Development of an ISFET-based enzyme assay for p-diphenol oxidase from locally isolated fungus Schizophyllum commune MF-O5

Abstract

p-Diphenol oxidases (PDO) are copper-containing enzymes that catalyze the oxidation of a wide range of non-phenolic substrates and phenolic compounds utilizing oxygen as the acceptor of electrons released during enzymatic reactions. In this study, we aimed to develop a simple method using ion sensing to measure the PDO activity obtained from locally isolated fungus Schizophyllum commune MF-O5 against an ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) substrate. The ion sensing system comprised ISFET, an Ag/AgCl reference electrode and a temperature sensor. The maximum PDO activity with this system was monitored for 10 min in citrate phosphate buffer (50 mM) at pH 3.0. A standard curve was generated to calculate the PDO units. The assay was validated by comparison with the spectrophotometric method. The ISFET-based assay could detect the PDO activity accurately with a fast response and was cost effective and in portable format. The developed method can be used to monitor PDO activities in processes where the enzyme is produced and can be useful in the development of PDO biosensors for environment monitoring.