Thermostability Enhancement of Tagatose 4-Epimerase through Protein Engineering and Whole-Cell Immobilization

d-Tagatose, a rare sugar endowed with a low-calorie property, superior taste quality, and probiotic functionality, has garnered significant research attention. However, the prevailing biological production methods relying on β-galactosidase and l-arabinose isomerase face challenges including high cost and suboptimal conversion efficiency. Consequently, it is of great research significance to find efficient alternative routes for d-tagatose synthesis. Previously, Thermotoga petrophila tagaturonate 3-epimerase was modified to function as tagatose 4-epimerase (T4E) enabling the direct conversion of d-fructose to d-tagatose. In this study, T4E was further engineered through directed evolution, specifically targeting the enhancement of its thermostability for application. This endeavor yielded promising T4E variants with superiority over those of the original enzyme. T4E I430P exhibits a half-life (t1/2) at 70 °C that is 1.83-fold that of T4E, with an increased melting temperature (Tm) of 5.1 °C compared to T4E. Additionally, T4E G90S/T272A/I430P demonstrated a 21.4% increase in specific activity compared to T4E. At 70 °C, its t1/2 was 1.69-fold that of T4E, and its Tm is 2.9 °C higher than T4E. Furthermore, whole-cell immobilization integrating these engineered T4E variants into a robust biocatalytic system was employed. This innovative approach not only underscores the practical feasibility of modifying enzymes through directed evolution but also establishes a foundation for the cost-effective, large-scale production of d-tagatose.