Optimizing Enzyme Immobilization in Mesoporous Silica - A Spectroscopic Study of the Dynamics and Spatial Distribution of the Confined Enzymes

Abstract: Enzymes as biological catalysts are immobilized in porous materials to improve the enzyme activity and simplify their purification from the reaction media in biocatalytic applications. Mesoporous silica (MPS) particles are used as a promising support material for the immobilization of enzymes. The focus of this thesis is placed on the dynamics and distribution of enzymes in confining environments, to gain further understanding of the immobilization mechanism. The work is mainly based on various spectroscopy techniques using enzyme-attached fluorescent probes. To elucidate the mechanistic steps of the immobilization process, high time resolutions methods are essential. In this thesis, a fluorescence spectroscopy assay is developed to monitor the translational dynamics of the enzymes in real time while immobilization occurs. It is shown that the size of enzyme for a given pore size can strongly affect the kinetics of enzyme immobilization. The rotational dynamics of the confined enzymes is studied using fluorescence anisotropy and it is shown that the rotation of the enzymes is slower inside the pores compared to the enzymes in free solution. In order to investigate if protein-protein or protein-wall hydrodynamic interactions have an impact on retarding the dynamics of the confined enzymes, the microenvironment inside the MPS particles needs to be studied. In this thesis, the microviscosity inside the MPS particles is measured using enzyme-attached carbocyanine dyes. The results show that the effective microviscosity is about ten times higher inside the MPS particles than in bulk water. The increase is stronger with smaller pores and higher enzyme concentration, and it is concluded that protein-wall hydrodynamic interactions probably have a more significant effect in retarding the confined enzymes. The distribution of two co-immobilized enzymes in a cascade reaction which converts carbon dioxide (CO2) to formaldehyde (CH₂O) is studied. The effect of the distance between the enzymes on the catalytic efficiency is investigated using Förster resonance energy transfer spectroscopy. The results demonstrate that the two immobilized enzymes are in close enough proximity resulting in substrate channeling between the active sites and four times more efficient conversion of CO2 to CH₂O. Finally the location of the immobilized enzymes is visualized using transmission electron microscopy and immunogold staining. Two types of MPS particles are used with different pore morphology, spherical and hexagonal. The results show that not only the size of the pores in the MPS particles is an important factor, but the morphology of the pores also plays a crucial role in optimizing the enzyme immobilization.