3D-printed alginate-based biocatalysts enable efficient myrosinase immobilization for sulforaphane production

Sulforaphane (SFN) is a biologically active isothiocyanate recognized by the U.S. National Cancer Institute as one of the most promising anticancer compounds. This study presents the first direct comparison between a 3D-printed biocatalyst and a conventionally immobilized system for SFN production. Myrosinase (MYR) was evaluated in three configurations: free enzyme, immobilization in alginate beads, and immobilization via 3D printing using alginate as the matrix. The 3D-printed biocatalyst exhibited the highest catalytic turnover (Vmax = 39.3 μM min −1 g −1 ) but also the highest apparent Km (56.2 μM), suggesting diffusional constraints within the printed matrix (effective diffusion coefficient: 6.8 × 10 −6 cm 2 s −1 ) compared with alginate beads (8.0 × 10 −6 cm 2 s −1 ). In contrast, the conventionally immobilized enzyme showed a more balanced kinetic profile (Vmax = 31.4 μM min −1 g −1 ; Km = 24.0 μM), indicating improved substrate accessibility. Despite diffusional limitations, the 3D-printed system achieved a sevenfold and threefold increase in SFN yield compared with conventional broccoli extraction and free MYR, respectively. SFN formation was confirmed by time-of-flight mass spectrometry (TOF-MS), and antioxidant activity assays demonstrated 35% inhibition, comparable to the highest values reported for plant-derived SFN. Overall, this work provides the first comparative assessment of 3D-printed and conventionally immobilized biocatalysts for SFN bioproduction, highlighting the potential of additive manufacturing to enhance functional metabolite production. • 3D-printed alginate biocatalysts were developed for myrosinase immobilization. • The 3D-printed system achieved a sevenfold increase of sulforaphane yield. • 3D-printed matrices demonstrated structural stability and reusability.