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Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea

Authors

  • Shicheng Chen,

    1. Department of Biology and the Centre for International Services to Mushroom Biotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China
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  • Wei Ge,

    1. Department of Biology and the Centre for International Services to Mushroom Biotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China
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  • John A. Buswell

    1. Department of Biology and the Centre for International Services to Mushroom Biotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China
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  • Enzyme: benzenediol:oxygen oxidoreductase (EC 1.10.3.2).

J.A. Buswell, Edible Fungi Institute, 35 Nanhua Road, Shanghai 201106, China.
Fax: + 86 21 62207544, Tel.: + 86 21 62208660,
E-mail: jbuswell@saas.sh.cn

Abstract

We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 µm CuSO4, by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to ≈ 20–30% of peak levels throughout the subsequent stages of sporophore development.

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