title: Laboratory evolution of high-redox potential laccases.
abstract: Thermostable laccases with a high-redox potential have been engineered through a strategy that combines directed evolution with rational approaches. The original laccase signal sequence was replaced by the α-factor prepro-leader, and the corresponding fusion gene was targeted for joint laboratory evolution with the aim of improving kinetics and secretion by Saccharomyces cerevisiae, while retaining high thermostability. After eight rounds of molecular evolution, the total laccase activity was enhanced 34,000-fold culminating in the OB-1 mutant as the last variant of the evolution process, a highly active and stable enzyme in terms of temperature, pH range, and organic cosolvents. Mutations in the hydrophobic core of the evolved α-factor prepro-leader enhanced functional expression, whereas some mutations in the mature protein improved its catalytic capacities by altering the interactions with the surrounding residues.
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, Madrid, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: Maté
authors.forename: Carlos
authors.initials: C
authors.lastname: García-Burgos
authors.forename: Eva
authors.initials: E
authors.lastname: García-Ruiz
authors.forename: Antonio O
authors.initials: AO
authors.lastname: Ballesteros
authors.forename: Susana
authors.initials: S
authors.lastname: Camarero
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
mesh_terms: D019020:Directed Molecular Evolution; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D042845:Laccase