Molecular and biochemical characterization of a beta-fructofuranosidase from Xanthophyllomyces dendrorhous.
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.
Linde
Dolores
D
Centro de Biología Molecular Severo Ochoa (CSIC/UAM), Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Macias
Isabel
I
Fernández-Arrojo
Lucía
L
Plou
Francisco J
FJ
Jiménez
Antonio
A
Fernández-Lobato
María
M
D001487:Basidiomycota / Q000201:enzymology*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D004271:DNA, Fungal; D005656:Fungal Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; D014312:Trisaccharides; C072433:1-kestose; C043600:fungitetraose; C072434:neokestose; D013395:Sucrose; D043324:beta-Fructofuranosidase