title: Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars.
abstract: 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.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, 28049, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Gimeno-Pérez
authors.forename: Dolores
authors.initials: D
authors.lastname: Linde
authors.forename: Lucía
authors.initials: L
authors.lastname: Fernández-Arrojo
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
authors.forename: María
authors.initials: M
authors.lastname: Fernández-Lobato
mesh_terms: D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D050260:Carbohydrate Metabolism; D015870:Gene Expression; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D056692:Prebiotics; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D056692:Prebiotics; D011994:Recombinant Proteins; D043324:beta-Fructofuranosidase