Hydrophobic-flexible rational modification strategy provides fructooligosaccharides activity from Aspergillus niger

Fructooligosaccharides (FOS), linear fructose oligomers with health benefits, are synthesized through sucrose transfructosylation using fructosyltransferases (FTases). This study employed the computer-aided design to modify the hydrophobic-flexible active domain of Aspergillus niger FTases. Mutant 142P exhibited 22.56 % (45.55 μkat) higher than wild-type FTases, with optimal conditions, i.e, 55 °C, pH 5.0, and CO 2 + . Kinetic analysis reduced Km value (6.174 ± 0.15 M), attributed to weakened active domain-sucrose interactions. FOS conversion by 142P enhanced up to 0.17–29.20 %, yielding 14.92 % 1-Kestose, and 14.27 % Nystose. Molecular docking enhanced active domain flexibility (structural shift from green to red) and reduced glucose affinity as key factors boosting activity and FOS conversion. Hydrophobic-flexible modification proves effective for optimizing FTases in industrial applications. This strategy provides a scalable solution for tailoring Aspergillus niger enzymes to meet the growing demands of the prebiotic and functional food industries. • Fructooligosaccharides are significantly useful for various purposes. • A 22.56 % increasing enzyme activity of FTase was achieved by rational design. • FOS yield improved from 0.17 to 29.20 %. • Mechanisms of enzyme activity enhanced by the energy changes and molecular docking.