New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

Abstract Background Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric...

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Autores: Pardo, Isabel, Chanagá, Xiomara, Vicente, Ana I., Alcalde Galeote, Miguel, Camarero, Susana
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2013
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/86547
Acceso en línea:http://hdl.handle.net/10261/86547
Access Level:acceso abierto
Palabra clave:High-throughput screening
Laccase
Lignocellulose
Dyes
Violuric acid
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spelling New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomassPardo, IsabelChanagá, XiomaraVicente, Ana I.Alcalde Galeote, MiguelCamarero, SusanaHigh-throughput screeningLaccaseLignocelluloseDyesVioluric acidAbstract Background Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast. Conclusions The high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.This work has been funded by the Spanish National Project EVOFACEL, BIO2010-19697. The internship of X. Chanagá at CIB-CSIC, Madrid, was supported by a grant from the UNAL-Medellín, Colombia. I. Pardo thanks Agencia Estatal CSIC for her JAE-Predoc Fellowship and A.I. Vicente thanks MINECO for her FPI Fellowship.Peer ReviewedBioMed CentralS-type phenolic mediators2013201320132013info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/86547reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttp://dx.doi.org/10.1186/1472-6750-13-90info:eu-repo/semantics/openAccessoai:digital.csic.es:10261/865472026-05-22T06:33:51Z
dc.title.none.fl_str_mv New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass
title New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass
spellingShingle New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass
Pardo, Isabel
High-throughput screening
Laccase
Lignocellulose
Dyes
Violuric acid
title_short New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass
title_full New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass
title_fullStr New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass
title_full_unstemmed New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass
title_sort New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass
dc.creator.none.fl_str_mv Pardo, Isabel
Chanagá, Xiomara
Vicente, Ana I.
Alcalde Galeote, Miguel
Camarero, Susana
author Pardo, Isabel
author_facet Pardo, Isabel
Chanagá, Xiomara
Vicente, Ana I.
Alcalde Galeote, Miguel
Camarero, Susana
author_role author
author2 Chanagá, Xiomara
Vicente, Ana I.
Alcalde Galeote, Miguel
Camarero, Susana
author2_role author
author
author
author
dc.contributor.none.fl_str_mv S-type phenolic mediators
dc.subject.none.fl_str_mv High-throughput screening
Laccase
Lignocellulose
Dyes
Violuric acid
topic High-throughput screening
Laccase
Lignocellulose
Dyes
Violuric acid
description Abstract Background Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast. Conclusions The high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.
publishDate 2013
dc.date.none.fl_str_mv 2013
2013
2013
2013
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/10261/86547
url http://hdl.handle.net/10261/86547
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv http://dx.doi.org/10.1186/1472-6750-13-90
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
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