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 68 kDa and showed stability at lower temperature (30 °C) for 1 h. The Km and Vmax values of the purified PDO recorded were 2.48 mM and 6.20 U/min. Thermal stability results showed that at 30 °C PDO had 119.17 kJ/K/mol Ea value and 33.64 min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0 mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1 mM showing 12.04% activity. The enzyme showed stability against 10 mM 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.

College of Food Sciences and Engineering Ocean University of China Shandong Province 266003 Qingdao China

Department of Biological Sciences Faculty of Basic and Applied Sciences International Islamic University H 10 Islamabad 44000 Pakistan

Department of Biosciences Comsats University Islamabad 45550 Pakistan

Department of Biotechnology Quaid i Azam University Islamabad Pakistan

Department of Botany Mirpur University of Science and Technology Mirpur Azad Kashmir 10250 Pakistan

Directorate of QEC University of Loralai Loralai 84800 Pakistan

Food and Biotechnology Research Centre Pakistan Council of Scientific and Industrial Research Laboratories Complex Ferozepur Road Lahore 54600 Pakistan

Insititute of Biological Sciences Khwaja Fareed University of Engineering and Information Technology Rahim Yar Khan 64200 Pakistan

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