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

Subject Area Biological Process Engineering
Term from 2019 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 407649267
 
Final Report Year 2023

Final Report Abstract

Enzyme molecules have become important tools in biotechnology, but their full potential as efficient and specific catalysts is nowhere near exploitation 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. Foam fractionation makes an attractive approach to overcome the bottleneck. Small bubbles are introduced to the enzyme solution. The enzymes selectively adsorb to the bubble surfaces, and the rising bubbles create a foam on the top of the liquid, which is collected for liquefaction and further processing. In principle, the method can achieve both enrichment from dilute solutions and separation of target enzymes. However, employment of surfactants or direct adsorption of the target enzyme to the gas-liquid interface results in enzyme denaturation, and thus severe loss of catalytic activity. In our collaborative project, we set to overcome these issues with the attachment of a molecular tag to the target enzyme, increasing the adsorption rate at the interface and diminishing direct contact between active moiety of the enzyme and interface. Promisingly, introduction of a molecular tag alone was already sufficient to create a foam with suitable stability for fractionation. We firstly fused the large, surface-active protein Ranaspumin-2 (Rsn-2) to a β-lactamase (Bla). This enabled both a surfactant-free formation of a stable foam and a directed enrichment of the enzyme while maintaining considerable catalytic activity. In the next step, we investigated the fusion of a range of proteins that might act as tag to Bla individually and evaluated their effects on the enzyme activity, and their performance in foam flotation. A problem was that most of the fused tags were insoluble in water. We solved this problem via the introduction of the Spy system, i.e. we produced Bla fused with SpyTag (SpyT) and F-Tags fused with SpyCatcher (SpyC) first separately and then covalently linked the two proteins by forming an isopeptide bond between SpyT and SpyC. Consequently, we could successfully separate the enzyme from the main solution with preserving the enzymatic activity to a great extent (Krause et al., 2023; under review). Moreover, we could demonstrate that the approach also works with other enzymes, namely Penicillin G acylase and a formate dehydrogenase. In parallel, we developed a numerical simulation of the foam fractionation process by solving the unsteady state drainage equation in the protein foam. We also performed flow-on-bubble experiments to evaluate the protein adsorption on the rising bubbles in the cell. The simulation results helped us optimize the foam fractionation process regarding the recovery and grade. In addition, we also designed an extended foam fractionation cell to remove the soluble impurities from the protein foam with addition of a wash-water. This cell was employed successfully for purification of tagged Bla enzyme from an EGFP protein solution.

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