The fungal laccase Lcc9 from Coprinopsis cinerea is a promising candidate for biotechnological applications due to its distinct biochemical properties. In the present work, Lcc9 cDNA was cloned from C. cinerea using reverse transcription polymerase chain reaction and heterologously expressed in Pichia pastoris GS115. The recombinant laccase was found to be a heavily hyperglycoprotein, with the molecular weight of 60.2 kDa as determined by MALDI-TOF. Laccase activity in the culture supernatant was 1750 ± 83 U/L and reached 3138 ± 62 U/L after expression condition optimization using orthogonal experiment. The biochemical property of the purified recombinant Lcc9 (rLcc9) was compared to that of wild-type Lcc9. rLcc9 shows a higher specific activity (315.3 U/mg) than Lcc9 (92.9 U/mg) when using ABTS (2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate)) as the substrate. Although rLcc9 and Lcc9 showed comparable optimal pH (6.5) and temperature (70 °C) toward syringaldazine, rLcc9 displayed higher activity and stability in the pH range of 6.5-8.5. rLcc9 showed improved ability to oxidize indigo carmine and 5 azo dyes when methyl syringate was used as the mediator, with the decolorization rate range from 71.9 ± 3.2% to 99.1 ± 1.6% for different dyes in a wide pH (4.5-9.0) and temperature (4-70 °C) ranges. In comparison, Lcc9 decolorized 50.3 ± 2.1% to 98.2 ± 2.0% of the dyes used. The improved activity and stability in alkaline pH of rLcc9 relative to Lcc9, and improved dye decolorization ability towards 6 dyes suggested greater application potential of rLcc9 in biotechnologies such as wastewater treatment.
Keywords: Alkaline pH; Dye decolorization; Glycoprotein; Heterologous expression; Laccase; Pichia pastoris.
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