New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme

Background: The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD + poo...

Descripción completa

Detalles Bibliográficos
Autores: Rocha-Martín, Javier, Vega, Daniel, Bolívar Bolívar, Juan Manuel, Godoy, César A., Hidalgo, Aurelio, Berenguer, José, Guisán, José Manuel, López-Gallego, Fernando
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2011
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/128050
Acceso en línea:http://hdl.handle.net/10261/128050
Access Level:acceso abierto
Palabra clave:Dehydrogenase
Immobilization
NAD+, extremophiles
id ES_4b3ebcf5b6137375aef3bdcf67a79e4d
oai_identifier_str oai:digital.csic.es:10261/128050
network_acronym_str ES
network_name_str España
repository_id_str
spelling New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzymeRocha-Martín, JavierVega, DanielBolívar Bolívar, Juan ManuelGodoy, César A.Hidalgo, AurelioBerenguer, JoséGuisán, José ManuelLópez-Gallego, FernandoDehydrogenaseImmobilizationNAD+, extremophilesBackground: The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD + pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD +. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.Results: The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.Conclusion: We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations. © 2011 Rocha-Martín et al; licensee BioMed Central Ltd.Madrid Region council CAM (grants S0505/PPQ/0344), CSIC (JAE-doc 108) and the Spanish Ministry of Science (Programa Ramón y Cajal and grants BIO-2008-01481, BIO2010-18875 and CTQ2009-07568) is gratefully acknowledged. An institutional grant from the Fundación Ramón Areces to the CBMSO is also acknowledgedPeer ReviewedBioMed CentralFundación Ramón ArecesComunidad de MadridMinisterio de Ciencia y Tecnología (España)Comunidad de MadridConsejo Superior de Investigaciones Científicas (España)Ministerio de Ciencia e Innovación (España)Fundación Ramón ArecesConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2016201620112016info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/128050reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésSíinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1280502026-05-22T06:33:51Z
dc.title.none.fl_str_mv New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
title New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
spellingShingle New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
Rocha-Martín, Javier
Dehydrogenase
Immobilization
NAD+, extremophiles
title_short New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
title_full New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
title_fullStr New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
title_full_unstemmed New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
title_sort New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
dc.creator.none.fl_str_mv Rocha-Martín, Javier
Vega, Daniel
Bolívar Bolívar, Juan Manuel
Godoy, César A.
Hidalgo, Aurelio
Berenguer, José
Guisán, José Manuel
López-Gallego, Fernando
author Rocha-Martín, Javier
author_facet Rocha-Martín, Javier
Vega, Daniel
Bolívar Bolívar, Juan Manuel
Godoy, César A.
Hidalgo, Aurelio
Berenguer, José
Guisán, José Manuel
López-Gallego, Fernando
author_role author
author2 Vega, Daniel
Bolívar Bolívar, Juan Manuel
Godoy, César A.
Hidalgo, Aurelio
Berenguer, José
Guisán, José Manuel
López-Gallego, Fernando
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Fundación Ramón Areces
Comunidad de Madrid
Ministerio de Ciencia y Tecnología (España)
Comunidad de Madrid
Consejo Superior de Investigaciones Científicas (España)
Ministerio de Ciencia e Innovación (España)
Fundación Ramón Areces
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Dehydrogenase
Immobilization
NAD+, extremophiles
topic Dehydrogenase
Immobilization
NAD+, extremophiles
description Background: The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD + pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD +. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.Results: The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.Conclusion: We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations. © 2011 Rocha-Martín et al; licensee BioMed Central Ltd.
publishDate 2011
dc.date.none.fl_str_mv 2011
2016
2016
2016
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/10261/128050
url http://hdl.handle.net/10261/128050
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv BioMed Central
publisher.none.fl_str_mv BioMed Central
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
_version_ 1869407547761360896
score 15,811543

Similares en LA Referencia