Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization |
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Authors: | René Ullrich Le Mai Huong Nguyen Lan Dung Martin Hofrichter |
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Affiliation: | (1) Unit of Environmental Biotechnology, International Graduate School of Zittau, Markt 23, Zittau, 02763, Germany;(2) Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam;(3) Vietnam Type Culture Collection, Department of Biotechnology, National University of Hanoi, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam |
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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 M, 21 s–1 for ABTS, 4 M, 5 s–1 for syringaldazine, 1,026 M, 15 s–1 for DMP and 4307 M, 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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