Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer

Abstract

Native horseradish peroxidase (HRP) on graphite has revealed ≈50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.