Abstract
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.
Highlights
Silica nanostructures can be fabricated using a room temperature sol-gel process that is compatible with biomolecules [1, 2]
The enzyme CPO was successfully entrapped inside a silica nanostructure prepared from the precursor TMOS with or without addition of the hydrophobic modifier MTMS
A combination of factors, such as enzyme leakage from the sol-gel host, insufficient recovery from inactivation caused by initial methanol exposure, hindered product release, or alternate reaction pathways, are most likely responsible for the decline in catalytic performance of CPO after sol-gel entrapment
Summary
Silica nanostructures can be fabricated using a room temperature sol-gel process that is compatible with biomolecules [1, 2]. We demonstrate the use of sol-gel technology to make a biocatalyst based on entrapment of the enzyme chloroperoxidase (CPO) inside a silica nanostructure. CPO (EC 1.11.1.10) is one of the most versatile heme enzymes known to date. CPO catalyzed oxidative transformations are based on hydrogen peroxide or an alkyl peroxide as oxidant, whereas the equivalent traditional chemical reactions require stoichiometric amounts of heavy metal salts [7]. Another advantageous feature of CPO is its ability to perform these reactions in a highly enantioselective manner [8]. The use of CPO as biocatalyst, is hampered by its loss of activity in the presence of organic solvents, deactivation at high concentrations of the oxidant H2O2, and instability at elevated temperatures [8, 12]
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