Active Pocket Engineering of d -Tagatose 4-Epimerase for Improved Catalytic Performance and Efficient Cascade Synthesis of d -Tagatose from d -Glucose

d -Tagatose is a rare hexose sugar with excellent properties, and its synthesis catalyzed by d -tagatose 4-epimerase (T4E) represents a competitive novel pathway. In this study, Eb T4E derived from the Eubacteriales bacterium was screened and systematically characterized. By reshaping the microenvironment of the active pocket, mutant M3(S131D/H410W/T279S) was constructed, which showed a 3.89-fold higher conversion rate compared with the wild-type (WT) enzyme. Kinetic parameter analysis and molecular dynamics (MD) simulations revealed that M3 had enhanced substrate affinity, hydrogen bond network, charge properties, and channel accessibility. Finally, the conversion rates of d -fructose to d -tagatose catalyzed by the purified M3 enzyme and M3 whole-cell catalysts reached 29.46% and 26.2%, respectively. Additionally, the dual-enzyme cascade reaction of M3 with glucose isomerase (GI) TEGI-M-L38M-V137L was constructed, achieving a 13.16% yield of d -tagatose from d -glucose. This study demonstrates that Eb T4E-M3 is a promising biocatalyst for d -tagatose production, laying the foundation for its subsequent industrial application.