Over the past few years the use of membrane-based bioreactors has become more widespread in various industrial processes. Membrane-based bioreactors or immobilized enzymes are one example of biofunctional membranes, defined as entities composed of biological molecules attached to micropores of polymeric membranes. Immobilized enzymes provide several advantages over their free soluble forms, including their relative ease of recoverability and reusability, their higher storage and thermal stability, and consequent lower costs. A detailed understanding of structure and function of enzymes upon immobilization is essential to develop a membrane bioreactor with optimal properties in order to get a best mix of enhanced stability/recoverability with minimal conformational changes at the active site of the enzyme. In this study, the high binding affinity between avidin and biotin has been utilized to create a non-covalent spacer for enzyme immobilization. NHS-LC-biotin, a derivative of biotin, was selected to enhance complex formation with the biotin-binding cleft of avidin. A sulfhydryl protease, papain (EC 3.4.22.2), was non-covalently immobilized onto the poly(ether)sulfone membrane via the avidin-biotin complex. Kinetic parameters for the amidase activity of non-covalently bound papain, using the substrate benzoyl arginine p-nitroanilide hydrochloride (BAPNA), were determined and the results were compared with those obtained from studies of papain in solution and papain directly immobilized onto the modified poly(ether)sulfone membrane. As expected, there was a decrease in the enzymatic activity upon direct immobilization. However, insertion of the avidin-biotin complex as a non-covalent spacer increased the apparent maximum enzymatic rate [V-max(app)], and decreased the apparent Michaelis-Menten constant [K-m(app)], relative to directly-immobilized papain. This non-covalently attached enzyme bioreactor also showed significant increase in stability and reusability as compared to the free enzyme. Electron paramagnetic resonance (EPR) spin labeling techniques were used for the first time to characterize the active site conformational changes of an enzyme immobilized on poly(ether)sulfone membranes through the avidin-biotin complex. Two EPR spectral subpopulations were seen corresponding to the active and denatured forms of papain. This paper reports the studies of pH dependence, reusability and storage stability of biofunctional membranes using this non-covalent spacer. There was a good correlation between the active site conformational changes and the amidase activity of papain upon non-covalent immobilization onto the poly(ether)sulfone membrane. All these findings indicate that the EPR spin labeling technique shows great promise as a powerful method for studying enzyme systems immobilized on polymeric membranes.