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Biotechnology Letters

, Volume 41, Issue 1, pp 159–169 | Cite as

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

  • Hua Shao
  • Xianmei Hu
  • Liping Sun
  • Wenshan Zhou
Original Research Paper
  • 112 Downloads

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.

Keywords

Proteus Lipase Inclusion body Refolding Biodiesel 

Notes

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

Compliance with ethical standards

Conflict of interest

The authors declares no financial or commercial conflict of interest.

Supplementary material

10529_2018_2625_MOESM1_ESM.doc (2.2 mb)
Supplementary material 1 (DOC 2285 kb)

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Hua Shao
    • 1
  • Xianmei Hu
    • 1
  • Liping Sun
    • 1
  • Wenshan Zhou
    • 1
  1. 1.School of Food and BioengineeringZhengzhou University of Light IndustryZhengzhouPeople’s Republic of China

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