Biocatalysis in Pickering emulsions

Abstract: Employing enzymes as catalysts in the production of chiral amines that are both used as building blocks and active pharmaceutical ingredients might be promising due to milder reaction conditions compared to non-biological catalysts, exclusion of heavy metals, high selectivity towards substrates and precision in chirality of the products. Transaminases are attractive for the use in this process. In an industrial setting, to obtain feasible yields, the amount of substrate supplied must be high. Most of the substrates are impolar organic solvents causing formation of two phases and the respective interphase which most of the transaminases are sensitive towards. This has as a consequence the deactivation of the enzymes in a usual biphasic system. In this project, a possible solution was investigated, namely the use of Pickering emulsions. The hypothesis was that the Pickering emulsion will protect the enzyme from deactivation. As biocatalyst, an ω-transaminase was employed, using the deactivated E. coli cells the enzymes were expressed in. Both the cells and the Pickering emulsion gave a colloidal system. The aim was the development of an assay using spectrophotometry and to investigate the hypothesis with the help of it. In each assay, a Pickering emulsion, a monophasic, and a biphasic system were compared. This was done by two reactions. The first reaction had the purpose to simulate a longterm exposure of the enzyme to the reactants, and the second reaction was used to determine the activity at the time point the sample was taken. Acetophenone was the reaction product of this second reaction. It has a characteristic absorbance maximum at 245 nm, so this wavelength was used in the spectrophotometric measurements. Transferring a sample from the first reaction into a 40X diluted solution containing the reactants for the second reaction inside the quartz cuvette without any processing of the suspension and subsequent measurement of the absorbance every 5 seconds lead to unreliable results. The particles in the cuvette disturbed the beam in a manner that the curves resulting from this measurement became very craggy. This lead to the conclusion that removing particles before absorbance measurement is necessary. To remove all particles, centrifugation was applied on samples taken from the second reaction at every second minute. After dilution, the absorbance was measured. The results were more reliable, so stop assay was the method of choice in all the following experiments. The assay was performed in two different scales, namely in 4.5 mL glass vials and in Eppendorf tubes. Here, it could be shown that when using Eppendorf tubes, employing the ones with a volume of 2 mL is necessary. An outcome of the project is that the geometry of the vials is crucial, so when applying 1000 rpm on 4.5 mL vials, the Pickering emulsion did not stay intact in contrast to applying this shaking speed on 2 mL Eppendorf tubes where the results seem to confirm the hypothesis, but this should be double-checked in future investigations. As well in future research, a method for sampling at t0 has to be developed, and the shaking speed that has to be applied for good mixing and an intact Pickering emulsion has to be found with respect to the vial used.