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首页> 外文期刊>Biomolecular engineering >Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro.
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Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro.

机译：甲烷磺酸酯基随机诱变在野木杆漆酶的分子进化。

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摘要

In order to improve the laccase activity, mutant libraries are constructed through ethyl methane sulfonate-based (EMS) random mutagenesis. Mutagenesis improved expression 3.7-fold to 144mgl(-1) laccase in yeast, together with a 1.4-fold increase in K(cat). Thus, the total activity is enhanced 5-fold for 2,2'-azino-bis 3-ethylbenzothiaoline-6-sulfonic acid (ABTS). In the presence of 0.6mM copper, the highest activity value reached 30Uml(-1) after a 3-day cultivation at a temperature of 30 degrees C(.) In comparison with the wild type, the best mutant enzymatic properties (K(m) for ABTS and guaiacol, thermo- and pH stability, optimal pH) are not changed. Moreover, amino acid sequence analysis indicates that there are four substitutions in the best mutant laccase (Gly160Asp, Ala167Thr, Gly174Asp, and Glu234Gly). The best mutant laccase model showed that the Gly160 and Ala167 are to be found near the water channel; especially the distance of Ala167 to the Cu3a is 14.46A. This implies that it is likely involved in the formation of water channel and that it helps facilitate the easy incoming and outgoing of water.
机译：为了提高漆酶活性，通过基于甲基磺酸乙酯（EMS）的随机诱变来构建突变体文库。诱变使酵母中的表达提高了3.7倍，至144mgl（-1）漆酶，而K（cat）的表达提高了1.4倍。因此，对于2，2'-叠氮基双3-乙基苯并噻吩啉-6-磺酸（ABTS），总活性提高了5倍。在存在0.6mM铜的情况下，在30摄氏度的温度下进行3天培养后，最高活性值达到30Uml（-1）。与野生型相比，最佳的突变型酶促特性（K（m ）对于ABTS和愈创木酚，热和pH稳定性，最佳pH不变。此外，氨基酸序列分析表明，在最佳突变漆酶（Gly160Asp，Ala167Thr，Gly174Asp和Glu234Gly）中存在四个取代。最佳的漆酶突变模型表明，在水道附近发现了Gly160和Ala167。特别是Ala167到Cu3a的距离是14.46A。这意味着它可能参与了水通道的形成，并有助于促进水的容易进出。

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