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International Journal of Medicinal Mushrooms
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ISSN Imprimer: 1521-9437
ISSN En ligne: 1940-4344

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International Journal of Medicinal Mushrooms

DOI: 10.1615/IntJMedMushr.v8.i1.90
pages 67-76

Cloning and Characterization of Polyphenoloxidase DNA from Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae)

Akiko Matsumoto-Akanuma
School of Pharmacy, Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Akihiko Yamagishi
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Masuro Motoi
Toei Pharmaceutical Co. Ltd., 2-5-3 Iguchi, Mitaka, Tokyo 181-0011; Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan
Naohito Ohno
Tokyo University of Pharmacy and Life Sciences School of Pharmacy, Lab Immunopharmacological and Microbiological Production, 1432-1 Horinouchi, Hachioji Tokyo, 192-0392, Japan

RÉSUMÉ

Polyphenoloxidases (PPOs), including tyrosinases and laccases, are ubiquitous metalloenzymes, which have several copper-binding sites. The enzymes cause undesirable browning in vegetables, mushrooms, and fruits, and melanin production of animals. Agaricus brasiliensis is an edible mushroom whose water extracts show biological response modifier effects. Since cold-water extracts of the mushroom fruiting body turn brown on incubation, it has been speculated that the mushroom contains significant amounts of PPOs. Our group has already shown that cold-water extracts indicated PPO activity. Therefore, we cloned a cDNA coding for a PPO from A. brasiliensis cultivated outside in the field. First, a partial sequence of the PPO gene was amplified by reverse transcription (RT)-PCR from the total RNA isolated from A. brasiliensis. Since two copper-binding motifs are well conserved among the PPO amino acid sequences from fungi, two degenerate primers used for the RT-PCR were designed and synthesized based on these sequences. The nucleotide sequence of the amplified fragment showed 83% identity with the corresponding sequence of the A. bisporus PPO gene. Full-length cDNA of the PPO gene was amplified from a cDNA library of A. brasiliensis. Obtained DNA sequences indicated that the amplified cDNA contained an open reading frame of 1731 nucleotides that encoded 576 amino acid residues. Homology search analysis showed that the deduced amino acid sequence was most similar to that of A. bisporus PPO, with 75% identity. The A. brasiliensisfull-length PPO gene was also amplified from the genomic DNA and then se-quenced. Five introns were found within the gene.