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Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris.

Abstract：

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.

journal_name

J Appl Microbiol

journal_title

Journal of applied microbiology

authors

Fonseca MI,Molina MA,Winnik DL,Busi MV,Fariña JI,Villalba LL,Zapata PD

doi

10.1111/jam.13720

subject

Has Abstract

pub_date

2018-06-01 00:00:00

pages

1454-1468

issue

eissn

1364-5072

issn

1365-2672

journal_volume

124

pub_type

杂志文章

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Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris.