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

Abstract

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications.

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

“…With two distinct types of agitated MNP-containing continuous-flow reactors in our hands, this work aimed to study the different modes of magnetic agitation of AAL-MNPs during the fluid flow and to compare these modes with batch reaction systems applying the same modes of agitation. Due to its robustness and easy in-line detectability, the enzymatic elimination of ammonia from l -aspartate catalysed by aspartate ammonia-lyase from Pseudomonas fluorescens R124 immobilized onto magnetic nanoparticles (AAL-MNPs) 32 was chosen as the model biotransformation for this work (Scheme 1). Magnetic saturation of MNPs obtained by co-precipitation method was within the range of 70–80 emu g −1 , while MNPs generated by thermal decomposition and hydrothermal methods could reach 80–90 emu g −1 at 100 nm.…”

“…Magnetic saturation of MNPs obtained by co-precipitation method was within the range of 70–80 emu g −1 , while MNPs generated by thermal decomposition and hydrothermal methods could reach 80–90 emu g −1 at 100 nm. 35 Since the magnetic cores of the AAL-MNPs were generated by hydrothermal method, 32 their magnetic saturation can be estimated as >50 emu g −1 .…”

“…The silica-covered MNPs (1.5 g) were then treated with 35% aqueous ammonia solution (120 μL) and ApTMOS (262 μL) in a solution of PEG 400 (675 mg) and ethanol (15 mL) at room temperature for 24 h. Next, to the suspension of amine-functionalized MNPs (150 mg) in 2-propanol (4 mL) were added GDE (44 μL) and glycidol (18 μL) and the mixture was shaken at 60 °C for 24 h. Finally, the GDE-functionalized MNPs (5 mg) were suspended in Tris buffer (500 μL, 50 mM, pH 8.8) and treated with AAL solution (500 μL, 100 μg mL −1 AAL) in an orbital shaker at 600 rpm and at 30 °C for 2 h to yield AAL-MNPs with ∼100% immobilization yield. 32…”

“…Preparation of epoxy-activated MNPs and immobilization of aspartate ammonia-lyase from Pseudomonas fluorescens R124 (AAL) onto epoxy-activated MNPs was performed exactly as described earlier. 29,32…”

“…The enzyme applied in this study was aspartate ammonia-lyase (AAL) from Pseudomonas fluorescens R124 catalysing the elimination of ammonia from l -aspartic acid in its MNP-bound form (Scheme 1). 32 Because the reaction catalysed by aspartate ammonia-lyase is reversible, AALs could be used to produce enantiopure l -aspartic acid (an important starting compound for the synthesis of food additives and artificial sweeteners) on a large scale. 33 AALs having a heteromeric structure have the potential to become promising biocatalysts with high catalytic efficiency.…”