Researchers from the University of California, Berkeley, and the University of Maryland School of Medicine have characterized a new cellulase (cellulose-digesting enzyme) that has optimal activity at 109 °C, a half-life of 5 h at 100 °C, and resists denaturation in strong detergents, high-salt concentrations, and ionic liquids. Cellulases active above 100 °C may assist in biofuel production from lignocellulosic feedstocks by hydrolyzing cellulose under conditions typically employed in biomass pretreatment, the authors noted in their paper in the journal Nature Communications.
Graham et al. identified the cellulase from a consortium of three hyperthermophilic archaea from a geothermal source in Nevada that were enriched by growth at 90 °C on crystalline cellulose, representing the first instance of Archaea able to deconstruct lignocellulose optimally above 90 °C. The cellulase is the most heat tolerant enzyme found in any cellulose-digesting microbe, including bacteria, the researchers said.
Many industrial processes employ natural enzymes, some of them isolated from organisms that live in extreme environments, such as hot springs. However, many of these enzymes are not optimized for industrial processes. For example, a fungal enzyme is currently used to break down cellulose into its constituent sugars so that the sugars can be fermented by yeast into alcohol. But the enzyme’s preferred temperature is about 50 °C, and it is not stable at the higher temperatures desirable to prevent other microbes from contaminating the reaction. Hence the need to look in extreme environments for better enzymes.
These are the most thermophilic Archaea discovered that will grow on cellulose and the most thermophilic cellulase in any organism. We were surprised to find this bug in our first sample.
Our hope is that this example and examples from other organisms found in extreme environments—such as high-temperature, highly alkaline or acidic, or high salt environments—can provide cellulases that will show improved function under conditions typically found in industrial applications, including the production of biofuels.
This discovery is interesting because it helps define the range of natural conditions under which cellulolytic organisms exist and how prevalent these bugs are in the natural world. It indicates that there are a lot of potentially useful cellulases in places we haven’t looked yet.—coauthor Douglas S. Clark, UC Berkeley professor of chemical and biomolecular engineering
Clark and coworkers at UC Berkeley are teaming with colleagues, led by Frank T. Robb, at the University of Maryland (U-Md) School of Medicine in Baltimore, to analyze microbes scooped from hot springs and other extreme environments around the United States in search of new enzymes that can be used in extreme industrial processes, including the production of cellulosic biofuels.
Their team is supported by a grant from the Energy Biosciences Institute (EBI), a public-private collaboration that includes UC Berkeley, in which bioscience and biological techniques are being applied to help solve the global energy challenge.
Robb and his colleagues collected sediment and water samples from the 95 °C Great Boiling Springs near the town of Gerlach in northern Nevada and grew microbes on pulverized Miscanthus gigas, a common biofuel feedstock, to isolate those that could grow with plant fiber as their only source of carbon.
After further growth on microcrystalline cellulose, the U-Md and UC Berkeley labs worked together to sequence the community of surviving microbes to obtain a metagenome, which indicated that three different species of Archaea were able to utilize cellulose as food. Using genetic techniques, they plucked out the specific genes involved in cellulose degradation, and linked the most active high-temperature cellulase, dubbed EBI-244, to the most abundant of the three Archaea.
Based on the structure of the enzyme, “this could represent a new type of cellulase or a very unusual member of a previously known family,” Clark said.
The enzyme is so stable that it works in hot solutions approaching conditions that could be used to pretreat feedstocks like Miscanthus to break down the lignocelluloses and liberate cellulose. This suggests that cellulases may someday be used in the same reaction vessel in which feedstocks are pretreated.
The newly discovered hyperthermophilic cellulase may actually work at too high a temperature for some processes, Clark said. By collecting more hyperthermophilic cellulases, protein engineers may be able to create a version of the enzyme optimized to work at a lower temperature, but with the robust structural stability of the wild microbe.
The EBI partnership, which is funded with $500 million for 10 years from the energy company BP, includes researchers from the UC Berkeley; the University of Illinois at Urbana-Champaign; and the Lawrence Berkeley National Laboratory.
Joel E. Graham, Melinda E. Clark, Dana C. Nadler, Sarah Huffer, Harshal A. Chokhawala, Sara E. Rowland, Harvey W. Blanch, Douglas S. Clark & Frank T. Robb (2011) Identification and characterization of a multidomain hyperthermophilic cellulase from an archaeal enrichment. Nature Communications 2, 375 doi: 10.1038/ncomms1373