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

用于多酶合成的细胞包膜：提高催化活性的方法

基本信息

批准号：
206669210
负责人：
Professor Dr.-Ing. Dirk Weuster-Botz
金额：
--
依托单位：
Lehrstuhl für Bioverfahrenstechnik (BVT) Erlangen
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2011
资助国家：
德国
起止时间：
2010-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/206669210?language=en
关键词：
Cell envelopes multi enzyme synthesis

项目摘要

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.

具有过度表达的酶的微生物经常用于生物催化反应。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，但通常必须将它们固定化以使工艺在经济上可行。一种基于细菌制成的细胞包膜的新型一步表达和固定方法结合了使用全细胞和分离酶的主要优点。催化所需反应的酶通过基因方式与膜锚融合，从而促进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，而固定化酶保留在空的细胞被膜内。除其他外，已经表明，具有固定化 β-半乳糖苷酶的细胞包膜明显优于具有过表达酶的相应全细胞，因为裂解孔减少了底物的传质限制，从而导致酶活性提高了 3 倍。到目前为止，仅细胞被膜的细胞质膜用于酶固定化。为了提高生物催化制剂的活性，应遵循两种策略：一方面，应使用表面展示将酶另外固定在细胞表面上。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。可能的候选者是细胞内膜、细菌表面层蛋白和细菌细胞骨架。作为反应系统，应研究由来自蓝藻的烯还原酶和辅因子再生酶（甲酸脱氢酶或葡萄糖脱氢酶）组成的双酶系统，该系统将用于将（R）-香芹酮还原为（2R， 5R)-二氢香芹酮。应使用不同的细胞包膜和固定化酶对不对称合成进行反应工程分析。具有最大酶活性的细胞被膜，也应尽可能平衡，将与在一相和两相系统中进行的生物转化中的全细胞和分离的酶进行比较。最后，应研究细胞包膜生产的可扩展性及其使用切向流过滤以及不对称合成的后处理。