Characteristics of magnetic nanoparticles-bound YADH in water/AOT/isooctane microemulsions

The yeast alcohol dehydrogenase (YADH) covalently bound onto Fe₃O₄ magnetic nanoparticles was performed in the NADH-containing water-in-oil microemulsions of water/AOT/isooctane. Both water and NADH were present in the aqueous phase of microemulsion solution and on particle surface. The ratio of the equilibrium water content on particle surface to that in the aqueous phase of microemulsion solution (i.e. aquaphilicty) was about 0.39 and independent of the molar ratio of water to AOT (ω₀). The thickness of aqueous film on particle surface was estimated to be 3.1-5.7nm, increasing with increasing the ω₀ value from 10 to 25. At a constant NADH amount, the concentration of NADH in the aqueous phase of microemulsion solution was not significantly affected by the ω₀ value. In addition, the bound YADH showed excellent storage stability and good thermal stability in the microemulsion system. At 25°C for 700h and 75°C for 30min, its residual activities were 78 and 56%, respectively. The specific activity of bound YADH in the microemulsion system was 40% of that in aqueous solution. The maximum specific activity of bound YADH (V_max), and the Michaelis constants for NADH (K_m^A) and 2-butanone (K_m^B) were determined to be 0.0306 μmol/minmg, 0.0589mM, and 0.1476M, respectively.