PROIMI   05436
PLANTA PILOTO DE PROCESOS INDUSTRIALES MICROBIOLOGICOS
Unidad Ejecutora - UE
artículos
Título:
Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris
Autor/es:
WINNIK, D.L.; VILLALBA, L.L.; MOLINA, M.A.; FARIÑA, J.I.; FONSECA, M.I.; BUSI, M.V.; ZAPATA, P.D.
Revista:
JOURNAL OF APPLIED MICROBIOLOGY
Editorial:
WILEY-BLACKWELL PUBLISHING, INC
Referencias:
Año: 2018 vol. 124 p. 1454 - 1468
ISSN:
1364-5072
Resumen:
Aims: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. Methods and Results: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat/Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. Conclusions: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. Significance and Impact of the Study: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.