Characterization of a tagatose 4-epimerase and optimizing its expression for efficient D-tagatose bioconversion from...

D-Tagatose is a promising functional sweetener due to its safety profile and versatile physiological effects. Recently, the bioconversion of D-tagatose from cost-effective natural resources has gained increasing attention. In this study, the hypothetical protein DRJ43_04605 from Thermoprotei archaeon, abbreviated as DRJ43-T4Ease, was identified as a tagatose 4-epimerase capable of efficiently converting d-fructose into D-tagatose. DRJ43-T4Ease exhibited optimal specific activity of 0.79 U/mg at pH 7.5 and 90 °C in the presence of 1 mM Ni²⁺. The denaturation temperature (T_m) of the enzyme was determined to be 105.9 °C, with a half-life of 7.19, 6.56, and 4.84 h at 85, 90, and 95 °C, respectively. The kinetic parameters for d-fructose conversion, including K_m, k_cat, and k_cat/K_m, were calculated to be 51.86 mM, 127.22 min^-1, and 2.45 mM^-1 min^-1, respectively. Furthermore, the expression of DRJ43-T4Ease in E. coli was optimized by engineering the translation initiation sequence. The engineered strain EcDRJ43-T4Ease-M1 achieved a volume activity of 98.4 U/L through shake-flask fermentation and a conversion efficiency of 17.2 % using 100 g/L d-fructose as the substrate. This study demonstrates the potential for D-tagatose production from d-fructose via whole-cell bioconversion, providing a promising approach for the industrial-scale production of this valuable sweetener.