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Heterologous Expression, Engineering and Characterization of a Novel Laccase of Agrocybe pediades with Promising Properties as Biocatalyst

Agaricomycetes fungi responsible for decay of wood and other lignocellulosic substrates constitute a valuable source of lignin-degrading enzymes. Among these enzymes, laccases (multi-copper oxidases) present remarkable biotechnological potential as environmentally friendly biocatalysts able to oxidi...

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Autores principales: Aza, Pablo, Molpeceres, Gonzalo, Ruiz-Dueñas, Francisco Javier, Camarero, Susana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147764/
https://www.ncbi.nlm.nih.gov/pubmed/34064437
http://dx.doi.org/10.3390/jof7050359
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author Aza, Pablo
Molpeceres, Gonzalo
Ruiz-Dueñas, Francisco Javier
Camarero, Susana
author_facet Aza, Pablo
Molpeceres, Gonzalo
Ruiz-Dueñas, Francisco Javier
Camarero, Susana
author_sort Aza, Pablo
collection PubMed
description Agaricomycetes fungi responsible for decay of wood and other lignocellulosic substrates constitute a valuable source of lignin-degrading enzymes. Among these enzymes, laccases (multi-copper oxidases) present remarkable biotechnological potential as environmentally friendly biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the only requirement. Laccases from saprotrophic Agaricales species have been much less studied than laccases from Polyporales, despite the fact that the former fungi are excellent sources of laccases. Here, the gene of a novel laccase of Agrocybe pediades, that is secreted by the fungus during lignocellulose degradation, was synthesised de novo and expressed in Saccharomyces cerevisiae using an improved signal peptide previously obtained and enzyme directed evolution. The characterization of the new laccase variants provided new insights on the contribution of different amino acid residues to modulate laccase production, catalytic activity or optimal pH. The selected double-mutated variant also showed interesting properties as a biocatalyst, such as the ability to oxidise a wide range of substrates, including high-redox potential mediators and recalcitrant organic dyes, improved activity at neutral pH and high tolerance to inhibitors. Finally, we demonstrate the existence of three N-glycosylation sites in the laccase and their distinct effect on the secretion or catalytic activity of the enzyme.
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spelling pubmed-81477642021-05-26 Heterologous Expression, Engineering and Characterization of a Novel Laccase of Agrocybe pediades with Promising Properties as Biocatalyst Aza, Pablo Molpeceres, Gonzalo Ruiz-Dueñas, Francisco Javier Camarero, Susana J Fungi (Basel) Article Agaricomycetes fungi responsible for decay of wood and other lignocellulosic substrates constitute a valuable source of lignin-degrading enzymes. Among these enzymes, laccases (multi-copper oxidases) present remarkable biotechnological potential as environmentally friendly biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the only requirement. Laccases from saprotrophic Agaricales species have been much less studied than laccases from Polyporales, despite the fact that the former fungi are excellent sources of laccases. Here, the gene of a novel laccase of Agrocybe pediades, that is secreted by the fungus during lignocellulose degradation, was synthesised de novo and expressed in Saccharomyces cerevisiae using an improved signal peptide previously obtained and enzyme directed evolution. The characterization of the new laccase variants provided new insights on the contribution of different amino acid residues to modulate laccase production, catalytic activity or optimal pH. The selected double-mutated variant also showed interesting properties as a biocatalyst, such as the ability to oxidise a wide range of substrates, including high-redox potential mediators and recalcitrant organic dyes, improved activity at neutral pH and high tolerance to inhibitors. Finally, we demonstrate the existence of three N-glycosylation sites in the laccase and their distinct effect on the secretion or catalytic activity of the enzyme. MDPI 2021-05-04 /pmc/articles/PMC8147764/ /pubmed/34064437 http://dx.doi.org/10.3390/jof7050359 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Aza, Pablo
Molpeceres, Gonzalo
Ruiz-Dueñas, Francisco Javier
Camarero, Susana
Heterologous Expression, Engineering and Characterization of a Novel Laccase of Agrocybe pediades with Promising Properties as Biocatalyst
title Heterologous Expression, Engineering and Characterization of a Novel Laccase of Agrocybe pediades with Promising Properties as Biocatalyst
title_full Heterologous Expression, Engineering and Characterization of a Novel Laccase of Agrocybe pediades with Promising Properties as Biocatalyst
title_fullStr Heterologous Expression, Engineering and Characterization of a Novel Laccase of Agrocybe pediades with Promising Properties as Biocatalyst
title_full_unstemmed Heterologous Expression, Engineering and Characterization of a Novel Laccase of Agrocybe pediades with Promising Properties as Biocatalyst
title_short Heterologous Expression, Engineering and Characterization of a Novel Laccase of Agrocybe pediades with Promising Properties as Biocatalyst
title_sort heterologous expression, engineering and characterization of a novel laccase of agrocybe pediades with promising properties as biocatalyst
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147764/
https://www.ncbi.nlm.nih.gov/pubmed/34064437
http://dx.doi.org/10.3390/jof7050359
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