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

离子液体(ILS)作为生物催化反应的溶剂引起了广泛的兴趣，这源于其特殊的溶剂性质，但在很大程度上取得了平淡无奇的结果。这种结果可以直接归因于目前对ILS如何在分子水平上与酶相互作用缺乏了解。科罗拉多大学的Joel Kaar教授假设，改变酶表面残基的电荷状态将提供一条途径来调节ILS和酶之间的静电相互作用。此外，改变酶的表面电荷也应该防止疏水相互作用，这种作用可以影响IL-酶结合和ILS中的酶聚集。美国国家科学基金会催化与生物催化计划将颁发一个热切的奖项，以支持这一高度探索性的实验，该实验将特别能够在分子水平上初步展示静电相互作用在酶和ILS关联中的作用。静电相互作用在酶和ILS结合中的作用将通过荧光猝灭分析作为酶表面电荷的函数来量化。还将展示利用电荷修饰来提高纤维素酶对ILS的耐受性的潜在用途。这对生物质转化技术具有重要意义。本研究将通过表征电荷修饰对IL诱导失活的影响的生物物理基础，来揭示ILS中酶失活的机制。这项工作的结果将最终支持使用酶电荷修饰作为一种手段来提高ILS在生物催化中的应用。这项研究将使学生有机会在提供实际实验室经验的同时，接受分子生物学、生化和生物物理技术方面的培训。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(国际基因工程机器)项目的学生，该项目是PI帮助培养的。这些技术可以在学生iGEM项目中实施，目的是开发在医学、能源和可持续发展领域具有实用价值的新生物系统。

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.