Analytical Sciences

Abstract − Analytical Sciences, 27(8), 823 (2011).

Estimation of Enzyme Kinetic Parameters of Cell Surface-displayed Organophosphorus Hydrolase and Construction of a Biosensing System for Organophosphorus Compounds
Katsumi TAKAYAMA,*1 Shin-ichiro SUYE,*2 Yoshikazu TANAKA,*3 Ashok MULCHANDANI,*4 Kouichi KURODA,*5 and Mitsuyoshi UEDA*5
*1 Department of Chemistry & Biology Engineering, Fukui National College of Technology, Geshi, Sabae, Fukui 916-8507, Japan
*2 Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan
*3 Research & Development Department, Wakasa Wan Energy Research Center, 64-52-1 Nagatani, Tsuruga, Fukui 914-0192, Japan
*4 Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA
*5 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan
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 (K′m) 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).