English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/190012
logo share SHARE logo core CORE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:


High-throughput screening assay for laccase engineering toward lignosulfonate valorization

AuthorsRodríguez-Escribano, David; De Salas, Felipe; Pardo, Isabel ; Camarero, Susana
KeywordsEnzyme directed evolution
High-throughput screening
Laccase Lignosulfonate
Phenolic content
Issue Date18-Aug-2017
PublisherMolecular Diversity Preservation International
CitationInternational Journal of Molecular Sciences 18(8): 1793 (2017)
AbstractThe 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.
Description10 p.- 7 fig.
Publisher version (URL)https://doi.org/10.3390/ijms18081793
Appears in Collections:(CIB) Artículos
Files in This Item:
File Description SizeFormat 
Rodriguez-Escudero-et-al MDPI-2017.pdf1,7 MBAdobe PDFThumbnail
Show full item record
Review this work

Related articles:

WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.