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

Abstract

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 2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 6C and stable up to 50 6C. 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. b-(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.

Senescence of the Lentinula edodes Fruiting Body After Harvesting

“…We expressed the lcc4 gene heterologously in Aspergillus oryzae, and observed Lcc activity of the recombinant enzyme (Yano et al 2009). These data suggest that lcc4 encodes Lcc2, as designated by Nagai et al (2003), and the gene lcc2 (Accession No. AB035410) does not.…”

“…Lcc in Aspergillus nidulans is considered to form L-DOPA to synthesize melanins in conidia (Aramayo and Timberlake, 1990). Activity of Lcc increases after harvest of the L. edodes fruiting body, and a Lcc in L. edodes purified from fruiting bodies after harvest can oxidize DOPA (Nagai et al, 2003). Therefore, it is considered that Tyr and Lcc are involved in melanin synthesis after harvest of L. edodes fruiting bodies.…”

“…Lcc1 is secreted from vegetative mycelia in culture (Nagai et al, 2002) and is encoded by lcc1 (Sakamoto et al, 2008). Lcc2 is expressed in the brown gills of fruiting bodies after harvesting (Nagai et al, 2003). The putative amino acid sequence of lcc4 includes identical amino acid sequences to the N-terminal amino acid sequence of an enzymatically digested peptide of Lcc2 .…”

“…However, postharvest spoilage, such as browning of the gills and softening of the fruiting body, results in loss of freshness and consequent loss of food value 1980a, b). Numerous studies addressing the mechanisms of quality loss during postharvest storage have revealed that browning of the L. edodes fruiting body is associated with increased activities of tyrosinase (Tyr; Kanda et al, 1996a, b) and laccase (Lcc; Nagai et al, 2003) after harvest. In addition to being an important fresh food source, shiitake mushrooms also have medicinal value.…”

Gene expression and metabolite changes during Tuber magnatum fruiting body storage

“…The regulation of TmaDHN1, which shows homology with T. borchii TbDHN1 and T. melanosporum TmelDHN1, which were previously observed to be differentially expressed during the reproductive stage as well as during the vegetative stage under stress conditions (Abbà et al 2006;Zampieri et al 2011), can be explained by the protective role of such a protein during cellular dehydration. Since tyrosinase and laccase activities have been shown to increase in L. edodes after harvest (Kanda et al 1996;Nagai et al 2003), T. magnatum sequences that code for a putative tyrosinase and laccase, which are involved in monophenols and polyphenols oxidation that can result in melanin synthesis, have been considered. Tyrosinase activity has also been reported in white truffle fruiting bodies, including T. magnatum (Miranda et al 1996).…”