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International Journal of Medicinal Mushrooms
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ISSN Druckformat: 1521-9437
ISSN Online: 1940-4344

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International Journal of Medicinal Mushrooms

DOI: 10.1615/IntJMedMushrooms.v16.i4.80
pages 375-393

Cloning and Characterization of Laccase DNA from the Royal Sun Medicinal Mushroom, Agaricus brasiliensis (Higher Basidiomycetes)

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
Satoshi Akanuma
Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 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
Akihiko Yamagishi
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Naohito Ohno
Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

ABSTRAKT

Laccase isozymes have been identified in several fungi. We report the cloning of 4 laccase genes from the medicinal mushroom Agaricus brasiliensis. The lac1 gene contained a 1560-base pair (bp) open reading frame (ORF) encoding 520 amino acids that was interrupted with 14 introns in genomic DNA. The deduced amino acid sequence indicated a multicopper oxidase signature 1 and 2 multicopper oxidase signature 2. The lac2 gene contained a 1566-bp ORF encoding 522 amino acids that was interrupted with 13 introns in genomic DNA. A number of different nucleotides were observed in whole regions containing the substitution of amino acid residues (lac2a and lac2b). The partial DNA fragments of lac3 and lac4 genes were subcloned using the semi-random two-step polymerase chain reaction method. The lac3 and lac4 genes contained coding sequences with a 1575-bp ORF encoding 525 amino acids and a 1584-bp ORF encoding 528 amino acids, respectively. However, the whole complementary DNA fragment of both laccases could not be amplified with polymerase chain reaction against the complementary DNA library; therefore, introns were deduced based on the GT-AG rule and multiple alignment of laccases from other fungi, which showed high identity. All laccases from A. brasiliensis conserved the fungal laccase signature sequence and suggest 2 subfamilies according to the location of introns and phylogenetic analysis. The genes lac2 and lac4 had a high degree of identity, and the lac2a gene was located upstream of the lac4 gene.


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