Magnetic field intensified bi-enzyme system with in situ cofactor regeneration supported by magnetic nanoparticles

Efficient dynamic interactions among cofactor, enzymes and substrate molecules are of primary importance for multi-step enzymatic reactions with in situ cofactor regeneration. Here we showed for the first time that the above dynamic interactions could be significantly intensified by exerting an external alternating magnetic field on magnetic nanoparticles-supported multi-enzymatic system so that the inter-particle collisions due to Brownian motion of nanoparticles could be improved. To that end, a multienzyme system including glutamate dehydrogenase (GluDH), glucose dehydrogenase (GDH) and cofactor NAD(H) were separately immobilized on silica coated Fe₃O₄ magnetic nanoparticles with an average diameter of 105nm, and the effect of magnetic field strength and frequency on the kinetics of the coupled bi-enzyme reaction was investigated. It was found that at low magnetic field frequency (25Hz and 100Hz), increasing magnetic field strength from 9.8 to 161.1Gs led to only very slight increase in reaction rate of the coupled bi-enzyme reaction expressed by glucose consumption rate. At higher magnetic field of 200Hz and 500Hz, reaction rate increased significantly with increase of magnetic field strength. When the magnetic field frequency was kept at 500Hz, the reaction rate increased from 3.89μM/min to 8.11μM/min by increasing magnetic field strength from 1.3 to 14.2Gs. The immobilized bi-enzyme system also showed good reusability and stability in the magnetic field (500Hz, 14.2Gs), that about 46% of original activity could be retained after 33 repeated uses, accounting for totally 34 days continuous operation. These results demonstrated the feasibility in intensifying molecular interactions among magnetic nanoparticle-supported multienzymes by using nano-magnetic stirrer for efficient multi-step transformations.