Polyacrylonitrile (PAN) nanofibrous membranes were prepared by electrospinning and activated by amidination reaction for immobilizing Candida rugosa lipase with covalent binding. The diameters of initial PAN nanofibers ranged from 150 to 300 nm. The lipase, which aggregated and formed small particles that attached onto the nanofiber surfaces, was observed using field emission scanning electron microscopy. The activity retention of the immobilized lipase was 87.5% of the free enzyme. The kinetic parameters, K-m and V-max of the immobilized lipase were determined to be 0.548 mM and 31.2 U/mg, respectively. The effects of oil-to-water ratio, temperature, and pH on hydrolysis of soybean oil were investigated to determine the optimal reaction conditions. Under these optimal reaction conditions, the hydrolysis conversion of soybean oil was 72% after 10 min and 85% after 1.5 h. In examining reusability, the immobilized lipase retained 65% of its initial conversion after 20 additional batch reactions. These results suggest that the proposed scheme for immobilization of lipase has potential in industrial applications for oil hydrolysis. (c) 2009 Elsevier B.V. All rights reserved.