The hydrolysis of cellobiose to glucose by β-glucosidase is the rate-limiting step in the enzymatic hydrolysis of cellulose to glucose, which is an industrially relevant step in the production of biofuels from lignocelluosic feedstock. In the present work, β-glucosidase was immobilized on silica-coated iron oxide particles with the goal of optimizing catalytic activity, and storage stability in the context of a reusable catalyst. The synthesized iron oxide particles were coated with silica and functionalized with various densities of (3-aminopropyl)triethoxysilane (APTES) to obtain particles with amine densities ranging from 0 to 3 × 10<sup>-5</sup> mol/g particle. β-glucosidase was immobilized to the particles using glutaraldehyde as a linker between amines on the particle and the enzyme. The initial activity, activity for subsequent hydrolysis cycles and activity under storage conditions were studied as a function of APTES density and concentration of enzyme used for immobilization. Two different relationships exist between retained specific activity post immobilization and APTES density, based upon the concentration of enzyme solution used for immobilization. When a dilute enzyme solution was used for immobilization, no difference in immobilized specific enzyme activity was observed for any APTES density and increasing the APTES lead to an increase in storage stability. When a concentrated enzyme solution was used for immobilization, the storage stability was not affected by the APTES density and the specific activity decreased with increasing APTES densities. The particles immobilized at a low enzyme concentration with high ligand density have high specific activity and retain this activity during storage. The results highlight the importance in studying both the ligand density on particles, and the density of immobilized enzyme for optimal enzymatic activity/stability.

β-glucosidase immobilized on iron oxide nanoparticle: Impacts of enzyme load and functional group density on specific activity and storage stability

ACS Spring 2022

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