Abstract
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.
Highlights
The β-fructofuranosidases (EC 3.2.1.26) catalyze the release of fructose from the non-reducing termini of different-size fructans and sucrose
As well as hydrolysing sucrose, β-fructofuranosidases may catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to a sucrose unit
Only a protein of ~170 kDa was detected by SDS-PAGE, which was initially processed for amino acid sequencing using tryptic and chymotryptic digestion followed by MALDI-TOF-MS analyses
Summary
The β-fructofuranosidases (EC 3.2.1.26) catalyze the release of fructose from the non-reducing termini of different-size fructans and sucrose (invertases). Different FOS series can be distinguished depending on the linkage type between the monosaccharide residues: 1 F-FOS, containing β-(2-1)-linked fructose units (inulin-type structure, e.g., 1-kestose); 6 F-FOS, containing β-(2-6)-linked fructose units (levan-type structure, e.g., 6-kestose); and 6 G-FOS, where a β-(2-6) linkage connects fructose to the glucosyl moiety of sucrose (neo-FOS, e.g., neokestose). The three series of FOS show beneficial effects, β-(2-6)-linked FOS appear to have improved prebiotic properties and increased chemical stability compared to the traditionally commercialized β-(2-1)-linked FOS [8,9]. Neo-FOS showed a potential inhibitory activity against melanoma cells and colon cancer [10,11]
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