Hitrap con a 4b column

The supernatant liquid from the culture of S. kanasensis ZX01 or mutant was filtrated using a 10 kDa ultrafiltration membrane. The filtrate was lyophilized and purified by DEAE-52 column (10 × 2 cm). The 0.1 M NaCl eluent was collected, dialyzed and freeze-dried after water elution. The freeze-dried sample was dissolved in water again and then purified by HiTrap™ ConA 4B column (GE, Branford, CT, USA). The eluent was collected, dialyzed and freeze-dried. Determination of glycoprotein GP-1 was performed by HPLC (Ailgent 1260, Palo Alto, CA, USA) with TSK-GEL G2000SWXL column (7.8 × 300 mm, 5 μm). Flowing phase: ultrapure water; flow rate: 0.5 mL/min; temperature of column: 30 °C; wavelength: 280 nm.

FBS was buffer-exchanged against 20 mM HEPES, 0.5 M NaCl, 1 mM MnCl₂, 1 mM CaCl₂ (binding buffer, pH 7.4) by Zeba Spin Desalting Columns (Thermo Fisher Scientific). The buffer-exchanged FBS (1 ml) was loaded onto a HiTrap Con A 4B column (1 ml, GE Healthcare) equilibrated with binding buffer using a manual syringe. After a 25-ml wash with binding buffer, the elution of proteins bound to the column was performed with 25 ml of 20 mM HEPES, 0.5 M NaCl, 0.5 M methyl-α-D-glucoside (pH 7.4), and 0.5 ml of fractions were collected. Aliquot of fractions from column unbound (Con A pass) and bound elutes (Con A elute) were subjected to the AOND assay and protein concentration analysis.

Crude enzymes were filtered with 0.22 µm pore size membrane and concentrated and ultra-diafiltrated using Pellicon cassettes (Merck Millipore, Darmstadt, Germany) and Amicon stirred cells (Merck Millipore, Darmstadt, Germany), both with a 10 kDa cutoff. The concentrated solution was dialyzed against 20 mM TrisHCl buffer pH 7 and immediately concentrated by Amicon Ultra (10 KDa) tubes at 5000 rpm, 10 min. Laccases were purified by FPLC (AKTA purifier system, GE Healthcare) in three steps: (i) anion exchange HiPrep QFF 16/10 column (GE Healthcare) using a salt gradient 0–40% (20 mM Tris-HCl, 1M NaCl, pH 7) to elute the enzyme; (ii) anion exchange Mono Q 5/50 GL column (GE Healthcare), using a 0–25% salt gradient (20 mM Tris-HCl, 1M NaCl, pH 7) to elute the laccase; (iii) molecular exclusion column HiLoad 16/600 Superdex 75pg (GE Healthcare). Between each purification step, fractions with laccase activity were collected, dialyzed and concentrated using Amicon^® Ultra (10 KDa) tubes. The purity of the enzyme was estimated in SDS-PAGE and N-deglycosylation was performed with Endoglycosidase H following manufacturer’s instructions. Additionally, a chromatographic affinity step with HiTrap Con A 4B column (GE Healthcare) was added for evaluating NGly laccases variants. The eluent buffer was 20 mM Tris-HCl, 0.5 M NaCl, 1M Glucose, pH 7.