Immobilized Enzymes in Functionalized Nanoporous Materials Exhibit Enhanced Activity and Stability

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. Hydrolysis of Organophosphorus by Immobilized OPH 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

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. Maintaining and Promoting Enzyme Activity Confinement can eliminate some expanded configurations of the unfolded chain, shifting the equilibrium from the unfolded state toward the native state. Denatured Enzyme, unfolded state in solution Renatured Enzyme, native state in a confined space Biochemistry2001, 40: 11289-11293.

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

7. Confined space for enzyme (protein): Functionalized Mesoporous silica (FMS) Schematic drawing of FMS. Feng, X.; Fryxell, G. E.; Wang, L. –Q.; Kim, A. Y.; Liu, J.; Kemner, K. M. Science1997, 276, 923-926.

8. 92 Å 56 Å 40 Å OPH structural dimensions & amino acid residues 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”.

9. Covalently linking protein in FMS Reaction of NH2-FMS with GDAH and subsequently with the enzyme.

10. Spontaneously entrapping protein in FMS

11. Organophosphorous Hydrolase (OPH) Structure of Organophosphorus Compounds: Toxicities of Organophosphorus compounds:

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

13. P ar aox on O O OPH C H C H O P O N O C H C H O P O H 3 2 2 3 2 H O O 2 O C H C H C H C H 2 3 2 3 Biosensin, Filtation, Decontamination

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

15. What’s next? • 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; • 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.