Enzymatic extract (10 mL) was dialyzed against 10 mM sodium phosphate pH 5.8 plus 0.2 mM CaCl2 (buffer A) for 12 h, and was applied to an ionic exchange chromatography on a hydroxyapatite column (1.5 cm × 12 cm, CHTTM Ceramic hydroxyapatite type I—Bio-Rad), previously equilibrated with buffer A in an AKTA-FPLC™ system (GE Healthcare Sciences). Elution was carried out using buffer B (400 mM sodium phosphate pH 5.8 plus 0.0075 mM CaCl2) in a 30-cv (column volume) linear gradient (0–100 % of buffer B) at a flow rate of 1.0 mL min−1, and the eluted fractions were tested for laccase activity using syringaldazine as substrate. Fractions with laccase activity were pooled, dialyzed against buffer C (50 mM sodium phosphate pH 7.0) and concentrated (10×) using ultrafiltration system (Amicon Ultra-15 Millipore). This sample was purified onto a size-exclusion chromatography on a superose 12 10/300 GL column (GE Healthcare Life Sciences) previously equilibrated with buffer C that was supplemented with 150 mM NaCl. The isocratic elution was performed with the same buffer at a flow rate of 0.3 mL min−1.
All purification steps were performed at 20 °C and were followed by enzymatic assays and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
All purification steps were performed at 20 °C and were followed by enzymatic assays and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).