Culture supernatant of T. polyzona KU-RNW027 was concentrated by an Amicon ultrafiltration system using a 30 kDa molecular weight cut off Millipore membrane at 4 °C. Concentrated enzyme was applied onto a Toyopearl® DEAE 650 M anion exchange chromatography column with 50 mM Tris-HCl (pH 7.5) as an elution buffer containing 0–1 M NaCl with an elution rate of 0.33 mL/min. Fractions of each MnP and laccase activities were collected separately and further subjected to a Toyopearl® HW-55 gel filtration chromatography column with 50 mM phosphate elution buffer (pH 7.0) at 0.33 mL/min.
It was noted that numbers of fraction collected would depend on the profiles of protein, activity, and heme. Non-denaturing polyacrylamide gel electrophoresis was used at the final step for laccase. Quantification of protein followed Lowry-Folin [30 (link)] or Bradford [31 (link)]. Bovine serum albumin (BSA) was used as the standard. Enzyme purification and molecular mass, as well as enzyme subunit, were determined using SDS-PAGE [32 (link)]. Molecular weight markers were obtained from Thermo Scientific (Waltham, MA). Protein bands were visualized with Coomassie brilliant blue R-250. After non-denaturing SDS-PAGE, the zymogram was visualized using a staining buffer consisted of 1 mM of 2,6-dimethoxyphenol (2,6-DMP), 1 mM of Mn2+, and 0.1 mM H2O2 in 50 mM malonate buffer, pH 4.5.
It was noted that numbers of fraction collected would depend on the profiles of protein, activity, and heme. Non-denaturing polyacrylamide gel electrophoresis was used at the final step for laccase. Quantification of protein followed Lowry-Folin [30 (link)] or Bradford [31 (link)]. Bovine serum albumin (BSA) was used as the standard. Enzyme purification and molecular mass, as well as enzyme subunit, were determined using SDS-PAGE [32 (link)]. Molecular weight markers were obtained from Thermo Scientific (Waltham, MA). Protein bands were visualized with Coomassie brilliant blue R-250. After non-denaturing SDS-PAGE, the zymogram was visualized using a staining buffer consisted of 1 mM of 2,6-dimethoxyphenol (2,6-DMP), 1 mM of Mn2+, and 0.1 mM H2O2 in 50 mM malonate buffer, pH 4.5.