The β-Fructofuranosidase from Rhodotorula dairenensis: Molecular Cloning, Heterologous Expression, and Evaluation of Its Transferase Activity

The β-fructofuranosidase from the yeast Rhodotorula dairenensis (RdINV) produces a mixture of potential prebiotic fructooligosaccharides (FOS) of the levan-, inulin- and neo-FOS series by transfructosylation of sucrose. In this work, the gene responsible for this activity was characterized and its functionality proved in Pichia pastoris. The amino acid sequence of the new protein contained most of the characteristic elements of β-fructofuranosidases included in the family 32 of the glycosyl hydrolases (GH32). The heterologous yeast produced a protein of about 170 kDa, where N-linked and O-linked carbohydrates constituted about 15% and 38% of the total protein mass, respectively. Biochemical and kinetic properties of the heterologous protein were similar to the native enzyme, including its ability to produce prebiotic sugars. The maximum concentration of FOS obtained was 82.2 g/L, of which 6-kestose represented about 59% (w/w) of the total products synthesized. The potential of RdINV to fructosylate 19 hydroxylated compounds was also explored, of which eight sugars and four alditols were modified. The flexibility to recognize diverse fructosyl acceptors makes this protein valuable to produce novel glycosyl-compounds with potential applications in food and pharmaceutical industries.