CONICET | Buscador de Institutos y Recursos Humanos

Abstract AimsIsolate 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 analyze the biotechnological application potential.Methods and Resultslac 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 in a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in Pichia 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 solvent and nine synthetic dyes could be degraded to different degrees. ConclusionsLac 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 a NaCl and various solvents and degraded some recalcitrant synthetic dyes.Significance and Impact of StudyThe 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.

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