caa a22 4500 605503907 CHVBK 20210128100613.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00253-014-6145-1 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6145-1 Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous , an enzyme producing prebiotic sugars [Elektronische Daten] [María Gimeno-Pérez, Dolores Linde, Lucía Fernández-Arrojo, Francisco Plou, María Fernández-Lobato] 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 1995bp, 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 1785bp 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 15U/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 (8-22g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date. Springer-Verlag Berlin Heidelberg, 2014 Extracellular β-fructofuranosidase nationallicence Xanthophyllomyces dendrorhous nationallicence Neo-fructooligosaccharides nationallicence Heterologous expression nationallicence Pichia pastoris nationallicence Blastose nationallicence Gimeno-Pérez María Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, 28049, Madrid, Spain aut Linde Dolores Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, 28049, Madrid, Spain aut Fernández-Arrojo Lucía Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049, Madrid, Spain aut Plou Francisco Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049, Madrid, Spain aut Fernández-Lobato María Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, 28049, Madrid, Spain aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3459-3467 0175-7598 99:8<3459 2015 99 253 https://doi.org/10.1007/s00253-014-6145-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6145-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Gimeno-Pérez María Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, 28049, Madrid, Spain aut NATIONALLICENCE 700 1- Linde Dolores Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, 28049, Madrid, Spain aut NATIONALLICENCE 700 1- Fernández-Arrojo Lucía Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049, Madrid, Spain aut NATIONALLICENCE 700 1- Plou Francisco Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049, Madrid, Spain aut NATIONALLICENCE 700 1- Fernández-Lobato María Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, 28049, Madrid, Spain aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3459-3467 0175-7598 99:8<3459 2015 99 253