A biocatalyst with high activity retention of lipase was fabricated by the covalent immobilization of Candida rugosa lipase on a cellulose nanofiber membrane. This nanofiber membrane was composed of nonwoven fibers with 200 nm nominal fiber diameter. It was prepared by electrospinning of cellulose acetate (CA) and then modified with alkaline hydrolysis to convert the nanofiber surface into regenerated cellulose (RC). The nanofiber membrane was further oxidized by NaIO(4). Aldehyde groups were simultaneously generated on the nanofiber surface for coupling with lipase. Response surface methodology (RSM) was applied to model and optimize the modification conditions, namely NaIO(4) content (2-10 mg/mL), reaction time (2-10 h), reaction temperature (25-35 degrees C) and reaction pH (5.5-6.5). Well-correlating models were established for the residual activity of the immobilized enzyme (R(2) = 0.9228 and 0.8950). We found an enzymatic activity of 29.6 U/g of the biocatalyst was obtained with optimum operational conditions. The immobilized lipase exhibited significantly higher thermal stability and durability than equivalent free enzyme. (C) 2011 Elsevier B.V. All rights reserved.