Use of an oxygen microsensor for the determination of intrinsic kinetic parameters of an immobilized oxygen reducing enzyme

Abstract

An oxygen microsensor was used to measure internal oxygen profiles in biocatalyst particles of different diameter and activity. The particles were made of agarose gel and contained an oxygen reducing enzyme, L-lactate mono-oxygenase. The kinetics of the enzyme could be well described by the Michaelis-Menten equation. From the internal substrate concentration profile the intrinsic kinetic parameters were determined by means of fitting a simulated profile to the measurements, using Marquardt's algorithm. The intrinsic kinetic parameters found following this procedure appeared to be independent of particle radius or enzyme loading used, proving the method to be reliable. These parameters were also compared with the kinetic parameters of the free enzyme which were determined in a biological oxygen monitoring system. The intrinsic kinetic parameters showed a decrease with a factor 2.3 for V(m) value and with a factor 2.7 for the K(m) value compared to the parameters for the free enzyme. From this the conclusion can be drawn that the immobilization as such or the carrier material not only can have an effect on the maximum intrinsic conversion rate (V(m)) but also on the affinity of the enzyme (K(m)) for oxygen.