Selected active mutants of HheC and HheG as well as wild-type enzymes were produced in larger scale in shake flasks using the same protocol as mentioned above but 500 mL TB medium. Cells were harvested by centrifugation (20 min, 3494 g, 4 °C) and resulting cell pellets were stored at -20 °C until further use. For purification via N-terminal His-tag, cell pellets were resuspended in 30 mL buffer A, supplemented with 1 mg mL -1 lysozyme and one Pierce Protease Inhibitor Mini Tablet, and disrupted by sonication on ice for 7 min (14 cycles of 10 s pulse and 20 s pause). Cell debris were removed by centrifugation (45 min, 18000 g, 4 °C) and resulting cell-free extracts were filtered through a 0.45 µm syringe filter. Cell-free extracts were loaded (2 mL min -1 flow rate) on a 5 mL HisTrap FF column (GE Healthcare, Freiburg, Germany), pre-equlibrated with buffer A, using an ÄktaStart FPLC system (GE Healthcare). Afterwards, the column was washed with 10 CV of buffer A to remove non-specifically bound proteins. His6-tagged target protein was eluted using a gradient from 0 to 100% buffer B in 60 mL while collecting fractions of each 1 mL. Fractions with highest UV absorbance at 280 nm were combined and concentrated to a volume of 2.5 mL using Vivaspin Turbo 15 centrifugation units (Sartorius, Göttingen, Germany) with 10 kDa molecular weight cut-off. For desalting, the concentrated protein solutions were loaded onto PD10 desalting columns (GE Healthcare), pre-equilibrated with TE buffer, and eluted with 3.5 mL TE-buffer. Respective yields of purified HheC and HheG variants are listed in Table .