Mining, enzymatic characterisation and mutagenesis of tagatose 4-epimerase

ᴅ-Tagatose is a functional sweetener with high safety and diverse physiological benefits. In recent years, ᴅ-tagatose bioconversion from economically accessible natural raw materials has emerged as a research focus in food and bioengineering. This study employed the amino acid sequence of UxaE from Thermotoga neapolitana as a template. Through a gene mining strategy, a novel tagatose 4-epimerase (SPI-T4Ease, accession number GHT70811.1) was successfully screened from Spirochaetia bacterium, which efficiently catalyzes the directed conversion of ᴅ-fructose into ᴅ-tagatose. Enzymatic characterisation revealed that the optimal reaction conditions for recombinant SPI-T4Ease are pH 10.5 and 65 °C, requiring 1.5 mmol/L Ni²⁺ for optimal catalytic activity. It exhibits excellent thermal stability with a half-life of 6.9 h at 65 °C. Kinetic analysis revealed the enzyme's K_m, k_cat, and k_cat/K_m values for ᴅ-fructose substrate to be 977 ± 116 mM, 44.76 ± 2.92 min^-1, and 0.0458 ± 0.0061 mM^-1·min^-1, respectively. Within a 100 g/L ᴅ-fructose reaction system, the maximum conversion of ᴅ-tagatose reached 25.3%. With homology modelling and molecular docking, virtual saturation mutagenesis was performed on residues within 5 Å of substrate. A set of virtual mutants with reduced binding energy were selected, along with a small number of mutants with increased binding energy for comparison. After preparation by site-directed mutagenesis, the resulting mutants exhibited relative activities ranging from approximately 16.67% to 86.73% of the wild-type enzyme. Molecular dynamics simulations revealed that mutations increased protein flexibility, destabilized hydrogen bonds and disrupted optimal catalytic conformation. This study enriches enzyme resources and reveals mutational impacts on substrate-binding pocket and hydrogen bond network.