Molecular Design of Nano-Carrier Materials for Reactions Catalyzed by Multi-Enzyme Complexes

这项提案的总体目标是设计和研究新型活性柔性和半柔性聚合物纳米载体平台，使多种活性酶能够在纳米尺度上空间共存。自然界中发现的几种多酶复合体的设计是为了确保反应中的每个中间体快速运输到下一个邻近的活性部位，因为中间体通常是不稳定的。因此，将这些酶分子共定位在纳米载体中是至关重要的，这样活性中间体才能为所需产品的形成找到下一个活性部位。虽然已经有很多关于酶固定化的研究，但还没有关于使用纳米材料来共定位多酶复合体的研究，特别是与活性中间体的研究。因此，这项提议的重点是创造活性纳米结构环境，通过在空间和时间尺度上限制活性催化功能来改变复杂转化的方向。我们将研究黄烷-3-醇的生物合成，它的产生是由两种具有高活性中间体的酶介导的。黄烷-3-醇，如(-)表儿茶素，是黄酮类天然产物，具有强大的抗氧化性能，是绿茶和黑巧克力等各种食物的心脏保护和抗癌活性的主要贡献者。该项目的具体目标是：1)设计和表征基于自组装离子和可降解共聚物的新型纳米载体平台，以实现多种酶与活性中间体的共定位和稳定；以及2)研究用于黄酮醇生物合成的纳米载体中的酶活性和通量。已经组建了一个不同的跨学科的研究团队来解决这个问题。智力上的优点：新的生物启发的强大的活性纳米载体平台将被设计成能够对多种酶进行纳米级的时空控制。这些材料形成了各种稳定的纳米隔间/纳米结构。这些平台被选择来研究纳米结构的灵活性如何影响酶的共定位和催化活性。这种限制也有望增加相对微妙的酶生物催化剂的稳定性。将使用实验纳米工具和计算方法来研究纳米隔间的结构、动力学、传输特性和与酶的热力学相互作用，以深入了解其活性机制。该方法将有助于有源纳米载体平台的合理设计。这些见解将被用来研究纳米胶囊对黄烷-3-醇生物合成中多酶复合体的酶活性和产物通量的影响。广泛影响：纳米载体平台可以扩展到有效地中介许多其他重要的与活性中间体的多酶反应(例如，三羧酸循环)和通向预期产品的通道反应，否则可能无法实现。环境友好的纳米载体平台提供的酶稳定和改善通量的双重功能将为酶生物催化剂在级联反应中的工业应用提供一般策略，包括在名义上苛刻的条件下高效(重复)使用。为阐明基本纳米结构功能关系而开发的纳米级探针可以很容易地应用于其他材料/生物分子系统。这一建议整合了研究和教育举措，为学生提供多方面和跨学科的学习体验。不同的学生群体将被主动招募。这项工作将通过传播解决问题的习语和生物伦理学案例研究，在全国范围内产生影响。这项建议将通过研究经验和几个外展机制影响K-12学生。该项目的成果将通过在专业会议、讲习班和档案出版物上举行的关于酶纳米催化的会议广泛传播。

0932517MallapragadaThe overall objective of this proposal is to design and investigate novel active flexible and semi flexible polymeric nano-carrier platforms that will enable nanoscale spatial co-localization of multiple active enzymes. Several multi-enzyme complexes found in Nature are designed to ensure rapid transport of each intermediate in the reaction to the next neighboring active site, since the intermediates are often unstable. Thus, it is critical to molecularly co-localize these enzymes in nano-carriers so that the reactive intermediates can find the next active site for the desired products to be formed. While there have been numerous studies dealing with enzyme immobilization, there are no studies of using nanomaterials to co-localize multi-enzyme complexes, especially with reactive intermediates. Thus, the focus of this proposal is to create active nanostructured environments that can modify the direction of complex conversions by confinement of the active catalytic functionality within both spatial and temporal scales. We will investigate the biosynthesis of flavan-3-ol, whose production is mediated by two enzymes with a highly reactive intermediate. Flavan-3-ols, such as (-) epicatechin, are flavonoid natural products with powerful antioxidant properties and are the major contributors to the cardioprotective and anticancer activity of various foods such as green tea and dark chocolate. The specific goals of the project are to: 1) Design and characterize novel nano carrier platforms based on self-assembling ionic and degradable copolymers to co-localize and stabilize multiple enzymes with reactive intermediates; and 2) Investigate enzymatic activity and flux in nano-carriers for flavon-3-ol biosynthesis. A diverse and interdisciplinary team of researchers has been assembled to address this problem.Intellectual Merit: New bioinspired robust active nano carrier platforms with tailored chemistries will be designed to enable nanoscale spatio-temporal control of multiple enzymes. These materials form various stable nano-compartments/nanostructures. These platforms have been chosen to investigate how the flexibility of the nanostructure affects the co-localization and catalytic activity of the enzymes. This confinement is also expected to increase the stability of relatively delicate enzymatic biocatalysts. The structure, dynamics, transport properties, and thermodynamic interactions of the nano-compartments with enzymes will be investigated using experimental nanoscale tools and computational methods to obtain insights into the mechanisms of activity. This approach will facilitate rational design of the active nano-carrier platforms. These insights will be used to investigate the effects of nano-encapsulation on the enzymatic activity of, and product flux through, a multi enzyme complex in flavan-3-ol biosynthesis.Broader Impact: The nano carrier platforms can be extended to effectively mediate many other important multi enzyme reactions with reactive intermediates (e.g., the tricarboxylic acid cycle) and channel reactions towards desired products that might not otherwise be possible. The dual functions of enzyme stabilization and improved flux provided by the environmentally responsive nano-carrier platforms will provide a general strategy for industrial use of enzymatic biocatalysts in cascade reactions, including their efficient (re)use under nominally harsh conditions. The nanoscale probes developed to elucidate fundamental nanostructure function relationships can be readily applied to other material/biomolecule systems. This proposal integrates research and educational initiatives to provide students multifaceted and interdisciplinary learning experiences. A diverse group of students will be proactively recruited. This work will have a nation-wide impact through the dissemination of problem-solving rubrics and bioethics case studies. This proposal will impact K-12 students through research experiences and several outreach mechanisms. Results from the project will be disseminated broadly through sessions on enzyme nanocatalysis at professional meetings, workshops, and archival publications.