title: Characterization of immobilized Escherichia coli alkaline phosphatase reactors in flow injection analysis.
abstract: The characterization of immobilized Escherichia coli alkaline phosphatase reactors used in flow injection analysis is reported for factors such as optimum pH, activity, ionic strength, product inhibition, and substrate specificity. The kinetics of the immobilized enzyme was studied, and mathematical descriptions were developed for the use of an immobilized enzyme packed-bed reactor to evaluate the kinetic parameters and the number of active sites on the immobilized enzyme. Suppression of phosphatase activity by orthophosphate was found to be significantly reduced, and the Michaelis-Menten constant increased when the enzyme was immobilized and packed in a reactor. Immobilized E. coli alkaline phosphatase exhibited similar activity at pH 8 in Tris-HCl, NaHCO3 and borate-HCl buffers but slightly lower activity in NH3H2O-NH4Ac buffer. The performance of the immobilized enzyme reactor was not affected by the presence of up to 10 M Mg(II), Ni(II), Cd(II), Co(II), Mn(II), Cu(II), or urea, 1 M Fe(II), or 0.1 M Fe(III) in the substrate stream. The chelating agent EDTA, however, gradually deactivated the immobilized enzyme. The periodic restoration of enzyme activity was achieved following the removal and addition of zinc ions. The immobilized E. coli alkaline phosphatase packed-bed reactor was used to measure the alkaline phosphatase available phosphorus content of a number of model organophosphorus compounds. p-Nitrophenyl phosphate showed a linear response in the range of 1.6 x 10(-7)-1.6 x 10(-4) M. This study forms part of a larger program to develop enzymatic systems for water quality measurement.
authors.affiliation: Water Studies Centre, Monash University, Victoria, Australia.
authors.forename: Y
authors.initials: Y
authors.lastname: Shan
authors.forename: I D
authors.initials: ID
authors.lastname: McKelvie
authors.forename: B T
authors.initials: BT
authors.lastname: Hart
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004492:Edetic Acid / Q000737:chemistry; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D017022:Flow Injection Analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D009994:Osmolar Concentration; D014508:Urea / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D008670:Metals; D014508:Urea; D004492:Edetic Acid; D000469:Alkaline Phosphatase