The enzyme chloroperoxidase (CPO) was immobilized in silica sol-gel beads prepared from tetramethoxysilane. The average pore diameter of the silica host structure (~3 nm) was smaller than the globular CPO diameter (~6 nm) and the enzyme remained entrapped after sol-gel maturation. The catalytic performance of the entrapped enzyme was assessed via the pyrogallol peroxidation reaction. Sol-gel beads loaded with 4 μg CPO per mL sol solution reached 9–12% relative activity compared to free CPO in solution. Enzyme kinetic analysis revealed a decrease inkcatbut no changes inKMorKI. Product release or enzyme damage might thus limit catalytic performance. Yet circular dichroism and visible absorption spectra of transparent CPO sol-gel sheets did not indicate enzyme damage. Activity decline due to methanol exposure was shown to be reversible in solution. To improve catalytic performance the sol-gel protocol was modified. The incorporation of 5, 20, or 40% methyltrimethoxysilane resulted in more brittle sol-gel beads but the catalytic performance increased to 14% relative to free CPO in solution. The use of more acidic casting buffers (pH 4.5 or 5.5 instead of 6.5) resulted in a more porous silica host reaching up to 18% relative activity.
Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase