Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Imaging, Molecular, and Cellular Analysis
100
Immobilized Enzymes in Nanoporous Materials Exhibit Enhanced Stability and Activity
Chenghong Lei1, Yongsoon Shin1, Jun Liu2, and Eric J. Ackerman1* (eric.ackerman@pnl.gov)
1Pacific Northwest National Laboratory, Richland, WA and 2Sandia National Laboratories, Albuquerque, NM
Enzymes (proteins) are the nano-machines of cells. In cells, molecular crowding provides enhanced protein stability and can induce order-of -magnitude enhancements in catalytic reaction rates compared to enzymes in solution. We recently demonstrated that enzymes can be artificially crowded through immobilization on surfaces to thereby increase their reaction rates and stability. Combining appropriately functionalized, nanoporous silica (FMS) with enzymes result in immobilizations at high enzyme concentrations that exhibit enhanced stability and activity compared to enzymes in the same solution. To date we have used either carboxylethyl- or aminopropyl- FMS to either entrap or covalently immobilize three different enzymes: glucose oxidase (GOD), glucose isomerase (GI), and organophosphorus hydrolase (OPH). The working buffer and its ionic strength affected the efficiency of protein entrapment. The data is consistent with electrostatic charges contributing an important parameter governing immobilization efficiency. Net negatively charged enzymes preferred entrapment in positively-charged FMS and vice versa. The optimal percent functionalization and pore sizes must be determined empirically. In general, pore sizes slightly larger than the enzymes appear optimal. Approaches that utilize spontaneously entrapping or covalently linking with heterobifunctional crosslinking agents produce immobilized enzymes that exhibit comparable Km and Vmax to the free enzyme in solution. The combination of FMS and proteins offers and excellent platform for biological reaction engineering. Our approach could be used to make more sensitive sensors, for decontamination, to develop advanced separations based on the high specificity of protein-mediated interactions, and to generate energy enzymatically provided that suitable enzymes and proteins could be identified and produced. A potential advantage of this approach is that non-living, yet efficient enzymatic chemical reactors could be deployed in environment-friendly and environment-compatible materials (e.g. silica) without the need to maintain complex biological communities or recombinantly-engineered microbes.
Figure 1: Depicts OPH immobilized in nanoporous material reacting with substrate molecules.
* Presenting author
