Magnetically Agitated Nanoparticle-Based Batch Reactors for Biocatalysis with Immobilized Aspartate Ammonia-Lyase

Abstract

In this study, we investigated the influence of different modes of magnetic mixing on effective enzyme activity of aspartate ammonia-lyase from Pseudomonas fluorescens immobilized onto epoxy-functionalized magnetic nanoparticles by covalent binding (AAL-MNP). The effective specific enzyme activity of AAL-MNPs in traditional shake vial method was compared to the specific activity of the MNP-based biocatalyst in two devices designed for magnetic agitation. The first device agitated the AAL-MNPs by moving two permanent magnets at two opposite sides of a vial in x-axis direction (being perpendicular to the y-axis of the vial); the second device unsettled the MNP biocatalyst by rotating the two permanent magnets around the y-axis of the vial. In a traditional shake vial, the substrate and biocatalyst move in the same direction with the same pattern. In magnetic agitation modes, the MNPs responded differently to the external magnetic field of two permanent magnets. In the axial agitation mode, MNPs formed a moving cloud inside the vial, whereas in the rotating agitation mode, they formed a ring. Especially, the rotating agitation of the MNPs generated small fluid flow inside the vial enabling the mixing of the reaction mixture, leading to enhanced effective activity of AAL-MNPs compared to shake vial agitation.

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

“…In the agitated MNP batch reactor with axial movement (ABR XM : see Fig. S1B†), the permanent magnets were moving along the Y -axis being perpendicular to the X -axis of a 1.5 mL (OD = 11.5 mm) vial with 8 mm of amplitude at the previously determined best frequency (160 mpm), 29 and the AAL-MNPs formed a cloud moving from one side of the vial to the other. Like in our previous study at higher substrate concentrations, 29 the conversion at 0.5 mM l -Asp concentration in the ABR XM system (9.4% at 2.5 min up to 28.8% at 7.5 min: see the 3rd group in Fig.…”

“…The reactions in ABR XM and ABR RM systems, with axial and rotational agitations, respectively, were performed at the previously determined optimal agitation frequencies [160 mpm (movements per minute) for ABR XM and 120 rpm (rotations per minute) for ABR RM ). 29…”

“…29 The reactions in ABR XM and ABR RM systems, with axial and rotational agitations, respectively, were performed at the previously determined optimal agitation frequencies [160 mpm (movements per minute) for ABR XM and 120 rpm (rotations per minute) for ABR RM ). 29 The AAL-MNP biotransformations in batch mode were performed at 25 °C in clean screw-capped vials (1.5 mL, 8-425 Vial Small Opening V813/V817, Aijiren Technology Inc., Zhejiang, China) using AAL-MNPs (5 mg) as a biocatalyst in the reaction mixture (1 mL) containing L-aspartic acid (0.5 mM) in Tris buffer (50 mM, pH 8.8). Samples (10 μL) were taken from the intermittently stopped reaction mixtures at 2.5 min, 5 min, and 7.5 min.…”

“…In this study, inspired by the beneficial properties of the previously investigated agitated AAL-MNP systems in batch mode biotransformations, 29 our goal was to extend the permanent magnet-based external agitation to continuous- flow systems by designing and implementing MNP-based reactors for continuous-flow biotransformations. Since the previous study already indicated that a repulsion arrangement of two permanent magnets is not suitable to keep the AAL-MNPs in a well-dispersed distribution within the desired reaction area, 29 the agitated MNP-based flow reactors (AFRs) were designed and investigated with only two permanent magnets in attraction arrangement. The first device applied axial agitation of the MNPs within the continuous-flow tube reactor (AFR XM ) by moving the two permanent magnets positioned at attraction mode along the y-axis being perpendicular to the reactor tube as shown in Fig.…”

Immobilization of the Aspartate Ammonia‐Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics

“…The enzyme has been characterized in its native and immobilized form. PfAAL turned out to be a stable, highly efficient enzyme, being applicable in further biocatalytic studies such as characterization of the efficiency of novel magnetically agitated batch reactors [32] …”