Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media

Myrto G Bellou; Elena Gkantzou; Anastasia Skonta; Dimitrios Moschovas; Konstantinos Spyrou; Apostolos Avgeropoulos; Dimitrios Gournis; Haralambos Stamatis

doi:10.3390/mi13111954

Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media

Micromachines (Basel). 2022 Nov 11;13(11):1954. doi: 10.3390/mi13111954.

Authors

Myrto G Bellou¹, Elena Gkantzou¹, Anastasia Skonta¹, Dimitrios Moschovas², Konstantinos Spyrou², Apostolos Avgeropoulos², Dimitrios Gournis², Haralambos Stamatis¹

Affiliations

¹ Laboratory of Biotechnology, Department of Biological Applications and Technologies, University of Ioannina, 45110 Ioannina, Greece.
² Department of Materials Science and Engineering, University of Ioannina, 45110 Ioannina, Greece.

Abstract

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.

Keywords: 3D printing; deep eutectic solvents; enzyme microreactor; immobilization; lipase.

Grants and funding

Part of this research has been co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH CREATE-INNOVATE (project code: Τ2ЕDΚ-01410). Financial support from the Empirikion Foundation (Greece) is gratefully acknowledged.