Simple and Rapid Immobilization of Firefly Luciferase on Functionalized Magnetic Nanoparticles; a Try to Improve Kinetic Properties and Stability

Abstract

We expressed and purified a recombinant P. pyralis luciferase with N-terminal His-tags. The silanized Ni or Cu-loaded magnetic particles were prepared and used to assemble the His-tagged P. pyralis luciferase. This enzyme immobilized on functionalized magnetic nanoparticles (MNPs) via electrostatic interactions of His-tag with Ni²⁺/Cu²⁺ ions on the surface of MNPs using simple one step method. These particles were also used for purification of recombinant luciferase from crude extract of cell lysate. Effect of incubation time and amount of MNPs in bioluminescent activity were investigated to determine optimum condition for immobilization. Several properties of immobilized luciferase were studied and compared with free enzyme. Immobilization has shown different effects on Km for ATP and luciferin. In both immobilized form, Km(ATP) was increased while Km(luciferin) was shown decreases. Optimal temperature of both immobilized luciferase increased to 30 ºC while thermal stabilities have not shown significant differences compared to free enzyme. Both immobilized form inactivated after five consecutive reaction cycles.