Dynamics of Enzyme Immobilization in Mesoporous Silica Particles

Licentiate thesis, 2015

The focus of this thesis is placed on the dynamic behavior of proteins in confining environments which is studied as a scientific challenge for a deeper understanding of the immobilization mechanism and a better designing of enzyme immobilization in porous materials. Enzymes are immobilized in porous materials to improve the enzyme activity and simplify their purification from the product solution in biocatalytic applications. Mesoporous silica particle is used as solid support material for immobilization of enzymes. By using various spectroscopic techniques it is possible to probe the environment that enzymes experience inside the pores and/or outer surface of solid porous materials in terms of pH, polarity and characterize the behavior of enzymes after attaching or during the immobilization process. The two papers presented in this thesis are an effort to get closer to the mechanistic steps of immobilization process. In the first paper, a fluorescence spectroscopy assay based on dye-labelled proteins was proposed to monitor the whole immobilization process into mesoporous silica in real time. The main aim was to quantify the kinetics of the enzyme immobilization into mesoporous particles. And secondly it was investigated how the rate of the immobilization depends on protein size for a given pore size, the larger the protein the slower the rate. The second paper described how the rotational motion of immobilized proteins is retarded compared to free proteins in solution by using steady state fluorescence anisotropy which was based on the intrinsic fluorescence of aromatic amino acids in proteins. The effect of the particle diameter and pore size on the mobility of immobilized enzymes were investigated and by calculating pore filling for each protein three possible mechanisms for decrease in rotational mobility of immobilized proteins have been discussed.