(575a) Immobilization Of Enzymes In Silica Nanomaterials By Incorporating An Autosilicification Domain

Enzymes are often immobilized in a matrix in order to improve stability and ensure re-usability. Encapsulation in inorganic silica matrices is an attractive option. These matrices are usually formed by sol-gel processing or by using polypeptides to recruit silica to the enzymes. These methods rely on random entrapment events during polymerization and may not ensure efficient or complete encapsulation. To overcome these limitations, we have developed a method of enzyme immobilization by incorporating autosilicification activity into the enzyme without affecting its original functionality. A large number of silica matrices produced in nature come from unicellular photosynthetic algae called diatoms. The silica cell walls of diatoms display fractal pores ranging in size from 10 to 1000 nm in diameter. In the diatom Cylindrotheca fusiformis, modified peptides called silaffins are responsible for silica deposition, which occurs at ambient conditions. Silaffins are heavily post-translationally modified. Polyamine chains are attached to the lysine residues of silaffins and the serine residues are phosphorylated. It was discovered that the unmodified silaffin peptide is also capable of precipitating silica in vitro and gets entrapped in the spheres during this process. If an enzyme or protein was tethered to the silaffin, it would lead to the encapsulation of these moieties in the spheres too. As proof of concept, fluorescent proteins and enzymes were produced as translational fusions with the silaffin (autosilicification domain) by linking the two proteins in frame with each other in an expression vector. Escherichia coli was used as the host for expressing the recombinant fusion proteins. These proteins had the capacity of precipitating silica on the order of minutes in the appropriate buffer at ambient conditions. Enzyme activity was retained in the silica spheres formed. These enzymatically-active nanobiomaterials displayed improved stability. The nanoscale feature dimensions and the morphology of the matrices can be tuned by varying the autosilicification domain.