Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies

Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan Correspondence Masaru Nagai nagai{at}ibrc.or.jp A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58·0 kDa. The enzyme had an isoelectric point of around pH 6·9. The optimum pH for enzyme activity was around 3·0 against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 °C and stable up to 50 °C. The enzyme contained 8·6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p -phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. -(3,4-Dihydroxyphenyl)alanine ( L -DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes , was also oxidized by Lcc 2, and the oxidative product of L -dopa was identified as L -DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain. Abbreviations: ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt; DHN, dihydroxynaphthalene; L -DOPA, -(3,4-dihydroxyphenyl)alanine; GDHB, -glutaminyl-3,4-dihydroxybenzene; GHB, -glutaminyl-4-hydroxybenzene; Lcc, laccase; Tyr, tyrosinase; PB, 10mM sodium phosphate buffer