Development of a biotechnological enzyme cascade process for the production of optically pure beta amino acids

本项目的目的是开发一种以（芳基-）取代的二氢嘧啶为起始原料，基于酶联反应合成手性β 3氨基酸的工艺。级联酶由二氢嘧啶酶和n -氨基甲酰- β - 3氨基酸脱氨基化酶组成。因此，从可以由简单前体（尿素和肉桂酸衍生物）合成的衍生物开始，提供了直接获得手性β -氨基酸的途径。多种不同的双氢嘧啶酶和具有双氢嘧啶酶活性的生物催化剂可用于该项目。此外，还将首次对β 3-氨基酸脱氨基化酶进行选择性筛选。到目前为止，还没有描述过催化这一反应的酶。然而，这是由于酶促脱氨基化最近才转移到生物催化研究的重点。预计在这个项目中会发现几种具有这种能力的酶。详细地说，二氢嘧啶酶和脱碳酰化酶将用分子生物学技术进行鉴定，广泛地进行生化表征，优化并最终作为重组酶提供给该过程。这些酶将配备适合于酶纯化以及在微流体系统中固定的亲和标签。此外，还对所提供的新的（芳基-）取代底物进行了详细的表征，并建立了相应的分析方法（高效液相色谱法、光度计、薄层色谱法、偏光仪）。有了这些结果，不同的新的和创新的工艺类型（茶包系统固定化全细胞和纯化酶在微流体系统的固定化）将被测试和比较。在项目结束时，可以使用不同的二氢嘧啶酶和脱碳酶工具箱。应用最优工艺类型，酶联反应可有效地制备手性β 3氨基酸。

The objective of this project is the development of a process for the synthesis of chiral beta3-amino acids based on an enzymatic cascade reaction starting from (aryl-) substituted dihydropyrimidines. The enzyme cascade is to be consisting of a dihydropyrimidinase and an enzyme for the decarbamoylation of N-carbamoyl-beta3-amino acids. Thus, starting from educts which can be synthesized from simple precursors (urea and cinnamic acid derivatives), a direct access to chiral beta3-amino acids is provided.A variety of different dihydropyrimidinases and biocatalysts with dihydropyrimidinase activity is available for this project. Additionally a selectively screening for enzymes able to decarbamoylize beta3-amino acids is to be conducted for the first time. Until now no enzymes catalyzing this reaction are described. However, this is due to the fact the enzymatic decarbamoylation only recently shifted in the focus of biocatalytic research. It is expected that several enzymes with this ability are discovered within this project.In detail dihydropyrimidinases and decarbamoylating enzymes are to be identified with molecular biological techniques, extensively biochemically characterized, optimized and finally provided for the process as recombinant enzymes. These enzymes are to be equipped with affinity-tags suitable for the enzyme purification as well as for the immobilization in microfluidic systems.Furthermore the provided new (aryl-) substituted substrates are to be characterized in detail and appropriate analytical methods are to be established (HPLC, photometer, thin layer chromatography, polarimeter).With these results different new and innovative prozess types (the Teabag-system for the immobilization of whole cells and the immobilization of purified enzymes in a microfludic system) are to be tested and compared.At the end of the project a toolbox of different dihydropyrimidinases and decarbamoylating enzymes is available. Applying the optimal process type the desired chiral beta3-amino acids can be efficiently produced with the enzymatic cascade process.