A team of researchers from Washington State University (WSU) and the Department of Energy’s Pacific Northwest National Laboratory (PNNL) has developed an artificial peptoid-based enzyme that can digest lignin, the tough polymer that helps woody plants hold their shape. Lignin stores tremendous potential for renewable energy and materials, but has stubbornly resisted previous attempts to develop it into an economically useful energy source. Chemists have tried and failed for more than a century to make valuable products from lignin. But the newly reported development could help to change that track record of frustration.

“Our bio-mimicking enzyme showed promise in degrading real lignin, which is considered to be a breakthrough,” said Xiao Zhang, PhD, associate professor in WSU’s Gene and Linda Voiland School of Chemical Engineering and Bioengineering. “We think there is an opportunity to develop a new class of catalysts and to really address the limitations of biological and chemical catalysts.”

Zhang, who also holds a joint appointment at PNNL, is corresponding author of the team’s published paper in Nature Communications, which is titled, “Highly stable and tunable peptoid/hemin enzymatic mimetics with natural peroxidase-like activities,” in which they concluded, “As far as we know, this is the only example showing the use of highly stable and tunable biomimetic enzymes for direct lignin depolymerization.”

Lignin is found in all vascular plants, forming cell walls and providing plants with rigidity. Lignin allows trees to stand, gives vegetables their firmness, and makes up about 20-35% of the weight of wood. Lignin is the second most abundant renewable carbon source on Earth, but mostly goes to waste as a fuel source.

When wood is burned for cooking, lignin byproducts help to impart the familiar smoky flavor to foods, but burning releases all the carbon into the atmosphere, instead of capturing it for other uses. Because lignin turns yellow when exposed to air, the wood products industry removes it as part of the fine papermaking process. Once removed, it is often inefficiently burned to produce fuel and electricity.

In nature, fungi and bacteria produce enzymes that can break down lignin. This is how a mushroom-covered log decomposes in the forest. Enzymes offer a much more environmentally benign process than chemical degradation, which requires high heat and consumes more energy than it produces. However, natural enzymes degrade over time, which makes them hard to use in an industrial process. They’re expensive, too.

“It’s really hard to produce these enzymes from microorganisms in a meaningful quantity for practical use,” said Zhang. “Then once you isolate them, they’re very fragile and unstable.” The authors commented, “Although enzymatic methods have been used for depolymerizing lignin via white-rot fungi or bacteria, this process often takes a long time (weeks) or is of low yield (from 7% to ~30%) … Natural peroxidases are composed of proteins; thus, one cannot overlook the deficiencies of natural peroxidases such as the low stability under elevated temperatures, narrow optimal pH range, and susceptibility to denaturing.”

While researchers have been unable to harness natural enzymes to work for them, they have over the decades learned a lot about how these enzymes work. A recent review article by Zhang’s research team outlined the challenges and barriers to the application of lignin-degrading enzymes. For their new study, the team aimed to generate synthetic enzymes that mimic natural peroxidase enzymes that can break down lignin, but address the challenges. Zhang noted, “Understanding these barriers provides new insights toward designing biomimetic enzymes.”

Their development of what they describe as tunable and stable peroxidase mimetics represents “a promising opportunity to improve and expand enzymatic catalysis in lignin depolymerization,” the team stated. In fact, scientists have previously generated a range of peroxidase mimetics that have broadened the scope of applications, but as Zhang and colleagues further noted, “Despite these advances, constructing highly efficient and robust peroxidase mimetics with natural enzyme-like flexibility in tuning active sites and microenvironments remains a grand challenge.” A key goal is therefore to explore materials that will mimic and tune the microenvironment of peroxidases, but remain stable under various conditions.

Researchers Xiao Zhang, PhD, (L) and Chun-long Chen, PhD, (R) examine the products of lignin digestion by their novel biomimetic peptoid catalyst. [Photo by Andrea Starr | Pacific Northwest National Laboratory]

To generate their new peroxidase bio-mimetics the researchers replaced the peptides that surround the active site of natural enzymes with protein-like molecules called peptoids. These peptoids then self-assembled into nanoscale crystalline tubes and sheets. Peptoids were first developed in the 1990s to mimic the function of proteins. They have several unique features, such as high stability, that allow scientists to address the deficiencies of natural enzymes.

In this case, they offer a high density of active sites, which is impossible to obtain with a natural enzyme. “By varying the structural features of peptoids including terminal ligands, side chain chemistries, and self-assembly behavior, we successfully develop a class of peptoid-based crystalline nanomaterials as peroxidase mimetics with tunable active sites and microenvironments,” the authors explained.

As expected, the resulting artificial enzymes were found to be much more stable and robust than the natural versions, so that they can work at temperatures up to 60°C, a temperature that would destroy a natural enzyme. “ … these enzymes offer a great opportunity to inspire models that copy their basic design,” Zhang pointed out.

“This is the first nature-mimetic enzyme which we know can efficiently digest lignin to produce compounds that can be used as biofuels and for chemical production,” added co-corresponding author Chun-Long Chen, PhD, a PNNL researcher, and affiliate professor in chemical engineering and chemistry at the University of Washington. “This work really opens up new opportunities … We can precisely organize these active sites and tune their local environments for catalytic activity, and we have a much higher density of active sites, instead of one active site … This is a significant step forward in being able to convert lignin into valuable products using an environmentally benign approach.”

The researchers further stated, “Our results showed that these peptoid-based enzyme mimetics can catalyze the oxidative depolymerization of organosolv lignin under much milder conditions with a shorter incubation time, in contrast, to peroxidase-based enzymic lignin depolymerization.”

If the new bio-mimetic enzyme can be further improved to increase conversion yield to generate more selective products, it has the potential for scale up to industrial scale. The technology offers new routes to renewable materials for aviation biofuel and biobased materials, among other applications.

As the researchers concluded in their paper, “Because peptoid-based crystalline nanomaterials are highly tailorable and stable, we expect the self-assembly of peptoids into hierarchically structured crystalline nanomaterials with the ordered alignment and organization catalytic sites will provide a fascinating strategy for the design and synthesis of robust enzyme mimetics for various applications including lignin depolymerization.”


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