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

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Engineering

Background:

• Bacterial laccases, like CotA, are studied for their simple structure-function relationship.
• Limited research exists on how altered structures affect heterologous expression in Escherichia coli.

Purpose of the Study:

• To investigate the effect of copper ion incorporation and mutations on CotA expression and activity in E. coli.
• To understand the role of amino acid residues in electron and water channels.

Main Methods:

• Site-directed mutagenesis of CotA, focusing on T466A.
• Heterologous expression in E. coli.
• Enzyme activity assays and structural modeling.
• Dye decolorization tests.

Main Results:

• Single-site mutations influence CotA soluble expression and copper-binding capacity.
• Mutants show varied laccase activity; T466A exhibits 2.29x enhanced catalytic efficiency.
• Reduced hydrogen bonds correlate with altered Cu2+ binding and water production.
• T466A mutant effectively decolorized Reactive Blue 19 and Eriochrome Black T.

Conclusions:

• Molecular mutations significantly impact CotA expression levels, enzyme activity, and dye decolorization capabilities.
• Structural modifications are key to optimizing bacterial laccase function for industrial applications.