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High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution

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Abstract

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 °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.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31200394), the Natural Science Foundation of Heilongjiang Province of China (C2017010) and the Fundamental Research Funds for the Central Universities (2572017CA22).

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Authors and Affiliations

  1. College of Life Sciences, Northeast Forestry University, No. 26, Hexing Road, Harbin, 150040, China

    Jiayi Wang, Xiaoyan Li, Fujuan Feng & Lei Lu

  2. College of Water Conservancy and Civil Engineering, Inner Mongolia Agricultural University, Hohhot, 010018, China

    Shuyu Yu

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  1. Jiayi Wang
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Wang, J., Yu, S., Li, X. et al. High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution. Bioprocess Biosyst Eng 43, 403–411 (2020). https://doi.org/10.1007/s00449-019-02236-0

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