Fe3O4@SiO2@NiAl-LDH microspheres implication in separation, kinetic and structural properties of phenylalanine dehydrogenase

Abstract

Fe3O4@SiO2@NiAl-LDH three-components microsphere contains a Fe3O4@SiO2 magnetic core and a layered double hydroxide with nickel cation provide the binding ability to (His)-tagged-protein and exhibits high performance in protein separation and purification. The morphology and chemistry of the synthesized Fe3O4@SiO2@NiAl-LDH microspheres were characterized by energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), vibrating sample magnetometer (VSM), Dynamic light scattering (DLS). Purified enzyme was assesed with SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis and intrinsic fluorescence spectroscopy. In this study, the separation of phenylalanine dehydrogenase (PheDH) by Fe3O4@SiO2@NiAl -LDH was performed and the effect of microsphere was investigated on the kinetic and structural properties of PheDH. After purification, kinetic parameters such as Km, Vmax, Kcat, kcat/Km, optimum temperature, thermal stability, and and activation energy were evaluated and compared according to the mentioned methods. The interaction between the enzyme and the microsphere displayed a high performance in protein binding capacity. The results also revealed that the kinetic parameters of the enzyme changed in a dose-dependent manner in the presence of a microsphere. Moreover, the results of intrinsic fluorescence and Circular Dichroism (CD) confirmed the structural changes of the protein in the interaction with the microsphere.

Keywords

• Phenylalanine dehydrogenase
• Fe3O4@SiO2@NiAl-LDH microsphere
• Kinetic parameters
• Protein purification
• Activation energy
• Thermal stability