A recombinant esterase from Pseudomonas putida IFO 12996 covalently bound to magnetic nanoparticles via glutaraldehyde coupling reaction was studied. Magnetic nanoparticles Fe3O4 prepared by coprecipitation of Fe2+ and Fe3+ ions in ammonia solution and they were subsequently treated with 3-aminopropyltriethoxysilane (APES) to obtain amino-silane coated particles. The amino functional group on the particle surface and the amino group of the esterase was coupled with glutaraldehyde. X-ray diffraction (XRD) patterns indicated the particle before and after binding to esterase were pure Fe3O4. Transmission electron microscopy (TEM) showed that the particle with mean diameter of 11.9 nm and binding to esterase did not significantly change its size. Fourier transform infrared (FTIR) spectroscopy confirmed the esterase bound to the particles and the measurement of protein content revealed that the weight ratio of the esterase bound to the magnetic nanoparticles was 0.058 which was about four esterase molecules per particle. The kinetic analysis data indicated that the immobilized esterase retained 63% of its original activity and it exhibited similar thermal and pH stability as free esterase. The immobilized esterase hydrolyzed methyl DL-beta-acetylthioisobutyrate (DL-MATI) to give D-beta-acetylthioisobutyric acid (DAT) with enatiometric excess value of 97.2% and enantioselectivity value (E-value) of 245. It retained 84% of activity and 40% of conversion after being used for 10 cycles. (c) 2006 Elsevier Inc. All rights reserved.