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

  • Jiayi Wang
  • Shuyu Yu
  • Xiaoyan Li
  • Fujuan Feng
  • Lei LuEmail author
Research Paper
  • 36 Downloads

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.

Keywords

Bacterial laccase Extracellular expression Domain substitution Chimeric enzyme 

Notes

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

449_2019_2236_MOESM1_ESM.docx (2.1 mb)
Supplementary file1 (DOCX 2195 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jiayi Wang
    • 1
  • Shuyu Yu
    • 2
  • Xiaoyan Li
    • 1
  • Fujuan Feng
    • 1
  • Lei Lu
    • 1
    Email author
  1. 1.College of Life SciencesNortheast Forestry UniversityHarbinChina
  2. 2.College of Water Conservancy and Civil EngineeringInner Mongolia Agricultural UniversityHohhotChina

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