Enzymology of multi-enzyme systems on self-assembled surfaces

1033222 Woodbury酶广泛用于从洗衣液到生物传感器的大量不同应用中。如在自然界中显而易见的，与表面相关的酶具有可溶性酶所缺乏的额外能力。有组织的，表面相关的酶系统可以催化整个反应途径比溶液中的相同酶更有效。为了将表面固定化酶系统纳入有用的应用中，存在需要克服的挑战。一种是通过优化表面结合酶的取向、构象和表面结合距离来提高其活性和稳定性。另一个是控制表面上多酶级联组分之间的空间关系，旨在最大限度地提高其催化效率。亚利桑那州立大学的首席研究员尼尔伍德伯里和郝燕认为，他们知道如何实现这一点，然后提供一种方法来提高机会。他们打算探索肽空间（从已知的库），以确定将结合到目标酶的配体，并优化其在表面上的取向，以实现活性功能。这将使他们能够在肽修饰的固体表面上创建和分析单酶和多酶系统。这些改进是通过下一步创建自组装DNA纳米支架表面来实现的，这将允许精确控制参数，包括酶间方向和间距以及酶与表面或支架的距离。在用这对酶证明这一点后，可以添加第三种酶以进一步增强。肽锚定物/调节剂与自组装DNA纳米结构的组合代表了一种独特的蛋白质固定化技术，可以显着提高表面固定化酶途径的活性和稳定性。这将导致基于多步酶系统的各种传感和生物催化应用的关键进展。这个建议的一个主要方面将是动力学分析和建模的酶系统组装在这样的合理设计的表面。这一建议的结果将是特别感兴趣的理解和设计多酶反应途径，其中一种酶的能力，直接通过一个产品到下一个是严格依赖于相关的酶的相对位置，从更广泛的背景下，有一个广泛的潜在应用工程，自组装酶系统。在可再生能源、二氧化碳固定和生物诊断应用中，酶介导的表面反应将发挥重要作用。自组装的复杂反应途径将在许多高价值化学品（包括治疗剂）的生产中引起极大的兴趣。因此，表面上的酶功能的改进将是一个重要的目标来实现。这项研究提供了机会，学生培训和推广到研究生，本科生，高中生和教师。PI计划参加亚利桑那州立大学生物设计研究所的暑期高中实习计划，让学生接触到高度跨学科的研究环境。该项目提供的跨学科培训机会将鼓励各种创造性思维，激发对科学和技术的更大兴趣。

1033222WoodburyEnzymes are widely used in a large number of diverse applications, from laundry detergents to biosensors. As apparent in nature, enzymes associated with surfaces have additional abilities that soluble enzymes lack. Organized, surface-associated enzyme systems can catalyze whole reactionpathways much more efficiently than the same enzymes in solution. In order to incorporate surface-immobilized enzyme systems into useful applications there are challenges that need to be overcome. One is to improve the activities and stabilities of surface-bound enzymes by optimizing their orientations and conformations as well as their surface attachment distances. The other is to control the spatial relationships between the components of multi-enzyme cascades on surfaces aiming to maximize their catalytic efficiencies.Principal Investigators Neal Woodbury and Hao Yan of Arizona State University believe they know how to accomplish this, and then offer a method to improve the opportunity. They intend to explore peptide space (from known libraries) to identify ligands that will bind to target enzymes and optimize their orientations on surfaces to achieve active functions. This will allow them to create and analyze single- and multi-enzyme systems on peptide-modified solid surfaces. The improvements come by next creating self-assembled DNA nanoscaffold surfaces, which will allow precise control over parameters including inter-enzyme orientation and spacing as well as the distances from enzymes to surfaces or scaffolds. After demonstrating this with the pair of enzymes, a third enzyme can be added for further enhancement.The combination of peptide anchors/modulators with self-assembled DNA nanostructures represents a unique protein immobilization technology that could significantly improve the activities and stabilities of surface-immobilized enzyme pathways. This will lead to critical advances in a variety of sensing and biocatalytic applications based on multi-step enzyme systems. A major aspect of this proposal will be the kinetic analysis and modeling of enzyme systems assembled on such rationally designed surfaces. The outcome of this proposal will be of particular interest for understanding and designing multi-enzyme reaction pathways in which the ability of one enzyme to directly pass a product to the next is critically dependent on the relative positions of the enzymes involved.From the broader context, there is an extensive set of potential applications for engineered, self-assembled enzyme systems. In renewable energy, carbon dioxide fixation and bio-diagnostic applications, enzyme-mediated reactions on surfaces will play a significant role. Self-assembled, complex reaction pathways will be of substantial interest in the production of many high-value chemicals, including therapeutics. Thus, improvements of enzyme functions on surfaces would be an important goal to achieve.This research provides opportunities for student training and outreach to graduates,undergraduates, high school students and teachers. The PIs plan to participate in the Summer High School Internship Program at the Biodesign Institute at ASU where students are exposed to a highly interdisciplinary research environment. The interdisciplinary training opportunity made possible by this project will encourage a spectrum of creative thinking and inspire a greater interest in science and technology.