High Enzyme Activity Screening Of Methyl Parathion Hydrolase And Study Of Nanowire Fluorescent Molecular Biosensor

A mature methyl parathion hydrolase gene from E.coli DH5a/pET5a-mph, an existing strain for methyl parathion hydrolase expression, was cloned into the plasmid pET20b (+) and transformed into E.coli BL21 (DE3).Thus an overexpression strain was obtained. The recombinantlipase was purified in a one-step procedure of His-tag affinity chromatography. Through one cycle of error-prone PCR coupled with a sensitive screening method, two mutants of MPH were obtained. The catalytic activity of one mutant, which named A7, was 2-fold higher than that of wild MPH, while the other, which named E6, with 3-fold higher catalytic activity than that of wild MPH.In this study, combining molecular recognition, MPH with a fluorophore, which acts as a signal transducer, enables a fluorescent molecular biosensor to be constructed based on a pH-sensitive fluorescence probe, E GFP. For a highly sensitive assay, E2GFP-MPH was further attached to a yeast prion protein Sup351-61 through gene fusion. The resultant nanowire fluorescent molecular biosensor could detect as low as 1 pmol/mL of methyl parathion (MP), which is about a 10,000-fold greater sensitivity than that of the free E2GFP-MPH molecular sensor.