One-step purification of a recombinant beta-galactosidase using magnetic cellulose as a support: Rapid immobilization and high thermal stability

For the first time, this work reported the one-step purification and targeted immobilization process of a 13-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after 13-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between 13-galactosidase and the substrate 1.2 x higher in the lactose hydrolysis of milk. 13-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 x . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of beta-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries.