Tangential flow filtration system

Fermentation medium was centrifuged at 30,000 g for 20 minutes at 4°C to pellet the yeast cells. The supernatant was filtered to remove any residual solids and concentrated, using a tangential flow filtration system from Sartorius, Göttingen, Germany (cut-off: 30 kDA). Affinity chromatography was carried out at 4°C with a Ni-NTA sepharose column (Qiagen, Hilden, Germany). The column was pre-equilibrated with 50 mM NaH₂PO₄-buffer pH 7.6, 300 mM NaCl, 5 mM imidazole. The enzyme was eluted with increasing imidazole concentrations. The fractions with the highest DP4-content were pooled and concentrated to 0.5 ml using an Amicon ultrafiltration cell (cut-off: 10 kDA). This volume was applied to a superdex 200 gel filtration column (Cytiva life sciences). The column was equilibrated with 50 mM NaH₂PO₄-buffer pH 7.6 containing 300 mM NaCl with a flow rate of 0.25 ml/min. His(6)-37-766 hDP4 containing fractions were concentrated to 1 ml as described above.

Alteromonas sp. Ni1-LEM [25 (link)] was grown for 4 days at 20 °C in a semi-continuous 5 L fermenter with M9 minimal saline medium (casamino acids 1 g/L, Na₂HPO₄ 6 g/L, KH₂PO₄ 3 g/L, NH₄Cl 1 g/L, NaCl 21 g/L, 0.1 M MgSO₄·7H₂O 10 mL, 0.01 M CaCl₂·2H₂O 10 mL, vitamin B1 (1%) 0.2 mL, and glucose (20%) 20 mL) until the stationary phase was reached. Afterward, the culture was centrifuged at 8500 rpm at 4 °C for 15 min. The supernatant was filtered in a tangential flow filtration system (Sartorius AG, Goettingen, Germany) with a 0.2 µm pore size (Hydrosart membrane, Sartorius AG) and stored at 4 °C until further use. For ROM cleaning treatments, the total protein concentration was determined using the Pierce BCA protein assay kit (Thermo scientific, Waltham, MA, USA).