Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins

At present, the end-of-life management of thermoset composite epoxy resins is limited to incineration and landfill storage, highlighting the demand for the development of more sustainable measures. Due to their broad spectrum of C-H oxyfunctionalization reactions, fungal unspecific peroxygenases (UP...

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Autores: Dolz, Mikel, Mateljak, Ivan, Méndez-Sánchez, Daniel, Sánchez-Moreno, Israel, Gómez de Santos, Patricia, Viña-González, Javier, Alcalde Galeote, Miguel
Formato: artículo
Estado:Versión publicada
Fecha de publicación:2022
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/416860
Acesso em linha:http://hdl.handle.net/10261/416860
Access Level:acceso abierto
Palavra-chave:Thermoset composites
Epoxy resin
N-dealkylation
High-throughput screening
Unspecific peroxygenase
Directed evolution
Saccharomyces cerevisiae
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spelling Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy ResinsDolz, MikelMateljak, IvanMéndez-Sánchez, DanielSánchez-Moreno, IsraelGómez de Santos, PatriciaViña-González, JavierAlcalde Galeote, MiguelThermoset compositesEpoxy resinN-dealkylationHigh-throughput screeningUnspecific peroxygenaseDirected evolutionSaccharomyces cerevisiaeAt present, the end-of-life management of thermoset composite epoxy resins is limited to incineration and landfill storage, highlighting the demand for the development of more sustainable measures. Due to their broad spectrum of C-H oxyfunctionalization reactions, fungal unspecific peroxygenases (UPOs) are becoming important biotechnological tools in organic synthesis while their potential use in biodegradation processes should not be underestimated. Here, we present a colorimetric screening assay aimed at engineering UPOs for the degradation of epoxy resins. We based our study on Hexflow® RTM-6, a commercial epoxy resin used extensively in the aeronautics sector. UPO mutants from the short and long families were initially benchmarked by GC/MS to determine their potential N-dealkylation activity on N,N-bis(2-hydroxypropyl)-p-toluidine (NNBT), the main structural scaffold of Hexflow® RTM-6. A reliable high-throughput colorimetric screening method was developed to quantify the lactaldehyde released by UPO attack on the tertiary amine of NNBT. Based on an evolved UPO from Psathyrella aberdarensis that was expressed by yeast, a small subset of mutant libraries with different mutational loadings was constructed and screened for NNBT N-dealkylation, thereby establishing a directed evolution platform as a vehicle to engineer UPO composite degrading variants.This work was supported by the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 Research and Innovation programme (grant agreement no. 886567-Bizente project), the I+D+I PID 2019-106166RB-100-OXYWAVE spanish project funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación/AEI/doi: 10.13039/501100011033/, the Comunidad de Madrid Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CM, and the CSIC program for the Spanish Recovery, Transformation and Resilience Plan funded by the Recovery and Resilience Facility of the European Union, established by the Regulation (EU) 2020/2094.Peer reviewedFrontiers MediaEuropean CommissionAgencia Estatal de Investigación (España)Comunidad de MadridConsejo Superior de Investigaciones Científicas (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2026202620222026info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/416860reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/EC/H2020/886567info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-106166RB-I00info:eu-repo/grantAgreement///info:eu-repo/grantAgreement///https://doi.org/10.3389/fctls.2022.883263Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/4168602026-05-22T06:33:51Z
dc.title.none.fl_str_mv Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
title Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
spellingShingle Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
Dolz, Mikel
Thermoset composites
Epoxy resin
N-dealkylation
High-throughput screening
Unspecific peroxygenase
Directed evolution
Saccharomyces cerevisiae
title_short Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
title_full Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
title_fullStr Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
title_full_unstemmed Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
title_sort Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
dc.creator.none.fl_str_mv Dolz, Mikel
Mateljak, Ivan
Méndez-Sánchez, Daniel
Sánchez-Moreno, Israel
Gómez de Santos, Patricia
Viña-González, Javier
Alcalde Galeote, Miguel
author Dolz, Mikel
author_facet Dolz, Mikel
Mateljak, Ivan
Méndez-Sánchez, Daniel
Sánchez-Moreno, Israel
Gómez de Santos, Patricia
Viña-González, Javier
Alcalde Galeote, Miguel
author_role author
author2 Mateljak, Ivan
Méndez-Sánchez, Daniel
Sánchez-Moreno, Israel
Gómez de Santos, Patricia
Viña-González, Javier
Alcalde Galeote, Miguel
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv European Commission
Agencia Estatal de Investigación (España)
Comunidad de Madrid
Consejo Superior de Investigaciones Científicas (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Thermoset composites
Epoxy resin
N-dealkylation
High-throughput screening
Unspecific peroxygenase
Directed evolution
Saccharomyces cerevisiae
topic Thermoset composites
Epoxy resin
N-dealkylation
High-throughput screening
Unspecific peroxygenase
Directed evolution
Saccharomyces cerevisiae
description At present, the end-of-life management of thermoset composite epoxy resins is limited to incineration and landfill storage, highlighting the demand for the development of more sustainable measures. Due to their broad spectrum of C-H oxyfunctionalization reactions, fungal unspecific peroxygenases (UPOs) are becoming important biotechnological tools in organic synthesis while their potential use in biodegradation processes should not be underestimated. Here, we present a colorimetric screening assay aimed at engineering UPOs for the degradation of epoxy resins. We based our study on Hexflow® RTM-6, a commercial epoxy resin used extensively in the aeronautics sector. UPO mutants from the short and long families were initially benchmarked by GC/MS to determine their potential N-dealkylation activity on N,N-bis(2-hydroxypropyl)-p-toluidine (NNBT), the main structural scaffold of Hexflow® RTM-6. A reliable high-throughput colorimetric screening method was developed to quantify the lactaldehyde released by UPO attack on the tertiary amine of NNBT. Based on an evolved UPO from Psathyrella aberdarensis that was expressed by yeast, a small subset of mutant libraries with different mutational loadings was constructed and screened for NNBT N-dealkylation, thereby establishing a directed evolution platform as a vehicle to engineer UPO composite degrading variants.
publishDate 2022
dc.date.none.fl_str_mv 2022
2026
2026
2026
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/416860
url http://hdl.handle.net/10261/416860
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/EC/H2020/886567
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-106166RB-I00
info:eu-repo/grantAgreement///
info:eu-repo/grantAgreement///
https://doi.org/10.3389/fctls.2022.883263
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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