title: Estimation of enzyme kinetic parameters of cell surface-displayed organophosphorus hydrolase and construction of a biosensing system for organophosphorus compounds.
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 (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 (R(2) = 0.9574) and a detection limit of 5 ppm (18 µM).
authors.affiliation: Department of Chemistry & Biology Engineering, Fukui National College of Technology, Geshi, Sabae, Fukui 916-8507, Japan. takayama@fukui-nct.ac.jp
authors.forename: Katsumi
authors.initials: K
authors.lastname: Takayama
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Suye
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Ashok
authors.initials: A
authors.lastname: Mulchandani
authors.forename: Kouichi
authors.initials: K
authors.lastname: Kuroda
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Ueda
mesh_terms: D043303:Aryldialkylphosphatase / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D015374:Biosensing Techniques / Q000379:methods*; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000032:analysis* / Q000378:metabolism
chemical_list: D009596:Nitrophenols; D009943:Organophosphorus Compounds; D043303:Aryldialkylphosphatase; C024836:4-nitrophenol