Directed evolution of a thermostable laccase from Geobacillus stearothermophilus for efficient reduction of oxygen

The growing demand for laccases with enhanced stability and high redox potential is driven by their potential applications in diverse bioelectrochemical processes. Bacterial laccases are generally more stable than their fungal counterparts but typically exhibit lower redox potentials. To address the need for a laccase with both high redox potential and enhanced stability, we identified a gene encoding a multicopper oxidase in the gram-positive, thermophilic bacterium Geobacillus stearothermophilus. Recombinant heterologous expression and characterization identified the enzyme as a putative laccase (GbsLac). GbsLac showed activity toward phenolic substrates, with a specific activity of 3.3 U/mg. It displayed thermophilic properties, with an optimum temperature of 70 °C at pH 7.5 and a high melting temperature of 91.8 °C, indicating strong thermostability. Electrochemical analysis of oxygen reduction reaction (ORR) revealed a lower onset potential compared to T. versicolor laccase (Tvl). Site-directed mutations-M503L and D438A-caused anodic shifts of ∼120 ± 25 mV and ∼40 ± 10 mV, respectively, indicating enhanced redox potential due to modifications in the T₁ copper coordination sphere. These effects are attributed to the removal of weak axial coordination (SMet-T₁Cu) via M503 deletion and reduced electron donation by H440 following disruption of its hydrogen bond with D438. Additionally, replacement with non-polar residues likely reduced reorganization energy, contributing to the increased E_T1^°. These findings highlight GbsLac as a promising biocatalyst for ORR applications.