Characterization of Gentisate 1,2-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and Its Stabilization by Immobilization on Nickel-Functionalized Magnetic Nanoparticles

The aim of this study was the biochemical and kinetic characterization gentisate 1,2-dioxygenase (GDO) from Pseudarthrobacter phenanthrenivorans Sphe3 development a nanobiocatalyst by its immobilization on Ni2+-functionalized Fe3O4-polydopamine magnetic nanoparticles (Ni2+-PDA-MNPs). This is first GDO to be immobilized. gene encoding cloned with an N-terminal His-tag overexpressed in E. coli. showed high purification efficiency crude cell lysates maximum activity recovery 97%. immobilized enzyme characterized Fourier transform infrared spectroscopy (FTIR). reaction product identified 1H NMR. Both free exhibited Michaelis–Menten kinetics Km values 25.9 ± 4.4 82.5 14.2 μM Vmax 1.2 0.1 0.03 0.002 mM*s−1, respectively. thermal stability enhanced at 30 °C, 40 50 compared GDO. Stored −20 retained more than 60% initial after d, while completely lost 10 d. Furthermore, nanoparticle–enzyme conjugate 50% up fifth cycle.