Particle-stabilized adsorptive bubble separation of tagged enzymes – a new way to efficient downstream processing?

酶分子已成为生物技术中的重要工具，但它们作为高效和特异性催化剂的全部潜力今天还远未被开发。这与缺乏从生长培养基或生物生产者的粗提取物进行活性保留和成本有效的酶下游加工的方法相关。从技术角度看，气泡吸附分离（ABS）过程中的气液界面分离是克服这一瓶颈的一条有吸引力的途径。在ABS中，酶吸附在气泡表面。气泡上升并在表面上产生泡沫。收集和溶解泡沫可以产生高纯度的酶。然而，驱动ABS朝着一个有效的和可推广的方法，酶纯化需要进一步的发展。在该项目中，为了克服某些问题，引入了两个新的概念：首先，将分子标签连接到目标酶上，旨在增加界面处的吸附速率，并减少酶的活性部分与界面之间的直接接触，因为这种接触通常会使酶变性。其次，添加颗粒代替表面活性剂以产生最佳稳定性的泡沫。颗粒优于通常使用的表面活性剂，因为表面活性剂通常促进酶变性并且几乎不能从最终产物中分离。泡沫稳定性的重要参数是粒度、表面疏水性和介质复杂性（纯缓冲液、发酵液、粗细胞提取物）。这两个概念将通过分子生物学家和工艺工程师的协调互动来整合。ABS方法的研究将集中在其在蛋白质分离和富集以及催化活性保留方面的效率。它将包括酶吸附动力学的实验测定，在气-液界面的剪切力下的蛋白质去折叠，以及考虑酶的标签结构，以获得分子特征和界面响应之间的相关性的理解。有了这些知识，纯化过程的参数将被定制为最高效率。

Enzyme molecules have become important tools in biotechnology, but their full potential as efficient and specific catalysts is nowhere near being exploited today. This correlates with a lack of methods for activity-preserving and cost-efficient enzyme downstream processing from growth media or crude extracts of biological producers. From a technological point of view, separation at gas-liquid interfaces in the course of adsorptive bubble separation (ABS) makes an attractive approach to overcome the bottleneck. In ABS, enzymes adsorb to bubble surfaces. The bubbles rise and create foam on the surface. Collecting and dissolving the foam can yield enzymes in high purity. However, driving ABS towards an efficient and generalizable method for enzyme purification requires further development. In the project, two novel concepts are introduced in order to overcome certain issues: Firstly, a molecular tag is attached to the target enzyme, aiming to increase the adsorption rate at the interface and diminish direct contact between active moiety of the enzyme and interface, because this contact often denatures the enzyme. Secondly, particles are added in place of surfactants in order to generate foam of optimum stability. Particles are advantageous over the usually employed surfactants because surfactants often promote enzyme denaturation and can hardly be separated from the final product. Important parameters for foam stability are particle size, surface hydrophobicity and media complexity (pure buffer, fermentation broth, crude cell extract). Both concepts will be integrated through concerted interaction of molecular biologists and process engineers. Investigation of the ABS-method will focus on its efficiency in protein separation and enrichment as well as retention of catalytic activity. It will include experimental determination of enzyme adsorption kinetics, protein defolding under shear forces at the gas-liquid interface, as well as consideration of the enzyme tag structure in order to gain understanding of the correlation between molecular features and interfacial response. With this knowledge, parameters of the purification process will be tailored for highest efficiency.