Multienzyme Cascade Coimmobilization on ZIF-8-Coated Magnetic Nanoparticles for Efficient <scp>d</scp>-Allulose Synthesis

This study develops a multienzyme coimmobilization strategy on NTA-functionalized ZIF-8-coated magnetic nanoparticles (NZMNPs) for efficient d-allulose synthesis. Under optimized immobilization conditions (enzyme-to-carrier ratio: 1:50 w/w, 30 min immobilization), the system achieved an immobilization efficiency of 93.7% along with 107.1% activity retention. Immobilization markedly enhanced the enzyme stability and reusability. The immobilized d-allulose 3-epimerase (DPE) retained 60% of its initial activity after 360 min at 55 °C─more than double the residual activity of the free enzyme (26.8%)─and maintained 65.2% activity after 20 cycles of reuse. Coimmobilization of DPE, l-rhamnulose kinase (RhaB), and polyphosphate kinase (PPK) achieved a 78.4% d-allulose conversion yield and retained 53.1% residual activity after five operational cycles. The poor thermostability of RhaB affected the overall catalytic efficiency of the multienzyme cascade. To address the thermal instability of RhaB, a PROSS strategy was employed. The resulting S44K mutant significantly improved cascade stability at 30 °C. Taken together, these findings confirm that NZMNPs serve as a robust and efficient platform for multienzymes coimmobilization, highlighting their potential for industrial d-allulose production.