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In this study, 3D printing technology was exploited for the development of immobilized enzyme microreactors that could be used for biocatalytic processes in Deep Eutectic Solvent (DES)-based media. 3D-printed polylactic acid (PLA) microwell plates or tubular microfluidic reactors were modified with polyethylenimine (PEI) and lipase from Candida antarctica (CALB) was covalently immobilized in the interior of each structure. DESs were found to have a negligible effect on the activity and stability of CALB, and the system proved highly stable and reusable in the presence of DESs for the hydrolysis of p-nitrophenyl butyrate (p-NPB). A kinetic study under flow conditions revealed an enhancement of substrate accessibility in the presence of Betaine: Glycerol (Bet:Gly) DES, while the system was not severely affected by diffusion limitations. Incubation of microreactors in 100% Bet:Gly preserved the enzyme activity by 53% for 30 days of storage at 60 °C, while the buffer-stored sample had already been deactivated. The microfluidic enzyme reactor was efficiently used for the trans-esterification of ethyl ferulate (EF) with glycerol towards the production of glyceryl ferulate (GF), known for its antioxidant potential. The biocatalytic process under continuous flow conditions exhibited 23 times higher productivity than the batch reaction system. This study featured an effective and robust biocatalytic system with immobilized lipase that can be used both in hydrolytic and synthetic applications, while further optimization is expected to upgrade the microreactor system performance.

3D printed PLA has already been demonstrated for several biotechnological applications, including enzymes immobilization. The prerequisites for an efficient screening assay include using small volumes of reagents, low cost, and rapid screening of large numbers of compounds and extracts. Hence, assays based on microtiter plates are predominant. Thus, designing and fabricating scaffolds on a similar scale, which could serve as immobilization carriers, and their recruitment in inhibitors screening studies is of great significance, adding both enzyme stability and reuse potentiality of the biocatalytic system in assay merits. In this work, pancreatic lipase was immobilized on 3D-printed PLA microwells for enzyme inhibitor screening. XPS analysis demonstrated the successful modification of the PLA scaffolds. The immobilized enzyme displayed high levels of operational, thermal, and storage stability under the tested conditions. The IC₅₀ values for PPL inhibition were calculated for Orlistat, a model lipase inhibitor, and olive leaf extract, a promising natural compound. This is the first study reporting the use of 3D-printed PLA wells with an immobilized enzyme for inhibitor screening assay.