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

酶分子已经成为生物技术的重要工具，但它们作为高效和特定催化剂的全部潜力在今天还远远没有被开发出来。这与缺乏从生长培养基或生物生产者的粗提取物中进行活性保存和低成本酶下游加工的方法有关。从技术角度看，吸附气泡分离（ABS）过程中气液界面分离是克服这一瓶颈的一种有吸引力的方法。在ABS中，酶吸附在气泡表面。气泡上升，在表面形成泡沫。收集和溶解泡沫可以产生高纯度的酶。然而，推动ABS成为一种高效、通用的酶纯化方法还需要进一步的发展。为了克服某些问题，本项目引入了两个新概念：首先，在目标酶上附着一个分子标签，旨在提高界面上的吸附速率，减少酶的活性部分与界面的直接接触，因为这种接触经常使酶变性。其次，添加颗粒代替表面活性剂，以产生最佳稳定性的泡沫。颗粒比通常使用的表面活性剂更有优势，因为表面活性剂通常会促进酶变性，并且很难从最终产物中分离出来。泡沫稳定性的重要参数是粒径、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整合。对abs方法的研究将集中在其对蛋白质分离和富集的效率以及催化活性的保留上。它将包括酶吸附动力学的实验测定，蛋白质在气液界面剪切力下的解折，以及酶标签结构的考虑，以了解分子特征与界面响应之间的关系。有了这些知识，净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.