Preparation and characterization of YADH-bound magnetic nanoparticles

The covalently binding of yeast alcohol dehydrogenase (YADH) to magnetic nanoparticles via carbodiimide activation was studied. The magnetic nanoparticles Fe₃O₄ with a mean diameter of 10.6 nm were prepared by co-precipitating Fe²⁺ and Fe³⁺ ions in an ammonia solution and treating under hydrothermal conditions. Transmission electron microscopy (TEM) micrographs showed that the magnetic nanoparticles remained discrete and had no significant change in size after binding YADH. X-ray diffraction (XRD) patterns indicated both the magnetic nanoparticles before and after binding YADH were pure Fe₃O₄. Magnetic measurement revealed the resultant magnetic nanoparticles were superparamagnetic characteristics, and their saturation magnetization was reduced only slightly after enzyme binding. The analysis of Fourier transform infrared (FTIR) spectroscopy confirmed the binding of YADH to magnetic nanoparticles and suggested a possible binding mechanism. In addition, the measurement of protein content revealed that the maximum weight ratio of YADH bound to magnetic nanoparticles was 0.125, below which the binding efficiency of YADH was almost 100%. The kinetic measurements indicated the bound YADH retained 62% of its original activity and exhibited a 10-fold improved stability than did the free enzyme. The maximum specific activities and Michaelis constants were also determined.