1. Immobilized Enzymes in Functionalized Nanoporous Materials Exhibit Enhanced Activity and Stability Eric J. Ackerman, Chenghong Lei,Yongsoon Shin (PNNL) Jon Magnuson, Glenn Fryxell, Linda Lasure, Doug Elliot (PNNL) Jun Liu (PNNL, now at Sandia)

2. Stable enzymes entrapped in nanopores may one day be routinely used for chemical reactions. Enzymes in this environment are stable for extended periods of time. J. Am. Chem. Soc. 2002, 124, 11242−3 Hydrolysis of Organophosphorus by Immobilized OPH

3. Potential Applications Enzymes are nano-machines of cells, catalyzing thousands of useful chemical reactions. Microscopic reversibility means that outside cells, reactions A --> B and B --> A are feasible. Unlike typical chemical catalysts, enzymatic reactions occur at ambient conditions; i.e. green technology. Enzyme fragility has been a primary limiting factor in applications. Our breakthrough is applicable at multiple scales: sensors to industrial reactions Focus areas: homeland security energy

4. Why is this a breakthrough? Decades of work immobilizing enzymes has yielded small amounts of mostly inactive enzyme. Previous approaches generally destroyed the enzymes activity as a consequence of the immobilization procedure. This occurred either by killing the enzyme or burying it inside a material so that substrates and products could not enter and leave. Specific activity (enzyme activity per amount of enzyme) is the important parameter. We immobilize larger quantities of active enzyme per amount of material than other methods. Our immobilized enzyme exhibits enhanced stability and, for the first time, enhanced activity.

5. Denatured Enzyme, unfolded state in solution Renatured Enzyme, native state in a confined space Biochemistry 2001, 40: 11289-11293. Confinement can eliminate some expanded configurations of the unfolded chain, shifting the equilibrium from the unfolded state toward the native state. Maintaining and Promoting Enzyme Activity

6. 60 nm 300 Å Mesoporous Silica Confined space: Mesoporous silica

7. Schematic drawing of FMS. Feng, X.; Fryxell, G. E.; Wang, L. –Q.; Kim, A. Y.; Liu, J.; Kemner, K. M. Science 1997, 276, 923-926. Confined space for enzyme (protein): Functionalized Mesoporous silica (FMS)

8. OPH structure with charged surface residues: lysine (red), arginine (green), glutamic acid (yellow) displayed by “ball and stick”. The majority of the protein is displayed by “backbone”. OPH structural dimensions & amino acid residues 92 Å 56 Å 40 Å

9. Reaction of NH 2 -FMS with GDAH and subsequently with the enzyme. Covalently linking protein in FMS

10. Spontaneously entrapping protein in FMS

11. CompoundsApprox. LD 50 (mg/kg, iv.) Diazinon150-600 Parathion13 Paraoxon0.5 Sarin0.01 Soman0.01 Tabun0.01 VX0.001 Palytoxin0.00015 Botulinum toxin0.000001 Toxicities of Organophosphorus compounds: Organophosphorous Hydrolase (OPH) Structure of Organophosphorus Compounds:

12. Enhanced Specific Activity & Stability of Immobilized OPH Comparison of different porous silica support for OPH immobilization

13. PO O ONO 2 CH 3 CH 2 O CH 2 CH 3 Paraoxon POH O OCH 3 CH 2 O CH 2 CH 3 OPH H 2 O Biosensin, Filtation, Decontamination

14. Electrochemical Biosensing of Immobilized OPH in FMS to Organophosphorus Paraoxon addition Buffer flush At 0.90V.

15. What’s next? (1)We will integrate our extensive experiments with modeling/computation approaches To understand how enzyme stability and catalytic activity are enhanced; To better design nanomaterials; To screen the desired enzymes by genetic engineering; (2) We will try other enzymes of strategic significance, such as hydrogenase; (3) We will also try alternative nanomaterials, especially conductive one instead of silica; (4) Design and fabrication of biosensing devices and filtration/decontamination systems for Homeland Security, Army, and Environmental Protection.