title: Characterization and cloning of laccase gene from Hericium coralloides NBRC 7716 suitable for production of epitheaflagallin 3-O-gallate.
abstract: Epitheaflagallin 3-O-gallate (ETFGg) is a minor polyphenol found in black tea extract, which has good physiological functions. It is synthesized from epigallocatechin gallate (EGCg) with gallic acid via laccase oxidation. Various basidiomycetes and fungi were screened to find a suitable laccase for the production of ETFGg. A basidiomycete, Hericium coralloides NBRC 7716, produced an appropriate extracellular laccase. The purified laccase produced twice the level of ETFGg compared with commercially available laccase from Trametes sp. The enzyme, termed Lcc2, is a monomeric protein with an apparent molecular mass of 67.2 kDa. The N-terminal amino acid sequence of Lcc2 is quite different from laccase isolated from the fruiting bodies of Hericium. Lcc2 showed similar substrate specificity to known laccases and could oxidize various phenolic substrates, including pyrogallol, gallic acid, and 2,6-dimethoxyphenol. The full-length lcc2 gene was obtained by PCR using degenerate primers, which were designed based on the N-terminal amino acid sequence of Lcc2 and conserved copper-binding sites of laccases, and 5'-, and 3'-RACE PCR with mRNA. The Lcc2 gene showed homology with Lentinula edodes laccase (sharing 77% amino acid identity with Lcc6). We successfully produced extracellular Lcc2 using a heterologous expression system with Saccharomyces cerevisiae. Moreover, it was confirmed that the recombinant laccase generates similar levels of ETFGg as the native enzyme.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan. Electronic address: nbito@pu-toyama.ac.jp.
authors.forename: Nobuya
authors.initials: N
authors.lastname: Itoh
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Takagi
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Miki
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Kurokawa
mesh_terms: D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D001567:Benzocycloheptenes; D002392:Catechin / Q000031:analogs & derivatives / Q000378:metabolism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005707:Gallic Acid / Q000378:metabolism; D005800:Genes, Fungal; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010636:Phenols / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000235:genetics; D059808:Polyphenols / Q000096:biosynthesis*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
chemical_list: D001567:Benzocycloheptenes; D005656:Fungal Proteins; D010636:Phenols; D059808:Polyphenols; D011993:Recombinant Fusion Proteins; C000611430:epitheaflagallin 3-O-gallate; D005707:Gallic Acid; D002392:Catechin; C045651:epigallocatechin gallate; D042845:Laccase; D010735:Phosphoglycerate Kinase
keywords: Enzymatic synthesis; Epitheaflagallin 3-O-gallate (ETFGg); Hericium coralloides NBRC 7716; Laccase lcc2