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



To obtain active lipases for biodiesel production by refolding Proteus sp. lipase inclusion bodies expressed in E. coli.


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%.


Preventing the formation of disulfide bond could enhance the refolding efficiency of rLipPN1 inclusion bodies.


Proteus Lipase Inclusion body Refolding Biodiesel 



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)


  1. Alnoch RC, Stefanello AA, Martini VP, Richter JL (2018) Co-expression, purification and characterization of the lipase and foldase of Burkholderia contaminans LTEB11. Int J Biol Macromol 116:1222–1231CrossRefGoogle Scholar
  2. Gao B, Su E, Lin J, Jiang Z, Ma Y, Wei D (2009) Development of recombinant Eshcerichia coli whole-cell biocatalyst expressing a novel alkaline lipase-coding gene from Proteus sp. for biodiesel production. J Biotechnol 139:169–175CrossRefGoogle Scholar
  3. Gupta S, Scott D, Prabha CR, Ashokkumar M (2017) Biodiesel synthesis assisted by ultrasonication using engineered thermo-stable Proteus vulgaris lipase. Fuel 208:430–438CrossRefGoogle Scholar
  4. Korman TP, Bowie JU (2012) Crystal structure of Proteus mirabilis lipase, a novel lipase from the Proteus/Psychrophilic subfamily of lipase family I.1. PLoS ONE 7:e52890CrossRefGoogle Scholar
  5. Korman TP, Sahachartsiri B, Charbonneau DM, Huang GL, Beauregard M, Bowie JU (2013) Dieselzymes: development of a stable and methanol tolerant lipase for biodiesel production by directed evolution. Biotech For Biofuels 6:70CrossRefGoogle Scholar
  6. Li S, Pang H, Lin K, Xu J, Zhao J, Fan L (2011a) Refolding, purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010. J Mol Catal B-Enzym 71:171–176CrossRefGoogle Scholar
  7. Li S, Zhang L, Wu Q, Xin A, Zhao J, Fan L (2011b) Increasing the refolding efficiency in vitro by site-directed mutagenesis of Cys383 in rat procarboxypeptidase B. Enzyme Microb Tech 49:139–145CrossRefGoogle Scholar
  8. Li K, Fan Y, He Y, Zeng L, Han X, Yan Y (2017) Burkholderia cepacia lipase immobilized on heterofunctional magnetic nanoparticles and its application in biodiesel synthesis. Sci Rep 7:16473CrossRefGoogle Scholar
  9. Lu Y, Lin Q, Wang J, Wu Y, Bao W, Lv F, Lu Z (2010) Overexpression and characterization in Bacillus subtilis of a positionally nonspecific lipase from Proteus vulgaris. J Ind Microbiol Biot 37:919–925CrossRefGoogle Scholar
  10. Natalia A, Kristiani L, Kim HK (2014) Characterization of Proteus vulgaris K80 lipase immobilized on amine-terminated magnetic micropariticles. J Microbiol Biotechnol 24:1382–1388CrossRefGoogle Scholar
  11. Peciak K, Tommasi R, Choi J, Brocchini S, Laurine E (2014) Expression of soluble and active interferon consensus in SUMO fusion expression system in E. coli. Protein Expres Purif 99:18–26CrossRefGoogle Scholar
  12. Quaas B, Burmeister L, Li Z, Nimtz M, Hoffmann A, Rinas U (2018) Properties of dimeric, disulfide-linked rhBMP-2 recovered from E. coli derived inclusion bodies by mild extraction or chaotropic solubilization and subsequent refolding. Process Biochem 67:80–87CrossRefGoogle Scholar
  13. Shao H, Xu L, Yan Y (2013) Isolation and characterization of a thermostable esterase from a metagenomic library. J Ind Microbiol Biot 40:1211–1222CrossRefGoogle Scholar
  14. Whangsuk W, Sungkeeree P, Thiengmag S, Kerdwong J, Sallabhan R, Mongkolsuk S, Loprasert S (2013) Gene cloning and characterization of a novel highly organic solvent tolerant lipase from Proteus sp. SW1 and its application for biodiesel production. Mol Biotechnol 53:55–62CrossRefGoogle Scholar
  15. Yang W, He Y, Xu L, Zhang H, Yan Y (2016) A new extracellular thermo-solvent-stable lipase from Burkholderia ubonensis SL-4: identification, characterization and application for biodiesel production. J Mol Catal B-Enzym 126:76–89CrossRefGoogle Scholar

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

Personalised recommendations