Protein production in 100 mL scale and purification via gravity-flow Selected HheG variants displaying improved enantioselectivity during screening as well as loop3947 deletion variants were produced in shaking flasks containing 100 mL TB medium supplemented with 50 µg mL -1 kanamycine and 0.2 mM IPTG. After inoculation with 10% (v/v) pre-culture, protein production was performed for 24 h at 22 °C and 220 rpm. Cells were harvested by centrifugation (3494 g, 20 min, 4 °C) and resulting cell pellets were stored at -20 °C until further use. For subsequent IMAC-based purification using gravity-flow columns, cells were resuspended in 20 mL lysis buffer (50 mM Tris•SO4 buffer, pH 7.9, 25 mM imidazole) supplemented with 1 mg mL -1 lysozyme and 1 pierce protease inhibitor tablet. Sonification was perfomed on ice (65% amplitude, 10 s pulse, 20 s pause). Cell debris was removed by centrifugation (18000 g, 45 min, 4 °C) and the resulting CFE was filtered through a 0.45 µm filter. Protein purification was performed using pierce gravity-flow columns (Thermo Fisher Scientific) containing 2 mL Ni-NTA sepharose (Cytiva, Freiburg, Germany). The column material was first equilibrated with 10 column volumes (CV) lysis buffer. The CFE was loaded on the column. After flow through, the column was washed with 10 CV lysis buffer. For elution of desired proteins, 10 CV elution buffer (50 mM Tris•SO4 buffer, pH 7.9, 500 mM imidazole) were used. The first 2.5 mL of elution containing the desired protein were collected and desalted using PD10 desalting columns in combination with TE buffer (10 mM Tris•SO4 buffer, pH 7.9, 4 mM ethylenediamine tetraacetic acid, 10% (v/v) glycerol) according to the manufacturer's instructions. Desalted proteins were stored at -20 °C until further use. Protein concentrations were determined by measuring the absorbance at 280 nm using an NP80 nanophotometer (Implen, München, Germany) and calculated via the Lambert-Beer law with molar extinction coefficients and molecular weights of the respective proteins obtained from Protparam .