FERULOYL ESTERASE IMMOBILIZATION IN MESOPOROUS SILICA: HYDROLYSIS AND TRANSESTERIFICATION REACTIONS.
Poster (konferens), 2015
Mesoporous silica materials (MPS) are an interesting choice as support to immobilize enzymes because MPS offer unique properties such as high enzyme loading and tunable pore size. They also provide the enzyme with a sheltered environment therefore reducing the risks of denaturation in industrial applications.
Immobilization parameters such as pH, buffer and pore size of the material were investigated. Among them, the chemical composition of the buffer as well as its pH proved to be critical resulting in enzyme loadings varying from nearly zero up to 0.025 mgenzyme.mgMPS-1.
Selectivity of the enzyme, a feruloyl esterase (FAE), was investigated by quantifying the molar ratio between the transesterification and hydrolysis products, namely butyl ferulate (BFA) and ferulic acid (FA). The reaction of interest was transesterification therefore hydrolysis was an unwanted side-reaction. The immobilization pH and the water content of the reaction were the most influent parameters inducing variation up to 4-fold of the BFA/FA molar ratio.
Optimal reaction conditions and kinetic parameters of the free and immobilized enzyme were determined for both hydrolysis and transesterification to determine in which conditions transesterification is prevailing. While optimal pHs were similar for all studied, temperature optimums varied from 25 to 50°C. Interestingly the Km of the FAE was not affected upon immobilization, but the kcat was decreased 10-fold resulting in a lower catalytic efficiency. Km was 100-fold higher for transesterification than for hydrolysis whereas kcat was 100-fold lower; this resulted in a drastic reduction of the catalytic efficiency of the FAE.
Stability of the enzyme was evaluated using the hydrolysis reaction. No significant improvement could be observed for the immobilized enzyme. Reusability of the immobilized biocatalyst was determined during 10-cycles of 48h. A decrease in activity was observed during the course of the experiment. In addition a decrease in the BFA/FA molar ratio indicating a shift in enzyme specificity happened.