One of the drawbacks of enzyme-based sensors is the rapid loss of activity because enzymes are very sensitive to their
environment and thermodynamically intrinsically unstable. Several studies have shown that covalent modification of
enzymes with glycans (Gly) can prevent activity loss and improve their long-term stability. To address this problem we glycosylate
horseradish peroxidase (HRP) with lactose to construct a simple and low cost amperometric horseradish peroxidase biosensor
based on a 4-mercaptobenzoic acid self-assembled monolayer on gold nano-particles (AuNPs) at the surface of a glassy carbon
(GC) electrode. We compared the sensitivity, linear range, and detection limit (LOD) of the biosensor using the native and
glycosylated form of the enzyme for this biosensor design. Circular dichroism (CD) spectra showed no change as result of
glycosylation with lactose. The highest sensitivity of the biosensor to hydroquinone was obtained for the Lac-HRP-4-MBAAuNPs-
GC biosensor with 370 nAμM-1cm-2 compared to 308 nAμM-1cm-2 for the HRP-4-MBA-AuNPs-GC biosensor; showing
that the glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly and efficiently than the native
form of the enzyme. The LOD observed for the native HRP biosensor was 0.74 and 0.83 for the Lac-HRP biosensor respectively.
The linear range for both biosensors was from 5- 300 μM. The Ea value obtained for native HRP biosensor was 3.49 kJmol-1 and
1.20 kJmol-1 for Lac-HRP biosensor. From this study we found that glycosylation of HRP does not affect the catalytic property
of the enzyme. Also, the glycosylated HRP showed an improvement on the biosensor sensitivity for detection of hydroquinone.
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