Preparation of multi-enzyme cocktail from antagonistic Pleurotus ostreatus mycelia grown in broth containing dead fungal tissue and its potential application for isolating protoplast of filamentous fungi.

From the paper

Abstract

Protoplast generation is a prerequisite for fungal genetic manipulation, yet dependence on costly and variable commercial lysing enzymes limits its accessibility. In this study, we developed a biologically derived multi-enzyme cocktail from antagonistic Pleurotus ostreatus KUFC114 grown in broth supplemented with heat-killed fungal tissue and evaluated its efficacy in isolation of the protoplast of Fusarium oxysporum f. sp. lycopersici conidia and subsequent transformation using the pCAMBIA1301 vector carrying the hygromycin-resistant gene. Substrate-induced cultivation significantly enhanced extracellular hydrolase production, with maximum activities recorded at 12 days. Kinetic analysis revealed low Km (0.26-0.67 mM) and high catalytic efficiency, particularly for chitinase (kcat/Km up to 1558.1 min-1 mM-1), indicating strong substrate affinity and rapid turnover. Zymographic profiling confirmed multiple active isoforms within the cocktail. Enzymatic treatment yielded 48.32 ± 1.17% viable protoplasts, and polyethylene glycol-mediated transformation achieved about 7% hygromycin-resistant colonies with stable growth and expression. This study demonstrates that P. ostreatus-derived hydrolases provide a cost-effective, sustainable alternative to commercial preparations, used in fungal biotechnology and molecular genetics applications.