Immobilizing enzymes for useful service
(Quicktime animated illustration (3572 KB). Courtesy of artist Mike Perkins and PNNL's Molecular Science Computing Facility.) |
January 6, 2003— For the first time, high concentrations of an active enzyme have been successfully immobilized in a mesoporous, functionalized silica structure. Researchers at the U.S. Department of Energy's Pacific Northwest National Laboratory (PNNL) have developed a technology that enhances the activity and prolongs the stability of enzymes.
Enzymes are the ubiquitous magicians of the biological world, catalyzing one substance into a material that is substantially different—for example, enzymes power the reaction that changes milk into cheese. Agriculture, manufacturing, pharmaceuticals, energy generation—all aspects of industry and human endeavor rely in some way on enzyme reactions. However, enzymes are fragile and operate within very specific temperatures and environments that reflect their cellular origins. This fragility has, until now, limited researchers' ability to precisely control enzyme reactions or to reuse the enzymes.
The discovery and the potential for follow-on research has PNNL molecular biologist Eric Ackerman and his team excited because the basic proof of principal research indicates that some enzymes can function with twice the activity and greatly enhanced stability when immobilized in an appropriately functionalized pore within a grain of silica versus a free enzyme in solution.
Ackerman and PNNL scientists Chenghong Lei and Yongsoon Shin are already investigating two potential applications for the test enzyme through PNNL's Homeland Security Initiative—a biosensor system to detect dangerous chemicals such as nerve agents, and another technology designed to neutralize these dangerous chemicals.
OPH—The Test Case
"An enzyme is like a very efficient, environmentally friendly chemical factory that doesn't require extreme conditions to operate," says Ackerman. "Cells have thousands of enzymes carrying out the chemical reactions that sustain life, and many of these enzymes can be tapped for useful applications."
The possibilities for using immobilized enzymes to carry out desirable targeted chemical reactions are endless. New and highly diverse areas of research such as generating energy more efficiently in hydrogen fuel cells, purifying chemical and biological materials for prescription drug use, and detecting and neutralizing dangerous chemical and biological agents are just a few of the possible applications of targeted enzyme reaction.
For their enzyme immobilization research, Ackerman and his team first selected the enzyme organophosphorus hydrolase—or OPH—which can inactivate chemical nerve agents and some pesticides.
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"We selected OPH for this work because it has been widely investigated for biosensing and decontaminating poisonous agents," explains Ackerman.
However, since OPH is not commercially available, Ackerman's team produced the enzyme by recombinant DNA technology. They were also familiar with other crucial information about OPH such as its shape, electrical charge, and dimensions.
The "Pore" House
For several years, scientists have been working to immobilize enzymes for the purpose of carrying out targeted chemical reactions. However, the work has not been very successful due to enzyme fragility—that is, its inability to retain activity in an incompatible environment. Immobilizing an enzyme in an optimal framework with a compatible environment could prevent its deterioration.
About two years ago, Ackerman's team began searching for an environment in which to immobilize their recombinant OPH enzyme, taking into consideration the need for the material to be porous and have good flow properties to allow a substrate to enter and its product to exit. In addition, because enzymes can be either net positively or negatively charged, the environment within the pore would need to contain the opposite net charge to attract and hold the enzyme in place. The enzyme would also need enough room within the pore to function normally and retain optimum activity to perform its work as the substrate flows through the pore.
Ackerman's team found the needed characteristics in a technology developed earlier by former PNNL scientist Jun Liu (now at Sandia National Laboratory). The technology—Self-Assembled Monolayer on Mesoporous Silica—was originally designed to sequester mercury. They found that mesoporous silica, with its open pore structure, could be modified to make the pore size larger to accommodate a typically sized enzyme such as OPH, and then "functionalized"—coated with a charge compatible with the enzyme's specific attributes.
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"The thought was that enlarged pores with the opposite charge to the enzyme might be suitable for immobilizing enzymes at high concentrations," says Ackerman. "Knowledge of the OPH enzyme's size, shape, net electrical charge, and reaction mechanism helped us to quickly find a compatible pairing of enzyme and silica that worked."
The immobilized OPH enzymes were twice as active within their silica, open-pore houses as the free enzyme in solution, and also displayed greatly enhanced stability.
They maintained high activity even after 145 days," says Lei. "The amount of substrate converted to product was faster than in solution and more efficient."
Applied Enzymes
"Enzymes have so many applications; tiny quantities can be used for sensors, while larger quantities can be used for decontamination or other applications," says Ackerman.
One of the important features of the OPH reaction is its propensity to generate electroactive products. With today's dime-sized electrochemical sensors, small amounts of this type of enzyme could be incorporated in these miniaturized technologies to detect poisonous airborne chemicals by recognizing the increased output of electrons.
Promising applications of enzymes in the national security arena, for example, could include infiltrating items such as air filters, masks, clothing, or bandages with the concentrated immobilized enzymes to neutralize dangerous chemical gases or vapors.
The DOE's Office of Science supported Ackerman and his team's discovery of immobilized enzymes and their continued research in using immobilized enzymes for biosensing and decontamination through PNNL's Homeland Security Initiative. — by Mary Ann Showalter
Media contact: Mary Ace, Communications Program Manager, 509-372-4277, Mary.Ace@pnl.gov
Technical Contact: Eric Ackerman, Staff Scientist, 509-373-3595, eric.ackerman@pnl.gov
Related Web Links
Entrapping Enzymes in a Functionalized Nanoporous Support," Chenghong Lei, Yongsoon Shin, Jun Liu, and Eric Ackerman, Journal of the American Chemical Society, 124(38): 11242 - 11243 August 2002,
The enzyme immobilization research was funded through the Laboratory-Directed Research & Development Program of the Fundamental Science Directorate at the Pacific Northwest National Laboratory. PNNL is one of 10 national laboratories supported by the U.S. Department of Energy's Office of Science.
Pacific Northwest National Laboratory is a DOE research facility and delivers breakthrough science and technology in the areas of environment, energy, health, fundamental sciences and national security. Battelle, based in Columbus, Ohio, has operated the Laboratory for DOE since 1965.
Author: Mary Ann Showalter is a science writer and electronic communications specialist at the Pacific Northwest National Laboratory. She is also the managing editor of PNNL's environment, safety, health, and quality newsletter, ESH&Q Exchange, and contributor to the award-winning former DOE Tanks Focus Area website. For more science news, see PNNL's News & Publications.