Thin layers of glucose oxidase and horseradish peroxidase have been prepared by covalent immobilization onto functionalized glass slides with COOH groups. Atomic Force Microscopy was utilized to characterize the immobilized enzymes. The AFM images show that both glucose oxidase and horseradish peroxidase were deposited as homogeneous monolayers, being their thickness 50 and 40 Å, respectively. The immobilized enzymes were also kinetically characterized by measuring the catalytic constants. In the case of glucose oxidase the apparent affinity of the immobilized protein for glucose was three times higher than that of the free enzyme, while in the case of horseradish peroxidase the KM for H2O2 decreased by a factor of two. This indicates that the immobilization of horseradish peroxidase on a negatively charged surface increases the affinity of the enzyme for the substrate. As regards the turnover number (kc) we found that the immobilization decreased the catalytic constants of both enzymes by about two orders of magnitude

Thin layers of Glucose Oxidase and Horseradish Peroxidase: preparation and characterization

DI PAOLO, MARIA LUISA;RIGO, ADELIO;VIANELLO, FABIO;ZENNARO, LUCIO
1998

Abstract

Thin layers of glucose oxidase and horseradish peroxidase have been prepared by covalent immobilization onto functionalized glass slides with COOH groups. Atomic Force Microscopy was utilized to characterize the immobilized enzymes. The AFM images show that both glucose oxidase and horseradish peroxidase were deposited as homogeneous monolayers, being their thickness 50 and 40 Å, respectively. The immobilized enzymes were also kinetically characterized by measuring the catalytic constants. In the case of glucose oxidase the apparent affinity of the immobilized protein for glucose was three times higher than that of the free enzyme, while in the case of horseradish peroxidase the KM for H2O2 decreased by a factor of two. This indicates that the immobilization of horseradish peroxidase on a negatively charged surface increases the affinity of the enzyme for the substrate. As regards the turnover number (kc) we found that the immobilization decreased the catalytic constants of both enzymes by about two orders of magnitude
III Convegno Nazionale Istituto Nazionale Biostrutture e Biosistemi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/177461
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