Enzyme specific activity in functionalized nanoporous supports.

Here we reveal that enzyme specific activity can be increased substantially by changing the protein loading density (P(LD)) in functionalized nanoporous supports so that the enzyme immobilization efficiency (I(e), defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in solution) can be much higher than 100%. A net negatively charged glucose oxidase (GOX) and a net positively charged organophosphorus hydrolase (OPH) were entrapped spontaneously in NH(2)- and HOOC-functionalized mesoporous silica (300 Å, FMS) respectively. The specific activity of GOX entrapped in FMS increased with decreasing P(LD). With decreasing P(LD), I(e) of GOX in FMS increased from<35% to>150%. Unlike GOX, OPH in HOOC-FMS showed increased specific activity with increasing P(LD). With increasing P(LD), the corresponding I(e) of OPH in FMS increased from 100% to>200%. A protein structure-based analysis of the protein surface charges directing the electrostatic interaction-based orientation of the protein molecules in FMS demonstrates that substrate access to GOX molecules in FMS is limited at high P(LD), consequently lowering the GOX specific activity. In contrast, substrate access to OPH molecules in FMS remains open at high P(LD) and may promote a more favorable confinement environment that enhances the OPH activity.