A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity

In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols f...

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Detalles Bibliográficos
Autores: Gimeno Pérez, María, Finnigan, James D., Echeverria, Coro, Charnock, Simon J., Hidalgo Huertas, Aurelio, Mate, Diana M.
Tipo de recurso: artículo
Fecha de publicación:2022
País:España
Institución:Universidad Autónoma de Madrid
Repositorio:Biblos-e Archivo. Repositorio Institucional de la UAM
Idioma:inglés
OAI Identifier:oai:repositorio.uam.es:10486/703084
Acceso en línea:http://hdl.handle.net/10486/703084
https://dx.doi.org/10.1002/cssc.202102750
Access Level:acceso abierto
Palabra clave:Fluorescence
High-throughput screening
Ketoreductase
Plastic biodegradation
Polyethylene terephthalate
Biología y Biomedicina / Biología
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spelling A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing ActivityGimeno Pérez, MaríaFinnigan, James D.Echeverria, CoroCharnock, Simon J.Hidalgo Huertas, AurelioMate, Diana M.FluorescenceHigh-throughput screeningKetoreductasePlastic biodegradationPolyethylene terephthalateBiología y Biomedicina / BiologíaIn the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described. It was based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that were able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase-type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methodsts This work was supported by a grant from the Universidad Autónoma de Madrid (Project for Young Researchers, Ref. SI1/PJI/ 2019-00394). D. M. M. was supported by a Research Talent Attraction contract from the Community of Madrid (Ref. 2016-T2/ BIO-1960). We thank Carmen Ortega (Center of Molecular Biology “Severo Ochoa”, UAM) for preliminary tests with KREDs. We are also grateful to Jose Luis González Alfonso (Institute of Catalysis and Petrochemistry, CSIC) and David Rodrigo Frutos (Analiza Calidad Madrid S.L.) for technical support on HPLC analysesWileyDepartamento de Biología MolecularFacultad de Ciencias20222022-03-22research articlehttp://purl.org/coar/resource_type/c_2df8fbb1VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10486/703084https://dx.doi.org/10.1002/cssc.202102750reponame:Biblos-e Archivo. Repositorio Institucional de la UAMinstname:Universidad Autónoma de MadridInglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:repositorio.uam.es:10486/7030842026-06-23T12:46:27Z
dc.title.none.fl_str_mv A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity
title A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity
spellingShingle A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity
Gimeno Pérez, María
Fluorescence
High-throughput screening
Ketoreductase
Plastic biodegradation
Polyethylene terephthalate
Biología y Biomedicina / Biología
title_short A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity
title_full A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity
title_fullStr A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity
title_full_unstemmed A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity
title_sort A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity
dc.creator.none.fl_str_mv Gimeno Pérez, María
Finnigan, James D.
Echeverria, Coro
Charnock, Simon J.
Hidalgo Huertas, Aurelio
Mate, Diana M.
author Gimeno Pérez, María
author_facet Gimeno Pérez, María
Finnigan, James D.
Echeverria, Coro
Charnock, Simon J.
Hidalgo Huertas, Aurelio
Mate, Diana M.
author_role author
author2 Finnigan, James D.
Echeverria, Coro
Charnock, Simon J.
Hidalgo Huertas, Aurelio
Mate, Diana M.
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Departamento de Biología Molecular
Facultad de Ciencias
dc.subject.none.fl_str_mv Fluorescence
High-throughput screening
Ketoreductase
Plastic biodegradation
Polyethylene terephthalate
Biología y Biomedicina / Biología
topic Fluorescence
High-throughput screening
Ketoreductase
Plastic biodegradation
Polyethylene terephthalate
Biología y Biomedicina / Biología
description In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described. It was based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that were able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase-type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methods
publishDate 2022
dc.date.none.fl_str_mv 2022
2022-03-22
dc.type.none.fl_str_mv research article
http://purl.org/coar/resource_type/c_2df8fbb1
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10486/703084
https://dx.doi.org/10.1002/cssc.202102750
url http://hdl.handle.net/10486/703084
https://dx.doi.org/10.1002/cssc.202102750
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Wiley
publisher.none.fl_str_mv Wiley
dc.source.none.fl_str_mv reponame:Biblos-e Archivo. Repositorio Institucional de la UAM
instname:Universidad Autónoma de Madrid
instname_str Universidad Autónoma de Madrid
reponame_str Biblos-e Archivo. Repositorio Institucional de la UAM
collection Biblos-e Archivo. Repositorio Institucional de la UAM
repository.name.fl_str_mv
repository.mail.fl_str_mv
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