Enzymatic reactions play an important role in numerous industrial processes, e.g., in food production, pharmaceuticals and the production of biofuels. However, a major challenge when using enzymes in industrial applications is maintaining their stability and activity, especially under harsh operating conditions. To solve this problem, enzyme immobilization techniques have been developed. Immobilization involves fixing the enzymes on solid supports, which increases their stability, enables their reusability and facilitates the easy separation of reaction mixtures. In addition, immobilized enzymes are ideal for continuous flow systems such as millireactors, where they allow better control of reaction conditions, improving efficiency and product consistency. Glucose dehydrogenase is an important enzyme in biotechnology, particularly in biosensors and the production of biofuels, as it catalyzes the oxidation of glucose to gluconolactone, reducing NAD+ to NADH. However, like many other enzymes, it tends to lose activity over time. The immobilization of glucose dehydrogenase in a millireactor provides a controlled environment that increases the stability and activity of the enzyme. The aim of this study was to investigate the effects of different immobilization strategies on the performance of glucose dehydrogenase in a 3D printed millireactor. The enzyme was immobilized in alginate gel in three immobilization strategies: as beads, on the bottom surface, and on both the top and bottom surfaces of the millireactor. The results showed that the application of the enzyme on both surfaces improved the glucose conversion two-fold compared to immobilization in beads and four-fold compared to immobilization only on the bottom surface. The dual-surface enzyme immobilization strategy showed the highest efficiency, achieving the highest conversion of 95.76 ± 1.01% (τ = 131 min) and NADH productivity of 0.166 ± 0.01 mmol/(L·min) (τ = 7.11 min) combined with operational stability over five days. Effective diffusion rates comparable to those of aqueous solutions confirmed the suitability of alginate gels for biocatalysis. These advancements highlight the potential of this modular and scalable platform for various biotechnological applications.
