Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150-300nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81 % that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained > 95% of its initial activity when stored in buffer at 30 degrees C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters. (c) 2007 Elsevier B.V. All rights reserved.