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 02 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 (similar to 94 000 clones) led to the identification of the 2B3 variant featuring a 2-order of magnitude higher catalytic efficiency (k(cat)/K-m) than the wild-type enzyme for the typical laccase substrate ABTS (2,2'-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid)) and additionally displaying a higher activity for phenolics and synthetic aromatic dyes. Notably, the recombinant 2B3 variant, unlike the wild-type, did not show temperature-dependent aggregation, exhibiting enhanced solubility and thus higher kinetic and thermodynamic thermostability. The structural basis of the altered substrate's catalytic efficiency and increased solubility/thermostability of the 2B3 variant are discussed on the basis of the biochemical analysis of single and double mutations of wild-type and its variants. The hyper-robustness of the evolved enzyme reported here shows clear advantages for current applications and provides a powerful tool for generation of more efficient biocatalysts for specific applications because it is widely acknowledged that thermostable proteins have an enhanced mutational robustness and evolvability.