Turning a Hyperthermostable Metallo-Oxidase into a Laccase by Directed Evolution

Multicopper oxidases are multifunctional enzymes that can be broadly divided into two functional classes: metallo-oxidases (with a robust activity toward metals, such as Cu+ or Fe2+) and laccases (with a superior catalytic efficiency for organic compounds). Laccases are green catalysts with an outstanding redox capability over a wide range of aromatic substrates using O2 as an electron acceptor and releasing water as reduced product. Hyperthermostable laccases are highly in demand for their robustness in biotechnological applications. In this study, a laboratory evolution approach was conducted to improve the specificity of the metallo-oxidase McoA from the hyperthermophilic bacterium Aquifex aeolicus for aromatic compounds. Four rounds of random mutagenesis of the mcoA-gene followed by high-throughput screening (∼94 000 clones) led to the identification of the 2B3 variant featuring a 2-order of magnitude higher catalytic efficiency (kcat/Km) than the wild-type enzyme for the typical laccase substrate ABT...