In the first two rounds, we were facing solubility issues with our designs since only three designs (AncDhaA1, AncDhaA10, and AncDhaA11) showed expression levels sufficient for in-depth characterization using in-house microfluidic devices : (i) secondary structure, (ii) thermostability, (iii) temperature profiles and (iv) substrate specificity. Surprisingly, we observed significant discrepancies between the CD spectra predicted based on the AlphaFold structures and collected experimental data. In particular, both the AlphaFold structures and derived spectra resembled those of the native proteins, contrary to clear signs of misfolding by experiments. This implies a bias in AlphaFold predictions towards native folds and stresses the need for caution when using these predictions for synthetic sequences generated by protein language models . Thermostability analysis showed that the variants neither improved nor compromised thermostability. AncDhaA10 exhibited above-average activity compared to other haloalkane dehalogenases.