Production of recombinant β-glucosidase and simultaneous immobilization and purification using immobilized metal ion affinity membrane

Various cellulolytic enzymes have been studied to date for their application in biofuels, food, cosmetics, beverages and pharmaceutical industries. However, due to the difficulty in recovery, the direct use of free enzymes is not economically viable in large-scale industrial applications. To preserve enzyme stability, recovery and reusability, immobilization is an effective approach. In this study, gene transformation was carried out using Escherichia coli DH5 host cells, and E. coli BL21(DE3), BW25113(DE3), ER2566(DE3), and JM109(DE3) were used to express recombinant β-glucosidase. The immobilized metal ion affinity membrane (IMAM) chromatography approach using modified regenerated cellulose membrane was applied to immobilize and purify recombinant β-glucosidase. Both free and immobilized β-glucosidases were analyzed for their pH/temperature stability, purification and kinetics. The reusability of immobilized β-glucosidase and regeneration of IMAM was also studied. The results showed that the immobilized β-glucosidase had higher activity and more stability at a wide range of pH and temperature. Immobilized β-glucosidase retained more than 80 % of relative activity at 4 ℃ after storage for 20 days. Moreover, it maintained 60 % of relative activity after 5 cycles of repeated use. IMAM membrane was successfully regenerated 6 times with 100 % β-glucosidase adsorption and activity.