Expression and purification of bacterial dye-decolorizing peroxidases using Escherichia coli

Dye-decolorizing peroxidases (DyPs) are heme-dependent enzymes that utilize peroxide as a co-substrate. These enzymes catalyze the oxidation of various compounds, inculding dyes and lignin, and their industrial potential has been widely recognized. Studying these enzymes necessitates the production of an active form suitable for detailed investigation. However, bacterial recombinant expression of DyPs frequently results in heme-deficient proteins. Our previous research demonstrated that codon-optimized genes significantly enhance both protein yields and heme content per monomer. In this chapter, we detail our strategy for expressing and purifying DyPs in Escherichia coli to obtain active enzymes for further analysis. We also outline a method to determine the concentration of the active enzyme.