Laboratorio de Biotecnología Molecular, Instituto de Biotecnología Misiones, Facultad de Ciencias Exactas Químicas y Naturales, Universidad Nacional de Misiones, Posadas, Misiones, Argentina.
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Rosario, Argentina.
J Appl Microbiol. 2018 Jun;124(6):1454-1468. doi: 10.1111/jam.13720. Epub 2018 Mar 23.
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.
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. K and highest k /K 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.
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.
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.
从栓菌（Phlebia brevispora BAFC 633）中分离和鉴定漆酶编码基因（lac I），并在巴斯德毕赤酵母（Pichia pastoris）中克隆和表达 lac I 的 cDNA。获得纯化和鉴定的重组漆酶，以分析其生物技术应用潜力。
通过 PCR 和长距离反向 PCR 克隆和测序了 lac I，它包含 2447 pb。在漆酶基因的结构区域上游，发现了金属、抗氧化剂、铜、氮和热休克等响应元件。编码区由一个编码 521 个氨基酸的 1563 pb ORF 组成。lac I 在巴斯德毕赤酵母中得到了功能表达，结果表明，使用α因子信号肽克隆的基因比天然信号序列更有效地指导重组蛋白的分泌。Lac I 对 ABTS、2,6-二甲基苯酚的 K 和最高 k / K 值与其他漆酶相似。Lac I 对 NaCl 和溶剂具有耐受性，能不同程度地降解 9 种合成染料。
成功测序了漆酶编码基因，发现其调控区存在顺式作用元件。使用α因子信号肽表达的 lac I cDNA 比天然信号序列更有效。纯化的 Lac I 对 NaCl 和各种溶剂具有较高的耐受性，并能降解一些难处理的合成染料。
顺式作用元件可能参与了漆酶基因表达的转录调控。这些结果可能为优化发酵工艺提供进一步的思路，也为工程化强启动子生产漆酶开辟了新的途径。Lac I 在氯化物和溶剂中的稳定性以及对合成染料的广泛脱色对其在有机合成工作中的应用和降解纺织废水染料分别具有重要意义。
