Creating Oxidase–Peroxidase Fusion Enzymes as a Toolbox for Cascade Reactions

A set of bifunctional oxidase-peroxidases has been prepared by fusing four distinct oxidases to a peroxidase. Although such fusion enzymes have not been observed in nature, they could be expressed and purified in good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. Peroxidase fusions of alditol oxidase and chitooligosaccharide oxidase could be used for the selective detection of xylitol and cellobiose with a detection limit in the low-micromolar range. The peroxidase fusions of eugenol oxidase and 5-hydroxymethylfurfural oxidase could be used for dioxygen-driven, one-pot, two-step cascade reactions to convert vanillyl alcohol into divanillin and eugenol into lignin oligomers. The designed oxidase-peroxidase fusions represent attractive biocatalysts that allow efficient biocatalytic cascade oxidations that only require molecular oxygen as an oxidant.