Engineering the Bacterial Laccase CotA for Functional Expression and Dye Decolorization Through Site-Directed Mutagenesis

The relationship between the structure and function of bacterial laccases has garnered significant research attention thanks to their straightforward molecular structure. Nevertheless, studies examining the impact of an altered molecular structure on the heterologous expression of bacterial laccases in <i>Escherichia coli</i> remain scarce. Our research focuses on elucidating the impact of incorporating copper ions into the molecular structure of modified CotA on its exogenous expression in <i>E. coli</i> as well as its impact on the significance of the amino acid residues surrounding the internal electron channels and water molecule channels of the enzyme molecule. The results show that single-site mutation may affect the expression of CotA by affecting its soluble expression with different binding capacities for copper ions. In addition, the mutants exhibited different laccase activity levels. The catalytic efficiency of T466A was found to be significantly enhanced, reaching 2.29 times that of the wild type. We used structural models to illustrate the correlation between molecular structure and function after the replacement of three mutation sites with alanine. The reduction of hydrogen bonds may be an important factor influencing Cu²⁺'s binding ability and the water molecule production rate. The T466A mutant exhibited strong decolorization ability for Reactive Blue 19 and Eriochrome Black T with 42.2% and 58.2% decolorization rates after one hour of reaction, respectively. This study demonstrates that the molecular mutation studied influences the CotA expression level, enzyme activity, and dye decolorization.