Cell envelopes for multi-enzyme synthesis: approaches for increasing the catalytic activity

具有过表达酶的微生物经常被用于生物催化反应。这些全细胞生物转化很有吸引力，因为它们不需要昂贵的酶纯化程序。然而，缺点是内源酶催化的副反应和细胞壁引起的传质限制。这些缺点可以通过使用分离的酶来避免，但它们通常必须被固定化，以使工艺在经济上可行。一种新的一步表达和固定化方法，基于细菌制成的细胞膜，结合了使用整个细胞和分离酶的关键优点。催化所需反应的酶在基因上融合到膜锚上，这有助于将表达的蛋白质固定在大肠杆菌细胞的细胞膜上。随后，表达噬菌体蛋白，导致形成裂解孔。细胞内部和周围介质之间的渗透压力的差异导致细胞质通过形成的孔释放，而固定化的酶保留在空细胞被膜中。其中，固定化β-半乳糖苷酶的细胞膜明显优于相应的高表达酶的整个细胞，因为裂解孔降低了底物的传质限制，从而使酶活性提高了3倍。到目前为止，只有细胞膜用于固定酶。为了提高生物催化制剂的活性，应采取两种策略：一方面，利用表面展示技术将酶额外固定化在细胞表面。另一方面，应利用蜂窝信封的内部空间。为此，必须使用在裂解过程中不会从细胞中排出的结构。可能的候选者是细胞内膜、细菌表层蛋白和细菌细胞骨架。作为反应体系，应研究由蓝藻烯烃还原酶和辅因子再生酶(甲酸脱氢酶或葡萄糖脱氢酶)组成的双酶体系，用于将(R)-香芹酮还原为(2R，5R)-二氢香芹酮。利用固定化酶利用不同的细胞包膜进行不对称合成的反应工程分析。在单相和两相体系中进行生物转化时，应将具有最大酶活性的细胞膜与整个细胞和分离的酶进行比较，酶活性也应该尽可能平衡。最后，应该研究利用切向流过滤和不对称合成生产细胞包膜及其工作的可扩展性。

Microorganisms with overexpressed enzymes are frequently employed in biocatalytic reactions. These whole-cell biotransformations are attractive because they do not require expensive enzyme purification procedures. Disadvantages are, however, side reactions catalyzed by endogeneous enzymes and mass transfer limitations caused by the cell wall. These disadvantages can be avoided by using isolated enzymes, but they usually have to be immobilized to make processes economically viable. A a novel one-step expression and immobilization method on the basis of cellular envelopes made from bacteria combines key benefits of using whole cells and isolated enzymes. The enzymes catalyzing the desired reaction are genetically fused to membrane anchors which facilitate the immobilization of the expressed proteins in the cytoplasmic membrane of E. coli cells. Subsequently, a phage protein is expressed, which results in the formation of a lysis pore. The difference in osmotic pressure between the interior of the cell and the surrounding medium leads to the release of the cytoplasm through the formed pore, whereas immobilized enzymes are retained within the empty cellular envelopes. Amongst others it has been shown that cellular envelopes with immobilized beta-galactosidase clearly outperform the corresponding whole cells with overexpressed enzymes because the lysis pore reduces the mass transfer limitation of the substrate, which leads to a 3-fold higher enzyme activity.So far, only the cytoplasmic membrane of the cellular envelopes was used for enzyme immobilization. To increase the activity of the biocatalytic preparations, two strategies should be followed: On the one hand, enzymes should be additionally immobilized on the cellular surface using surface display. On the other hand, the inner space of the cellular envelopes should be utilized. For this purpose, structures have to be used that are not expelled from the cells during the lysis. Possible candidates are intracellular membranes, bacterial surface-layer proteins and bacterial cytoskeletons. As reaction system, a two-enzyme system consisting of an ene reductase from cyanobacteria and a cofactor-regenerating enzyme (formate dehydrogenase or glucose dehydrogenase) should be investigated, which will be used for the reduction of (R)-carvone to (2R,5R)-dihydrocarvone. A reaction engineering analysis of asymmetric syntheses using the different cellular envelopes with immobilized enzymes should be made. The cellular envelopes with maximum enzyme activities, which should also be as balanced as possible, are to be compared with whole cells and isolated enzymes in biotransformations performed in one- and two-phase systems. Finally, the scalability of the production of cellular envelopes and their work-up using tangential flow filtration as well as of the asymmetric synthesis should be investigated.