In the present work, the immobilized footprint of β-glucosidase (BGL) on silica-coated iron oxide was explored to produce reusable catalysts with flexible active sites for high activity and heightened storage stability. 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 × 10-5 mol/g particle. The amine-modified particles were activated with glutaraldehyde, and subsequently, BGL was immobilized using either a 0.1 or 1 mg/mL enzyme solution to produce biomolecules with a large or small footprint on the particle surface. The initial activity, activity for subsequent hydrolysis cycles, activity after extended storage, and biomolecule footprint were studied as a function of APTES density and concentration of enzyme used for immobilization. At high immobilization amounts, the specific activity and footprint were reduced, but the immobilized biomolecules were stable during storage. However, at low enzyme immobilizations, the activity of the enzymes was retained, the immobilized enzymes adopted large footprints, and the storage stability increased with APTES density relative to the free enzyme. These results highlight how controlling both the protein load and functional group density can yield immobilized enzymes possessing high activity, which are stable during storage.
Keywords: enzyme activity; immobilized enzyme footprint; immobilized enzyme stability; lignocellulosic ethanol; magnetic particles.