DETERMINATION OF PHOSPHOLIPASE-D ACTIVITY WITH A CHOLINE BIOSENSOR

A choline biosensor for the determination of the phospholipase D activ ity in rape seeds was prepared. Choline oxidase was co-immobilized wit h catalase on a partially hydrolysed nylon net using glutaraldehyde an d cyclohexyl isocyanide. The nylon net with immobilized enzymes was fi xed on the tip of a Clark-type oxygen sensor. The hydrogen peroxide fo rmed was removed by catalase to prevent inactivation of the choline ox idase. The prepared choline biosensor was characterized by the determi nation of the specific activity of immobilized choline oxidase (3.24 I U mg-1 at pH 8.0), pH and temperature effects on the enzyme activity, the apparent Michaelis constant (1.02 mM at pH 8.0) and the range of t he linear biosensor response to the choline concentration (3.34 x 10(- 3) to 0.167 mM). The long-term biosensor stability is shown by its use for 600 assays over an 18-month period under different pH conditions. The determination of phospholipase D activity was performed in parall el with radiochemical assay and the results obtained demonstrated the priority of the biosensor application.