Molecular basis of enzyme-catalyzed pehydrolysis of acetate esters for pretreatment of lignocellulosic biomass

用木质纤维素生物质如木材、柳枝稷或农业废料代替当前的生物燃料原料-玉米是生物能源研究的关键当前目标。从木质纤维素生物质中低效释放糖的顽拗性是制造这种替代品必须解决的关键问题。所提出的解决方案是一种非天然的酶催化反应，以制备强氧化剂过氧乙酸。本申请的目的是在分子水平上理解非天然酶催化的反应-酯的过水解形成过氧乙酸。中心假设是氢键供体或受体在活性位点内的精确定位选择性地使水失活，从而有利于过氧化氢。PI基于对具有不同能力的酶的X射线晶体结构的比较来阐述这一假设。对这一假设的检验将是关联过水解的结构和动力学，并设计新的酶变体，使高浓度的过乙酸用于木质纤维素生物质的预处理。智力优势：智力优势首先包括对如何设计酶催化的非自然反应的分子水平的理解。这将有助于研究前沿催化混杂酶催化几个不同的化学反应。了解如何促进一种或另一种反应，可以深入了解不同酶进化的自然过程，并加深我们对酶催化的微妙之处的理解。更广泛的影响：拟议的研究将在酶的催化活性如何演变为新的催化活性的分子基础上产生新的基础知识，并有助于理解蛋白质氧化的机制。该研究将有助于发现过乙酸去除木质素的有效途径，从而将木质纤维素生物质转化为燃料。拟议的研究将教育和培训本科生、研究生和博士后研究员，包括来自化学科学领域代表性不足群体的学生。在过去的三年里，PI参加了圣保罗公立学校的共同X变化计划，该计划在小学一级教授实验科学。在过去的四年里，这个项目的科学教师合作伙伴，Henriette Ngo-Bissoy女士，一位非洲裔美国妇女，和班上的许多学生都来自科学领域代表性不足的群体。共同PI，Tschirner，教一个为期两周的全天夏季研讨会题为“自然资源的主题：可再生能源和生物产品”为20名高中教师。课堂上约有一半是动手实验，探究式教学适合课堂教学。拟议的研究还将支持来自欧洲和亚洲的访问者到PI实验室的国际合作。

0932762KazlauskasSummary 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.