Boosting multi-enzyme cascade activity for glucose biosynthesis by kinetics-oriented grouped immobilization

The biosynthesis of glucose (C6 compound) from CO₂ (C1 compound) represents a highly promising pathway toward sustainable carbon neutrality, requiring ordered multi-enzyme cascade catalysis. However, conventional co-immobilization of multiple enzymes follows an all-in-one approach that struggles to reconcile enzyme compatibility and efficiency. Based on kinetic decoupling, this study introduces a novel strategy of grouped enzyme immobilization to construct a rational multi-enzyme cascade catalytic system. Five enzymes were divided into two groups—upstream (DHAK, TPI, and FSA) and downstream (PGI and G6PP)—and immobilized on the D301 resin to reduce random substrate diffusion and improve cascade efficiency. Compared to all-in-one co-immobilization, this strategy led to a 6.65-fold improvement in glucose yield (508.5 mg L⁻¹) within 2 h. Molecular dynamics simulations revealed that enzymes with higher surface charge form stronger electrostatic bonds with the resin, whereas larger enzymes exhibit weaker binding and greater desorption tendencies, leading to reduced operational stability upon repeated use. The integration of the immobilized enzymes into a packed-bed microreactor enabled stable production of glucose for 12 h of continuous-flow, achieving a space–time yield of 105.9 mg h⁻¹ L⁻¹. These findings highlight the potential of grouped immobilization on inexpensive carriers for scalable and continuous sugar biomanufacturing.