Enhanced activity and stability of papain immobilized on CNBr-activated sepharose

Immobilization of papain was carried out by covalent attachment on Sepharose 6B activated by using cyanogen bromide. Immobilization process brought about significant enhancement of storage and thermal stability, stability at extreme pHs, and resistance against the inhibitory effects of various bivalent metal ions with respect to papain. The optimum temperature of papain increased by 20 °C (from 60 to 80 °C) and its optimum pH was shifted from 6.5 to 8.0 upon immobilization. The activation energy of the enzymatic reaction for immobilized papain showed a significant increase as compared with its free form (1.87 kcal mol−1 K−1 for free and 4.69 kcal mol−1 K−1 for immobilized enzyme). The kinetic parameters, Km and kcat, were estimated to be 0.62 μM and 162 × 10−4 s−1 for free and 0.79 μM and 102 × 10−4 s−1 for immobilized papain, respectively.