EAGER: Rational Modification of Enzyme Charge for Enhanced Biocatalyst Stability in Ionic Liquids

EAGER：合理修饰酶电荷以增强离子液体中生物催化剂的稳定性

• 批准号: 1347737
• 负责人: Joel Kaar
• 金额: $ 8.45万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1347737&HistoricalAwards=false
• 关键词: EAGER; Rational; Modification; Enzyme; Charge

Widespread interest in the use of ionic liquids (ILs) as solvents for biocatalytic reactions, which stems from their exceptional solvent properties, has largely been met with underwhelming results. Such results can be directly attributed to the current lack of understanding of how ILs interact with enzymes at the molecular level. The PI, Prof. Joel Kaar of the University of Colorado, hypothesizes that altering the charge state of residues on the enzyme surface will provide a route to mediate electrostatic interactions between ILs and enzymes. Moreover, altering the surface charge of an enzyme should also prevent hydrophobic interactions that can influence IL-enzyme binding as well as enzyme aggregation in ILs. The National Science Foundation Catalysis & Biocatalysis Program will offer an EAGER Award to support this highly exploratory experimentation which will specifically enable preliminary demonstration of the role of electrostatic interactions in the association of enzymes and ILs at the molecular level. The role of electrostatic interactions in the association of enzymes and ILs will be quantified as a function of enzyme surface charge via fluorescence quenching assays. The potential use of charge modification to increase the tolerance of cellulases to ILs will also be demonstrated. This has implications in biomass conversion technology.This research will begin to uncover the mechanisms by which enzymes are inactivated in ILs by characterizing the biophysical basis for the impact of charge modification on IL-induced inactivation. The results of this work will ultimately support the use of enzyme charge modification as a means to enhance the utility of ILs for biocatalysis in general.This research will enable opportunities to train students in molecular biology, biochemical, and biophysical techniques while providing practical laboratory experience. Training in new protein engineering techniques developed in this work will be extended to students in the University of Colorado iGEM (international Genetically Engineered Machines) program, which the PI has helped grow. These techniques may be implemented in student iGEM projects for the purpose of developing novel biological systems with utility in areas of medicine, energy, and sustainability.

离子液体由于其特殊的溶剂性质而引起了人们对离子液体作为生物催化反应溶剂的广泛兴趣，但结果却差强人意。这样的结果可以直接归因于目前缺乏对IL如何在分子水平上与酶相互作用的理解。主要研究者、科罗拉多的Joel Kaar教授假设，改变酶表面残基的电荷状态将提供一种介导离子液体和酶之间静电相互作用的途径。此外，改变酶的表面电荷还应防止可影响IL-酶结合以及IL中的酶聚集的疏水相互作用。美国国家科学基金会催化生物催化计划将提供EAGER奖，以支持这一高度探索性的实验，该实验将特别能够初步证明静电相互作用在分子水平上酶和离子液体缔合中的作用。静电相互作用在酶和IL的缔合中的作用将通过荧光猝灭测定作为酶表面电荷的函数来量化。电荷修饰的潜在用途，以增加耐受性的纤维素酶离子液体也将被证明。本研究将开始揭示酶在IL中失活的机制，通过表征电荷修饰对IL诱导的失活的影响的生物物理基础。这项工作的结果将最终支持使用酶的电荷修饰作为一种手段，以提高在生物catalysis.This研究将使机会，培养学生在分子生物学，生物化学和生物物理技术，同时提供实际的实验室经验的IL的效用。在这项工作中开发的新蛋白质工程技术的培训将扩展到科罗拉多大学iGEM（国际遗传工程机器）计划的学生，PI帮助发展。这些技术可以在学生iGEM项目中实施，以开发在医学，能源和可持续发展领域具有实用性的新型生物系统。