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Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris

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Abstract

Laccases are strong oxidizing enzymes that oxidize chlorinated phenols, synthetic dyes, pesticides, polycyclic aromatic hydrocarbons as well as a very wide range of other compounds with high redox potential. Based on the bias of genetic codons between fungus and yeast, we synthesized a laccase gene GlLCCI, originated from Ganoderma lucidum using optimized codons and a PCR-based two-step DNA synthesis method. The recombinant laccase, GlLCCI was successfully over-expressed in yeast, Pichia pastoris, with an alcohol oxidase1 promoter. The recombinant GlLCCI has a molecular mass of approximately 58 kDa. The K m values of GlLCCI for 2-2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol were 0.9665, and 1.1122 mM, respectively. The V max of GlLCCI for both substrates was 3,024 and 82.13 μM mg−1 min−1. When ABTS was used as a substrate, the enzyme had an optimal temperature of approximately 55°C. The enzyme was detected over pH values from 2 to 8. The enzyme was strongly activated by K+, Na+, Cu2+ and mannitol. Six amino acids (alanine, histidine, glycine, arginine, aspartate and phenylalanine) increased the catalytic ability of the enzyme. The activity of laccase was obviously inhibited by Fe2+, Fe3+, sodium hydrosulphite, and sodium azide. Additionally, under optimal conditions, GlLCCI decolorized 37.62 mg l−1 of azo dye methyl orange (MO) in cultural medium. With a high MO degradation ability, GlLCCI may have potential in the treatment of industrial effluent containing azo dye MO.

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Acknowledgments

This research study was supported with funding from the Science and Technology Commission of Shanghai Municipality (09dz2200800). Another financial support was the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, 225009, China

    Jing Sun, Hu Xu, Da-Tong Liu & Jian-Min Chen

  2. Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnological Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China

    Jing Sun, Ri-He Peng, Ai-Sheng Xiong, Yongsheng Tian, Wei Zhao & Quan-Hong Yao

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  1. Jing Sun
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  2. Ri-He Peng
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Correspondence to Jian-Min Chen or Quan-Hong Yao.

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Sun, J., Peng, RH., Xiong, AS. et al. Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris . Mol Biol Rep 39, 3807–3814 (2012). https://doi.org/10.1007/s11033-011-1158-7

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