Design, construction and characterization of laccase-xylanase chimeras by insertional fusion

The broad substrate specificity of laccases makes these enzymes suitable for a wide range of applications. The use of protein engineering strategies to modulate the catalytic properties of these enzymes is a promising strategy to expand their use in the sustainable economy. Here we describe the construction of laccase-xylanase bifunctional enzyme by insertional fusion using a procedure based on the rational design starting with the analysis of the 3D-structure of laccase to select positions for the insertion of the xylanase domain, followed by the creation of the fusion construct by ligation of overlapping fragments generated by PCR. Finally, the heterologous expression and biochemical characterization of the laccase and xylanase activities of the fusion protein is described and demonstrate significant increase in the laccase activity. These protocols can be applied to the fusion of any pair of proteins.