Consensus Design of an Evolved High-Redox Potential Laccase Gomez-Fernandez, Bernardo J. Risso, Valeria Alejandra Sánchez Ruiz, José Manuel Consensus design High-redox potential laccase Ancestor mutation Thermostability Activity Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion. 2020-06-23T11:41:31Z 2020-06-23T11:41:31Z 2020-05 journal article Gomez-Fernandez BJ, Risso VA, Sanchez-Ruiz JM and Alcalde M (2020) Consensus Design of an Evolved High-Redox Potential Laccase. Front. Bioeng. Biotechnol. 8:354. [doi: 10.3389/fbioe.2020.00354] http://hdl.handle.net/10481/62664 10.3389/fbioe.2020.00354 eng http://creativecommons.org/licenses/by/3.0/es/ open access Atribución 3.0 España Frontiers Media