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

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.

Figure 1

3D-printed models made of natural polylactic acid (PLA). The well plate can be designed with a customized number of wells depending on the experimental needs, while the microreactor bares a serpentine-like internal shape in order to hold more volume in a smaller space. The models are ready-to-use after printing (no post-printing treatment needed).

Figure 2

C1s photoelectron spectra for the modified 3D-printed PLA structures (a) before enzyme immobilization, (b) after enzyme immobilization.

Figure 3

SEM micrographs of modified PLA structures. (a) modified PLA, (b) modified PLA incubated in 100% DES for 2 h, (c) modified PLA incubated in 100% DES for 24 h.

Figure 4

Relative activity of immobilized CALB on PLA well plates (a) in various 10% v/v DES:buffer solutions and (b) in increased concentrations of certain ones. 100% is defined as the activity of the immobilized enzyme in buffer (0% v/v DES). Reaction conditions: 0.25 mM pNPB in 50 mM phosphate buffer pH 7.5, 10, 20, or 50% v/v DESs, 40 °C, reaction time 5 min.

Figure 5

Stability of immobilized CALB on PLA wells after 1h of incubation at different concentrations (0–100% v/v) of particular DESs at 40 °C. One hundred percent is defined as the initial activity (before the incubation) in buffer (0% v/v DES). Reaction conditions: 0.25 mM pNPB in 50 mM phosphate buffer pH 7.5, 40 °C, reaction time 5 min.

Figure 6

Storage stability of immobilized CALB on PLA wells, at 4 °C. One hundred percent is defined as the initial activity (activity before the storage). Reaction conditions: 0.25 mM pNPB in 50 mM phosphate buffer pH 7.5, 40 °C, reaction time 5 min.

Figure 7

Effect of enzyme concentration on the microreactor’s biocatalytic performance and immobilization yield. Reaction conditions: 0.25 mM pNPB in 50 mM phosphate buffer pH 7.5, 40 °C, flow rate 300 μL/min (t_R = 30 s).

Figure 8

Operational stability of immobilized CALB in PLA microreactors in the absence and the presence of 10% v/v Bet:Gly for 100 cycles of use. Reaction conditions: 0.25 mM pNPB in 50 mM phosphate buffer pH 7.5, 0 or 10% v/v Bet:Gly, 40 °C, flow rate 300 μL/min (t_R = 30 s).

Figure 9

Thermal stability of immobilized CALB in PLA microreactor, at 60 °C, after incubation in 100% DES Bet:Gly and 100% buffer. Reaction conditions: 0.25 mM pNPB in 50 mM phosphate buffer pH 7.5, 40 °C, flow rate 300 μL/min (t_R = 30 s).

Figure 10

Effect of flow rate on the hydrolysis of p-NPB in CALB immobilized microreactors under continuous flow conditions (enzyme concentration 43 μg/mL, T = 40 °C, pH = 7.5). (a) Application of the Lilly–Hornby model on the data collected for different enzyme concentrations and flow rates in a buffer system (0% v/v DES), and (b) in 10% Bet:Gly system. (c) Linear fitting of the K_m(app) values against flow rate in a buffer system, and (d) in 10% Bet:Gly system.

Figure 11

Reaction scheme for the transesterification of ethyl ferulate with glycerol in 100% Bet:Gly DES acting both as solvent and substrate, towards the production of glyceryl ferulate. The reaction was performed in a microreactor system with immobilized CALB, at 60 °C for a total retention time of 4 h (24 runs × 10 min).