Activity and stability improvement: structure-function insights into CotA from Bacillus subtilis.

CotA, a bacterial multicopper oxidase, exhibits exceptional thermal stability, alkali resistance, and substrate versatility, making it valuable for industrial biocatalysis, environmental remediation, and dye degradation. Extensive research has enhanced its activity and stability through directed evolution, semi-rational design, and rational design, yet the underlying structural features remain largely unclear. In this study, we performed site-directed mutagenesis on CotA from Bacillus subtilis and analyzed its effects on stability. Among the CotA single and double mutants we constructed, the T377I/T418G variant exhibited a 6.12-fold increase in activity compared to the wild type and a 50 % improvement in thermal stability at 80 °C. To uncover the relationship between the enzyme activity, stability, and the spatial structure of the CotA mutants, we conducted molecular dynamics (MD) simulations to analyze the catalytic structural features. The results indicated that the increase in ABTS-specific enzyme activity was linked to an expanded binding pocket, while the increased thermal stability was attributed to a higher proportion of random coils in the secondary structure. Our study provides new insights into the catalytic activity and thermostability structural features of CotA, laying the groundwork for its applications in industrial and environmental fields.