Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris

Autores
Fonseca, Maria Isabel; Molina, Melisa Antonella; Winnik, D. L.; Busi, María Victoria; Fariña, Julia Ines; Villalba, Laura; Zapata, Pedro Dario
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Aims: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. Methods and Results: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat/Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. Conclusions: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. Significance and Impact of the Study: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.
Fil: Fonseca, Maria Isabel. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Molina, Melisa Antonella. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Winnik, D. L.. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina
Fil: Busi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina
Fil: Fariña, Julia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina
Fil: Villalba, Laura. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Zapata, Pedro Dario. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
CHARACTERIZATION
GENE ISOLATION
HETEROLOGOUS EXPRESSION
LACCASE
PHLEBIA BREVISPORA
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/86423
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/86423
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastorisFonseca, Maria IsabelMolina, Melisa AntonellaWinnik, D. L.Busi, María VictoriaFariña, Julia InesVillalba, LauraZapata, Pedro DarioCHARACTERIZATIONGENE ISOLATIONHETEROLOGOUS EXPRESSIONLACCASEPHLEBIA BREVISPORAhttps://purl.org/becyt/ford/2.8https://purl.org/becyt/ford/2Aims: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. Methods and Results: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat/Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. Conclusions: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. Significance and Impact of the Study: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.Fil: Fonseca, Maria Isabel. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Molina, Melisa Antonella. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Winnik, D. L.. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; ArgentinaFil: Busi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; ArgentinaFil: Fariña, Julia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Villalba, Laura. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zapata, Pedro Dario. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaWiley Blackwell Publishing, Inc2018-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/86423Fonseca, Maria Isabel; Molina, Melisa Antonella; Winnik, D. L.; Busi, María Victoria; Fariña, Julia Ines; et al.; Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris; Wiley Blackwell Publishing, Inc; Journal of Applied Microbiology; 124; 6; 6-2018; 1454-14681364-5072CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://onlinelibrary.wiley.com/doi/10.1111/jam.13720info:eu-repo/semantics/altIdentifier/doi/10.1111/jam.13720info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-11-05T10:13:44Zoai:ri.conicet.gov.ar:11336/86423instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-11-05 10:13:44.331CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
title Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
spellingShingle Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
Fonseca, Maria Isabel
CHARACTERIZATION
GENE ISOLATION
HETEROLOGOUS EXPRESSION
LACCASE
PHLEBIA BREVISPORA
title_short Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
title_full Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
title_fullStr Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
title_full_unstemmed Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
title_sort Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
dc.creator.none.fl_str_mv Fonseca, Maria Isabel
Molina, Melisa Antonella
Winnik, D. L.
Busi, María Victoria
Fariña, Julia Ines
Villalba, Laura
Zapata, Pedro Dario
author Fonseca, Maria Isabel
author_facet Fonseca, Maria Isabel
Molina, Melisa Antonella
Winnik, D. L.
Busi, María Victoria
Fariña, Julia Ines
Villalba, Laura
Zapata, Pedro Dario
author_role author
author2 Molina, Melisa Antonella
Winnik, D. L.
Busi, María Victoria
Fariña, Julia Ines
Villalba, Laura
Zapata, Pedro Dario
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv CHARACTERIZATION
GENE ISOLATION
HETEROLOGOUS EXPRESSION
LACCASE
PHLEBIA BREVISPORA
topic CHARACTERIZATION
GENE ISOLATION
HETEROLOGOUS EXPRESSION
LACCASE
PHLEBIA BREVISPORA
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.8
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv Aims: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. Methods and Results: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat/Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. Conclusions: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. Significance and Impact of the Study: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.
Fil: Fonseca, Maria Isabel. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Molina, Melisa Antonella. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Winnik, D. L.. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina
Fil: Busi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina
Fil: Fariña, Julia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina
Fil: Villalba, Laura. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Zapata, Pedro Dario. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Aims: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. Methods and Results: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat/Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. Conclusions: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. Significance and Impact of the Study: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.
publishDate 2018
dc.date.none.fl_str_mv 2018-06
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/86423
Fonseca, Maria Isabel; Molina, Melisa Antonella; Winnik, D. L.; Busi, María Victoria; Fariña, Julia Ines; et al.; Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris; Wiley Blackwell Publishing, Inc; Journal of Applied Microbiology; 124; 6; 6-2018; 1454-1468
1364-5072
CONICET Digital
CONICET
url http://hdl.handle.net/11336/86423
identifier_str_mv Fonseca, Maria Isabel; Molina, Melisa Antonella; Winnik, D. L.; Busi, María Victoria; Fariña, Julia Ines; et al.; Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris; Wiley Blackwell Publishing, Inc; Journal of Applied Microbiology; 124; 6; 6-2018; 1454-1468
1364-5072
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://onlinelibrary.wiley.com/doi/10.1111/jam.13720
info:eu-repo/semantics/altIdentifier/doi/10.1111/jam.13720
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Wiley Blackwell Publishing, Inc
publisher.none.fl_str_mv Wiley Blackwell Publishing, Inc
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.087074

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