Elucidating Water-Ionic Liquid-Enzyme Mixtures Interactions for Enhanced Deconstruction of Cellulosic Biomass

PI：Maupin，Christopher 提案编号：1337044 机构：科罗拉多矿业学院 标题：阐明水-离子液体-酶混合物的相互作用，以增强纤维素生物质的解构 纤维素解构的工程增强系统对于经济有效地利用生物质生产液体燃料和其他增值化学品至关重要。不幸的是，结晶纤维素底物的不溶性以及由多相催化引起的限制导致反应时间长，不适合大规模工业过程。特定任务的离子液体（IL）是一种有前途的“绿色”？超级溶剂能够快速溶解结晶纤维素生物质，同时还保留纤维素酶活性，从而为纤维素生物质的单步均相降解提供了一条有前途的途径。然而，在了解影响酶结构、活性和稳定性的潜在 IL-酶相互作用方面，还有很多工作要做。在这个计算和实验相结合的项目中，我们提出了一种双管齐下的方法来评估三种 IL 与两种纤维素酶之间的相互作用。正在研究的纤维素酶在 IL-H2O 混合物中表现出不同程度的活性，从而可以鉴定功能所需的特定酶特征，而不同的 IL-H2O 混合物则可以鉴定有利于酶功能的特定 IL 特征。导致酶功能和活性改变的特定 IL-酶相互作用的鉴定将通过以下目标来完成：(1) IL-H2O-酶系统的 MD 模拟。这些模拟将揭示特定的IL-酶相互作用及其对动态波动和酶整体结构的影响。（2）对IL-H2O-酶和IL-H2O-纤维素低聚物系统进行热力学积分计算。这些计算探讨了 IL 进入纤维素酶活性位点的能力，并阐明了竞争溶剂化能的作用，(3) 对 IL-H2O-酶系统进行 CpHMD 模拟。这些模拟将揭示 IL 混合物对活性位点关键残基 pKa 的影响，以及 (4) 对 IL-H2O-酶降解纤维素进行动力学分析。这些实验将揭示 IL-H2O 混合物对观察到的酶活性的影响。计算和实验研究将提供对 IL-酶相互作用的分子水平理解，以及由此产生的对结构、动态波动、pKa 值和溶剂化的影响，将阐明溶剂环境如何影响酶和与工业过程相关的其他大分子系统的功能。此外，对促进结构稳定性和活性的酶和 IL 特性的评估将使得能够利用合理的设计为广泛的行业创建定制的酶和 IL。与研究紧密结合的是跨多个教育层次的下一代科学家和工程师的教育。通过与丹佛高中 Martin Luther King Jr. Early ollege 合作开展的大学预科学生研究体验 (REPS) 外展计划，让弱势群体参与 STEM 领域。研究成果将通过研究文章、区域和国家会议广泛传播，此外还将纳入 AAAS 太平洋分会区域会议和博伊西州立大学（主要是本科院校）的计算研讨会。

PI: Maupin, ChristopherProposal Number: 1337044Institution: Colorado School of MinesTitle: Elucidating Water-Ionic Liquid-Enzyme Mixtures Interactions for Enhanced Deconstruction of Cellulosic BiomassEngineering enhanced systems for the deconstruction of cellulose is essential to the cost-effective utilization of biomass for production of liquid fuels and other value added chemicals. Unfortunately, the insolubility of the crystalline cellulose substrate and resulting limitations incurred by heterogeneous catalysis leads to high reaction times that are not suitable for large scale industrial processes. Task specific ionic liquids (IL) are a promising ?green? super solvent that enable the rapid dissolution of crystalline cellulosic biomass while also retaining cellulase enzyme activity thereby providing a promising route to a single step, homogeneous phase degradation of cellulosic biomass. However, much remains in regards to understanding the underlying IL-enzyme interactions affecting enzyme structure, activity, and stability.In this combined computational and experimental project we propose a two pronged approach to evaluate the interactions between three ILs on two cellulase enzymes. The cellulase enzymes being studied have shown varying degrees of activity in IL-H2O mixtures, allowing for the identification of specific enzyme features necessary for functionality while the different IL-H2O mixtures enable the identification of specific IL features that favorably affect enzyme functionality. The identification of specific IL-enzyme interactions leading to altered enzyme functionality and activity will be accomplished through the following objectives: (1) MD simulations on IL-H2O-enzyme systems. These simulations will reveal specific IL-enzyme interactions and their impact on dynamic fluctuations and the overall structure of the enzyme, (2) Conduct Thermodynamic Integration calculations on IL-H2O-enzyme and IL-H2O-cellulose oligomer systems. These calculations probe the ability of the IL to enter the active site of cellulose enzymes and illuminate the role of competing solvation energies, (3) Conduct CpHMD simulations on IL-H2O-enzyme systems. These simulations will reveal the impact of IL mixtures on the pKa of critical residues in the active site, and (4) Conduct kinetic analysis of IL-H2O-enzyme degradation of cellulose. These experiments will reveal the impact of IL-H2O mixtures on the observed enzyme activity.The computational and experimental studies will provide an increased molecular-level understanding of the IL-enzyme interactions and the resulting effect on structure, dynamical fluctuations, pKa values, and solvation will elucidate how the solvent environment impacts the functionality of enzymes and other macromolecule systems relevant to industrial processes. In addition, evaluation of the enzyme and IL properties that facilitate structural stability and activity will enable the use of rational design to create tailored enzymes and ILs for a wide range of industries. Closely integrated with the research is the education of the next generation of scientists and engineers across multiple education levels. Engaging underrepresented groups in STEM fields is accomplished through the Research Experience for Pre-Collegiate Students (REPS) outreach program with Martin Luther King Jr. Early ollege, a Denver high school. The research effort will be broadly disseminated through research articles, regional and national meetings, in addition to the incorporation into computational workshops at the regional AAAS Pacific Division meetings and Boise State University, a primarily undergraduate institution.