Replacing the current biofuels feedstock - corn - with lignocellulosic biomass such as wood, switchgrass or agricultural wastes is a key current goal of bioenergy research. Recalcitrance - inefficient release of sugars from lignocellulosic biomass - is the key problem that must be solved to make this replacement. The proposed solution is an unnatural enzyme-catalyzed reaction to make a strong oxidant peracetic acid. The objective for this application is a molecular-level understanding of an unnatural enzyme-catalyzed reaction - perhydrolysis of esters to form peracetic acid. The central hypothesis is that precise positioning of hydrogen bond donors or acceptors within the active site selectively deactivates water, thus favoring hydrogen peroxide. The PIs have formulated this hypothesis based on the comparison of x-ray crystal structures of enzymes with different abilities to make peracetic acid. The test for this hypothesis will be to correlate the structure and kinetics of perhydrolysis and to design new enzyme variants that make high concentrations of peracetic acid for pretreatment of lignocellulosic biomass. Intellectual Merit: The intellectual merit includes first the molecular-level understanding of how to design enzyme catalyzed non-natural reactions. It will contribute to the research frontier catalytic promiscuity where enzymes catalyze several distinct chemical reactions. Learning how to favor one or another reaction gives insight into the natural process of divergent enzyme evolution and will deepen our understanding of the subtleties of enzymatic catalysis.
Broader Impact: The proposed research will generate new fundamental knowledge on the molecular basis of how catalytic activity of enzymes can evolve to new catalytic activities and contribute to the understanding of the mechanisms of oxidation of proteins. The proposed research will contribute to the discovery of efficient routes to peracetic acid to remove lignin to convert lignocellulosic biomass to fuels. The proposed research will educate and train undergraduate students, graduate students and postdoctoral fellows, including students from underrepresented groups in chemical sciences. For the last three years, the PI participates in the Common X-Change program in Saint Paul public schools, which teaches experimental science at the elementary school level. The science teacher partner in this project over the last four years, Ms. Henriette Ngo-Bissoy, an African-American woman, and many of the students in the classes are from underrepresented groups in science. The co-PI, Tschirner, teaches a two week all day summer workshop titled "Topics in Natural Resources: Renewable energy and Bioproducts" for twenty high school teachers. About half of the class is hands-on experiments, inquiry based teaching that is suitable for classroom teaching. The proposed research will also support international cooperation of visitors from Europe and Asia to the PI's laboratories.
Replacing the current biofuels feedstock - corn - with lignocellulosic biomass such as wood, switchgrass or agricultural wastes is a key current goal of bioenergy research. Recalcitrance - the inefficient release of sugars from lignocellulosic biomass - is the key problem that must be solved to make this replacement. For centuries, applications of biocatalysis were scale-ups of naturally occurring processes involving nature's substrates. For example, conversion of sugars to ethanol and fungal manufacture of antibiotics are scaled up versions of natural processes. In the last few decades, biocatalysis has expanded to unnatural substrates. For example, enzymes are used to make some pharmaceuticals. The next frontier is unnatural reactions: reactions that nature does not catalyze, but that are needed to solve society’s problems in energy, chemicals, and materials. This research focused on such an unnatural reaction - the enzyme-catalyzed synthesis of peracetic acid. Peracetic acid is a strong oxidant that can be used to pretreat biomass to increase the release of sugars. Our research identified the differences between the natural reaction, which uses water, and the unnatural reaction, which uses hydrogen peroxide. By modifying the natural enzyme we increased the efficiency of the unnatural reaction. This research gave an unusually detailed understanding of how subtle differences in structure change the reactivity of an enzyme. We revised the previously proposed mechanism and found a new enzyme variant that uses a completely different mechanism. Although researchers have studied this class of enzymes for decades and felt they were well understood, this research showed that subtle differences in the reaction site can change how they work. It helps understand how new enzymes evolve in nature and how researchers can design enzymes for new industrial applications. We also optimized the enzyme-catalyzed production of peracetic acid and demonstrated that treating wood with enzyme-generated peracetic acid dramatically increased the amount of sugars released. This research trained students and international visitors in state-of-the-art research methods and critical thinking. The researchers also participated in outreach activities explaining biocatalysis and biofuels to middle school students and teachers.
