Improving the decolorization for textile dyes of a metagenome-derived alkaline laccase by directed evolution
Improving the decolorization for textile dyes of a metagenome-derived alkaline laccase by directed evolution
复制标题
通过定向进化改善宏基因组衍生的碱性漆酶的纺织染料脱色
DOI：
10.1007/s00253-011-3292-5
复制
发表时间：
2011-08-01
影响因子：
5
通讯作者：
Li, Gang
中科院分区：
文献类型：
--
作者：
Liu, Yu Huan;Ye, Mao;Li, Gang
为了获得性能较好的染料脱色漆酶，对宏基因组衍生的碱性漆酶Lac591进行了随机诱变。经过三轮容易出错的PCR和测定对酚类底物2,6-二甲氧基苯酚（2,6- dmp）酶活性的高通量筛选，获得了酶活性显著提高的突变体Lac3T93。序列分析显示，Lac3T93中积累了N40S、V55A、F62L和E316V 4个氨基酸取代位点。与野生型酶相比，Lac3T93对2,6- dmp的比活性提高了4.8倍（61.22 U/mg），其最适温度和pH分别由野生型酶的55℃和7.5℃改变为60℃和8.0℃。此外，研究了Lac3T93对纺织染料的降解能力，发现该酶对4种苯基结构复杂的工业染料（碱性蓝3、亚甲基蓝、溴酚蓝和结晶紫）的脱色率有所提高，对靛蓝胭脂红的脱色率也比亲本酶高。在不添加羟基苯并三唑（hydroxybenzotrizole， HBT）的情况下，Lac3T93对5种染料的脱色率明显高于添加1 mM HBT的野生型酶，HBT可以进一步提高其脱色能力。
To obtain better performing laccases for textile dyes decolorization, random mutagenesis of Lac591, a metagenome-derived alkaline laccase, was carried out. After three rounds of error-prone PCR and high-throughput screening by assaying enzymatic activity toward the phenolic substrate 2,6-dimethoxyphenol (2,6-DMP), a mutant (Lac3T93) with remarkably improved enzymatic activity was obtained. Sequence analysis revealed that four amino acid substitutions (N40S, V55A, F62L, and E316V) were accumulated in the Lac3T93. Compared to the wild-type enzyme, the specific activity of Lac3T93 toward 2,6-DMP was increased to 4.8-fold (61.22 U/mg), and its optimal temperature and pH were changed to 60A degrees C and 8.0 from 55A degrees C and 7.5 of the wild-type enzyme, respectively. Furthermore, the degradation ability of Lac3T93 for textile dyes was investigated, and the new variant represented improved decolorization percentage for four industrial dyes with complex phenyl structure (Basic Blue 3, Methylene Blue, Bromophenol Blue, and Crystal Violet) and higher decolorization efficiency for Indigo Carmine than that of the parent enzyme. Furthermore, the decolorization percentage of Lac3T93 for five dyes in the absence of hydroxybenzotrizole (HBT) is clearly higher than those of the wild-type enzyme with 1 mM HBT, and HBT can further improve its decolorization ability.