Development of 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 which 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. This study was aimed to develop a simple method using ion sensing to measure the PDO activity obtained from locally isolated fungus Schizophyllum commune MF-O5 against ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) substrate. The ion sensing system comprises of an 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 spectrophotometric method. The ISFET based assay can detect the PDO activity accurately with fast response, cost effective and in portable format. The developed method can be used to monitor PDO activities in the processes where the enzyme is produced and can be useful in the development of PDO biosensor for environment monitoring. Keywords p-Diphenol Oxidase (Laccases), ISFET, Schizophyllum commune MF-O5, enzyme assay, activity measurement