This study implements a convenient microreactor for biocatalysis with enzymes immobilized on magnetic nanoparticles (MNPs). The enzyme immobilized onto MNPs by adsorption or by covalent bonds was lipase B from Candida antarctica (CaLB). The MNPs for adsorption were obtained by covering the magnetite core with a silica shell and later with hexadecyltrimethoxysilane, while for covalent immobilization, the silica-covered MNPs were functionalized by a layer forming from mixtures of hexadecyl- and 3-(2-aminoethylamino)propyldimethoxymethylsilanes in 16:1 molar ratio, which was further activated with neopentyl glycol diglycidyl ether (NGDE). The resulting CaLB-MNPs were tested in a convenient continuous flow system, created by 3D printing to hold six adjustable permanent magnets beneath a polytetrafluoroethylene tube (PTFE) to anchor the MNP biocatalyst inside the tube reactor. The anchored CaLB-MNPs formed reaction chambers in the tube for passing the fluid through and above the MNP biocatalysts, thus increasing the mixing during the fluid flow and resulting in enhanced activity of CaLB on MNPs. The enantiomer selective acylation of 4-(morpholin-4-yl)butan-2-ol (±)-1, being the chiral alcohol constituent of the mucolytic drug Fedrilate, was carried out by CaLB-MNPs in the U-shape reactor. The CaLB-MNPs in the U-shape reactor were compared in batch reactions to the lyophilized CaLB and to the CaLB-MNPs using the same reaction composition, and the same amounts of CaLB showed similar or higher activity in flow mode and superior activity as compared to the lyophilized powder form. The U-shape permanent magnet design represents a general and easy-to-access implementation of MNP-based flow microreactors, being useful for many biotransformations and reducing costly and time-consuming downstream processes.

The production of active pharmaceutical ingredients (APIs) and fine chemicals is accelerating due to the advent of novel microreactors and new materials for immobilizing customized biocatalysts that permit long-term use in continuous-flow reactors. This work studied the scalability of a tunable U-shape magnetic nanoparticles (MNPs)-based microreactor. The reactor consisted of a polytetrafluoroethylene tube (PTFE) of various inner diameters (ID = 0.75 mm, 1.50 mm, or 2.15 mm) and six movable permanent magnets positioned under the tube to create reaction chambers allowing the fluid reaction mixture to flow through and above the enzyme-loaded MNPs anchored by permanent magnets. The microreactors with various tube sizes and MNP capacities were tested with the preparative scale kinetic resolution of the drug-like alcohols 4-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol (±)-1a and 4-(3,4-dihydroquinolin-1(2H)-yl)butan-2-ol (±)-1b, utilizing Lipase B from Candida antarctica immobilized covalently onto MNPs, leading to highly enantioenriched products [(R)-2a,b and (S)-1a,b]. The results in the U-shape MNP flow reactor were compared with reactions in the batch mode with CaLB-MNPs using similar conditions. Of the three different systems, the one with ID = 1.50 mm showed the best balance between the maximum loading capacity of biocatalysts in the reactor and the most effective cross-section area. The results showed that this U-shaped tubular microreactor might be a simple and flexible instrument for many processes in biocatalysis, providing an easy-to-set-up alternative to existing techniques.

In lipase-catalyzed kinetic resolutions (KRs), the choice of immobilization support and acylating agents (AAs) is crucial. Lipase B from Candida antarctica immobilized onto magnetic nanoparticles (CaLB-MNPs) has been successfully used for diverse KRs of racemic compounds, but there is a lack of studies of the utilization of this potent biocatalyst in the KR of chiral amines, important pharmaceutical building blocks. Therefore, in this work, several racemic amines (heptane-2-amine, 1-methoxypropan-2-amine, 1-phenylethan-1-amine, and 4-phenylbutan-2-amine, (±)-1a–d, respectively) were studied in batch and continuous-flow mode utilizing different AAs, such as diisopropyl malonate 2A, isopropyl 2-cyanoacetate 2B, and isopropyl 2-ethoxyacetate 2C. The reactions performed with CaLB-MNPs were compared with Novozym 435 (N435) and the results in the literature. CaLB-MNPs were less active than N435, leading to lower conversion, but demonstrated a higher enantiomer selectivity, proving to be a good alternative to the commercial form. Compound 2C resulted in the best balance between conversion and enantiomer selectivity among the acylating agents. CaLB-MNPs proved to be efficient in the KR of chiral amines, having comparable or superior properties to other CaLB forms utilizing porous matrices for immobilization. An additional advantage of using CaLB-MNPs is that the purification and reuse processes are facilitated via magnetic retention/separation. In the continuous-flow mode, the usability and operational stability of CaLB-MNPs were reaffirmed, corroborating with previous studies, and the results overall improve our understanding of this potent biocatalyst and the convenient U-shape reactor used.

Two magnetically agitated continuous-flow tube reactors (AFRs)—applying external permanent magnets to move magnetic nanoparticles coated with a biocatalyst within a stream of the reaction medium—were developed and studied with aspartate ammonia-lyase (AAL) from Pseudomonas fluorescens immobilized onto epoxy-functionalized magnetic nanoparticles (MNPs) by covalent binding. The biotransformation of L-aspartate to fumarate by the AAL-MNPs (5 mg, D = 420 nm, 6 μg g⁻¹ AAL) took place in the reaction tube (PTFE, ID 2.15 mm) of both AFRs in a space containing the AAL-MNPs agitated within the flow of the reaction medium (0.5 mM L-aspartate solution, 4.7–14 μL min⁻¹, 25 °C) by two permanent ring magnets (N48 neodymium, 10 × 5 × 5 mm) positioned at a fixed distance in attraction mode. In the first version (AFR_XM), the two magnets positioned at opposite sides of the reaction tube (distance: 20 mm) performed axial movement (amplitude: 8 mm, frequency: 40–140 mpm) along the Y-axis, being perpendicular to the X-axis of the tube. In the second version (AFR_RM), the two magnets (distance: 10 mm from each other, 5 mm from the X-axis) performed rotation movement (frequency: 40–140 rpm) around the X-axis. Whereas in the AFR_XM the AAL-MNPs formed a cloud moving back and forth, they created a ring-shaped cloud rotating within the tube in the AFR_RM. The efficient internal mixing in the AFR_RM at the best frequency (80 rpm) resulted in the highest apparent specific activity (U_b = 354–469 U g⁻¹, at residence times of 2.5–7.5 min) of the AAL-MNPs in the reactors studied. In the other continuous-flow systems, significantly lower U_b values were achieved (135–290 U g⁻¹ at 120 mpm in AFR_XM; or 142–273 U g⁻¹ and 64–129 U g⁻¹ in tubular reactors anchoring MNPs in static mode with double or single magnets, respectively), whereas more than a magnitude of order lower values could be realised in the batch mode reactors (11.4–14.9 U g⁻¹ with rotational magnetic agitation at 120 rpm; 5.0–5.8 U g⁻¹ with axial magnetic agitation at 160 mpm; or 4.6–5.2 U g⁻¹ in an orbital shaker at 600 rpm) at comparable reaction times (2.5–7.5 min).