As a next step, this part can be implemented in the directed evolution approach of phage-assisted continuous evolution (PACE) or in the phage-related discontinuous evolution (PREDCEL) approach to improve organosilicon synthesis by cytochrome engineering. For the characterization of this part, we conducted experiments for the production of organosilicons and therefore used a previously described triple mutant, already engineered by [Arnold et al., 2016]. According to the protocol, we obtained the matured cytochrome c and performed conversion experiments analyzed via GC-MS method.
The following figures show the results obtained with the previously engineered enzyme. First, we tested the enzyme on the conversion of the compound (1) with (3) to the final product of (5) (Fig. 1). The gas chromatogram shows one product peak which emerges at 9.2 min retention time. Analysis of the gas chromatogram indicates a conversion rate of >99% on the basis of the non-existence of the respective educt peak. As we performed GC-MS analysis, mass spectrometry data were also available which can be seen in Fig. 3 for the product peak that emerges at 9.2 minutes. It shows the fission of this product and can clearly be identified as our desired product (5). Furthermore, we performed experiments on the compound (1) which differs from compound (2) in its amino functional group. The product is eluted after 11.7 minutes retention time (Fig. 4) and also confirmed as the right product via mass spectrometry in Fig. 5. Worth mentioning, there is still educt (1) present which emerges at 6.9 minutes retention time, indicating incomplete conversion. Calculations showed a conversion rate of 47.5% for the product (4).As these results only show activity for a previously engineered cytochrome c, we are convinced that our part is still functional despite not as effective as the mutant. This part as our best basic part is intended as a platform for future teams to evolve their own cytochrome c for their specific target molecule.