Functional screening pipeline to uncover laccase-like multicopper oxidase enzymes that transform industrial lignins

Laccase-like multicopper oxidases are recognized for their potential to alter the reactivity of lignins for application in value-added products. Typically, model compounds are employed to discover such enzymes; however, they do not represent the complexity of industrial lignin substrates. In this work, a screening pipeline was developed to test enzymes simultaneously on model compounds and industrial lignins. A total of 12 lignin-active fungal multicopper oxidases were discovered, including 9 enzymes active under alkaline conditions (pH 11.0). Principal component analysis revealed the poor ability of model compounds to predict enzyme performance on industrial lignins. Additionally, sequence similarity analyses grouped these enzymes with Auxiliary Activity-1 sub-families with few previously characterized members, underscoring their taxonomic novelty. Correlation between the lignin-activity of these enzymes and their taxonomic origin, however, was not observed. These are critical insights to bridge the gap between enzyme discovery and application for industrial lignin valorization.