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

Abstract

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