Immobilization of feruloyl esterases in mesoporous silica

Doctoral thesis, 2014

Feruloyl esterases are a class of enzymes that can be used as biocatalysts for the modification of bioactive hydroxycinnamic acids. In industry, enzyme immobilization is often necessary to stabilize the enzymes and to allow their reuse. Mesoporous materials have become a popular immobilization support due to advantages such as large surface area, high mechanical stability and the possibility of adjusting the pore size to the dimensions of the enzyme. The hypothesis investigated in this work was that mesoporous material would provide a robust immobilization support for feruloyl esterases, and would not suffer from the problem of enzyme inactivation reported in previous studies. As well as excellent reusability and high yields in the transesterification of methyl ferulate to butyl ferulate, the feruloyl esterases immobilized in mesoporous silica exhibited altered product selectivity. This indicated the need for deeper studies into how enzymes are affected by the immobilization process, and which factors and material properties influence enzyme activity. A novel method for real-time studies of the immobilization process was therefore developed, based on quartz crystal microbalance with dissipation monitoring. The immobilization process was found to occur in two stages, with initial covering of the outer surface followed by filling of the pores. Proof that the enzymes indeed enter the pores was also found, which is important as it is usually assumed that the enzymes are located inside the pores. Furthermore, modeling of the enzyme structure gave new insights on the structure-activity relationship as a function of pH, and was linked to an apparent structural memory effect from the adsorption to the mesoporous material. Differences in the microenvironment inside the pores were characterized by develop¬ing a new method for measuring the local pH through fluorescent labeling of the enzymes, which showed a buffering effect towards neutral pH inside the pores. The aim of the present work was to link the new knowledge obtained on the microenvironment, immobilization conditions and material properties of the mesoporous material to their effects on enzyme activity. The use of immobilization as a means of controlling enzyme activity is important for the development of more rationally designed biocatalysts based on enzymes immobilized in mesoporous materials.