Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales

204 páginas, 64 figuras y 29 tablas

Detalles Bibliográficos
Autor: Zumárraga, Miren
Tipo de recurso: tesis doctoral
Fecha de publicación:2007
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/113356
Acceso en línea:http://hdl.handle.net/10261/113356
Access Level:acceso abierto
Palabra clave:Lacasas
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spelling Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionalesZumárraga, MirenLacasas204 páginas, 64 figuras y 29 tablasMimicking the darwinist algorithm of natural selection, through several rounds of random mutagenesis and/or DNA recombination coupled to massive high-throughput screenings, directed molecular evolution allows to tailor enzymes more robust, stable, efficient and in general with many improved features. In this evolutionary scenario, semi-rational analysis - where researches are taking advantage from protein structural information to create and explore libraries constructed by saturation mutagenesis- constitutes also a powerful methodology. Laccases from white-rot fungi are remarkable biocatalysts due to their broad substrate specificity, with potential applications in bioremediation, lignocellulose processing, organic synthesis and more. Most of these transformations must be carried out at high concentrations of organic cosolvents where laccase undergoes unfolding, therefore loosing its catalytic performance. We have evolved the thermostable laccase from the Ascomycete Myceliophthora thermophila to tolerate high concentrations of cosolvents. The genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae, variant R2, was capable to resist a wide array of miscible cosolvents of biotechnological significance at concentrations as high as 50% (v/v). Intrinsic electrochemical laccase features such as the redox potential at the T1 and T2/T3 sites and the geometry and electronic structure of the catalytic coppers were altered during the course of the in vitro evolution experiment. Some mutations located at the surface of protein contributed to the reinforcement of the protein architecture at different key-denaturation regions by establishing new hydrogen bonds and salt bridges. Moreover, the mutations introduced at the C-terminal extension affected the protein folding at the post-translational maturation steps. Additionally, we have developed a new methodology named in vivo overlap extension (IVOE) which is based on the high homologous recombination frequency of Saccharomyces cerevisiae. This methodology provides a simple manner to build combinatorial saturation mutagenesis libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. Several positions that seem to be related with the redox potential at the T1 site were targeted for combinatorial saturation mutagenesis. After exploring over 170000 clones, the best variant revealed a direct relationship between the highly conserved tripeptide 509VSG511, located in the vicinity of the T1 site, and the C-terminal plug. The Km O2 value of the mutant was increased 1.5-fold and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, thus improving catalytic efficiencies about 3- and 8-fold towards nonphenolic and phenolic substrates, respectively. Although the copper geometry at the T1 site was perturbed upon mutation, paradoxically the laccase redox potential was not significantly altered. Taking together, the present study suggests that 509VSG511 tripeptide may play a hithertounrecognized role in regulating the traffic of O2 to the T2/T3 copper cluster, in combination with the C-terminal plug, which is dependent on the binding of the reducing substrate at the copper T1.Peer reviewedAlcalde Galeote, MiguelPlou Gasca, Francisco JoséConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201520152007info:eu-repo/semantics/doctoralThesishttp://purl.org/coar/resource_type/c_db06http://hdl.handle.net/10261/113356reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)EspañolSíinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1133562026-05-22T06:33:51Z
dc.title.none.fl_str_mv Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales
title Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales
spellingShingle Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales
Zumárraga, Miren
Lacasas
title_short Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales
title_full Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales
title_fullStr Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales
title_full_unstemmed Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales
title_sort Evolución molecular dirigida de lacasas fúngicas en Saccharomyces cerevisiae: tolerancia a disolventes orgánicos y estudios semiracionales
dc.creator.none.fl_str_mv Zumárraga, Miren
author Zumárraga, Miren
author_facet Zumárraga, Miren
author_role author
dc.contributor.none.fl_str_mv Alcalde Galeote, Miguel
Plou Gasca, Francisco José
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Lacasas
topic Lacasas
description 204 páginas, 64 figuras y 29 tablas
publishDate 2007
dc.date.none.fl_str_mv 2007
2015
2015
dc.type.none.fl_str_mv info:eu-repo/semantics/doctoralThesis
http://purl.org/coar/resource_type/c_db06
format doctoralThesis
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/113356
url http://hdl.handle.net/10261/113356
dc.language.none.fl_str_mv Español
language_invalid_str_mv Español
dc.relation.none.fl_str_mv Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
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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score 15,811543

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