Kinetics of glucose oxidase immobilized in p(HEMA)-hydrogel microspheres in a packed-bed bioreactor

Glucose oxidase (GOx) was immobilized via both physical entrapment and covalent linkage to crosslinked poly(hydroxyethyl methacrylate-co-dimethylaminoethyl methacrylate, (p(HEMA-DMEMA)) hydrogel microspheres (20–150 μm in diameter) that were synthesized by inverse suspension polymerization. Loading capacities of 7–8 mg GOx per gram of hydrogel were achieved with physical entrapment, compared to <1.8 mg GOx per gram of gel with the covalent technique. The microspheres containing physically entrapped enzyme were packed into bioreactors and the kinetics of the immobilized enzyme investigated under various flow conditions. Flow rate dependence of Km(app) and Cmax, when extrapolated to near diffusion-free conditions, resulted in values of 13.2 mM and 2.7×10−3 mol min−1, respectively, for the immobilized enzyme. Studies of pH-dependence of Km(app) and Cmax suggest that the imidazolium and sulphydryl groups may be involved at the active site of the immobilized GOx. Studies of the temperature dependence of C and Cmax confirm lower activation energies for the oxidation of glucose at temperatures >35 °C, suggesting the influence of diffusional limitations within the hydrogel.