Biological Sciences Division
Nanobiotechnology Research Featured in Prominent Journals
Functionalized mesoporous silica wakes up inactive enzymes
Results: A Research Focus article commenting on recently published nanobiotechnology work at Pacific Northwest National Laboratory appears in the May 2007 issue of the journal Trends in Biotechnology. In their abstract, authors Keith Dunker and Ariel Fernandez state that:
Pacific Northwest National Laboratory scientists Chenghong Lei (left) and Eric Ackerman with a poster of their enzyme immobilization work using functionalized mesoporous silica.
"[PNNL scientist Eric] Ackerman and colleagues showed that an entrapping environment consisting of functionalized mesoporous silica actually enhances enzyme activity beyond the test-tube levels of free enzymes in solution. These findings provide an approach for dissecting the effect of various contributors to enzyme activity and thereby provide a means for fine-tuning the entrapping matrices to optimize enzyme performance in a rational way."
This article is in response to a recent spate of journal articles published by Ackerman's team on their characterization of functionalized mesoporous silica (FMS) for protein confinement and enzyme immobilization. Their article that was featured in the November 28 issue of Nanotechnology was highlighted on the journal's webpage. A PNNL news release about their work, "Night of the living enzyme," generated a flurry of media interest and appeared in thousands of publications.
In their recently published paper in Nano Letters, they report that an immobilized enzyme in FMS can still work better in the presence of high concentrations of urea (a strong denaturant). "Rather than losing all activity in the highly concentrated denaturant solution, the specific activity of the enzyme entrapped in FMS remained higher than the highest specific activity of free enzyme in solution," says lead author Chenghong Lei.
Why it matters: Mesoporous materials have a very high surface-to-volume ratio, large nanoporosity and ordered, uniform pore structure. Enzymes and proteins are the nanomachines of cells and are required to sustain life. These nanomachines synthesize and degrade cellular components and generate energy. The only ways to use these molecular machines are either inside cells or by separating them from cells. For many applications, it is not feasible to use entire cells, especially if the necessary molecular machines are scattered among many incompatible species.
The barrier to harnessing enzymes and molecular machines outside cells is that they are fragile and lose their activities once removed from cells. The results from the work by the PNNL team mean that it may be possible to mimic the crowded and stabilizing environment of cells by using FMS. This could in turn lead to environmentally friendly, efficient, chemical reactors based on cellular molecular machines.
The team found that a dramatic increase of enzyme loading occurred when using FMS compared to using unfunctionalized mesoporous silica and normal porous silica. Interestingly, the inactive or less active enzymes in solution become active, or more active once entrapped in FMS. These results are noteworthy because the specific activity of immobilized enzymes using conventional methods is usually far less than that of free enzymes in solution before immobilization.
In the work with urea, the team's results mean that immobilized enzymes in FMS can work well under harsh conditions that would kill cells or non-immobilized enzymes. "This can lead to combining solubilizing reagents with immobilized enzymes to chew up targets that are usually very resistant to degradation (e.g., lignocellulose waste site environmental contaminants) and convert them to energy or harmless byproducts. Under the correct reactions conditions, enzymes can drive reactions in both directions. Instead of an enzyme helping to convert substance A to B, it can catalyze the reverse reaction from B to A. Exploiting these reverse reactions would greatly extend the utility of enzymes, but the required reaction conditions are often harmful to living cells. Our immobilization approach means that biological molecular machines might be harnessed, and even optimized through high-throughput expression methods, to work under reaction-favorable, nonbiological conditions," says Ackerman.
Methods: The general approach the scientists took was to incubate the enzymes with an appropriate FMS, so it should be applicable to many enzymes, proteins, and protein complexes, because both pore sizes and functional groups of FMS are controllable. Unlike traditional approaches to immobilize enzymes, here it is unnecessary to expose the enzymes to activity-killing reagents or conditions during the immobilization procedure because all harsh chemical synthesis conditions involved in immobilization material production were completed before introduction of the enzyme.
Next steps: The team wants to understand how much farther the activity and stability of immobilized enzymes can be enhanced by rationally altering the enzymes and the nanopore sizes and functional groups. They also hope to expand the repertoire of enzymes and proteins under investigation to include those that can generate energy from sugars, provide extremely sensitive and stable sensors, and promote environmental remediation.
Research team: Eric Ackerman, Chenghong Lei, Yongsoon Shin, Jun Liu, Jon Magnuson, Glen Fryxell, Linda Lasure, Doug Elliott, Michelle Valenta and K Prasad Saripalli, all PNNL.
Funding: This work was supported by the U.S. Department of Energy's Office of Biological and Environmental Research and PNNL's Laboratory Directed Research and Development program.
Acknowledgments: This work was supported by the U.S. Department of Energy's Office of Biological and Environmental Research, Office of Basic Energy Sciences and PNNL's Laboratory Directed Research and Development program.
Dunker AK and A Fernandez. 2007. "Engineering productive enzyme confinement." Trends in Biotechnology 25(5):189-190. doi:10.1016/j.tibtech.2007.03.009.
Lei C, Y Shin, J Liu, and EJ Ackerman. 2007. "Synergetic effects of nanoporous support and urea on enzyme activity." Nano Letters 7(4):1050-1053.
Lei C, Y Shin, JK Magnuson, GE Fryxell, LL Lasure, DC Elliott, J Liu and EJ Ackerman. 2006. "Characterization of functionalized nanoporous supports for protein confinement." Nanotechnology 17(22):5531-5538.
Lei C, MM Valenta, K Prasad Saripalli, and EJ Ackerman. 2007. "Biosensing paraoxon in simulated environmental samples by immobilized organophosphorus hydrolase in functionalized mesoporous silica." Journal of Environmental Quality 36: 233-238. doi:10.2134/jeq2006.0216.
Lei C, TA Soares, Y Shin, J Liu and EJ Ackerman. 2008. "Enzyme specific activity in functionalized nanoporous supports." Nanotechnology 19:125102 (1-9). doi:10.1088/0957-4484/19/12/125102.