Scientists Gain New Molecular-Level Insights into Breaking Down Plant Material for Biofuels

A specialized microscope allows investigations of single molecules of cellulase enzymes.

The Science

Compared to biofuel crops like corn, breaking down cellulose from nonedible plant parts or grasses grown on marginal lands is slow and inefficient. Cellulose is the building block of plant cell walls. However, using these non-food plants could help avoid concerns about using food sources for fuel. Cellulase is an enzyme produced by microbes that breaks down plant material into glucose. Scientists and engineers can then ferment this glucose to generate bioethanol, which can be used as a substitute for fossil fuels. In recent work, researchers used a specialized optical microscope to visualize single cellulase enzymes interacting with different forms of cellulose. By investigating enzyme function in the presence of the product of the reaction and other components of plant biomass, the researchers gained new insights into how the enzyme binds and processes cellulose.

The Impact

The researchers found that the product of cellulose breakdown, cellobiose, can inhibit that breakdown process in two ways. It can bind to the “front door” of the enzyme where a cellulose strand threads into the enzyme. Alternately, it can bind to the “back door” of the enzyme where the product of the breakdown is discharged. This finding provides new avenues for developing modified enzymes that can tolerate working at high concentrations of cellobiose. Bioenergy researchers need enzymes with improved function to make biofuel more cost effective and reduce dependence on fossil fuels.

Summary

This study involved visualizing the model cellulase Cel7A using single-molecule fluorescence microscopy on a specialized “SCATTIRSTORM” microscope. By investigating the enzymes binding to and moving along the cellulose substrate one at a time, the researchers revealed enzyme behavior in much more detail than normal bulk studies. They found that the product of cellulose breakdown, cellobiose, slows the movement of Cel7A along cellulose, which was expected, but also that cellobiose blocks binding of Cel7A to cellulose, which was a surprise. This activity can be explained as a ‘front door’ inhibition, in which the cellobiose plugs the opening of a tunnel through the enzyme where the cellulose strand normally is threaded.

In related work, researchers modified the cellulose with components of normal plant cell walls, lignin and xylan, and the binding and movement of Cel7A on these modified cellulose substrates was characterized. Xylan reduced the proportion of the enzymes that can bind to and move cellulose, whereas lignin inhibited the enzyme’s ability to bind to cellulose as well as the velocity and distance of its movement.

Contact

Funding

This research, including the construction of the SCATTIRSTORM microscope, was funded by the Department of Energy (DOE) Office of Science, Biological and Environmental Research program’s Biological Systems Science Division and by the U.S. National Science Foundation. Additional support was provided by the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by DOE.

Publications

Zexer, N., et al., Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution. RSC Sustainability 2, 1118-1127 (2024). [DOI: 10.1039/D4SU00006D]

Nong, D., et al., Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose. Proceedings of the National Academy of Sciences 121, e2322567121 (2024). [DOI: 10.1073/pnas.2322567121]

Haviland, Z. K., et al., Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink. Biotechnology for Biofuels and Bioproducts 17, 7 (2024). [DOI: 10.1186/s13068-023-02456-3]

Related Links

Why is breaking down plant material for biofuels so slow?  Penn State news

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