Structure-Guided Iterative Mutagenesis Drives Dual-Functional Evolution of α-1,3-Fucosyltransferase

α-1,3-Fucosyltransferase (α-1,3-FucT), the rate-limiting enzyme in the biosynthesis of the human milk oligosaccharide 3-fucosyllactose (3-FL), suffers from limited industrial applicability due to its low catalytic activity and poor thermostability. Herein, we systematically optimized the catalytic performance and thermostability of M32-H10 using computer-aided design coupled with a greedy combinatorial strategy. Four computational tools identified 107 potential mutation sites, and subsequent library screening yielded six beneficial single mutants (S98R, K132I, E134M, D147P, N199F and K301P), exhibiting 28%–84% higher specific activity and improved thermostability (ΔTm increased by 0.22 ∼ 4.87 °C). A stepwise combinatorial approach further generated a quadruple mutant (K132I-E134M-N199F-K301P), which demonstrated synergistic effects: a 116% increase in enzymatic activity over the wild-type, an extended half-life (8.05 h at 40 °C), and robust catalytic efficiency across a broad pH range (5.0 ∼ 8.5). Structural analysis revealed that the mutations optimized substrate binding and conformational stability by remodeling the hydrophobic cluster (I132-M134-F199–P301), strengthening the hydrogen-bond network, and fine-tuning the local rigidity-flexibility balance. This study provides an efficient strategy for enzyme rational design and highlights the critical role of combinatorial mutations in industrial enzyme engineering.