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Applied and Environmental Microbiology, February 2009, p. 1065-1073, Vol. 75, No. 4
0099-2240/09/$08.00+0 doi:10.1128/AEM.02061-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous
,
Dolores Linde,1
Isabel Macias,1
Lucía Fernández-Arrojo,2
Francisco J. Plou,2
Antonio Jiménez,1 and
María Fernández-Lobato1*
Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, Cantoblanco, 28049 Madrid, Spain,1 Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Cantoblanco, 28049 Madrid, Spain2
Received 5 September 2008/ Accepted 6 December 2008
An extracellular β-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°C) and thermostability (with a T50 in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2
1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the kcat/Km ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-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 β-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 β-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.
* Corresponding author. Mailing address: Centro de Biología Molecular Severo Ochoa (CSIC/UAM), Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-1964492. Fax: 34-91-1924420. E-mail: mfernandez{at}cbm.uam.es
Published ahead of print on 16 December 2008.
Supplemental material for this article may be found at http://aem.asm.org/.
Applied and Environmental Microbiology, February 2009, p. 1065-1073, Vol. 75, No. 4
0099-2240/09/$08.00+0 doi:10.1128/AEM.02061-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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