ABSTRACT:Invertase was immobilized onto poly(p-chloromethylstyrene) (PCMS) beads that were produced by a suspension polymerization with an average size of 186 m. The beads had a nonporous but reasonably rough surface. Because of this, a reasonably large external surface area (i.e., 14.1 m 2 /g) could be achieved with the proposed carrier. A two-step functionalization protocol was followed for the covalent attachment of invertase onto the bead surface. For this purpose, a polymeric ligand that carried amine groups, polyethylenimine (PEI), was covalently attached onto the bead surface by a direct chemical reaction. Next, the free amine groups of PEI were activated by glutaraldehyde. Invertase was covalently attached onto the bead surface via the direct chemical reaction between aldehyde and amine groups. The appropriate enzyme binding conditions and the batch-reactor performance of the immobilized enzyme system were investigated. Under optimum immobilization conditions, 19 mg of invertase was immobilized onto each gram of beads with 80% retained activity after immobilization. The effects of pH and temperature on the immobilized invertase activity were determined and compared with the free enzyme. The kinetic parameters K M and V M were determined with the Michealis-Menten model. K M of immobilized invertase was 1.75 folds higher than that of the free invertase. The immobilization caused a significant improvement in the thermal stability of invertase, especially in the range of 55-65°C. No significant internal diffusion limitation was detected in the immobilized enzyme system, probably due to the surface morphology of the selected carrier. This result was confirmed by the determination of the activation energies of both free and immobilized invertases. The activity half-life of the immobilized invertase was approximately 5 times longer than that of the free enzyme.
Ferrocene-functionalized polyethylenimine and multiwalled carbon nanotubes were attached covalently by glutaraldehyde onto a carbon cloth to develop an immobilized enzyme (glucose oxidase) electrode for biofuel cell applications. Developed enzymatic anode was characterized by electrochemical methods to determine electrochemical performance. Anodic open-circuit potential was measured as within 0-20 mV range. Cyclic voltammetry showed anodic peak for glucose oxidation around 400-600 mV (vs. sat. Ag/AgCl) varying with scan rate. An enzyme fuel cell with 2.5 mg/cm 2 glucose oxidase-loaded bioanode and 0.70 mg/cm 2 Pt-loaded cathode attached to Nafion™ 115 membrane has provided around 2.5 mA/cm 2 current density at short-circuit conditions. Enzymatic kinetic parameters of prepared anode were determined by electrochemical methods that surprisingly indicated less K M (i.e., better substrate affinity) than that of determined by conventional enzymatic methods. Enzymatic stability determined by electrochemical methods moreover indicated longer enzyme half-life.
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