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.
Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising b-1,3-glucan with b-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited b-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.
In this paper, we propose a multicriteria decision making (MCDM) method by using a genetic algorithm (GA). The system consists of three phases. In the first phase, a rough set of Pareto optimal solutions is obtained using Kohonen's self organizing map (SOM). In the second phase, the decision maker (DM) selects his preferred sclutions among the obtained set, where the mechanism of GA is used with the DM's preference assisted by radial basis function network (RBFN). In the third phase, the DM can explore the solution space further for the final decision. 0-7803-2559-1/9 § $4.00 0 1995 IEEE
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