Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars

© 2014, Springer-Verlag Berlin Heidelberg. The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially imp...

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Detalles Bibliográficos
Autores: Gimeno-Pérez, María, Linde, Dolores, Fernández Arrojo, Lucía, Plou Gasca, Francisco José, Fernández Lobato, María
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2015
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/139327
Acceso en línea:http://hdl.handle.net/10261/139327
Access Level:acceso abierto
Palabra clave:Extracellularβ-fructofuranosidase
Blastose
Pichia pastoris
Heterologous expression
Neo-fructooligosaccharides
Xanthophyllomyces dendrorhous
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spelling Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugarsGimeno-Pérez, MaríaLinde, DoloresFernández Arrojo, LucíaPlou Gasca, Francisco JoséFernández Lobato, MaríaExtracellularβ-fructofuranosidaseBlastosePichia pastorisHeterologous expressionNeo-fructooligosaccharidesXanthophyllomyces dendrorhous© 2014, Springer-Verlag Berlin Heidelberg. The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially improved prebiotic properties. The Xd-INV gene comprises an open reading frame of 1995 bp, which encodes a 665-amino acid protein. Initial N-terminal sequencing of Xd-INV pointed to a majority extracellular protein of 595 amino acids lacking the first 70 residues (potential signal peptide). Functionality of the last 1785 bp of Xd-INV gene was previously proved in Saccharomyces cerevisiae but only weak β-fructofuranosidase activity was quantified. In this study, different strategies to improve this enzyme level in a heterologous system have been used. Curiously, best results were obtained by increasing the protein N-terminus sequence in 39 amino acids, protein of 634 residues. The higher β-fructofuranosidase activity detected in this study, about 15 U/mL, was obtained using Pichia pastoris and represents an improvement of about 1500 times the level previously obtained in a heterologous organism and doubles the best level of activity obtained by the natural producer. Heterologously expressed protein was purified and characterized biochemically and kinetically. Except by its glycosylation degree (10 % lower) and thermal stability (4–5 °C lower in the 60–85 °C range), the properties of the heterologous enzyme, including ability to produce neo-FOS, remained unchanged. Interestingly, besides the neo-FOS referred before blastose was also detected (8–22 g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date.Spanish Ministry of Economy and Competitiveness supported this research. We thank Fundación Ramón Areces for the institutional grant to the Centro de Biologia Molecular Severo OchoaPeer ReviewedSpringer NatureMinisterio de Economía y Competitividad (España)Fundación Ramón ArecesConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2016201620152016info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/139327reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésSíinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1393272026-05-22T06:33:51Z
dc.title.none.fl_str_mv Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
title Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
spellingShingle Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
Gimeno-Pérez, María
Extracellularβ-fructofuranosidase
Blastose
Pichia pastoris
Heterologous expression
Neo-fructooligosaccharides
Xanthophyllomyces dendrorhous
title_short Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
title_full Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
title_fullStr Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
title_full_unstemmed Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
title_sort Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars
dc.creator.none.fl_str_mv Gimeno-Pérez, María
Linde, Dolores
Fernández Arrojo, Lucía
Plou Gasca, Francisco José
Fernández Lobato, María
author Gimeno-Pérez, María
author_facet Gimeno-Pérez, María
Linde, Dolores
Fernández Arrojo, Lucía
Plou Gasca, Francisco José
Fernández Lobato, María
author_role author
author2 Linde, Dolores
Fernández Arrojo, Lucía
Plou Gasca, Francisco José
Fernández Lobato, María
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
Fundación Ramón Areces
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Extracellularβ-fructofuranosidase
Blastose
Pichia pastoris
Heterologous expression
Neo-fructooligosaccharides
Xanthophyllomyces dendrorhous
topic Extracellularβ-fructofuranosidase
Blastose
Pichia pastoris
Heterologous expression
Neo-fructooligosaccharides
Xanthophyllomyces dendrorhous
description © 2014, Springer-Verlag Berlin Heidelberg. The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially improved prebiotic properties. The Xd-INV gene comprises an open reading frame of 1995 bp, which encodes a 665-amino acid protein. Initial N-terminal sequencing of Xd-INV pointed to a majority extracellular protein of 595 amino acids lacking the first 70 residues (potential signal peptide). Functionality of the last 1785 bp of Xd-INV gene was previously proved in Saccharomyces cerevisiae but only weak β-fructofuranosidase activity was quantified. In this study, different strategies to improve this enzyme level in a heterologous system have been used. Curiously, best results were obtained by increasing the protein N-terminus sequence in 39 amino acids, protein of 634 residues. The higher β-fructofuranosidase activity detected in this study, about 15 U/mL, was obtained using Pichia pastoris and represents an improvement of about 1500 times the level previously obtained in a heterologous organism and doubles the best level of activity obtained by the natural producer. Heterologously expressed protein was purified and characterized biochemically and kinetically. Except by its glycosylation degree (10 % lower) and thermal stability (4–5 °C lower in the 60–85 °C range), the properties of the heterologous enzyme, including ability to produce neo-FOS, remained unchanged. Interestingly, besides the neo-FOS referred before blastose was also detected (8–22 g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date.
publishDate 2015
dc.date.none.fl_str_mv 2015
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/10261/139327
url http://hdl.handle.net/10261/139327
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 Springer Nature
publisher.none.fl_str_mv Springer Nature
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 15.81155

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