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A colorimetric determination of fatty acids as a new assay of lipases in reverse micelles

  • Surfactants & Detergents
  • Published:
Journal of the American Oil Chemists’ Society

Abstract

A new simple spectrophotometric method is described for the determination of the free fatty acid content in triglyceride oils or other lipophilic samples. The method utilizes phenol red as fatty acid indicator, which is solubilized phenol red as fatty acid indicator, which is solubilized in reverse micelles formed by AOT [sodiumbis(2-ethylhexyl) sulfosuccinate] in isooctane. Fatty acid determinations in vegetable oils can be carried out rapidly with oil samples of less than 100 mg. The acid value of three different oils tested agreed quite well with the acid value obtained for the same samples with another colorimetric determination using cupric acetate.

The method can be extended to a continuous determination of fatty acids which are released during the initial stage of a lipase catalyzed hydrolysis of triglyceride substrates in reverse micelles. This new sensitive lipase assay has been applied for a lipase ofPseudomonas bacteria with lipase concentrations as low as 0.1 μg/ml. Using trioctanoylglycerol as substrate in 50 mM AOT/isooctane with wo=[H2O]/[AOT]=11.1 (pH 9.0), the apparent overall Michaelis-Menten constant (Km′app,ov) is 27 mM and the turnover number (kcat) 44 sec−1.

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References

  1. Sonntag, N.O.V., inFatty Acids in Industry, edited by R. W. Johnson and E. Fritz, Marcel Dekker, New York and Basel, 1989, pp. 23–72.

    Google Scholar 

  2. Linfield, W.M., R.A. Barauskas, L. Sivieri, S. Serota and R.W. Stevenson Sr.,J. Am. Oil Chem. Soc. 61: 191 (1984).

    CAS  Google Scholar 

  3. Klibanov, A.M., inProtein Engineering, Applications in Science, Medicine, and Industry, edited by M. Inouye and R. Sarma, Academic Press, Orlando, Florida, 1988, pp. 341–349.

    Google Scholar 

  4. Wang, Y.-F., J.J. Lalonde, M. Momongan, D.E. Bergbreiter and C.-H. Wong,J. Am. Chem. Soc., 110: 7200 (1988).

    Article  CAS  Google Scholar 

  5. Brockman, H.L., inLipases, edited by B. Borgström and H.L. Brockman, Elsevier, Amsterdam, 1987, pp. 3–46.

    Google Scholar 

  6. Chapus, C., M. Rovery, L. Sarda and R. Verger,Biochimie 70: 1223 (1988).

    Article  CAS  Google Scholar 

  7. Benzonana, G., and P. Desnuelle,Biochim. Biophys. Acta 105: 121 (1965).

    CAS  Google Scholar 

  8. Eicke, H.-F.,Chimia 36: 241 (1982).

    CAS  Google Scholar 

  9. Nicholson, J.D., and J.H.R. Clarke, inSurfactants in Solution, Volume 3, edited by K.L. Mittal and B. Lindman, Plenum Press, New York and London, 1984, pp. 1663–1674.

    Google Scholar 

  10. Luisi, P.L.,Angew. Chem. 97: 449 (1985).

    CAS  Google Scholar 

  11. Martinek, K., A.V. Levashov, N. Klyachko, Y.L. Khmelnitski and I.V. Berezin,Eur. J. Biochem. 155: 453 (1986).

    Article  CAS  Google Scholar 

  12. Walde, P. inReactions in Compartmentalized Liquids, edited by W. Knoche and R. Schomäcker Springer-Verlag, Heidelberg, 1989, pp. 11–19.

    Google Scholar 

  13. Han, D., and J.S. Rhee,Biotechnol. Bioeng. 28: 1250 (1986).

    Article  CAS  Google Scholar 

  14. Oesterberg, E., C. Ristoff and K. Holmberg,Tens. Surf. Det. 25: 293 (1988).

    CAS  Google Scholar 

  15. Walde, P., and P.L. Luisi,Biochemistry 28: 3353 (1989).

    Article  CAS  Google Scholar 

  16. Scartazzini, R., and P.L. Luisi,Biocatalysis, in press.

  17. Sugiura, M., T. Oikawa, K. Hirano and T. Inukai,Biochim. Biophys. Acta 488: 353 (1977).

    CAS  Google Scholar 

  18. Sugiura, M., and T. Oikawa,Biochim. Biophys. Acta 489: 262 (1977).

    CAS  Google Scholar 

  19. Yamamoto K., and N. Fujiwara,Agric. Biol. Chem. 52: 3015 (1988).

    CAS  Google Scholar 

  20. Fletcher, P.D.I., N.M. Perrins, B.H. Robinson and C. Toprakcioglu, inReverse Micelles, edited by P.L. Luisi and B.E. Straub, Plenum Press, New York, London, 1984, pp. 69–72.

    Google Scholar 

  21. The Lipid Handbook, edited by F.D. Gunstone, J.L. Harwood and F.B. Padley, Chapman & Hall, London, New York, 1986, p. 260.

    Google Scholar 

  22. Lowry, R.R., and I.J. Tinsley,J. Am. Oil Chem. Soc. 53: 470 (1976).

    CAS  Google Scholar 

  23. Fujii, H., T. Kawai, H. Nishikawa and G. Ebert,Colloid Polymer Sci., 260: 697 (1982).

    Article  CAS  Google Scholar 

  24. Kratzert, W., and R. Peichert,Farbstoffe, Quelle & Meyer, Heidelberg, 1981.

    Google Scholar 

  25. Smith, R.E., and P.L. Luisi,Helv. Chim. Acta 63: 2302 (1980).

    Article  CAS  Google Scholar 

  26. Baker, D.,J. Am. Oil Chem. Soc., 41: 21 (1964).

    Google Scholar 

  27. Bains, G.S., S.V. Rao and D.S. Bhatia,Ibid., 41: 831 (1964).

    CAS  Google Scholar 

  28. Kwon, D.Y., and J.S. Rhee,Ibid. 63:89 (1986).

    CAS  Google Scholar 

  29. Malakhova, E.A., B.I. Kurganov, A.V. Levashov, I.V. Berezin and K. Martinek,Dokl. Akad. Nauk. SSSR, 270: 474 (1983).

    CAS  Google Scholar 

  30. Schmidli, P.K., and P.L. Luisi,Biocatalysis, in press.

  31. Leser, M.E., P.L. Luisi and S. Palmieri,Bioctechnol. Bioeng., 34:1140 (1989).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Institut für Polymere, Eidgenössische Technische Hochschule, CH-8092, Zürich, Switzerland

    Peter Walde

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  1. Peter Walde
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Walde, P. A colorimetric determination of fatty acids as a new assay of lipases in reverse micelles. J Am Oil Chem Soc 67, 110–115 (1990). https://doi.org/10.1007/BF02540637

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  • DOI: https://doi.org/10.1007/BF02540637

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