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

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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.