Analytical Sciences
Online ISSN : 1348-2246
Print ISSN : 0910-6340
ISSN-L : 0910-6340
Original Papers
Estimation of Enzyme Kinetic Parameters of Cell Surface-displayed Organophosphorus Hydrolase and Construction of a Biosensing System for Organophosphorus Compounds
Katsumi TAKAYAMAShin-ichiro SUYEYoshikazu TANAKAAshok MULCHANDANIKouichi KURODAMitsuyoshi UEDA
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2011 Volume 27 Issue 8 Pages 823

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Abstract

Yeast cells displaying organophosphorus hydrolase (OPH), which was anchored using a lectin-like cell-wall protein (Flo1p), were used as a biocatalyst for the detection of organophosphorus compounds (OPs). The concentration of p-nitrophenol produced by the hydrolysis of the OPs paraoxon was calculated from the absorbance change at 415 nm during a 30-min reaction. The apparent Michaelis–Menten constant (Km) for surface-displayed OPH was estimated to be approximately 50 μM, which is consistent with the value of purified OPH, and shows that cell-surface expression is a useful strategy to overcome the mass-transport resistance of substrates across the cell membrane. Notably, the long-term storage stability of the enzyme activity exceeded 40 days when cells displaying OPH were preserved at temperatures below –4°C. A fiber-optic biosensing system was also constructed using a commercial optical-fiber detection device and yeast cells with surface-displayed OPH. A linear relationship was obtained for paraoxon concentrations of up to 50 ppm (182 μM), with a detection sensitivity of 0.0043 A.U. per ppm (R2 = 0.9574) and a detection limit of 5 ppm (18 μM).

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© 2011 by The Japan Society for Analytical Chemistry
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