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.

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Citation 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 K m (0.26-0.67 mM) and high catalytic efficiency, particularly for chitinase (k cat /K m 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.