Two-Step Production of Neofructo-Oligosaccharides Using Immobilized Heterologous Aspergillus terreus 1F-Fructosyltransferase Expressed in Kluyveromyces lactis and Native Xanthophyllomyces dendrorhous G6-Fructosyltransferase

Fructo-oligosaccharides (FOS) are prebiotic low-calorie sweeteners that are synthesized by the transfer of fructose units from sucrose by enzymes known as fructosyltransferases. If these enzymes generate &beta
(2,6) glycosidic bonds, the resulting oligosaccharides belong to the neoseries (neoFOS). Here, we characterized the properties of three different fructosyltransferases using a design of experiments approach based on response surface methodology with a D-optimal design. The reaction time, pH, temperature, and substrate concentration were used as parameters to predict three responses: The total enzyme activity, the concentration of neoFOS and the neoFOS yield relative to the initial concentration of sucrose. We also conducted immobilization studies to establish a cascade reaction for neoFOS production with two different fructosyltransferases, achieving a total FOS yield of 47.02 &plusmn
3.02%. The resulting FOS mixture included 53.07 &plusmn
1.66 mM neonystose (neo-GF3) and 20.8 &plusmn
1.91 mM neo-GF4.