Modulation of Fructose Transfer Process for Promoting Apparent Isomerization Activity of Amylosucrase from <i>Bifidobacterium thermophilum</i>

amylosucrase through saturation mutagenesis at Gly374 to enhance product selectivity. Among 19 variants, G374H and G374T demonstrated remarkable improvements in turanose and trehalulose production, respectively. G374H exhibited a 9.1-fold increase in catalytic efficiency for turanose formation, achieving 88.64% selectivity. G374T showed a 7.8-fold enhancement in trehalulose production efficiency. Molecular dynamics simulations revealed that these substitutions induced coordinated structural changes, specifically by modulating the flexibility of active-site loops (loops 3 and 7) and optimizing the local hydrogen-bond network, which collectively determine product specificity. G374H showed enhanced conformational flexibility for optimal substrate positioning, while G374T displayed efficient product release mechanisms. This work demonstrates that strategic modification of substrate-binding residues enables selective control of product distribution for functional food ingredient production.