In recent years, great efforts have been focused on providing information about the domains implicated in the substrate binding and catalytic mechanisms of the glycosyl hydrolase (GH) families; to date, three-dimensional structures of at least a representative member of 52 of the 115 GH families have been reported. This information is continuously updated in the CAZy (Carbohydrate-Active Enzymes) database (Cantarel et al.
The GH32 family (EC 3.2.1.–) includes enzymes such as invertases or β-fructofuranosidases that catalyze the release of β-fructose from the nonreducing termini of various β-d
-fructofuranoside substrates. These proteins contain two acidic residues, Asp or Glu, that act as the nucleophile and the acid/base catalyst responsible for the cleavage of glycosidic bonds and are included in the GH-J clan together with the GH68 (inulosucrase) family. To date, the three-dimensional structures of the β-fructofuranosidases from the bacterium Thermotoga maritima
(Alberto et al.
) and the yeast Schwanniomyces occidentalis
(Polo et al.
; Álvaro-Benito et al.
), as well as of an exoinulinase from the fungus Aspergillus niger
(Nagem et al.
) and of a fructan exohydrolase and a cell-wall invertase from the plants Chicorium intybus
(Verhaest et al.
) and Arabidopsis thaliana
(Lammens et al.
), respectively, which are all members of the GH32 family, have been solved. Very recently, the structure of the fructosyltransferase from Aspergillus japonicus
has also been reported (Chuankhayan et al.
). These studies have shown a common bimodular folding for the GH32 family, with an N-terminal fivefold β-propeller catalytic domain and a C-terminal β-sandwich domain, the function of which is still not well known. Interestingly, this β-sandwich domain was shown to be involved in dimerization of the S. occidentalis
β-fructofuranosidase, being directly implicated in shaping its active site (Álvaro-Benito et al.
In addition to releasing β-fructose from the nonreducing termini of various β-d
-fructofuranoside substrates, microbial β-fructofuranosidases may catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the oligosaccharide used as the biosynthetic reaction substrate, which is generally sucrose, by different glycosidic bonds depending on the enzyme source (Sangeetha et al.
). FOS act as prebiotics, which are nondigestible food ingredients that improve the health of the consumer by stimulation of the growth of beneficial bifid bacteria in the digestive tract (Gibson & Roberfroid, 1995
). At present, Aspergillus
enzymes are the main industrial producers of FOS (Ghazi et al.
), giving a mixture of FOS with an inulin-type structure containing β-(2→1)-linked fructose oligomers (1
F-FOS; 1-kestose, nystose or 1F-fructofuranosylnystose). However, there is an interest in the development of novel molecules with improved prebiotic and physiological properties. In this context, β-(2→6)-linked FOS, in which the link exists between two fructose units (6
F-FOS; 6-kestose) or between fructose and the glucosyl moiety (6
G-FOS; neokestose, neonystose and neofructofuranosylnystose), have enhanced prebiotic properties compared with commercial FOS (Marx et al.
; Kilian et al.
). The basidiomycetous yeast Xanthophyllomyces dendrorhous
produces an extracellular β-fructofuranosidase (XdINV) which shows broad substrate specificity and hydrolyzes sucrose, 1-kestose and nystose. Unlike other microbial β-fructofuranosidases, it produces neokestose as the main transglycosylation product (Linde et al.
). Therefore, structural analysis of XdINV is necessary in order to fully understand its particular biological specificity and to improve its biotechnological potential. In this study, we describe an abbreviated purification protocol to overproduce the enzyme, its crystallization and a preliminary X-ray crystallographic analysis.