Purification and characterization of a novel laccase from Fomitopsis pinicola mycelia

• Laccase produced from F. pinicola is a glycoprotein with high pH stability and belongs to blue-protein family containing copper.
• Laccase from F. pinicola was slightly larger and exhibited a very different N-terminal amino acid sequence compared to laccases from other basidiomycetes.
• The enzyme showed a relatively higher decolorization activity of blue color. Therefore, our results show that laccase possesses important properties for industrial applications.
• Further research should focus on analyzing the laccase encoded gene, catalytic mechanism, and other biotechnological applications.

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 °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 °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 °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 Km value of the enzyme for ABTS substrate was 0.28 mM with a Vmax 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.

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