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Functional and Biochemical Characterization of Polysaccharide Monooxygenases

多糖单加氧酶的功能和生化表征

基本信息

批准号：
1904540
负责人：
Michael Marletta
金额：
$ 50万
依托单位：
University of California-Berkeley
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2022-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1904540&HistoricalAwards=false
关键词：
Functional Biochemical Characterization Polysaccharide Monooxygenases

项目摘要

Cellulose is the major component of biomass in nature and a potential source of biofuels. Despite years of study, the industrial production of biofuels from cellulose is limited by the efficiency and ease of breaking down (degrading) cellulose. Polysaccharide monooxygenases (PMOs) are enzymes important in cellulose degradation and biofuel development that have been recently discovered. PMOs may help to overcome the high costs associated with cellulose degradation. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Michael Marletta from the University of California, Berkeley to continue his studies of PMOs. The research is focused on understanding the chemical reactions and the physiological functions of PMOs found in fungi and bacteria. Preliminary data indicates that PMOs play important roles beyond cellulose degradation. The research may contribute to the understanding PMO chemical reactions as well as to the discovery of new biological uses for these enzymes. Beyond the chemistry impact and relevance to sustainable energy, the project also creates training opportunities for students. The project continues Dr. Marletta's commitment to training of scientists and to advancing Science, Technology, Engineering, and Mathematics (STEM) fields. Students in the College of Chemistry Scholars Program (COSIP) are directly engaged in the research. Engagement of students from the Berkeley Chapter of the Society for the Advancement of Chicanos/Hispanics and Native Americans in Science (SACNAS) is also underway.The objective of this project is to gain insight into the mechanism by which the oxygen- and copper-dependent fungal PMOs catalyze the regioselective hydroxylation and oxidation of glycosidic bonds of crystalline cellulose. A multi-disciplinary approach is used to gain insight to the PMO active site chemistry and to probe the functional and biochemical roles of selected PMOs. Requisite recombinant expression systems for both fungal (Neurospora crassa and Pichia pastori) and bacterial PMOs (Escherichia coli) as well as methods for the isolation of PMOs from the native hosts have been developed. The chemistry of the active site of the PMO is examined because this enzyme utilizes soluble substrates and thus makes possible in-depth kinetic and spectroscopic studies. Assays have been developed to unravel the complex kinetics and chemical mechanism of PMOs. The kinetics of oxygen (O2) activation and the copper active site species used to hydroxylate substrate are characterized. Through bioinformatic analysis, unique subsets of PMOs have been identified that are may have important roles in the natural lifecycle of those organisms. The structure and function of these novel PMOs are being studied.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

纤维素是自然界生物质的主要成分，也是生物燃料的潜在来源。尽管多年的研究，从纤维素的生物燃料的工业生产是有限的效率和容易分解（降解）纤维素。多糖单加氧酶是近年来发现的一类在纤维素降解和生物燃料开发中起重要作用的酶。PMO可能有助于克服与纤维素降解相关的高成本。有了这个奖项，化学系的生命过程化学项目资助了加州大学伯克利分校的Michael Marletta博士继续他对PMO的研究。该研究的重点是了解真菌和细菌中发现的PMO的化学反应和生理功能。初步数据表明，PMO除了降解纤维素外还发挥着重要作用。该研究可能有助于了解PMO化学反应以及发现这些酶的新生物用途。除了化学的影响和可持续能源的相关性，该项目还为学生创造了培训机会。该项目延续了Marletta博士对科学家培训和推进科学，技术，工程和数学（STEM）领域的承诺。化学学者计划（COSIP）的学生直接参与研究。该项目的目的是深入了解依赖氧和铜的真菌PMO催化结晶纤维素糖苷键的区域选择性羟基化和氧化的机制。一个多学科的方法是用来深入了解PMO活性位点的化学和探测选定的PMO的功能和生化作用。已经开发了用于真菌（粗糙脉孢菌和巴斯德毕赤酵母）和细菌PMO（大肠杆菌）的所需重组表达系统以及用于从天然宿主分离PMO的方法。PMO的活性位点的化学检查，因为这种酶利用可溶性底物，从而使深入的动力学和光谱研究成为可能。已经开发了分析来解开PMO的复杂动力学和化学机制。氧（O2）活化的动力学和铜的活性位点物种用于羟基化基板的特点。通过生物信息学分析，已经确定了PMO的独特子集，它们可能在这些生物体的自然生命周期中发挥重要作用。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。