(567bj) Stabilization of Proteins by Sol-Gel Encapsulation

Utilization of biological molecules such as proteins in engineering applications is one of the most innovative research fields. Porous silica material made by the sol-gel process can provide a promising host matrix for the encapsulation of proteins. The main objective of this study is to explore encapsulation techniques and parameters for the stabilization of proteins. In the present study, hen egg white lysozyme and cytochrome c were used as model proteins. We have developed a novel vapor exposure sol-gel process to encapsulate proteins using tetra methyl orthosilicate (TMOS) as a precursor. The encapsulation is carried out by two different methods: (I) in-situ protein exposure method, where a buffer containing a protein is directly exposed to TMOS vapor, and (II) buffer exposure method, where a buffer was first exposed to TMOS vapor and then a protein was added. Circular dichroism (CD) and high resolution derivative UV spectroscopic techniques were used to evaluate the structure and thermal stability of encapsulated proteins. The effect of protein concentration and sucrose (a model osmolyte) on the structure and stability of encapsulated proteins were characterized. We saw that cytochrome c retained native-like structures while lysozyme became partly unfolded when encapsulated in the silica matrix. We demonstrated that silica entrapment generally increases the thermal stability of proteins. Proteins encapsulated via the in situ technique were found to have higher thermal stability compared to those encapsulated using method II. In general, encapsulation method II gave rise to proteins with more secondary structure. However, proteins in this matrix were less resistant to thermal denaturation. In addition, there was concentration dependent decrease in protein secondary structure when encapsulated by method II. We believe that the trends are due to protein adsorption onto silica which causes denaturation. Osmolytes shift the protein native state ensemble towards more compact conformations, thereby increasing the conformational stability of proteins. We observed enhanced secondary structure of cytochrome c with the addition of 0.5 M sucrose and this enhanced structure and stability was preserved when the protein was encapsulated in silica gel in the presence of sucrose. Our future work includes characterization of the silica matrix and the elucidation of its interactions with proteins.