Immobilization of a tagatose 4-epimerase on amino-functionalized macroporous resin and its application in bioconversion of D-fructose to D-tagatose.

The biocatalytic production of D-tagatose, a valuable low-calorie sweetener, is often hindered by poor enzyme stability and limited reusability. To address this issue, a thermostable tagatose-4-epimerase (TpT4Ease) from Thermotoga petrophila was crosslinked with glutaraldehyde followed by immobilization on an amino-functionalized macroporous resin (LX-1000HA), resulting in TpT4Ease@LX-1000HA-GA.The effective synthesis of TpT4Ease@LX-1000HA-GA was verified by CD and SEM after optimization of the immobilization procedure. Kinetic analysis revealed that the conformational constraint within the porous support slightly compromised intrinsic turnover but effectively prevented thermal unfolding. Consequently, TpT4Ease@LX-1000HA-GA displayed improved thermal and storage stability, with its half-life at 70 °C extended to 15.4 h compared to 3.3 h for the free enzyme. Under optimized continuous-flow conditions (200 g/L fructose, 70 °C, 0.5 mL/min), TpT4Ease@LX-1000HA-GA achieved an efficient bioconversion of D-fructose to D-tagatose with a yield of 17.3%. After five consecutive cycles, TpT4Ease@LX-1000HA-GA maintained 90.6% of its original catalytic activity in continuous-flow bioconversion, indicating an excellent operational stability. Overall, this study establishes an efficient immobilization-continuous-flow integrated system that significantly enhances TpT4Ease stability and reusability, providing a practical strategy for the continuous biomanufacturing of D‑tagatose.