Nylon membrane was used to immobilize Mucor miehei lipase. Variables that affect this immobilization procedure were studied by experimental design. A 23 full factorial design was employed for this purpose. The protein retention and hydrolytic activity of the immobilized lipase were used as response variables. The rapid loss of enzyme activity was the main problem during repetitive use. Two strategies were used to improve the low operational stability: nylon treated with HCl and nylon coated with polyvinyl alcohol (PVA). Lipase-nylon-PVA was the best enzyme derivative, allowing performance of five consecutive assays, with a retained activity of 0.5 U mg of protein−1 g of support−1.