-
[show abstract]
[hide abstract]
ABSTRACT: The extracellular β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous mainly synthesizes the neo-fructooligosaccharides (neo-FOS) neokestose and neonystose. This enzyme is a glycoprotein with a content of 59-67% N-linked carbohydrates and an estimated molecular mass of 160-200 kDa. The extent level of glycosylation affects the thermal behaviour of the enzyme but not its hydrolase and transferase activities, which are optimal at 60-70 °C. The neo-FOS yield of this enzyme was increased from 40 to 168 g/L when the sucrose concentration increased from 420 to 600 g/L and when the reaction was carried out at 60 °C. The neo-FOS levels obtained (168 g/L) in this work are the largest reported for any microbial β-fructofuranosidase.
Bioresource technology 04/2012; 109:123-30. · 4.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Xanthophyllomyces dendrorhous invertase is an extracellular enzyme that releases β-fructose from the nonreducing termini of various β-D-fructofuranoside substrates. Its ability to produce neokestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which is highly glycosylated, failed to crystallize. Therefore, it was submitted to EndoH deglycosylating treatment and crystals were grown by vapour-diffusion methods. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 75.29, b = 204.93, c = 146.25 Å. Several diffraction data sets were collected using a synchrotron source. Self-rotation function and gel-filtration experiments suggested that the enzyme is a dimer with twofold symmetry.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2010; 66(Pt 11):1441-4. · 0.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An extracellular beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70 degrees C) and thermostability (with a T(50) in the range 66 to 71 degrees C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-beta-(2-->1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k(cat)/K(m) ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial beta-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter(-1). In addition, we isolated and sequenced the X. dendrorhous beta-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant beta-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.
Applied and environmental microbiology 12/2008; 75(4):1065-73. · 3.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Xanthophyllomyces dendrorhous grown in different media shows amylolytic activity, consisting in an extracellular exo-acting enzyme able to hydrolysed alpha,1-4 glycosidic bonds from soluble starch, which also cleaves maltose and malto-oligosaccharides. The enzyme was purified, using basically a couple of chromatography process on DEAE-Sephacel. It is a glycoprotein with a molecular weight estimated to be 60.2 kDa based on its mobility in SDS-PAGE and 115 kDa based on gel filtration. N-linked carbohydrate accounts for 12% of the total mass. It exhibited optimum activity at pH 5.5 and 45 degrees C. Thermostability analysis indicated that it was stable to thermal treatment up to 50 degrees C; 50% of the activity was maintained after 3 h. The rate parameters measured for the hydrolysis of starch and various chain length malto-oligosaccharides shows high catalytic efficiency, calculated by the relationship V(cat)/K(m), for malto-oligosaccharides, such as maltotriose (873 mM(-1) min(-1)), or maltoheptose (698 mM(-1) min(-1)). The new enzyme hydrolysed soluble starch with nearly 3.5- and 1.4-fold lower efficiency than that for maltotriose and maltose, respectively. No activity was found on heterogeneous substrates, such as sucrose and aryl alpha-glucoside, or on isomalto-oligosaccharides. In accordance to substrate specificity profile, the new enzyme was classified as an alpha-glucosidase.
Yeast 02/2006; 23(2):117-25. · 1.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The transglycosylation activity of a novel α-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with α-(1 → 2), α-(1 → 4) and α-(1 → 6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one α-(1 → 6) linkage (panose, 6-O-α-glucosyl-maltotriose and 6-O-α-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the α-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri- and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial α-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides.
Process Biochemistry.
