Monolithic titania could offer significant potential as a support for bioaffinity chromatography because
of its stability, unlike silica, to a wide range of pH conditions and its ability to selectively bind
phosphorylated proteins and peptides. However, traditional routes to monolithic titania utilize harsh
conditions incompatible with most biomolecules. To address this, titania monoliths were prepared in a
biocompatible sol−gel process from Ti(OiPr)4 and glycerol. Varied porosities could be introduced by
the additional use of high-molecular-weight poly(ethylene oxide) in the sol, which led to the formation
of two phases prior to gelation. Morphologies, including bimodal meso- and macroporous structures,
and the polymerization of either the dispersed or condensed phases could be controlled by the fraction
and molecular weight of PEO in the sol. The roles of glycerol and PEO are to retard hydrolysis and
condensation reactions so that phase separation of titanium-rich species precedes gelation processes. PEO
also facilitates aggregation of growing TiO2 oligomers and particles.
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.
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