Engineering a Bifunctional Tag for Enhancing Thermostability and Immobilization Efficiency of <scp>d</scp> -Allulose 3-Epimerase

d-allulose, a low-calorie rare sugar produced by d-allulose 3-epimerase (DAE), has broad application potential, but its production is limited by the poor thermostability and immobilization efficiency of DAE. In this study, a multimerization-promoting tag (Mp-tag) was fused to the N-terminus of DAE from <i>Ruminococcus</i> sp. (RDAE), targeting the dimer-dimer interface of its tetrameric structure. The optimized triple mutant Mp-tag enhanced interdimer interactions, increasing the melting temperature from 64.9 °C (without Mp-tag) to 71.8 °C. Molecular dynamics simulations confirmed that Mp-tag stabilized tetramer formation by lowering interdimer binding free energy. To improve immobilization, a glutamate-rich immobilization-promoting tag (Ip-tag) was fused to the N-terminus of the Mp-tag, enhancing site-specific binding and increasing immobilized activity recovery by 30.69%. The resulting immobilized bifunctional-tagged enzyme retained over 90% activity after 500 reaction cycles. This strategy also proved effective for other tetrameric DAEs, demonstrating its general applicability for enhancing enzyme stability and immobilization in biocatalytic processes.