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

酶过表达的微生物常用于生物催化反应。这些全细胞生物转化是有吸引力的，因为它们不需要昂贵的酶纯化程序。然而，缺点是内源性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酶来避免，但它们通常必须固定化以使工艺在经济上可行。一种基于细菌细胞包膜的新型一步表达和固定化方法结合了使用全细胞和分离酶的主要优点。催化所需反应的酶被基因融合到膜锚点上，这有助于将表达的蛋白质固定在大肠杆菌细胞的细胞质膜上。随后，噬菌体蛋白被表达，导致裂解孔的形成。细胞内部和周围介质之间的渗透压差异导致细胞质通过形成的孔释放，而固定化酶则保留在空的细胞包膜内。其中有研究表明，固定化β -半乳糖苷酶的细胞包膜明显优于酶过表达的相应全细胞，因为裂解孔减少了底物的传质限制，从而导致酶活性提高3倍。到目前为止，只有细胞质膜的包膜被用于酶固定。为了提高生物催化制剂的活性，应采取两种策略：一方面，利用表面显示技术将酶额外固定在细胞表面。另一方面，应该利用细胞包膜的内部空间。为此，必须使用在裂解过程中不会从细胞中排出的结构。可能的候选物质有胞内膜、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，将(R)-香芹酮还原为(2R,5R)-二氢香芹酮。对不同细胞包膜固定化酶的不对称合成进行了反应工程分析。具有最大酶活性的细胞包膜，也应尽可能平衡，将与在一相和两相系统中进行生物转化的全细胞和分离酶进行比较。最后，应该研究使用切向流过滤和不对称合成生产细胞包膜及其加工的可扩展性。

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.