Development of Macroporous Titania Monoliths by a Biocompatible Method. Part 2: Enzyme Entrapment Studies

Although sol−gel-derived silica materials have been extensively used as a matrix to immobilize enzymes and other proteins, the poor pH stability and fragility of silica limits its utility in applications that require operation at pH >8. Herein, we report an alternative matrix, sol−gel-derived monolithic titania, for protein entrapment. The material is prepared from biocompatible precursors using aqueous processing conditions involving the formation of a glycerol−titania composite sol followed by titania condensation and can be made macroporous by the addition of poly(ethylene oxide). The clinically relevant protein γ-glutamyl transpeptidase (γ-GT) was entrapped in monolithic titania, and the effects of the titania sol−gel processing parameters on the retention (leaching), catalytic constant ( k cat ), Michaelis constant ( K M ), and long-term stability of entrapped γ-GT were investigated. It was found that the retention of γ-GT within the monolith was strongly related to the glycerol and PEO concentrations in the starting sol. Under optimal conditions, up to 70% of enzyme initially added to the titania sol was retained in the gel even after copious washing. Entrapped γ-GT demonstrated a higher K M and lower k cat value than in solution, indicating that substrate turnover was limited by partitioning effects and/or diffusion through the titania matrix. The entrapped enzyme demonstrated better long-term stability than in solution, likely because of protection from unfolding within a rigid titania pocket as well as the liberation of the biocompatible reagent glycerol during the sol−gel process. The entrapped enzyme did not show any loss of activity after storage at 4 °C for 3 weeks, but did show a loss in activity beyond this time. Potential applications of protein-doped titania are described.