Developing Electroanalytical Methods for Enzymology Applications

开发酶学应用的电分析方法

• 批准号: 2406605
• 负责人: Shelley Minteer
• 金额: $ 42万
• 依托单位: Missouri University of Science and Technology
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2406605&HistoricalAwards=false
• 关键词: Developing; Electroanalytical; Methods; Enzymology; Applications

With support from the Chemical Measurement & Imaging (CMI) program in the Division of Chemistry (CHE) and the Biosensors program in the CBET Division, Professor Shelley Minteer at the University of Utah is developing new electroanalytical tools for investigating enzymes, biological catalysts that play critical roles in the chemistry of life processes but that are also very useful in biotechnology. Biochemists and molecular biologists have keen interest in how enzyme structure controls function. In addition, enzymes have inspired the design of selective catalysts for challenging transformations such as reduction of carbon dioxide to methanol and nitrogen (N2) to ammonia. These attributes have motivated extensive enzymology research over the last couple of decades, prompting interest in developing better analytical tools for investigating enzyme function. The Minteer group is both developing such tools and extending their impact through educational opportunities offered to both college and high school students. For example, Professor Minteer is providing unique high school student research internships to inspire student interest in STEM (science, technology, engineering and mathematics) disciplines. In order to develop new electroanalytical methods for enzymology, it is critical to be able to immobilize enzymes on electrode surfaces in a manner whereby they can undergo direct electron transfer with the electrode without the need for exogenous redox mediators. The Minteer group is utilizing two strategies to enable direct electron transfer: 1) incorporation of non-natural amino acids as orienting tags for conjugation of proteins to electrode surfaces, thereby ensuring that the active site is within tunneling distance of the electrode surface; and 2) "wiring" enzymes to electrode surfaces by incorporating metal-binding peptide sequences that promote in-situ formation of metal nanoparticles within the protein. Bioelectrodes modified by either approach will be used to study both single enzymes and enzyme cascades using new galvanostatic titration methods, with an aim of improved understanding of how the rate of electron injection into the active site of a protein (i.e. nitrogenase) controls the selectivity of the products formed (ammonia versus hydrogen). Additionally, rotating ring-disk electroanalytical chemistry methods are being developed to study substrate channeling and to quantitate the leaking of intermediates to the bulk solution from the supercomplex of an enzyme cascade.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.

在化学系（CHE）化学测量与成像（CMI）项目和化学系（CBET）生物传感器项目的支持下，犹他大学的Shelley Minteer教授正在开发新的电分析工具，用于研究酶和生物催化剂，它们在生命过程的化学过程中起着关键作用，但在生物技术中也非常有用。生物化学家和分子生物学家对酶的结构如何控制酶的功能有着浓厚的兴趣。此外，酶还启发了选择性催化剂的设计，用于具有挑战性的转化，如将二氧化碳还原为甲醇和将氮（N2）还原为氨。在过去的几十年里，这些特性激发了广泛的酶学研究，激发了人们对开发更好的分析工具来研究酶功能的兴趣。Minteer小组正在开发这些工具，并通过向大学和高中生提供教育机会来扩大它们的影响。例如，Minteer教授提供独特的高中生研究实习，以激发学生对STEM（科学、技术、工程和数学）学科的兴趣。为了开发新的酶学电分析方法，至关重要的是能够将酶固定在电极表面，使它们能够在不需要外源氧化还原介质的情况下与电极直接进行电子转移。Minteer团队利用两种策略来实现直接电子转移：1)结合非天然氨基酸作为定向标签，将蛋白质偶联到电极表面，从而确保活性位点在电极表面的隧道距离内；2)通过结合金属结合肽序列将酶“连接”到电极表面，从而促进蛋白质内金属纳米颗粒的原位形成。通过这两种方法修饰的生物电极将用于研究单酶和酶级联，使用新的恒电流滴定方法，目的是提高对电子注射到蛋白质（即氮酶）活性位点的速度如何控制形成产物（氨与氢）的选择性的理解。此外，正在开发旋转环盘电分析化学方法来研究底物通道和定量中间体从酶级联的超络合物泄漏到体溶液。该奖项反映了美国国家科学基金会的法定使命，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。