*3.3. Purification Strategy*

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.
