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.

An efficient continuous flow process for the synthesis of a non-conventional mixture of fructooligosaccharides

“…In the last decade the concept of ''prebiotic'', a substrate that selectively stimulates the growth and activity of health-promoting Lactobacilli and Bifidobacteria, has assumed much interest in terms of improving human host health (Gimeno-Pérez, Linde, Fernández-Arrojo, Plou, & Fernández-Lobato, 2015). In order to investigate the ability of probiotic strains to use blastose as the sole carbon source and thus its suitability for a potential application in symbiotic-prebiotic mixtures, an in vitro fermentation study was carried out on 5 well characterized probiotic Lactobacillus strains, easier to cultivate than Bifidobacteria: L. paracasei DG (Ferrario et al, 2014), L. rhamnosus GG (Segers & Lebeer, 2014); L. paracasei SHIROTA (Aoki et al, 2014); L. johnsonii LC1 (Isobe et al, 2012); L. reuteri ATCC55730 (Di Nardo et al, 2014;Valeur, Engel, Carbajal, Connolly, & Ladefoged, 2004).…”

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts

“…Initially, the potential hydrolytic activity on sucrose of M. reukaufii was assessed using a solid rich medium for yeast including sucrose and bromothymol blue (BTB), a pH indicator (yellow, greenish and blue in acidic, neutral and basic solutions respectively). Schwanniomyces occidentalis and Pichia pastoris were used as positive and negative controls in this assay due to their proven or absent capacity to hydrolyse sucrose respectively (Álvaro‐Benito et al , ; Gimeno‐Pérez et al , ). As expected, all yeast species were able to grow on this rich medium.…”

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity

“…Active fractions were concentrated using Microcon YM-10 (Amicon) filters and stored at Ϫ70°C. ␤-Fructofuranosidase variants from X. dendrorhous (wild type and mutants) expressed in P. pastoris were purified as described elsewhere (25). ProtoBlue Safe Colloidal Coomassie-stained (National Scientific) denaturing gel electrophoresis (SDS-PAGE; 8% polyacrylamide) of the samples confirmed the purity of the protein fractions.…”

“…Deglycosylated protein was subjected to a 1-min ultrafiltration using an Amicon Ultra-4 50K device (Millipore) to remove the Endo H present in the sample and was subsequently concentrated to about 8 mg ml Ϫ1 using a 10-kDa cutoff membrane. ␤-Fructofuranosidase Activity and Kinetics Assays-␤-Fructofuranosidase hydrolytic activity was determined by the dinitrosalicylic acid method adapted to a 96-well microplate scale as described previously (25). Standard enzymatic assays were performed using 2% sucrose in 100 mM sodium phosphate buffer, pH 5.5, at 60°C.…”