Immobilization of a thermostable enzyme using a sol-gel preparation method

Abstract

A thermostable β-galactosidase was immobilized with tetramethoxysilane (TMOS) using a sol-gel preparation method. The thermal stability of free and immobilized enzymes changed with enzyme concentration, suggesting an effect of contaminants in the enzyme solution. The thermal stability of the immobilized enzyme was affected by preparation conditions and was much lower than that of free enzyme. The use of high concentrations of magnesium in the preparation enhanced the thermal stability of the immobilized enzyme, although high concentrations of magnesium in the incubation buffer reduced the thermal stability. A cell-free extract of E. coli B cells exhibiting no β-galactosidase activity was found to be most effective in improving the thermal stability of both the free and immobilized enzyme. The pH of incubation buffer during thermal treatment and preparation of immobilized enzyme significantly affected enzyme activity and thermal stability. The highest activity and stability were obtained at pH 6. Immobilization and thermal treatment had no effect on the Michaelis constants independent of the enzyme concentration and this suggests that immobilizing support and the cell-free extracts have insignificant effect on the kinetic properties of the immobilized enzyme.