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首页> 外文期刊>Bioprocess and Biosystems Engineering >High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution
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High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution

机译：淀粉芽孢杆菌漆酶的高表达及其通过结构域取代提高稳定性的嵌合变体的构建

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摘要

Large-scale application of bacterial laccases is usually limited by their low production, and their recombinant expression in Escherichia coli is prone to form inactive aggregates in the cytoplasm. In this work, we optimized the expression conditions of Bacillus amyloliquefaciens laccase (LacA) in E. coli, and obtained high yield for the extracellular production of LacA. The final activity reached 20,255 U/L for LacA, which is among one of the highest activities for recombinant bacterial laccases. Moreover, a chimeric enzyme (Lac3A/S) was designed based on LacA by domain substitution with a stable laccase from B. subtilis. The hybrid laccase could also be secreted into the culture medium with high expression level, and had higher thermal and alkaline stabilities than those of LacA. It was fully active after 10-day incubation at pH 9.0, and retained 47% of its initial activity after incubation at 70 degrees C for 5 h. Homology analysis of protein structure indicated Lac3A/S had a more packed structure in the copper-binding sites than LacA, which might lead to an enhancement in stability under harsh conditions.
机译：细菌漆酶的大规模应用通常受到其低产量的限制，并且它们在大肠杆菌中的重组表达易于在细胞质中形成无活性的聚集体。在这项工作中，我们优化了解淀粉芽孢杆菌漆酶（LacA）在大肠杆菌中的表达条件，并获得了细胞外生产LacA的高产量。 LacA的最终活性达到20,255 U / L，是重组细菌漆酶的最高活性之一。此外，通过用来自枯草芽孢杆菌的稳定漆酶进行结构域取代，基于LacA设计了嵌合酶（Lac3A / S）。杂漆酶还可以分泌到高表达水平的培养基中，并且具有比LacA更高的热稳定性和碱性稳定性。在pH 9.0下孵育10天后，它具有完全活性，并在70摄氏度下孵育5小时后，保留了其初始活性的47％。蛋白质结构的同源性分析表明，Lac3A / S在铜结合位点比LacA具有更多的堆积结构，这可能导致在苛刻条件下稳定性增强。

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