Kinetics and Efficacy of an Organophosphorus Hydrolase in a Rodent Model of Methyl-parathion Poisoning

ABSTRACT Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.

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Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System

Applied and Environmental Microbiology ◽

10.1128/aem.02162-09 ◽

2009 ◽

Vol 76 (2) ◽

pp. 434-440 ◽

Cited By ~ 29

Author(s):

Chao Yang ◽

Roland Freudl ◽

Chuanling Qiao ◽

Ashok Mulchandani

Keyword(s):

Escherichia Coli ◽

Methyl Parathion ◽

Ice Nucleation ◽

Organophosphorus Hydrolase ◽

Display System ◽

Ice Nucleation Protein ◽

Methyl Parathion Hydrolase ◽

E Coli ◽

Tat Pathway ◽

Parathion Hydrolase

ABSTRACT A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell.

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Diversity of organophosphorus pesticide-degrading bacteria in a polluted soil and conservation of their organophosphorus hydrolase genes

Canadian Journal of Microbiology ◽

10.1139/w05-010 ◽

2005 ◽

Vol 51 (4) ◽

pp. 337-343 ◽

Cited By ~ 58

Author(s):

Ruifu Zhang ◽

Zhongli Cui ◽

Jiandong Jiang ◽

Jian He ◽

Xiangyang Gu ◽

...

Keyword(s):

Southern Blot ◽

Methyl Parathion ◽

Organophosphorus Pesticide ◽

Polluted Soil ◽

Organophosphorus Hydrolase ◽

Rrna Gene ◽

Gene Probe ◽

Degrading Bacteria ◽

Gene Libraries

Seven methyl parathion-degrading bacteria were isolated from a long-term methyl parathion contaminated soil and were found to belong to the genera Pseudaminobacter, Achromobacter, Brucella, and Ochrobactrum. Southern blot analysis using an mpd gene probe revealed that their hydrolase genes were similar to the mpd gene from Plesiomonas sp. strain M6 and were all located on the chromosome. Gene libraries were constructed from genomic DNA of each of the 7 organophosphorus pesticide-degrading bacteria, and their mpd genes were cloned and sequenced. Sequence analysis revealed that their hydrolase genes were conserved, and that the G+C content of the mpd genes were distinctly different from that of the chromosome-located 16S rRNA gene, suggesting that the mpd gene could be transferred and expressed among a variety of bacterial hosts.Key words: methyl parathion, degrading bacteria, Southern blot, mpd gene.

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Human methyl parathion poisoning

Clinical Toxicology ◽

10.1080/15563650701232745 ◽

2007 ◽

Vol 45 (8) ◽

pp. 956-960 ◽

Cited By ~ 18

Author(s):

Geoffrey K. Isbister ◽

Katie Mills ◽

Lena E. Friberg ◽

Mary Hodge ◽

Enda O'Connor ◽

...

Keyword(s):

Methyl Parathion ◽

Parathion Poisoning

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Utility of organophosphorus hydrolase for the remediation of mutagenicity of methyl parathion

Environmental Toxicology and Chemistry ◽

10.1002/etc.5620190810 ◽

2000 ◽

Vol 19 (8) ◽

pp. 2022-2028 ◽

Cited By ~ 10

Author(s):

Tae-Hyeon Cho ◽

James R. Wild ◽

Kirby C. Donnelly

Keyword(s):

Methyl Parathion ◽

Organophosphorus Hydrolase

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Preparation of Electrochemical Biosensor for Detection of Organophosphorus Pesticides

International Journal of Analytical Chemistry ◽

10.1155/2014/303641 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Ashish Gothwal ◽

Puneet Beniwal ◽

Vikas Dhull ◽

Vikas Hooda

Keyword(s):

Methyl Parathion ◽

Electrochemical Biosensor ◽

Incubation Temperature ◽

Electrochemical Cell ◽

Organophosphorus Pesticides ◽

Evaluation Study ◽

Organophosphorus Hydrolase ◽

Corrosion Resistant ◽

Sodium Phosphate Buffer ◽

Optimum Response

Polyvinyl chloride (PVC) can be used to develop reaction beaker which acts as electrochemical cell for the measurement of OP pesticides. Being chemically inert, corrosion resistant, and easy in molding to various shapes and size, PVC can be used for the immobilization of enzyme. Organophosphorus hydrolase was immobilized covalently onto the chemically activated inner surface of PVC beaker by using glutaraldehyde as a coupling agent. The carbon nanotubes paste working electrode was constructed for amperometric measurement at a potential of +0.8 V. The biosensor showed optimum response at pH 8.0 with incubation temperature of 40°C.KmandImaxfor substrate (methyl parathion) were 322.58 µM and 1.1 µA, respectively. Evaluation study showed a correlation of 0.985, which was in agreement with the standard method. The OPH biosensor lost 50% of its initial activity after its regular use for 25 times over a period of 50 days when stored in 0.1 M sodium phosphate buffer, pH 8.0 at 4°C. No interference was observed by interfering species.

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