A highly stable laccase obtained by swapping the second cupredoxin domain

Abstract 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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Isabel Pardo, David Rodríguez-Escribano, Pablo Aza, Felipe de Salas, Angel T. Martínez, Susana Camarero
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Cupredoxin Domains Kraft Lignin High Redox Potential Laccase Surface Salt Bridges Laccase Activity Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d95467d880f942c59297af612d813c18
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:d95467d880f942c59297af612d813c18
record_format	dspace
spelling	oai:doaj.org-article:d95467d880f942c59297af612d813c182021-12-02T15:08:39ZA highly stable laccase obtained by swapping the second cupredoxin domain10.1038/s41598-018-34008-32045-2322https://doaj.org/article/d95467d880f942c59297af612d813c182018-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-34008-3https://doaj.org/toc/2045-2322Abstract 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.Isabel PardoDavid Rodríguez-EscribanoPablo AzaFelipe de SalasAngel T. MartínezSusana CamareroNature PortfolioarticleCupredoxin DomainsKraft LigninHigh Redox Potential LaccaseSurface Salt BridgesLaccase ActivityMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-10 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cupredoxin Domains Kraft Lignin High Redox Potential Laccase Surface Salt Bridges Laccase Activity Medicine R Science Q
spellingShingle	Cupredoxin Domains Kraft Lignin High Redox Potential Laccase Surface Salt Bridges Laccase Activity Medicine R Science Q Isabel Pardo David Rodríguez-Escribano Pablo Aza Felipe de Salas Angel T. Martínez Susana Camarero A highly stable laccase obtained by swapping the second cupredoxin domain
description	Abstract 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.
format	article
author	Isabel Pardo David Rodríguez-Escribano Pablo Aza Felipe de Salas Angel T. Martínez Susana Camarero
author_facet	Isabel Pardo David Rodríguez-Escribano Pablo Aza Felipe de Salas Angel T. Martínez Susana Camarero
author_sort	Isabel Pardo
title	A highly stable laccase obtained by swapping the second cupredoxin domain
title_short	A highly stable laccase obtained by swapping the second cupredoxin domain
title_full	A highly stable laccase obtained by swapping the second cupredoxin domain
title_fullStr	A highly stable laccase obtained by swapping the second cupredoxin domain
title_full_unstemmed	A highly stable laccase obtained by swapping the second cupredoxin domain
title_sort	highly stable laccase obtained by swapping the second cupredoxin domain
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/d95467d880f942c59297af612d813c18
work_keys_str_mv	AT isabelpardo ahighlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT davidrodriguezescribano ahighlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT pabloaza ahighlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT felipedesalas ahighlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT angeltmartinez ahighlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT susanacamarero ahighlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT isabelpardo highlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT davidrodriguezescribano highlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT pabloaza highlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT felipedesalas highlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT angeltmartinez highlystablelaccaseobtainedbyswappingthesecondcupredoxindomain AT susanacamarero highlystablelaccaseobtainedbyswappingthesecondcupredoxindomain
_version_	1718388092166995968

A highly stable laccase obtained by swapping the second cupredoxin domain

Ejemplares similares