Laccase Functional Analysis: Substrates, Activity Assays, Challenges, and Prospects

Abstract Enzyme functional analysis is a multifaceted process that can be used for various purposes, such as screening for specific activities, as well as developing, optimising, and validating processes or final products. Functional analysis methods are crucial for assessing enzyme performance and catalytic properties. Laccase, a well‐known blue multi‐copper oxidase, holds immense potential in diverse industries such as pharmaceuticals, paper and pulp, food and beverages, textiles, and biorefineries due to its clean oxidation process and versatility in handling a wide range of substrates. Despite its prominence, the use of laccase encounters challenges in selecting appropriate functional analysis substrates and methods. This review delves into the substrates utilised in qualitative and quantitative techniques for laccase activity analysis. Although laccase catalyses mono‐electron oxidation of aromatic hydroxyl, amine, and thiol compounds efficiently, using molecular oxygen as an electron acceptor, the review identifies limitations in the specificity of the commonly employed substrates, concerns regarding the stability of certain compounds and highlights potential strategies.