Water-insoluble nucleases were prepared by immobilizing the endonuclease from S. aureus onto the surface of nylon-66 and polystyrene spheres. The activation phase of the synthetic supports was optimized to define optimal conditions of pH, temperature, and Ca2+ concentration for using immobilized enzymes. The activity, evaluated by hydrolysis of high-molecular-weight and supercoiled DNA, indicates that both derivatives are highly stable for storage and further use. Immobilization of the enzyme is much more effective when the covalent binding is performed on polystyrene. By using different activation methods with these matrices, a set of immobilized nucleases with various levels of enzymatic activity can be prepared. The possibility of working in a wide range of enzymatic activity and at low temperature and Ca2+ concentrations in different buffers makes these immobilized nucleases very useful for investigating accessible DNA regions in chromatin structure.
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