Enhancing the thermostability of lignin peroxidase: Heme as a keystone cofactor driving stability changes in heme enzymes.

Heme-containing enzymes, critical across life's domains and promising for industrial use, face stability challenges. Despite the demand for robust industrial biocatalysts, the mechanisms underlying the thermal stability of heme enzymes remain poorly understood. Addressing this, our research utilizes a 'keystone cofactor heme-interaction approach' to enhance ligand binding and improve the stability of lignin peroxidase (LiP). We engineered mutants of the white-rot fungus LiP () to increase thermal stability by 8.66 °C and extend half-life by 29 times without losing catalytic efficiency at 60 °C, where typically, wild-type enzymes degrade. Molecular dynamics simulations reveal that an interlocked cofactor moiety contributes to enhanced structural stability in LiP variants. Additionally, a stability index developed from these simulations accurately predicts stabilizing mutations in other LiP isozymes. Using milled wood lignin, these mutants achieved triple the conversion yields at 40 °C compared to the wild type, offering insights for more sustainable white biotechnology through improved enzyme stability.