Rational Design of Tagatose 6-Phosphate 4-Epimerase for Efficient D -Tagatose Production Under Mg ²⁺ -Supplemented Conditions

As the demand for d-tagatose increases in food and pharmaceutical applications, in vitro multienzyme cascades based on the phosphorylation-isomerization-dephosphorylation pathway offer great potential for its production from maltodextrin. However, differences in metal ion requirements among the three metal-dependent enzymes in this system restrict overall catalytic efficiency. In this study, we rationally engineered the key tagatose 6-phosphate 4-epimerase CaT6PE, generating two high-performance mutants, N195K and V221S, which exhibited markedly enhanced Mg2+-dependent catalytic activity. In multienzyme cascades using 50 g/L maltodextrin under Mg2+-only conditions, N195K and V221S achieved d-tagatose conversion rates of 43.64% and 46.62%, respectively─1.9- and 2.0-fold those of the wild type (22.82%). Molecular dynamics simulations further elucidated the structural mechanisms underlying their improved catalytic activity in the presence of Mg2+. This work provides a universal strategy for enhancing Mg2+-dependent catalytic performance and advances the industrial biocatalytic potential of d-tagatose production.