Non-Covalent Immobilization of Papain Onto Modified Polysulfone Membrane Using Avidin-Biotin Coupling: EPR and Kinetic Studies
Membrane-based bioreactors or immobilized enzymes are one example of biofunctional membranes, defined as entities composed of biological molecules attached to a polymeric matrix cast in the form of porous polymeric membranes. A detailed understanding of the structure and function of enzymes upon immobilization is essential to develop a membrane bioreactor with optimal properties involving minimal conformational changes at the active site of the enzyme. In this study, a sulfhydryl protease, papain (EC 18.104.22.168), was noncovalently immobilized onto the modified polysulfone membrane through the avidinbiotin complex. Various kinetic parameters for the amidase activity of noncovalently 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 polysulfone 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 resulted in an enzyme bioreactor which was more stable and reusable than the free enzyme.
Electron paramagnetic resonance (EPR) spin labeling techniques were used to characterize the active site conformational changes of papain immobilized on the modified polysulfone membranes through the avidin-biotin complex. This paper reports the pH dependence, reusability and storage stability studies of biofunctional membranes using avidin-biotin complex as a non-covalent spacer. A good correlation was found between the active site conformational changes and the amidas activity of papain upon noncovalent immobilization onto the modified polysulfone membrane.
KeywordsElectron Paramagnetic Resonance Electron Paramagnetic Resonance Spectrum Immobilize Enzyme Spin Label Amidase Activity
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