Consensus Design of an Evolved High-Redox Potential Laccase
Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved h...
Main Authors: | , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Frontiers Media S.A.
2020-05-01
|
Series: | Frontiers in Bioengineering and Biotechnology |
Subjects: | |
Online Access: | https://www.frontiersin.org/article/10.3389/fbioe.2020.00354/full |
id |
doaj-69192d5580c04ee0be7655f55b6854ae |
---|---|
record_format |
Article |
spelling |
doaj-69192d5580c04ee0be7655f55b6854ae2020-11-25T02:02:36ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852020-05-01810.3389/fbioe.2020.00354487663Consensus Design of an Evolved High-Redox Potential LaccaseBernardo J. Gomez-Fernandez0Valeria A. Risso1Jose M. Sanchez-Ruiz2Miguel Alcalde3Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, SpainFacultad de Ciencias, Departamento de Química Física, Universidad de Granada, Granada, SpainFacultad de Ciencias, Departamento de Química Física, Universidad de Granada, Granada, SpainDepartment of Biocatalysis, Institute of Catalysis, CSIC, Madrid, SpainAmong the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion.https://www.frontiersin.org/article/10.3389/fbioe.2020.00354/fullconsensus designhigh-redox potential laccaseancestor mutationthermostabilityactivity |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Bernardo J. Gomez-Fernandez Valeria A. Risso Jose M. Sanchez-Ruiz Miguel Alcalde |
spellingShingle |
Bernardo J. Gomez-Fernandez Valeria A. Risso Jose M. Sanchez-Ruiz Miguel Alcalde Consensus Design of an Evolved High-Redox Potential Laccase Frontiers in Bioengineering and Biotechnology consensus design high-redox potential laccase ancestor mutation thermostability activity |
author_facet |
Bernardo J. Gomez-Fernandez Valeria A. Risso Jose M. Sanchez-Ruiz Miguel Alcalde |
author_sort |
Bernardo J. Gomez-Fernandez |
title |
Consensus Design of an Evolved High-Redox Potential Laccase |
title_short |
Consensus Design of an Evolved High-Redox Potential Laccase |
title_full |
Consensus Design of an Evolved High-Redox Potential Laccase |
title_fullStr |
Consensus Design of an Evolved High-Redox Potential Laccase |
title_full_unstemmed |
Consensus Design of an Evolved High-Redox Potential Laccase |
title_sort |
consensus design of an evolved high-redox potential laccase |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Bioengineering and Biotechnology |
issn |
2296-4185 |
publishDate |
2020-05-01 |
description |
Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion. |
topic |
consensus design high-redox potential laccase ancestor mutation thermostability activity |
url |
https://www.frontiersin.org/article/10.3389/fbioe.2020.00354/full |
work_keys_str_mv |
AT bernardojgomezfernandez consensusdesignofanevolvedhighredoxpotentiallaccase AT valeriaarisso consensusdesignofanevolvedhighredoxpotentiallaccase AT josemsanchezruiz consensusdesignofanevolvedhighredoxpotentiallaccase AT miguelalcalde consensusdesignofanevolvedhighredoxpotentiallaccase |
_version_ |
1724951908416749568 |