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Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization

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Abstract

The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l−1) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (<100 units l−1) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the “blue copper oxidases”. The enzyme’s pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20°C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40°C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined (Km, kcat): 63 μM, 21 s−1 for ABTS, 4 μM, 5 s−1 for syringaldazine, 1,026 μM, 15 s−1 for DMP and 4307 μM, 159 s−1 for guaiacol. The results show that—in addition to the wood-colonizing white-rot fungi—the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.

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Acknowledgements

This work was supported financially by the German Federation of Industrial Cooperative Research Associations “Otto von Guericke” (AiF), the German Academic Exchange Service (DAAD, for L.M.H.), the Academy of Finland (grant 52063, for M.H.) and the administration of the International Graduate School Zittau (R. Konschak, Zittau, Germany). We thank U. Schneider (Zittau) for excellent technical assistance.

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Authors and Affiliations

  1. Unit of Environmental Biotechnology, International Graduate School of Zittau, Markt 23, Zittau, 02763, Germany

    René Ullrich & Martin Hofrichter

  2. Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

    Le Mai Huong

  3. Vietnam Type Culture Collection, Department of Biotechnology, National University of Hanoi, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

    Nguyen Lan Dung

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  1. René Ullrich
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  2. Le Mai Huong
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  3. Nguyen Lan Dung
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  4. Martin Hofrichter
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Correspondence to René Ullrich.

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Ullrich, R., Huong, L.M., Dung, N.L. et al. Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization. Appl Microbiol Biotechnol 67, 357–363 (2005). https://doi.org/10.1007/s00253-004-1861-6

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  • DOI: https://doi.org/10.1007/s00253-004-1861-6

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