Coordinated Engineering of Noncatalytic and Active-Site Regions in a Thermophilic D-Tagatose 4-Epimerase Enhances D-Tagatose Bioconversion from D-Fructose

D-Tagatose is a low-calorie rare sugar with glycemic control and obesity prevention benefits, making it a promising functional sweetener. One-step epimerization of D-fructose to D-tagatose by D-tagatose 4-epimerase (T4E) is an attractive approach, but current T4Es suffer from low activity and poor thermostability. To address this, a novel T4E from the Thermoproteales archaeon (TaDt4e) was identified and engineered. A truncation variant Δ104–123 increased activity by 76.9%, and directed evolution produced the double mutant S46A/I113M with a 153.8% increase over wild-type enzyme. Using 100 g/L D-fructose, whole-cell biotransformation with S46A/I113M achieved 12.1% conversion within 10 min, and the purified enzyme reached 23.8% within 90 min. These results show that coordinated engineering of noncatalytic and active-site regions can substantially improve the catalytic performance of TaDt4e. This study not only identifies TaDt4e as a promising candidate for D-tagatose production, but also provides insights into the semirational or rational engineering of T4Es and related enzymes.