Cofactor-dependent multi-step enzymatic reactions generally require dynamic interactions among cofactor, enzyme and substrate molecules. Maintaining such molecular interactions can be quite challenging especially when the catalysts are tethered to solid state supports for heterogeneous catalysis for either biosynthesis or biosensing. The current work examines the effects of the pattern of immobilization, which presumably impacts molecular interactions on the surface of solid supports, on the reaction kinetics of a multienzymic system including glutamate dehydrogenase, glucose dehydrogenase and cofactor NAD(H). Interestingly, particle collision due to Brownian motion of nanoparticles successfully enabled the coupled reactions involving a regeneration cycle of NAD(H) even when the enzymes and cofactor were immobilized separately onto superparamagnetic nanoparticles (124 nm). The impact of particle motion and collision was evident in that the overall reaction rate was increased by over 100% by applying a moderate alternating magnetic field (500 Hz, 17 Gs), or using additional spacers, both of which could improve the mobility of the immobilized catalysts. We further observed that integrated immobilization, which allowed the cofactor to be placed in the molecular vicinity of enzymes on the same nanoparticles, could enhance the reaction rate by 1.8 fold. These results demonstrated the feasibility in manipulating molecular interactions among immobilized catalyst components by using nanoscale fabrication for efficient multienzymic biosynthesis. (C) 2011 Elsevier B. V. All rights reserved.