Nickel-Functionalized Chitosan for the Oriented Immobilization of Histidine-Tagged Enzymes: A Promising Support for Food Bioprocess Applications

Recombinant protein biosynthesis and oriented immobilization of enzymes can expand the possibilities of applying biocatalysts to industrial processes. Using β-galactosidase as a model, this study aimed to develop a support based on chitosan functionalized with glutaraldehyde, Nα,Nα-Bis(carboxymethyl)- l -lysine hydrate , and nickel for the oriented immobilization of histidine-tagged (His-tagged) recombinant enzymes. The His-tagged enzyme from Kluyveromyces sp. was produced in Escherichia coli BL21(DE3). With an enzyme loading of 2.0 U enzyme /bead, in 16 h of immobilization, the yield and efficiency obtained were approximately 70%, and the recovered activity was above 50%. The oriented immobilization process increased enzyme affinity for substrate, and thermal stability also improved with His-tag immobilization, showing an increase in half-time ( t 1/2 ). In milk lactose hydrolysis, the operational stability of immobilized β-galactosidase was greater in the continuous process than in the batch process. When continuously processed for 24 h, approximately 90% of the lactose in 1.5 L of milk was converted into glucose and galactose. In the batch process, 52% of the lactose in 0.17 L of milk was hydrolyzed after 35 reuse cycles and about 16 h of reaction. The support obtained for immobilizing His-tagged recombinant enzymes may enable the development of solid, competitive industrial bioprocesses. Graphical Abstract