High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution
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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.
KeywordsBacterial laccase Extracellular expression Domain substitution Chimeric enzyme
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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Conflict of interest
The authors declare that they have no conflict of interest.
- 10.Wang TN, Lu L, Wang JY, Xu TF, Li J, Zhao M (2015) Enhanced expression of an industry applicable CotA laccase from Bacillus subtilis in Pichia pastoris by non-repressing carbon sources together with pH adjustment: recombinant enzyme characterization and dye decolorization. Process Biochem 50:97–103CrossRefGoogle Scholar
- 15.Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, Henriques AO (2002) Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277:18849–18859CrossRefPubMedCentralGoogle Scholar