3D-Printed Platforms for Enzyme Immobilisation Screening and Direct Translation into Continuous-Flow Biocatalysis

The development of immobilised biocatalysts for continuous-flow processes is still largely based on laborious trial-and-error screening, and conditions optimised on conventional carriers are often not directly transferable to structured reactors. Here, we introduce a 3D-printed methodology that integrates high-throughput screening of enzyme immobilisation with implementation in a continuous-flow reactor using the same photopolymeric formulation. The surface of the printed objects was modified through imidazolium-based supported ionic liquid phases. Immobilisation conditions for an enzymatic solution with alcohol dehydrogenase (ADH-200) were rapidly evaluated in a 96-well format using a colorimetric assay that enables quantitative comparison of activity and loading across multiples of conditions in a parallel fashion. Methyl-imidazolium-modified supports showed markedly higher enzymatic activity and immobilisation efficiency than other imidazolium-based ionic liquids, and the best-performing formulation was directly applied to functionalise the 3D-printed honeycomb-structured reactor. The resulting heterogeneous biocatalyst catalysed the oxidation of 1-phenylethanol to acetophenone under continuous flow with high conversion at moderate residence times and sustained performance over several hundred hours on stream. This integrated 3D-printed platform facilitates the identification of effective immobilisation chemistries and their translation to robust flow reactors, providing a general strategy for the rapid development of immobilised biocatalysts.