TY - JOUR
T1 - Kinetics of glucose oxidase immobilized in p(HEMA)-hydrogel microspheres in a packed-bed bioreactor
AU - Brahim, Sean
AU - Narinesingh, Dyer
AU - Guiseppi-Elie, Anthony
N1 - Funding Information:
The authors gratefully acknowledge the VCU Center for Bioelectronics, Biosensors and Biochips (C3B), the Virginia Center for Innovative Technology (CIT BIO-99–010) and ABTECH Scientific Inc. for financial support. Sean Brahim thanks Dow Maharajh and Lebert Grierson for useful discussions.
PY - 2002/9/13
Y1 - 2002/9/13
N2 - 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 (20-150μm in diameter) that were synthesized by inverse suspension polymerization. Loading capacities of 7-8mg GOx per gram of hydrogel were achieved with physical entrapment, compared to <1.8mg 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 13.2mM and 2.7×10-3molmin-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.
AB - 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 (20-150μm in diameter) that were synthesized by inverse suspension polymerization. Loading capacities of 7-8mg GOx per gram of hydrogel were achieved with physical entrapment, compared to <1.8mg 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 13.2mM and 2.7×10-3molmin-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.
KW - Enzyme kinetics
KW - Glucose oxidase
KW - Hydrogel microspheres
KW - Packed-bed bioreactor
KW - p(HEMA)
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U2 - 10.1016/S1381-1177(02)00061-9
DO - 10.1016/S1381-1177(02)00061-9
M3 - Article
AN - SCOPUS:0037073003
SN - 1381-1177
VL - 18
SP - 69
EP - 80
JO - Journal of Molecular Catalysis B: Enzymatic
JF - Journal of Molecular Catalysis B: Enzymatic
IS - 1-3
ER -