Layered Co-Immobilization of β-Glucosidase and Cellulase on Polymer Film by Visible-Light-Induced Graft Polymerization.

Exploring a suitable immobilization strategy to improve catalytic efficiency and reusability of cellulase is of great importance to lowering the cost and promoting the industrialization of cellulose-derived bioethanol. In this work, a layered structure with a thin PEG hydrogel as the inner layer and sodium polyacrylate (PAANa) brush as the outer layer was fabricated on low density polyethylene (LDPE) film by visible-light-induced graft polymerization. Two enzymes, β-glucosidase (BG) and cellulase, were separately coimmobilized onto this hierarchical film. As supplementary to cellulase for improving catalytic efficiency, BG was in situ entrapped into the inner PEG hydrogel layer during the graft polymerization from the LDPE surface. After graft polymerization of sodium acrylate on the PEG hydrogel layer was reinitiated, cellulase was covalently attached on the outer PAANa brush layer. Owing to the mild reaction condition (visible-light irradiation and room temperature), the immobilized BG could retain a high activity after the graft polymerization. The immobilization did not alter the optimal pH and temperature of BG or the optimal temperature of cellulase. However, the optimal pH of cellulase shifts to 5.0 after immobilization. Compared with the original activity of single cellulase system and isolated BG/cellulase immobilization system, the dual-enzyme system exhibited 82% and 20% increase in catalytic activity, respectively. The dual-enzyme system could maintain 93% of carboxymethylcellulose sodium salt (CMC) activity after repeating 10 cycles of hydrolysis and 89% of filter paper activity after 6 cycles relative to original activity, exhibiting excellent reusability. This layer coimmobilization system of BG and cellulase on the polymer film displays tremendous potential for practical application in a biorefinery.