Molecular cloning and characterization of a novel laccase gene from a white-rot fungus Polyporus grammocephalus TR16 and expression in Pichia pastoris

• Published in: Letters in applied microbiology Vol. 52; no. 3; pp. 290 - 297
• Main Authors: Huang, S.J, Liu, Z.M, Huang, X.L, Guo, L.Q, Lin, J.-F
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.03.2011; Blackwell
• Subjects: Amino Acid Sequence; anchored PCR; Biological and medical sciences; Cloning, Molecular; DNA, Complementary - genetics; DNA, Complementary - metabolism; DNA, Fungal - genetics; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; heterologous expression; Laccase - genetics; Laccase - metabolism; laccase gene; Microbiology; molecular cloning; Molecular Sequence Data; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Polymerase Chain Reaction; Polyporus; Polyporus - enzymology; Polyporus - genetics; Protein Sorting Signals - genetics; Transformation, Genetic; Ascomycota; Enzyme; Applied microbiology; anchored PCR; heterologous expression; Pichia pastoris; Gene expression; Characterization; Fungi; Polymerase chain reaction; Gene; laccase gene; Oxidoreductases; Molecular cloning; Laccase
• ISSN: 0266-8254; 1472-765X
• DOI: 10.1111/j.1472-765X.2010.02997.x

Aims: To isolate a novel laccase gene from white-rot fungus Polyporus grammocephalus TR16 and heterologous expression in Pichia pastoris. The characteristics of the heterologously expressed laccase are also studied. Methods and Results: Anchored PCR and 3′ RACE protocol were applied to obtain the full length of the laccase gene, which comprised 12 introns and an opening frame of 1769 bp. The deduced amino acid sequence of the laccase gene had an identity of 45-66% with the laccases reported previously. The cDNA was expressed in Pi. pastoris GS115 with native and α-factor secretion signal peptides. The laccase activity obtained with the native signal peptide is threefold higher than that obtained with the α-factor secretion signal peptide. The highest activity of the heterologously expressed laccase reached 893·3 U ml⁻¹, with its molecular mass estimated to be 65·4 kDa by SDS-PAGE. The purified heterologously expressed laccase was stable at a pH range of 7·0-10·0. The optimum pH and temperature were 4·5 and 50°C, respectively; the Km value for ABTS (3-ethylbenzthiazoline-6-sulphonate) was 0·66 mmol l⁻¹. Conclusion: The novel laccase gene is cloned successfully and heterologously expressed in Pi. pastoris. Significance and Impact of the Study: A novel laccase gene isolated from a tropical fungus serves as a good source for pulp bleaching and wood processing.