Heterologous expression of a thermostable laccase from Chrysoporthe cubensis in Komagataella phaffii improves thermal stability and dye decolorization.

The phytopathogenic fungus Chrysoporthe cubensis was cultivated on agro-industrial residues to evaluate laccase production, with coffee husks inducing the highest activity. The enzyme, identified as laccase MCO12, was purified and biochemically characterized, showing acidic activity (pH 2.2-5.0), optimum at 55 °C, and stability between 45 and 60 °C, with half-lives of 290, 22, and 3 min at 45, 55, and 65 °C, respectively. To improve yield and stability, the MCO12 gene was codon-optimized and overexpressed in Komagataella phaffii, resulting in 246 U/L of recombinant enzyme. The recombinant laccase displayed superior thermal performance, retaining >80% activity from 55 to 70 °C, with an optimum at 60 °C, and extended half-lives of 142, 45, and 11 h at 50, 60, and 70 °C, respectively. Molecular docking with ABTS revealed stronger binding affinity and stabilization for the recombinant form, supporting enhanced substrate specificity. Functionally, recombinant MCO12 achieved high dye decolorization efficiency, removing 99% of Malachite Green and 71% of Congo Red at 50 °C after 72 h, without chemical mediators. Overall, these findings demonstrate that recombinant MCO12 is a robust and thermostable laccase, with promising potential for industrial and environmental applications, particularly in the bioremediation of dye-contaminated effluents from the textile industry.