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Título: | Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars |
Autor: | Gimeno-Pérez, María CSIC ORCID; Linde, Dolores CSIC ORCID ; Fernández Arrojo, Lucía; Plou Gasca, Francisco José CSIC ORCID ; Fernández Lobato, María CSIC ORCID | Palabras clave: | Extracellularβ-fructofuranosidase Blastose Pichia pastoris Heterologous expression Neo-fructooligosaccharides Xanthophyllomyces dendrorhous |
Fecha de publicación: | 2015 | Editor: | Springer Nature | Citación: | Applied Microbiology and Biotechnology 99: 3459- 3467 (2015) | Resumen: | © 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. | URI: | http://hdl.handle.net/10261/139327 | DOI: | 10.1007/s00253-014-6145-1 | Identificadores: | doi: 10.1007/s00253-014-6145-1 issn: 1432-0614 |
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