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

(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.