Immobilization of TtAlaDH on magnetic nanoparticles: effects on stability and reuse

L-Alanine dehydrogenase (L-AlaDH) is a microbial enzyme that catalyzes the reversible oxidative deamination of L-Alanine to pyruvate. This enzyme catalyzes the reversible conversion of L-Alanine to pyruvate, while concurrently maintaining redox homeostasis through the NAD + /NADH coenzyme system. Despite the critical role of the L-AlaDH enzyme in biotechnological amino acid production, its implementation is hampered by poor operational stability, specifically difficulties in maintaining activity at high temperatures and ensuring adequate reusability. In this study, the recombinant plasmid encoding TtAlaDH from Thermus thermophilus was transformed into E. coli BL21 (DE3) for heterologous expression and was successfully immobilized on the surface of functionalized magnetic nanoparticles (F-NH₂ and FS-NH₂) (immobilized TtAlaDHs). The chemical characterization of magnetic nanoparticles and immobilized TtAlaDHs was validated using FTIR, SEM, EDS, TGA, and XRD investigations. This immobilization conferred significant advantages, including enhanced activity at elevated temperatures, improved long-term storage stability (at 4 °C) and reusability. Specifically, the TtAlaDH enzyme immobilized on the surface of the F-NH₂ magnetic nanoparticles demonstrated superior thermal stability, retaining approximately 85% of its initial activity at the elevated temperature of 85 °C. Furthermore, the F-NH₂-E magnetic nanoparticle system (F-NH₂-E) maintained 50% of the initial TtAlaDH activity following ten (10) repeated catalytic cycles. Crucially, immobilized TtAlaDHs demonstrated excellent storage stability, maintaining nearly 100% of its activity at 4 °C. Consequently, the demonstrated superior thermal and operational stability of immobilized TtAlaDHs, coupled with its magnetic separability, establishes this biocatalyst as highly promising for continuous flow biocatalysis. This enhancement provides a clear path toward the economic viability and scalability of L-Alanine production in industrial biotechnological applications. KEY POINTS: F-NH₂-E retains 85% activity at 85 °C 50% activity after 10 cycles with F-NH₂-E Near 100% storage stability at 4 °C.