Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris.
Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae alpha-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated.
Otterbein
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Laboratoire de Biotechnologie des Champignons Filamenteux, INRA, Faculté des Sciences de Luminy, Marseille, France.
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Longhi
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Asther
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Moukha
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D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D042845:Laccase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D010843:Pichia / Q000378:metabolism; D011124:Polyporaceae / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013045:Species Specificity
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D042845:Laccase