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

Abstract

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.

Polydopamine‐assisted immobilization of trypsin onto monolithic structures for protein digestion

“…Recently there has been an increased use of monolithic supports for the immobilization enzymes in the development of protein digestion bioreactors []. Monoliths are a relatively new type of chromatographic media that have rapidly generated great interest in recent years, due their improved mass transfer kinetics and separation performance over a wide range of flow rates, as compared to conventional particle‐based chromatographic media []. Their ease of fabrication and surface modification, combined with ability to be coupled to separation and detection systems, make monoliths highly attractive tools for fully automated and high‐throughput chemical and biological analytical systems [].…”

Flow‐through immobilized enzyme reactors based on monoliths: I. Preparation of heterogeneous biocatalysts

“…Besides silica‐based materials, some other inorganic monoliths have also been developed for immobilization of enzyme. The macroporous titania monolith preparation from biocompatible precursors at aqueous conditions was reported by Yi et al . The procedure involved the formation of glycerol‐titania composite sol followed by titania condensations.…”

Monolith‐based immobilized enzyme reactors: Recent developments and applications for proteome analysis

“…Yi et al [77] prepared a kind of titania monolith from biocompatible precursors using aqueous conditions involving the formation of glycerol -titania composite sol followed by condensation. By the addition of poly(ethylene oxide) (PEO), macroporous titania monolith was obtained.…”