Skip to main content
SpringerLink
Log in

Purification and characterization of an extracellular lipase from Geotrichum marinum

  • Articles
  • Published:
Lipids

Abstract

An extracellular lipase (EC 3.1.1.3) from Geotrichum marimum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDSPAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V max and K m were 1000 μmol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40°C. At 6 mM, Na+, K+, Ca2+, and Mg2+ stimulated activity, but Na+, and K+ at 500 mM and Fe2+ and Mn2+ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

1,2(2,3)-DG:

1,2(2,3)-dioleoyl-sn-glycerol

1,3-DG:

1,3-diacylglycerol (1,3-diolein)

BCA:

bicinchoninic acid

CHAPS:

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

IEF:

isoelectric focusing

TG:

triacylglycerol

References

  1. Jaeger, K.E., and Reetz, M.T. (1998) Microbial Lipases Form Versatile Tools for Biotechnology, Trends Biotechnol. 16, 396–403.

    Article  PubMed  CAS  Google Scholar 

  2. Vulfson, E.N. (1994) Industrial, Applications of Lipases, in Lipases (Woolley, P., and Peterson, S.B., eds.), pp. 271–288, Cambridge University Press, New York.

    Google Scholar 

  3. Bjorkling, F., Godtfredsen, S.E., and Kirk, O. (1991) The Future Impact of Industrial Lipases, Trends Biotechnol. 9, 360–363.

    Article  Google Scholar 

  4. Persson, M., Svensson, I., and Adlercreutz, P. (2000) Enzymatic Fatty Acid Exchange in Digalactosyldiacylglycerol, Chem. Phys. Lipids 104, 13–21.

    Article  PubMed  CAS  Google Scholar 

  5. Rice, K.E., Watkins, J., and Hill, C.G., Jr. (1999) Hydrolysis of Menhaden Oil by a Candida cylindracea Lipase Immobilized in a Hollow-Fiber Reactor, Biotechnol. Bioeng. 63, 33–45.

    Article  PubMed  CAS  Google Scholar 

  6. Jonzo, M.D., Hiol, A., Zagol, I.I., Druet, D., and Comeau, L. (2000) Concentrates of DHA from Fish Oil by Selective Esterification of Cholesterol by Immobilized Isoforms of Lipase from Candida Rugosa, Enzyme Microb. Technol. 27, 443–450.

    Article  PubMed  CAS  Google Scholar 

  7. Garcia, H.S., Arcos, J.A., Ward, D.J., and Hill, C.G., Jr. (2000) Synthesis of Glycerides Containing n−3 Fatty Acids and Conjugated Linoleic Acid by Solvent-Free Acidolysis of Fish Oil, Biotechnol. Bioeng. 70, 587–591.

    Article  PubMed  CAS  Google Scholar 

  8. Litthauer, D., Louw, C.H., and Du Toit, P.J. (1996) Geotrichum Candidum P-5 Produces an Intracellular Serine Protease Resembling Chymotrypsin, Int. J. Biochem. Cell Biol. 28, 1123–1130.

    Article  PubMed  CAS  Google Scholar 

  9. Sugihara, A., Hata, S., Shimada, Y., Goto, K., Tsunasawa, S., and Tominaga, Y. (1993) Characterization of Geotrichum Candidum Lipase III with Some Preference for the Inside Ester Bond of Triglyceride, Appl. Microbiol Biotechnol. 40, 279–283.

    Article  PubMed  CAS  Google Scholar 

  10. Veeraragavan, K., Colpitts, T., and Gibbs, B.F. (1990) Purification and Characterization of Two Distinct Lipases from Geotrichum candidum, Biochim. Biophys. Acta 1044, 26–33.

    PubMed  CAS  Google Scholar 

  11. Jacobsen, T., and Poulsen, O.M. (1995) Comparison of Lipases from Different Strains of the Fungus Geotrichum candidum, Biochim. Biophys. Acta 1257, 96–102.

    PubMed  Google Scholar 

  12. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson B.J., and Klenk, D.C. (1985) Measurement of Protein Using Bicinchoninic Acid, Anal. Biochem. 150, 76–85.

    Article  PubMed  CAS  Google Scholar 

  13. Mozaffar, Z., and Weete, J.D. (1993) Purification and Properties of an Extracellular Lipase from Pythium Ultimum, Lipids 28, 377–382.

    CAS  Google Scholar 

  14. Bollag, D.M., and Edelstein, S.J. (1991) Staining a Gel with Coomassie Blue, in Protein Methods (Bollag, D.M., and Edelstein, S.J., eds.), pp. 114–116, Wiley-Liss, New York.

    Google Scholar 

  15. Lineweaver, H., and Burk, D. (1934) The Determination of Enzyme Dissociation Constants, J. Am. Chem. Soc. 56, 658–666.

    Article  CAS  Google Scholar 

  16. Torossian, K., and Bell, A.W. (1991) Purification and Characterization of an Acid-Resistant Triacylglycerol Lipase from Aspergillus niger, Biotechnol. appl. Biochem. 13, 205–211.

    CAS  Google Scholar 

  17. Baillargeon, M.W., and McCarthy, S.G. (1991) Geotrichum candidum NRRL Y 553 Lipase: Purification, Characterization and Fatty Acid Specificity, Lipids 26, 831–836.

    PubMed  CAS  Google Scholar 

  18. Baillargeon, M.W. (1990) Purification and Specificity of Lipase from Geotrichum candidum, Lipids, 25, 841–848.

    CAS  Google Scholar 

  19. Sugihara, A., Shimada, Y., and Tominaga, Y. (1990) Separation and Characterization of Two Molecular Forms of Geotrichum candidum Lipase, J. Biochem. (Tokyo) 107, 426–430.

    CAS  Google Scholar 

  20. Jacobsen, T., and Poulsen, O.M. (1992) Separation and Characterization of 61- and 57-kDa Lipases from Geotrichum Candidum ATCC 66592, Can. J. Microbiol. 38, 75–80.

    Article  PubMed  CAS  Google Scholar 

  21. Sidebottom, C.M., Charton, E., Dunn, P.P., Mycock, G., Davies, C., Sutton, J.L., Macrae, A.R., and Slabas, A.R. (1991) Geotrichum candidum Produces Several Lipases with Markedly Different Substrate Specificities, Eur. J. Biochem. 202, 485–491.

    Article  PubMed  CAS  Google Scholar 

  22. Sugihara, A., Shimada, Y., Nakamura, M., Nagao, T., and Tominaga, Y. (1994) Positional and Fatty Acid Specificities of Geotrichum candidum Lipases, Protein Eng. 7, 565–588.

    Article  Google Scholar 

  23. Jensen, R.G. (1974) Characteristic of the Lipase from the Mold, Geotrichum candidum: A Review, Lipids 9, 149–157.

    PubMed  CAS  Google Scholar 

  24. Bertolini, M.C., Schrag, J.D., Cygler, M., Ziomek, E., Thomas, D.Y., and Vernet, T. (1995) Expression and Characterization of Geotrichum candidum Lipase I Gene. Comparison of Specificity Profile with Lipase II, Eur. J. Biochem. 228, 863–869.

    Article  PubMed  CAS  Google Scholar 

  25. Muderhwa, J.M., and Ratomahenina, R. (1985) Purification and Properties of the Lipase from Candida deformans (Zach) Langeron and Guerra, J. Am. Oil Chem. Soc. 62, 1031–1036.

    Article  CAS  Google Scholar 

  26. Omar, I.C., Hayashi, M., and Nagai, S. (1987) Purification and Some Properties of a Thermostable Lipase from Humicola lanuginosa No. 3, Agric. Biol. Chem. 51, 37–45.

    CAS  Google Scholar 

  27. Yadav, R.P., Saxeena, R.K., Gupta, R., and Davidson, S. (1998) Lipase Production by Aspergillus and Penicillium species, Folia Microbiol. (Praha) 43, 373–378.

    CAS  Google Scholar 

  28. Ruiz, B., Farres, A., Langley, E., Masso, F., and Sanchez, S. (2001) Purification and Characterization of an Extracellular Lipase from Penicillium candidum, Lipids 36, 283–289.

    Article  PubMed  CAS  Google Scholar 

  29. Haas M.J., Cichowicz, D.J., and Bailey, D.G. (1992) Purification and Characterization of an Extracellular Lipase from the Fungus Rhizopus delemar, Lipids, 27 571–576.

    CAS  Google Scholar 

  30. Stocklein, W., Sztajer, H., Menge, U., and Schmid, R.D. (1993) Purification and Properties of a Lipase from Penicillium expansum, Biochim. Biophys. Acta 1168, 181–189.

    PubMed  CAS  Google Scholar 

  31. Krieger, K., Taipa, M.A., Melo, E.H.M., Lima-Filho, J.L., Aires-Barros, M.R., and Carbral, J.M.S. (1999) Purification of a Penicillium citrinum Lipase by Chromatographic Processes, Bioprocess Eng. 20, 59–65.

    CAS  Google Scholar 

  32. Olama, Z.A., and el-Sabaeny, A.H. (1993) Lipase Production by Aspergillus niger Under Various Growth Conditions Using Solid State Fermentation, Microbiologia 9, 134–141.

    PubMed  CAS  Google Scholar 

  33. Commenil, P., Belingheri, L., Sancholle, M., and Dehorter, B. (1995) Purification and Properties of an Extracellular Lipase from the Fungus Botrytis cinerea, Lipids 30, 351–356.

    PubMed  CAS  Google Scholar 

  34. Weete J.D. (1998) Microbial Lipases, in Food Lipids (Akoh, C.C., and Min, D.B., eds.), pp. 641–664, Marcel Dekker, New York.

    Google Scholar 

  35. Simons, J.W.F.A., Van Kampen, M.D., Ubarretxena-Belandia, I., Cox, R.C., Alves Dos Santos, C.M., Egmond, M.R., and Verheij, H.M. (1999) Identification of a Calcium Binding Site in Staphylococcus hyicus Lipase: Generation of Calcium-Independent Variants, Biochemistry 38, 2–10.

    Article  PubMed  CAS  Google Scholar 

  36. Wang, Y.J., Sheu, J.Y., Wang, F.F., and Shaw, J.F. (1988) Lipase-Catalyzed Oil Hydrolysis in the Absence of Added Emulsifier, Biotechnol. Bioeng. 31, 628–633.

    Article  CAS  PubMed  Google Scholar 

  37. Benzonana G., and Desnuelle, P. (1968) Action of Some Effectors on the Hydrolysis of Long-Chain Triglycerides by Pancreatic Lipase, Biochim. Biophys. Acta 164, 47–58.

    PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Youliang Huang

    Present address: Research Office, West Virginia University, 886 Chestnut Ridge Rd. Suite 201, P.O. Box 6216, Morgantown, WV, 26506

Authors and Affiliations

  1. Department of Animal Sciences, Auburn University, 209 Upchurch Hall, 36849, Auburn, AL

    Youliang Huang, Robert Locy & John D. Weete

Authors
  1. Youliang Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert Locy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John D. Weete
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Youliang Huang.

About this article

Cite this article

Huang, Y., Locy, R. & Weete, J.D. Purification and characterization of an extracellular lipase from Geotrichum marinum . Lipids 39, 251–258 (2004). https://doi.org/10.1007/s11745-004-1227-1

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-004-1227-1

Keywords

Search

Navigation