d-Tagatose is a highly promising functional
sweetener
known for its various physiological functions. In this study, a novel
tagatose 4-epimerase from Thermoprotei archaeon (Thar-T4Ease),
with the ability to convert d-fructose to d-tagatose,
was discovered through a combination of structure similarity search
and sequence-based protein clustering. The recombinant Thar-T4Ease
exhibited optimal activity at pH 8.5 and 85 °C, in the presence
of 1 mM Ni2+. Its k
cat and k
cat/K
m values toward d-fructose were measured to be 248.5 min–1 and 2.117 mM–1·min–1, respectively.
Notably, Thar-T4Ease exhibited remarkable thermostability, with a t
1/2 value of 198 h at 80 °C. Moreover,
it achieved a conversion ratio of 18.9% using 100 g/L d-fructose
as the substrate. Finally, based on sequence and structure analysis,
crucial residues for the catalytic activity of Thar-T4Ease were identified
by molecular docking and site-directed mutagenesis. This research
expands the repertoire of enzymes with C4-epimerization activity and
opens up new possibilities for the cost-effective production of d-tagatose from d-fructose.