Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16
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.
KeywordsRalstonia eutropha Lipase Chaperone Triacylglycerol Palm oil Emulsification
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.
- Godtfredsen SE (1990) Application of lipases for synthesis of new chemicals. Opp Biotransform 1:17–22Google Scholar
- Kok RG, Nudel CB, Gonzalez RH, NugterenRoodzant IM, Hellingwerf KJ (1996) Physiological factors affecting production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413: fatty acid repression of lipA expression and degradation of LipA. J Bacteriol 178:6025–6035Google Scholar
- Macrae AR, Hammond RC (1985) Present and future applications of lipases. Biotechnolo Genet Eng Rev 3:193–217Google Scholar
- Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol VT (1999) The realm of microbial lipases in biotechnology. Biotechnol Appl Biochem 29:119–131Google Scholar
- Park H, Lee KS, Chi YM, Jeong SW (2005) Effects of methanol on the catalytic properties of porcine pancreatic lipase. J Microbiol Biotechnol 15:296–301Google Scholar
- Slater S, Houmiel KL, Tran M, Mitsky TA, Taylor NB, Padgette SR, Gruys KJ (1998) Multiple beta-ketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J Bacteriol 180:1979–1987Google Scholar