Ligninolytic peroxidases: A guide for their heterologous expression in E. coli and protocols to evaluate their...

Ligninolytic peroxidases are specialized enzymes involved in the degradation of lignin, the most recalcitrant component of lignocellulosic biomass. They are part of the enzymatic machinery deployed by white-rot basidiomycetes fungi to decompose lignocellulose in nature, allowing these organisms to subsequently mineralize all its components, including lignin. Despite their environmental importance and biotechnological potential, the study of ligninolytic peroxidases has received little attention compared to other enzymes also contributing to the degradation of lignin, such as laccases. This is partly due to pitfalls associated with their expression in heterologous hosts and the difficulties of carrying out kinetic studies using the lignin polymer as a substrate. To address these challenges and facilitate the study of ligninolytic peroxidases, this chapter provides: (i) detailed methods for optimizing the in vitro folding and purification of these enzymes after being produced in bacteria, (ii) a protocol to perform kinetic studies and quantify the oxidative capabilities of these enzymes acting on lignin by stopped-flow spectrophotometry, and (iii) a straightforward approach for assessing the changes caused in the lignin macromolecule using size-exclusion chromatography. By addressing these critical aspects, we aim to motivate the scientific community studying fungal lignin degradation to further explore the wide diversity of ligninolytic peroxidases unveiled by recent advances in omics technologies.