Abstract
Objective
To obtain active lipases for biodiesel production by refolding Proteus sp. lipase inclusion bodies expressed in E. coli.
Results
A lipase gene lipPN1 was cloned from Proteus sp. NH 2-2 and expressed in E. coli BL21(DE3). Non-reducing SDS-PAGE revealed that recombinant LipPN1(rLipPN1) were prone to form inclusion bodies as disulfide-linked dimers in E. coli. Site-directed mutagenesis confirmed that Cys85 in LipPN1 was involved in the dimer formation. After optimizing the inclusion body refolding conditions, the maximum lipase activity reached 1662 U/L. The refolded rLipPN1 exhibited highest activity toward p-nitrophenyl butyrate at pH 9.0 and 40 °C. It could be activated by Ca2+ with moderate tolerance to organic solvents. It could also convert soybean oil into biodiesel at a conversion ratio of 91.5%.
Conclusion
Preventing the formation of disulfide bond could enhance the refolding efficiency of rLipPN1 inclusion bodies.
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Acknowledgements
The authors acknowledge the financial support of the National Natural Science Foundation of P. R. China (NSFC No. 31501420 and No. 31500442) and the Doctoral Scientific Research Foundation of Zhengzhou University of Light Industry (No. 13501050027).
Supporting information
Supplementary Table 1–Primers used in this study
Supplementary Fig. 1–A lipase-producing strain isolated from a sediment sample collected from the South China Sea
Supplementary Fig. 2–a Effect of temperature on rLipPN1 expression; b Effect of the concentration of IPTG on rLipPN1 expression
Supplementary Fig. 3–a The refolding of rLipPN1 inclusion bodies in different buffers; b The refolding of rLipPN1_C85S inclusion bodies in different buffers
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Shao, H., Hu, X., Sun, L. et al. Gene cloning, expression in E. coli, and in vitro refolding of a lipase from Proteus sp. NH 2-2 and its application for biodiesel production. Biotechnol Lett 41, 159–169 (2019). https://doi.org/10.1007/s10529-018-2625-1
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DOI: https://doi.org/10.1007/s10529-018-2625-1