Protein Engineering of Tagatose 4-Epimerase for D-Tagatose Production

D-Tagatose, a promising sugar substitute with various functional properties and commercial applications, can be enzymatically converted from D-fructose by tagatose 4-epimerase. The development of an efficient tagatose 4-epimerase that catalyzes the conversion of D-fructose into D-tagatose is essential to make the production technology of D-tagatose applicable. In this study, tagatose 4-epimerase from Thermotogae (TsT4Ease) was engineered through semi-rational design and directed evolution, resulting in a 2.8-fold improvement in catalytic activity compared to the wild type (WT). The production of D-tagatose reached 42 g/L in 2 h. Crystal structure analysis determined the structural features with a common (α/β)₈-TIM barrel and a Zn²⁺-binding architecture at the active center. Subsequent molecular dynamics (MD) simulations revealed that the substitutions improved substrate binding energy and stabilized the active pocket. This study offers new insights into the structure-function relationship of TsT4Ease and provides a candidate tagatose 4-epimerase.