Enzyme Immobilization in Porous Silicon: Quantitative Analysis of the Kinetic Parameters for Glutathione-S-transferases

摘要

Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (～6.5 μm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro-2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is ～4× higher than the immobilized enzyme, for which we measured an apparent K′_(m)~(GSH) value of 1.0±0.3. The maximum velocity, V′_(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is ～20 times lower than that in solution. Results suggest ～25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented.