Immobilization of Enzymes in Mesoporous Silica for the Conversion of CO2 to Methanol

Abstract: Enzymes are powerful biomolecules able to catalyse chemical reactions under mild conditions and with high selectivity. However, their use in industrial processes is many times hindered due to their high costs and low stability. To overcome these problems the enzymes can be immobilized in an inert support material. The enzyme immobilization is also an interesting approach to increase enzyme activity, especially in the case of cascade reactions where more than one enzyme is involved. One extremely relevant cascade reaction is the conversion of CO2 to methanol that can be performed by three enzymes: formate dehydrogenase (FateDH) that converts CO2 to formate; formaldehyde dehydrogenase (FaldDH) for the reduction of formate to formaldehyde; and alcohol dehydrogenase (ADH) that reduces formaldehyde to methanol. This reaction can help to mitigate the environmental impact caused by the high emissions of CO2. However, the catalytic activity of the cascade reaction needs to be improved. In this work we investigate if an improvement can be achieved by the immobilization of the enzymes in a type of mesoporous silica particles (MPS), called siliceous mesostructured cellular foams (MCF), which physical and chemical properties can be specifically designed for the immobilization. MCFs with different pores sizes containing the functional groups octyl (OC), mercaptopropyl (MP), chloromethyl (CM) or aminopropyl (AP) were synthesized. They were initially used to study the immobilization and reaction of each enzyme separately. However, the reaction performed by FateDH is thermodynamically unfavourable, thus it was necessary to add FaldDH in the system to drive the reaction towards the reduction pathway. The co-immobilization of these two enzymes in MCF-MP resulted in a specific activity of about 4 times higher than for the free enzymes in solution. Förster resonance energy transfer measurements suggested that the enzymes were in closer proximity inside this material which potentially contributed to the higher activity. For FaldDH, specifically, MCF with large pores were required for the enzyme to remain active upon immobilization and its activity was enhanced using MCF-MP. The catalytic activity of the last enzyme, ADH, was higher upon immobilization in MCF-OC. ADH showed to be sensitive to pressure, and high concentrations of formaldehyde were required to achieve high enzymatic activity. Combining the knowledge from those studies we co-immobilized the three enzymes in MCF-MP and obtained methanol yields about 4.5 times higher than with the free enzymes in solution. One complementary study in this thesis was the comparison of two mesoporous silica materials, MCF and SBA-15, for the immobilization of ADH. From nitrogen sorption analysis we could observe that in MCF a larger fraction of the enzymes become immobilized inside the pores than on the outer surface in comparison to SBA-15, confirming that MCF is a good support material for this type of enzyme.  The findings of this work contribute to a better understanding of enzyme immobilization in MPS and the improvement of the rate of the reactions involved in the conversion of CO2 to methanol.