Rational design of 3-D electroenzymatic electrodes comprising novel enzymatic cascade and NADP(H) co-factor regeneration

电酶技术在精细化工和日用化学品的生物技术新工艺开发方面具有巨大的潜力，但在产品范围、辅因子再生和生产率等方面存在一些主要瓶颈，阻碍了其技术应用。在这个项目中，通过生物催化(Bornscheuer)和电化学工程(Vidakovic-Koch)小组的共同努力来解决这些问题。本文将研究酶催化与电化学再生相结合所面临的一些典型问题的模型--酶催化级联反应。它由烯酸还原酶(ERED)和Baeyer-Villiger单加氧酶(BVMO)组成。这两种类型的酶都需要可溶的NAD(P)H辅助因子，这是一种广泛的酶还原反应所需的多功能辅助因子，对全球药物市场具有重要意义。我们的目标是在电极表面直接再生NAD(P)H，而不涉及任何其他成分，除了电子。为了将辅因子再生与单加氧酶/烯酸还原酶级联反应相结合，将开发新型三维多孔电极。在基于模型的分析中，将评估辅因子再生的酶促级联反应的宏观动力学。合理的蛋白质设计和定向进化将用于使酶适应高效电酶反应器所需的条件。

Electroenzymatic processes have enormous potential for the development of new biotechnological processes for fine and commodity chemicals, but there are some major bottlenecks, like product scope, co-factor regeneration and productivity which impede their technical applications. In this project these issues are addressed by joint efforts between the biocatalysis (Bornscheuer) and electrochemical engineering (Vidakovic-Koch) groups. A model enzymatic cascade which exemplifies some typical problems to be faced by the integration of enzymatic catalysts and electrochemical regeneration will be studied. It comprises the combination of enoate reductases (ERED) and Baeyer-Villiger monooxygenases (BVMO). Both enzyme types require the soluble NAD(P)H co-factor which is a versatile co-factor required for a broad range of enzymatic reduction reactions of significant importance for global pharmaceutical market. We aim for a direct NAD(P)H regeneration at the electrode surface, without any further components, except electrons, involved. To combine the co-factor regeneration with a monooxygenase / enoate reductase enzymatic cascade, novel 3-D porous electrodes will be developed. The macro kinetics of the enzymatic cascade reaction with co-factor regeneration will be assessed in model-based analysis. Rational protein design and directed evolution will be used to adapt the enzymes to conditions required for efficient electroenzymatic reactor.