Production, purification, and characterization of p-diphenol oxidase (PDO) enzyme from lignolytic fungal isolate Schizophyllum commune MF-O5.

Abstract

Fungi are producers of lignolytic extracellular enzymes which are used in industries like textile, detergents, biorefineries, and paper pulping. This study assessed for the production, purification, and characterization of novel p-diphenol oxidase (PDO; laccase) enzyme from lignolytic white-rot fungal isolate. Fungi samples collected from different areas of Pakistan were initially screened using guaiacol plate method. The maximum PDO producing fungal isolate was identified on the basis of ITS (internal transcribed spacer sequence of DNA of ribosomal RNA) sequencing. To get optimum enzyme yield, various growth and fermentation conditions were optimized. Later PDO was purified using ammonium sulfate precipitation, size exclusion, and anion exchange chromatography and characterized. It was observed that the maximum PDO producing fungal isolate was Schizophyllum commune (MF-O5). Characterization results showed that the purified PDO was a monomeric protein with a molecular mass of 68kDa and showed stability at lower temperature (30°C) for 1h. The Km and Vmax values of the purified PDO recorded were 2.48mM and 6.20 U/min. Thermal stability results showed that at 30°C PDO had 119.17kJ/K/mol Ea value and 33.64min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1mM showing 12.04% activity. The enzyme showed stability against 10mM concentration oxidizing reducing agents like DMSO, EDTA, H2O2, NaOCl, and urea and retained more than 75% of relative activity. The characterization of purified PDO enzyme confirmed its tolerance against salt, metal ions, organic solvents, and surfactants indicating its ability to be used in the versatile commercial applications.