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. 2019 Jan;41(1):159-169. doi: 10.1007/s10529-018-2625-1. Epub 2018 Nov 16.

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: Biodiesel; Inclusion body; Lipase; Proteus; Refolding.

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Biofuels*
  • Escherichia coli* / genetics
  • Escherichia coli* / metabolism
  • Inclusion Bodies / enzymology
  • Inclusion Bodies / genetics
  • Lipase / biosynthesis
  • Lipase / chemistry
  • Lipase / genetics
  • Mutagenesis, Site-Directed
  • Protein Refolding*
  • Proteus* / enzymology
  • Proteus* / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics

Substances

  • Bacterial Proteins
  • Biofuels
  • Recombinant Proteins
  • Lipase