authors.initials: A
authors.lastname: Mulchandani
authors.forename: Wilfred
authors.initials: W
authors.lastname: Chen
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D010755:Organophosphates / Q000493:pharmacokinetics*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008565:Membrane Proteins; D010755:Organophosphates; D011994:Recombinant Proteins; C063113:ice nucleation protein; C495424:methyl parathion hydrolase, Pseudomonas; D010744:Phosphoric Monoester Hydrolases