Enzyme immobilization using two processing methods onto silica core-shell particles

Abstract

Two methods of enzyme immobilization onto silica core-shell particles were developed. The first method involved the immobilization of Candida rugosa lipase inside a previously synthesized mesoporous silica layer (deposited at 80°C) surrounding a dense silica core. To prevent lipase leakage from the support, an outer mesoporous silica layer was deposited at 40°C around the first silica layer containing the immobilized lipase. The deposition of the second layer was performed at a relatively lower temperature, to prevent thermal inactivation of the immobilized enzyme. The internal silica layer was obtained by assembling primary silica nanoparticles generated from highly basic sodium silicate solution at 80°C on the surface of poly (diallyldimethylammonium chloride) (PDDA) functionalized silica core particles. The average shell thickness and pore size of the internal silica layer was ∼60nm and 24nm, respectively. The effect of process parameters on generation and aggregation of silica nanoparticles prepared from highly basic sodium silicate solution was also investigated. The aggregation of silica particles generated at 40°C and 80°C took place after 840s and 570 of reactions, respectively. The immobilization efficiency of lipase on the mesoporous silica monolayer was 80%. A decline of immobilized lipase activity was approximately 6 times after 10 reaction cycles due to lipase leakage from the monolayered shell. An outer mesoporous silica layer was deposited at 40°C onto the surface of previously PDDA-functionalized monolayered silica core-shell particles containing the immobilized lipase. The average thickness and pore size of outer mesoporous silica layer was ∼60nm and 17nm, respectively. The activity of lipase immobilized inside the bilayered shell was further reduced due to diffusion resistance within the outer silica layer and PDDA layer however, it was retained for the next reaction cycles.The pore size of mesoporous silica layer obtained at 80°C was insufficient to allow invertase immobilization. Thus, the second method for the immobilization of invertase was developed. It involved the preparation of the mesoporous silica layer simultaneously with invertase immobilization at 40°C. The immobilized invertase showed decreased activity, but it was not hampered by substrate inhibition, as in the case of the free enzyme, due to the location of the enzyme inside the mesoporous silica layer, where the mass transfer resistance for the substrate to the enzyme active site was present.