Enzyme, Electrode and Reactor Design for Enzymatic Cascade Reactions

拟议项目的目标是开发一种基于酶的生物电催化硝酸盐氨化系统（也称为异化硝酸盐还原为铵，DNRA），该系统将硝酸盐定量转化为亚硝酸盐，并进一步转化为铵，首先单独进行，但最终在一个集成的环境中进行。为此，硝酸盐还原酶和亚硝酸盐还原酶将与定制的3D多孔电极相结合，并集成到先进的生物电化学反应器中，以实现高转化率。酶将被工程化以获得高活性和稳定性以及有效的电极附着（静电、配位、共价结合）。定制的三维多孔电极材料和电极兼容的原位光谱电化学方法将随后开发和微调的酶固定化（吸附和解吸研究）和蛋白质膜伏安法（直接与介导的电子转移）。最后，高性能的电化学零间隙电池将被设计为允许高电流密度和电极集成将实现有前途的酶电极组合，连续操作的单反应器反应进行评估。一旦材料、固定化策略和反应器设计得到优化，将开发用于酶级联反应的集成系统。取决于单个酶的相应反应动力学，诸如酶负载、共相对于分层固定化和电极厚度的参数将变化，目的在于完全的底物转化和长期稳定性。

The objective of the proposed project is to develop an enzyme-based bioelectrocatalytic nitrate ammonification system (also known as dissimilatory nitrate reduction to ammonium, DNRA) that quantitatively transforms nitrate into nitrite and further into ammonium, at first separately but ultimately in an integrated setting. For this, nitrate reductases and nitrite reductases will be coupled with tailored 3D-porous electrodes and integrated into advanced bioelectrochemical reactors to achieve high conversion rates. The enzymes will be engineered for high activity and stability as well as efficient electrode attachment (electrostatics, coordination, covalent binding). Tailored 3D-porous electrode materials and electrodes compatible with in-situ spectroelectrochemistry methods will subsequently be developed and finetuned in terms of enzyme immobilization (adsorption and desorption studies) and protein film voltammetry (direct versus mediated electron transfer). Finally, high performance electrochemical zero-gap cells will be designed that allow for high current densities and electrode integration will be realized with promising enzyme-electrode combinations that are evaluated for continuously operated single reactor reactions. Once the materials, immobilization strategies and reactor designs are optimized, integrated systems for enzymatic cascade reactions will be developed. Depending on the respective reaction kinetics of the single enzymes, parameters such as enzyme loading, co- versus layered immobilization, and electrode thicknesses will be varied, aiming for complete substrate conversion and long-term stability.