Phenyl sepharose fast flow column

Ten g of lyophilized mycelium were re-suspended in 400 mL buffer A (50 mM Bis-Tris, pH 6.0, 1 M (NH₄)₂SO₄) and extracted for 1 h at 4 °C in horizontal position in an orbital shaker. Insoluble components were removed by centrifugation (5000× g, 4 °C, 15 min) followed by filtration (PES filter, 0.45 µm, Merck). Subsequently, 80 mL filtered supernatant were applied on a Phenyl Sepharose fast flow column (20 mL, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) pre-equilibrated with buffer A. After the column was washed with buffer A, the active enzyme was eluted with a linear gradient (130 mL, 100–0% buffer A) with 100% distilled water at a constant flow rate of 2 mL/min. Active fractions were pooled, desalted and concentrated by ultrafiltration (3 kDa cut off, polyethersulfone (PES), Sartorius, Göttingen, Germany). Concentrate (20 mL) was diluted two times with 20 mM sodium acetate pH 4.0 (buffer B) and loaded onto three linked HiTrap SP Sepharose columns (1 mL, GE Healthcare Bio-Sciences AB) pre-equilibrated with buffer B. Proteins were eluted with a stepwise ionic strength gradient (0, 20, 100% buffer C: 20 mM sodium acetate pH 4.0, 1 M NaCl) with 100% buffer C at a constant flow rate of 1 mL/min.

AmPDH1, AmPDH2 and AmPDH3 were essentially produced in cultivations in a 7-L laboratory fermenter as described for AmPDH1 (Graf et al. 2015 (link)), using basal salts fermentation medium. A purification scheme as described previously (Graf et al. 2015 (link)) was used. Briefly, this scheme is based on hydrophobic interaction chromatography (HIC) using a Phenyl Sepharose Fast Flow column (GE Healthcare, Little Chalfont, UK), equilibrated with 50 mM potassium phosphate buffer (pH 6.5, 40% saturation (NH₄)₂SO₄), and immobilized metal affinity chromatography (IMAC) with a Ni²⁺-charged Chelating Sepharose Fast Flow column (GE Healthcare), equilibrated with 100 mM potassium phosphate buffer pH 7.0, 1 M NaCl and 5 mM imidazole. An additional ion exchange chromatography (IEX) step using a DEAE-Sepharose Fast Flow column (GE Healthcare) equilibrated with 100 mM potassium phosphate buffer pH 7.0 was added for AmPDH3 as a polishing step.