Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris

W. Liu, Y. Chao, S. Liu, H. Bao, S. Qian

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82 Scopus citations

Abstract

A cDNA encoding for a laccase was isolated from the white-rot fungus Pome lignosus by RT-PCR. It contained an open reading frame of 1,557 bp. The deduced mature protein consisted of 497 amino acids and was preceded by a signal peptide of 21 amino acids. The genomic DNA of the laccase; containing 11 introns, was cloned by PCR. The cDNA was cloned into the vectors pGAPZaA and pGAPZA, and expressed in the Pichia pastoris GS115. Laccase-secreting transformants were selected by their ability to oxidize the substrate 2′2-azinobis-(3-ethylbenzthiaoline-6-sufonic acid) (ABTS). The laccase activity obtained with the native signal peptide was found to be fivefold higher than that obtained with the α-factor secretion signal peptide. The presence of 0.4 mM copper was necessary for optimal activity of the enzyme. The highest activity value reached 9.03 U ml-1, and the optimal secreting time was 2-3 days at 20°C. The crude laccase was stable in a pH range from 6.0 to 10.0 and at temperatures lower than 30°C in pH4.5 for 24 h. The molecular mass of the enzyme was estimated to be 66.5 kDa by SDS-PAGE. The optimum pH and temperature were 2.4 and 55°C. The Km and Vmax values for ABTS were 177 μM and 23.54 μmol min -1 respectively. The extent of glycosylation of the purified enzyme was 58.6%.

Original languageEnglish (US)
Pages (from-to)174-181
Number of pages8
JournalApplied Microbiology and Biotechnology
Volume63
Issue number2
DOIs
StatePublished - Dec 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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