The Effect of Enzyme Immobilization on the Activity and Stability of Papain

Papain (EC2. 22. 4. 3) is a thiol protease with high level of activity that has widespread industrial applications. The use of immobilized papain provides many advantages over its free form. In many applications، cysteine must be added as an activator. On the other hand، certain bivalent metal ions including Ca2+ behave as the inhibitors of papaein. In the present study، after preparation of Sepharose 6B with CNBr، a 5 mg/ml-protein solution was added to activate the gel for covalent attachment of enzyme and، subsequently، 2M glycine solution was added to block the remaining active groups on the gel. The immobilization process brought about significant enhancement of storage، thermal stability، stability at extreme pHs، and resistance against the inhibitory effect of bivalent metal ions with respect to papain. The optimum temperature of papain was increased by 20 °C (from 60 to 80 °C) and its optimum pH was shifted from 7 to 8. 0 upon immobilization. Also km and kcat of the enzyme altered due to the immobilization process. These results are important in particular if one considers that the major problem in enzyme immobilization is the loss of enzyme activity and catalytic efficiency.