Enzymology of multi-enzyme systems on self-assembled surfaces

从洗衣洗涤剂到生物传感器，woodbury酶被广泛应用于大量不同的应用中。在自然界中，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相同的酶在溶液中更有效。为了将表面固定化酶系统整合到有用的应用中，需要克服一些挑战。一是通过优化表面结合酶的取向和构象以及表面附着距离来提高酶的活性和稳定性。另一个是控制多酶级联在表面上的组分之间的空间关系，以最大化其催化效率。亚利桑那州立大学的首席研究员尼尔·伍德伯里（Neal Woodbury）和郝彦（Hao Yan）认为，他们知道如何实现这一目标，并提供了一种改善机会的方法。他们打算探索肽空间（从已知的文库中），以确定将与靶酶结合的配体，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。下一步是创造自组装DNA纳米支架表面，这将允许精确控制参数，包括酶间的方向和间距，以及酶到表面或支架的距离。在用这对酶证明了这一点之后，可以添加第三种酶以进一步增强。肽锚/调节剂与自组装DNA纳米结构的结合代表了一种独特的蛋白质固定化技术，可以显著提高表面固定化酶途径的活性和稳定性。这将导致基于多步骤酶系统的各种传感和生物催化应用的关键进展。这一建议的一个主要方面将是在这种合理设计的表面上组装酶系统的动力学分析和建模。这一提议的结果将对理解和设计多酶反应途径特别感兴趣，其中一种酶直接将产物传递给下一种酶的能力严重依赖于所涉及的酶的相对位置。从更广泛的背景来看，工程的、自组装的酶系统有广泛的潜在应用。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对许多高价值化学品（包括治疗药物）的生产具有重大意义。因此，改善酶在表面上的功能将是一个重要的目标。本研究为毕业生、本科生、高中生和教师提供了培训和拓展的机会。这些学生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，在那里学生将接触到高度跨学科的研究环境。这个项目提供的跨学科培训机会将鼓励各种创造性思维，并激发人们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.