A highly stable laccase obtained by swapping the second cupredoxin domain

• Published in: Scientific reports Vol. 8; no. 1; pp. 15669 - 10
• Main Authors: Pardo, Isabel, Rodríguez-Escribano, David, Aza, Pablo, de Salas, Felipe, Martínez, Angel T., Camarero, Susana
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.10.2018; Nature Publishing Group; Nature Portfolio
• Subjects: 38/22; 38/23; 38/77; 42/44; 42/70; 631/45/603; 631/45/607/1168; 631/61/338/469; 82/80; 82/81; 82/83; Amino acid sequence; Cupredoxin Domains; Enzymes; Ethanol; Glycosylation; High Redox Potential Laccase; High temperature; Humanities and Social Sciences; Kraft Lignin; Laccase; Laccase Activity; Lignin; multidisciplinary; Organic solvents; pH effects; Redox potential; Science; Science (multidisciplinary); Secretion; Site-directed mutagenesis; Surface Salt Bridges; Thermal stability; Surface Salt Bridges; Kraft Lignin; Laccase Activity; High Redox Potential Laccase; Cupredoxin Domains
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-34008-3

The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2–9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50–70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times.