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Biological Sciences Division
Research Highlights

February 2013

Protein Probes for Biomass

New protein probes find enzymes for biofuel production

Ethanol and cellulose structure
New protein probes are helping find the best microbial enzymes to break down cellulose, shown here as a line of clusters, as part of the process to convert biomass into biofuels, such as ethanol, the large molecule.

Results: New protein probes developed at Pacific Northwest National Laboratory are now helping scientists find the best biomass-to-biofuel production enzymes that nature has to offer. To help find these microbe-made enzymes, called glycoside hydrolases (GHs), a team of PNNL scientists built a suite of chemical probes purposefully designed to bind to known GH active sites.

The probes are special activity-based protein profiling (ABPP) probes that contain a handle to which a reporter can be attached. This enables further analysis, such as visualizing probe-bound proteins using fluorophores or isolating probe-bound proteins for mass spectrometry characterization using biotin. Most important, because GHs share close catalytic similarities, the probes bind not just to known GHs but to previously undiscovered GHs.

Why It Matters: Finding biofuel alternatives to liquid fossil fuels has kindled worldwide interest in converting cellulosic biomass to fuels and other small carbon compounds. Cellulosic biomass is the most abundant renewable source of carbon and energy on the planet: 180 million tons of feedstocks from agriculture and forest litter are estimated to be available per year.

However, turning biomass into biofuel hinges on the breakdown of the energy-rich primary component, cellulose. Cellulose is a polysaccharide, or "many sugars" bonded together. For biofuel production, the bonds between the sugars must be broken so that they can then be further processed; for example, fermented to make ethanol. But breaking these strong bonds is no small feat. The best-known candidates to do so are the GHs.

Methods: The PNNL team demonstrated the effectiveness of the probes by incubating them with the secretome of, or all of the proteins secreted by, Clostridium thermocellum, a biofuel-relevant bacterium that has remarkably effective cellulose degradation machinery. It can even convert cellulose into ethanol directly.

Mass spectrometry tools at EMSL were used to identify the secretome proteins that were bound by the probes and proved the probes to be GH selective and specific.

What's Next? This novel approach is a high-throughput way to find biofuel-relevant enzymes in complex mixtures and can be used to study any microbe.

"Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems," said PNNL biochemist Dr. Aaron Wright, senior author of the paper, which appears in the Journal of the American Chemical Society. "It facilitates greater understanding of an organism's role in biofuel development."

Future efforts are focused on further optimizing the selectivity of the probe suite and expanding its application to fungi.


Sponsors: This work was supported by the Laboratory Directed Research and Development Program at PNNL and used instrumentation and capabilities developed with support from the National Institutes of Health National Center for Research Resources and the National Institute of General Medical Sciences as well as the U.S. Department of Energy Biological and Environmental Research (DOE-BER) program. Mass spectrometry-based proteomic measurements were performed at EMSL, a DOE-BER national scientific user facility located at PNNL.

Research Area: Biological Systems Science

User Facility: Environmental Molecular Sciences Laboratory

Research Team: Lacie Chauvigné-Hines, Aaron Wright, Lindsey Anderson, Holly Weaver, Joseph Brown, Phillip Koech, Carrie Nicora, Beth Hofstad, Richard (Dick) Smith, Mike Wilkins, and Stephen Callister, PNNL.

Reference: Chauvigné-Hines LM, LN Anderson, HM Weaver, JN Brown, PK Koech, CD Nicora, BA Hofstad, RD Smith, MJ Wilkins, SJ Callister, and AT Wright. 2012. "Suite of Activity-Based Probes for Cellulose-Degrading Enzymes."  Journal of the American Chemical Society 134(50):20521-20532. DOI: 10.1021/ja309790w.

Related Highlight: Chemical Probe Finds Fungal Organism Function

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