Surface display of MPH on Pseudomonas putida JS444 using ice nucleation protein and its application in detoxification of organophosphates.
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.
Yang
Chao
C
State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Cai
Ning
N
Dong
Ming
M
Jiang
Hong
H
Li
Jinming
J
Qiao
Chuanling
C
Mulchandani
Ashok
A
Chen
Wilfred
W
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
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