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

本项目的目标是开发一种以(芳基)取代的二氢嘧啶为原料，通过酶促级联反应合成手性β-3-氨基酸的方法。该酶系由一个二氢嘧啶酶和一个用于N-氨基甲酰-β-3-氨基酸脱氨甲酰的酶组成。因此，从可以从简单的前体(尿素和肉桂酸的衍生物)合成的导出物开始，提供了直接获得手性β3-氨基酸的途径。该项目提供了各种不同的二氢嘧啶酶和具有二氢嘧啶酶活性的生物催化剂。此外，将首次选择性地筛选能够使β-3-氨基酸脱碳氨基化的酶。到目前为止，还没有对催化这一反应的酶进行描述。然而，这是由于生物催化研究的焦点最近才转移到酶的脱氨酰化。预计在该项目中将发现几种具有这种能力的酶。详细地说，二氢嘧啶酶和脱氨基甲酸酶将通过分子生物学技术进行鉴定，进行广泛的生化表征，并进行优化，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和微流控系统中固定化的亲和标签。此外，提供的新的(芳基)取代底物的详细表征和适当的分析方法将被建立(高效液相、光度计、薄层色谱、旋光计)。利用这些结果，将测试和比较不同的新的和创新的过程类型(用于固定化整个细胞的茶袋系统和在微流控系统中固定化纯化的酶)。项目结束时，可以使用不同的二氢嘧啶酶和脱氨基酶的工具箱。应用优化的工艺类型，采用酶促串联工艺可以有效地生产所需的手性β3-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.