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Applied Microbiology and Biotechnology

, Volume 97, Issue 6, pp 2443–2454 | Cite as

Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

  • Jingnan Lu
  • Christopher J. Brigham
  • ChoKyun Rha
  • Anthony J. Sinskey
Biotechnologically relevant enzymes and proteins

Abstract

Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid–water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg2+, Ca2+, and Mn2+ were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.

Keywords

Ralstonia eutropha Lipase Chaperone Triacylglycerol Palm oil Emulsification 

Notes

Acknowledgments

The authors thank Dr. Charles F. Budde for his helpful ideas and discussions; Mr. John W. Quimby and Sebastian Riedel, Dipl.-Ing. (FH) for critical review of the manuscript. This work was funded by the Malaysia-MIT Biotechnology Partnership Program (MMBPP). We thank our MMBPP collaborators for their helpful discussions and support throughout the course of this study.

Supplementary material

253_2012_4115_MOESM1_ESM.doc (1.3 mb)
ESM 1 (DOC 1,286 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Jingnan Lu
    • 1
  • Christopher J. Brigham
    • 2
  • ChoKyun Rha
    • 3
  • Anthony J. Sinskey
    • 2
    • 4
    • 5
  1. 1.Department of ChemistryMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA
  3. 3.Biomaterials Science & Engineering LaboratoryMassachusetts Institute of TechnologyCambridgeUSA
  4. 4.Division of Health Sciences and TechnologyMassachusetts Institute of TechnologyCambridgeUSA
  5. 5.Engineering Systems DivisionMassachusetts Institute of TechnologyCambridgeUSA

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