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Purification and characterization of an extracellular lipase from Clostridium tetanomorphum

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Summary

The strictly anaerobic bacterium Clostridium tetanomorphum formed an extracellular lipase when the growth medium contained glycerol in addition to fermentable substrates such as l-glutamate or glucose. The lipase was purified from the concentrated culture supernatant and exhibited a final specific activity of 900 U/mg. The purified lipase had a Stokes’ radius of 5.0 nm and a sedimentation coefficient of 5.7S. The native molecular mass calculated from these values was 118,000 Da, which is considerably higher than the molecular mass calculated by PAGE (70,000 Da). With p-nitrophenyl esters of different fatty acids as substrates enzyme activity was highest when the acyl chain was short (C2). The purified lipase showed no protease or thioesterase activity.

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References

  1. Andersson L.O., Borg H., Mikaelsson. M., 1972. Molecular weight estimation of proteins by electrophoresis in polyacrylamide gel of graded porosityFEBS Letters 20: 199–202

  2. Barker H.A., 1939 The use of glutamic acid for the isolation and identification of Clostridium cochlearium and C. tetanomorphumArchives of Microbiology 10: 376–384

  3. Blum H., Beier H., Gross H.J., 1987 Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gelsElectrophoresis 8: 93–99

  4. Choo D.-W., Kurihara T., Suzuki T., Soda K., Esaki N., 1998 A cold-adapted lipase of an alaskan psychrotroph Pseudomonas sp. strain B11–1: gene cloning and enzyme purification and characterizationApplied and Environmental Microbiology 64: 486–491

  5. Eggstein, M. & Kuhlmann, E. 1974 Triglycerides and glycerol determination after alkaline hydrolysis. In Methoden der enzymatischen Analyse, ed. Bergmeyer H.U., 2nd edn, New York: Academic Press: ISBN 0-120-91301-1

  6. Feller G., Thiry M., Arpigny J.L., Gerday C., 1991 Cloning and expression in Escherichia coli of three lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144 Gene 102: 111–115

  7. Ghanem E.H., Al-Sayeed H.A., Saleh K.M., 2000 An alkalophilic thermostable lipase produced by new isolate of Bacillus alcalophilusWorld Journal of Microbiology and Biotechnology 16: 459–464

  8. Gilbert E.J., Drozd J.W., Jones C.W., 1991 Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2Journal of General Microbiology 137: 2215–2221

  9. Gupta R., Gupta N., Rathi P., 2004 Bacterial lipases: an overview of production, purification and biochemical propertiesApplied Microbiology and Biotechnology 64: 763–781

  10. Henne A., Schmitz R.A., Bömeke M., Gottschalk G., Daniel R., 2000 Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coliApplied and Environmental Microbiology 66: 3113–3116

  11. Jaeger K.-E., Reetz M.T., 1998 Microbial lipases form versatile tools for biotechnologyTrends in Biotechnology 16: 396–403

  12. Jaeger K.-E., Dijkstra B.W., Reetz M.T., 1999 Bacterial biocatalysts: molecular biology, three-dimensional structure and biotechnological applications of lipasesAnnual Reviews of Microbiology 53: 315–351

  13. Jaeger K.-E., Ransac S., Dijkstra B.W., Colson C., van Heuvel M., Misset O., 1994 Bacterial lipasesMicrobiology Reviews 15: 29–63

  14. Kouker G., Jaeger K.-E., 1987 Specific and sensitive plate assay for bacterial lipasesApplied and Environmental Microbiology 53: 211–213

  15. Laurent T.C., Killander J., 1964 A theory of gel filtration and its experimental verificationJournal of Chromatography 14: 317–330

  16. Martin R.G., Ames B.N., 1961 A method for determining the sedimentation behavior of enzymes: application to protein mixturesJournal of Biological Chemistry 236: 1372–1379

  17. McKellar R.C., Shamsuzzaman K., San Jose C., Cholette H., 1987 Influence of iron(III) and pyoverdine on extracellular proteinase and lipase production by Pseudomonas fluorescens B52Archives of Microbiology 147: 225–230

  18. Siegel L.M., Monty K.J., 1966 Determinations of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductasesBiochimica et Biophysica Acta 112: 346–362

  19. Sztajer H., Lünsdorf H., Erdmann H., Menge U., Schmid R., 1992 Purification and properties of lipase from Penicillium simplicissimumBiochimica et Biophysica Acta 1124: 253–261

  20. Wilde E., Hippe N., Tosunoglu N., Herwig K., Gottschalk G., 1989 Clostridium tetanomorphum sp. nov., nom. revInternational Journal of Systematic Bacteriology 39: 127–134

  21. Winkler U.K., Stuckmann M., 1979 Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipases by Serratia marcescensJournal of Bacteriology 138: 663–670

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Acknowledgement

We thank Prof. G. Gottschalk for generous support.

Author information

Correspondence to Rolf Daniel.

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Petersen, M., Daniel, R. Purification and characterization of an extracellular lipase from Clostridium tetanomorphum . World J Microbiol Biotechnol 22, 431–435 (2006). https://doi.org/10.1007/s11274-005-9052-x

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Keywords

  • Clostridium tetanomorphum
  • enzyme purification
  • extracellular enzymes
  • lipase
  • triolein