Purification and characterization of a novel laccase from Fomitopsis pinicola mycelia

A novel laccase was isolated from the culture filtrate of the brown-rot fungus, Fomitopsis pinicola. Enzyme production reached its highest level after cultivation for 8 days at 25 degrees C. The enzyme was purified by ultrafiltration, ion exchange chromatography, gelfiltration chromatography, and hydrophobic interaction chromatography. Zymography analysis of the purified enzyme showed a laccase band with a molecular mass of 92 kDa. The molecular weight of the enzyme was 92 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and gel filtration chromatography. The enzyme also had an isoelectric point of 3.8. The optimum temperature and pH for enzyme activity were 80 degrees C and 3.0, respectively. Enzyme activity was relatively stable in the pH range from 1.5 to 11.0 and at temperatures below 40 degrees C. The N-terminal amino acid sequence of the enzyme was DTHKAEIACRFKDLG. Enzyme activity was potently inhibited by NaN3 and SDS. The enzyme showed the highest specific activity for 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) as a substrate. The K-m value of the enzyme for ABTS substrate was 0.28mM with a V-max value of 4.5 U/min. The enzyme degraded several recalcitrant dyes at different time intervals during decolorization. Therefore, the novel laccase from F. pinicola may be potentially useful in industry. (C) 2014 Elsevier B.V. All rights reserved.