Biochemical characterization and biocatalytic application of a hyperthermostable tagatose 4-epimerase from Infirmifilum uzonense.

D-Tagatose is a representative rare sugar with the physiochemical properties of low energy and high sweetness, as well as excellent physiological functions such as blood sugar regulation, enhancement of intestinal flora, and prevention of dental caries. At present, D-tagatose production involves lactose hydrolysis and D-galactose isomerization processes, resulting in high production costs that hinder its industrial advancement. Tagatose 4-epimerase (T4Ease) has the capability to directly convert d-fructose into D-tagatose through C-4 epimerization, providing a new approach for D-tagatose production. In this study, a hyperthermostable T4Ease from Infirmifilum uzonense (Inuz-TE4ase) was identified from the Foldseek clustered AlphaFold database and its biochemical properties were characterized in detail. Under the optimal reaction conditions of 90 °C and pH 8.5 (Tris-HCl) with the addition of 1 mM Ni2+, the maximum catalytic activity towards d-fructose was determined to be 0.680 U/mg. Inuz-TE4ase exhibited exceptional thermostability, with half-life (t1/2) values of 19.3 h at 85 °C and 8.9 h at 90 °C, respectively. Inuz-TE4ase was strictly metal-dependent, and its stability could be enhanced by Ni2+ with an increase in the melting temperature (Tm) value from 101.1 °C to 105.7 °C. When 100 g/L d-fructose was used as the substrate, Inuz-TE4ase could catalyze the production of 21.67 g/L D-tagatose, indicating its significant potential for D-tagatose bioproduction.