UNS: Engineered Protein-Inorganic Self-Assembly to Control Enzyme Performance and Recovery

将酶固定在不同的支撑结构上可以提高酶的稳定性、活性、回收率和经济性。这项工作的目标是创造一种新的酶固定模式，这种模式含有每体积的高酶水平，最重要的是，保留或提高酶的活性。在生物相容性材料存在的情况下，通过使用蛋白质自组织，将产生含有活性酶的多孔、高表面积颗粒。作为一个测试案例，一种产生药物前体的酶和它的再生伙伴酶都将被固定在颗粒中。这些颗粒将被测试用于产品的生产，随时间的稳定性，以及在多个反应周期中收集和再利用的能力。这项研究的结果将不仅是一种改进的酶固定化方法，而且将产生关于如何在固定化结构中放置伙伴酶可以提高其性能和生产力的基本知识。这里开发的颗粒平台是模块化的，可以很容易地应用于制造重要产品的各种酶。pi将把研究与中学到研究生院的活动结合起来，以招募和留住STEM领域的女性。这项研究的目标是创造生物催化、自组装材料，能够提供所需的微纳米结构环境，以支持和增强工业相关应用的酶活性。传统上，酶通过共价相互作用或吸附固定在固体合成载体上，以提高活性、恢复和/或寿命。在这里，酶被设计成与互补的蛋白质构建块组装，以创造生物催化材料。这提供了一个局部蛋白质拥挤的环境，消除了共价固定或非特异性吸附的需要，这通常会导致结构和功能的丧失，以及对特定酶的放置或密度缺乏控制。本项目结合蛋白质工程，生产含有酶和自组装结构域的蛋白质，以及蛋白质-无机自组装，以构建适当的结构来控制酶的性能和恢复。自组装首先发生在纳米尺度上，然后在微观尺度上产生具有内部结构复杂性的分层结构超粒子，以实现高表面积和高孔隙率。提出的研究将提供基本的知识和证明蛋白质-无机自组装的概念与耦合的两酶系统生产手性胺与辅助因子再生。所采用的设计策略是模块化的，因此多种酶可以同时在同一个超颗粒中使用，无论是均匀分散还是空间分离。模块化设计还设置了独立于酶的物理性质的材料结构，并允许使用几乎任何酶。将评估超颗粒固定化对酶活性和协同作用的影响，并对该系统在工业反应过程中的实用性进行实际评估。此外，这项工作还为蛋白质与无机组分的自组装提供了基础知识，并深入了解了酶放置的空间控制如何控制动力学参数和活性。这项工作将证明，蛋白质-无机自组装酶可以提供一个通用模板，以及所需的化学和物理环境，以支持复杂的工业生物催化。该奖项由CBET部门的生物技术和生化工程项目颁发，由材料研究部的生物材料项目共同资助。

1510551 Champion, Julie Immobilization of enzymes on various support structures is used to enhance enzyme stability, activity, recovery, and economics. The goal of this work is to create a new mode of enzyme immobilization that contains high enzyme levels per volume, and most importantly, retains or improves enzyme activity. Through the use of protein self-organization in the presence of biocompatible materials, a porous, high surface area particle containing active enzyme will be created. As a test case, an enzyme that produces pharmaceutical precursors and its regenerating partner enzyme will both be immobilized in the particles. The particles will be tested for production of product, stability over time, and the ability to collect and reuse over multiple reaction cycles. The results of this research will not only be an improved method of enzyme immobilization, but will generate fundamental knowledge of how placement of partner enzymes in immobilized structures can improve their performance and productivity. The particle platform developed here is modular and can be easily applied to a wide variety of enzymes that make important products. The PIs will integrate the research with activities for middle to graduate school to recruit and retain women in STEM.The goal of this research is to create bio-catalytic, self-assembled materials capable of providing the required micro and nano-structured environment to support and enhance enzymatic activity for industrially relevant applications. Traditionally, enzymes are immobilized on solid synthetic supports via covalent interactions or adsorption in order to enhance activity, recovery, and/or lifetime. Here, enzymes are engineered to assemble with complementary protein building blocks to create biocatalytic materials. This provides a local protein-crowded environment and eliminates the need for covalent immobilization or non-specific adsorption which often leads to loss of structure and function, and lack control over specific enzyme placement or density. This project combines protein engineering, to produce proteins containing both enzymatic and self-assembly domains, and protein-inorganic self-assembly, to build appropriate structures to control enzyme performance and recovery. Self-assembly occurs first at the nanoscale and then at the microscale to produce hierarchically structured supraparticles that have internal structural complexity to achieve high surface area and porosity. The proposed research will provide basic knowledge and proof of concept of protein-inorganic self-assembly with a coupled two enzyme system for production of chiral amines with co-factor regeneration. The employed design strategy is modular, so that multiple enzymes can be used simultaneously in the same supraparticles, either dispersed homogenously or segregated spatially. The modular design also sets the material structure independently of the physical properties of the enzyme(s), and allows almost any enzyme(s) to be used. The impact of supraparticle immobilization on enzyme activity and synergy will be evaluated, and also a practical assessment of the utility of this system to be used in industrial reaction processes will be provided. Additionally, this work also generates fundamental knowledge on protein self-assembly with inorganic components and insight as to how spatial control of enzyme placement can give control of kinetic parameters and activity. This work will demonstrate that protein-inorganic self-assembly of enzymes can provide a general template, as well as the required chemical and physical environment, to support complex industrial biocatalysis.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Biomaterials Program of the Division of Materials Research.