Department of Biological Engineering, Nanjing Forestry University, Nanjing, 210037, Jiangsu, People's Republic of China.
Mol Biol Rep. 2013 Feb;40(2):1927-36. doi: 10.1007/s11033-012-2249-9. Epub 2012 Oct 18.
Two new laccase genes, named lac1 and lac2, were cloned from the edible basidiomycete Coprinus comatus. Comparison of the deduced amino acid sequences revealed two laccases showed 66.12 % identity and clustered with lac2 and lac3 from Coprinopsis cinerea in same phylogenetic group. Lac1 and lac2 encode proteins of 517 and 523 amino acids preceded by 18 and 21-residue signal peptides, respectively. Lac1 was functionally expressed in Pichia pastoris. The optimum pHs of recombinant Lac1 were 3.0, 6.0, 5.5 and 6.0 and the optimum temperatures were 65, 55, 70 and 50 °C for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The Km values of Lac1 were 34, 4,317, 7,611 and 14 μM, and the corresponding kcat values were 465.79, 7.67, 1.15 and 0.60 (s(-1) mM), for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The enzyme activity was completely inhibited by sodium azide (NaN(3)) and 1,4-dithiothreitol (DTT) at the concentration of 5 mM. Laccase activity was also inhibited by several metal ions, especially Fe(2+), while K(+) and NH(4) (+) slightly enhanced laccase activity. Twelve synthetic dyes belonging to anthraquinone, azo and triphenylmethane dyes were decolorized by the recombinant Lac1 at different extents. The recombinant Lac1 decolorized azo dye Reactive Dark Blue KR up to 90 % without any mediator and increasing to 96 % with mediator, indicating its potential in the treatment of industrial effluent containing some recalcitrant synthetic dyes.
两个新的漆酶基因，命名为 lac1 和 lac2，从可食用担子菌蘑菇 Coprinus comatus 中克隆得到。推导的氨基酸序列比较表明，两种漆酶显示出 66.12%的同一性，并与来自 Coprinopsis cinerea 的 lac2 和 lac3 聚类在同一进化群中。lac1 和 lac2 分别编码 517 和 523 个氨基酸的蛋白质，前面分别有 18 和 21 个残基的信号肽。Lac1 在巴斯德毕赤酵母中得到功能表达。重组 Lac1 的最适 pH 值分别为 3.0、6.0、5.5 和 6.0，最适温度分别为 65、55、70 和 50°C，用于 ABTS、愈创木酚、2,6-二甲基苯酚和桑色素。Lac1 的 Km 值分别为 34、4、317、7、611 和 14μM，相应的 kcat 值分别为 465.79、7.67、1.15 和 0.60（s(-1)mM），用于 ABTS、愈创木酚、2,6-二甲基苯酚和桑色素。该酶的活性完全被 5mM 浓度的叠氮化钠（NaN(3)）和 1,4-二硫苏糖醇（DTT）抑制。几种金属离子，特别是 Fe(2+)，也抑制漆酶的活性，而 K(+)和 NH(4) (+) 则轻微增强漆酶的活性。十二种属于蒽醌、偶氮和三苯甲烷染料的合成染料在不同程度上被重组 Lac1 脱色。重组 Lac1 在没有任何介体的情况下可将偶氮染料 Reactive Dark Blue KR 脱色至 90%，而在有介体的情况下可将其脱色至 96%，表明其在处理含有某些难处理合成染料的工业废水方面具有潜力。
