Heterologous expression, in vitro refolding and steady-state kinetics of fungal aryl-alcohol oxidase.

Aryl-alcohol oxidase (AAO) is an FAD-dependent enzyme belonging to the glucose-methanol-choline oxidoreductase superfamily. AAOs are secreted by fungi and play a fundamental role as auxiliary enzymes in the lignocellulolytic process. On the one hand, they produce H₂O₂ that activates peroxidases, which directly oxidize lignin, and triggers Fenton reactions to produce reactive oxygen species that attack lignin and carbohydrates. On the other, it is now known that they can also produce hydroquinones that promote hydroxyl radical formation to foster lignin decay. Genomic studies have revealed the significance of this class of enzymes for ligninolytic fungi, being produced by both white and brown-rot species. In this chapter, we deal with the methodology for the expression of AAO in the heterologous host E. coli as inclusion bodies, its subsequent in vitro refolding to produce active and soluble enzyme, as well as the estimation of their bi-substrate steady-state kinetic parameters.