Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 99, Issue 8, Pages 3459-3467Publisher
SPRINGER
DOI: 10.1007/s00253-014-6145-1
Keywords
Extracellular beta-fructofuranosidase; Xanthophyllomyces dendrorhous; Neo-fructooligosaccharides; Heterologous expression; Pichia pastoris; Blastose
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Funding
- Spanish Ministry of Economy and Competitiveness [BIO2010-20508-C4-1/-4, BIO2013-48779-C4-1/-4]
- Fundacion Ramon Areces
- Ministry of Education, Culture and Sports
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The beta-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 beta-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 beta-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 A degrees C lower in the 60-85 A degrees 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.
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