Magnetically agitated continuous-flow tube reactors with aspartate ammonia-lyase immobilized on magnetic nanoparticles

Abstract

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).