Characterization of Lipoxygenase in AOT/Isooctane Reversed Micelles

AOT/isooctane 역미셀계내에서의 lipoxygenase 반응 특성

  • Published : 2002.04.01


An investigation was carried out to study the characteristics of lipoxygenase in dioctyl sulfosuccinate (aerosol-OT, AOT)/isooctane revered micelles of microaqueous system containing infinitesimal water. ${\alpha}-Linoleic$ acid as a substrate could be analyzed by the colorimetric methodology using 5%(w/v) cupric acetate-pyridine solution and the activity of lipoxygenase was able to be assayed by the degree of ${\alpha}-linoleic$ acid consumption per minute. Optimal pH, temperature, and R-value ([water]/[AOT]) were determined as the value of 5.0, $25^{\circ}C$, and 10.0, respectively. Kinetic analysis of the enzyme reaction under the optimal conditions showed that the values of $K_m$ and $V_{max}$ were 0.31 mM of ${\alpha}-linoleic$ acid and $384.16{\mu}mol$ of ${\alpha}-linoleic$ acid decomposed/min, respectively. The results indicate the reaction to be lipoxygenase-catalyzed oxidation of ${\alpha}-linoleic$ acid in AOT/isooctane reversed micellar system. The inhibitory effect of natural antioxidants on lipoxygenase showed little inhibitory effect of L-ascrobic acid while ${\alpha}-tocopherol$ showed 72% of inhibitory effect.


lipoxygenase;reversed micelles;kinetic analysis;inhibition


  1. Rho, Y.T., Kim, Y.S., Cho, H.Y. and Pyun, Y.R. A study on pro-tein separation with AOT-isooctane reverse micellar system.Korean J. Food Sci. Technol. 21: 542-547 (1989)
  2. Han, D. and Rhee, J.S. Characteristics of lipase-catalyzed hydrol-ysis of olive oil in AOT-isooctane reversed micelles. Biotechnol. Bioeng. 28: 1250-1255 (1986)
  3. Eskin, N.A. Grossman, S. and Pinsky, A. Biochemistry of lipoxy-genase in relation to food quality. CRC Crit. Rev. Food Sci. Nutr.9: 1-40 (1977)
  4. Chang, M.J., Cho, I.Y. and Lee, S.K. Characteristics of hydroper-oxide lyase and lipoxygenase activity in cucumber (Cucumis Sati-vus) fruit. Korean J. Food Sci. Technol. 29: 314-319 (1997)
  5. Choi, S.W. and Osawa, T. Isolation and idendification of lipoxy-genase inhibitor from nutmeg spice. Food Sci. Biotechnol. 5:156-160(1996)
  6. Galliard, T. and Chan, H.W. A comprehensive treatise, In: The Biochemistry of Plants, Vol. 4, p. 131. Stumpf, P.K. (ed.), Aca-demic Press, New York, USA (1980)
  7. Choi, S.W., Kang, W.W., Chung, S.K. and Cheon, S.H. Antioxi-dative activity of flavonoids in persimmon leaves. Food Sci. Bio-technol.5:119-123(1996)
  8. Seoud, A.A.E. Acidities and basicities in reversed micellar sys-tems. In: Reverse Micelles, p. 81. Luisi, P.L. and Straub, B.E.(eds.), Plenum Press, New York, USA (1984)
  9. Boyes, S., Perera, C. and Young, H. Kiwifruit lipoxygenase:Preparation and characteristcs. J. Food Sci. 57: 1390-1395 (1992)
  10. Fletcher, P.D.L, Freedman, R.B., Robinson, B.H., Rees, G.D., and Schmacker, R. Lipase-catalyzed ester synthesis in oil-continuous microemulsions. Biochim. Biophys. Acta 912: 278-282 (1987)
  11. Eicke, H.F. and Shepherd, J.C.W. Dielectric properties of apolar micelle solutions containing solubilized water. Helv. Chim. Acta 57: 1951-1963 (1974)
  12. Im, H.S., Cho, Y.H. and Rhee, C.O. Effect of inhibitor on lipoxy-genase inactivation in soybean homogenates. Korean J. Food Sci. Technol. 27: 19-24 (1995)
  13. Zakut, R. Grossman, S. Pinsky, A. and Wilchek, M. Evidence for an essential methionine residue in lipoxygenase. FEBS Lett. 72:107-110(1976)
  14. Chang, P.S., Kang, S.T. and Rhee, J.S. A general kinetic model for lipase-catalyzed glycerolysis of triolein. Food Sci. Biotechnol 1:46-49(1992)
  15. Zaks, A. and Klibanov, A.M. Enzymatic catalysis in organic media at $100^\circ C$. Science 224: 1249-1251 (1984)
  16. Nakayama, T., Takeura, Y. and Ueda, U. Visible spectrophotomet-ric assay, Purification and molecular properties of a lipoxygenase from egg plant (Solanum melongena Linne) fruits. Biochem. Bio-phys. Res. Commun. 214: 1067-1072 (1995)
  17. Kim, D.K., Han, K.Y. and Noh, B.S. Characteristics of crude lipoxygenase in Chinese cabbages. Korean J. Food Sci. Technol.29: 710-715 (1997)
  18. Shipe, W.F, Senyk, G.F. and Fountain, K.B. Modification of cupric sulfate method for the analysis of free fatty acids, J. Dairy Sci. 63: 193-198 (1980)
  19. Eicke, H. F. On the stabilization of micelles in apolar solvents by solubilized ions and polar liquids. J. Coll. Interface Sci. 52: 65-76 (1975)
  20. Lee, Y.C. Lipoxygenase and off-flavor development in some fro-zen foods. Korean J. Food Sci. Technol. 17: 53-58 (1981)
  21. Grossman, S., Trop, M., Avtalion, R. and Pinsky, A. Properties of agarose-bound soya-bean lipoxygenase. Biochem. J. 127: 909-910(1972)
  22. Barbaric, S. and Luisi, P.L. Micellar solubilization of biopolymers in organic solvents. 5. Activity and conformation of $\alpha$-chymot-rypsin in isooctane-AOT reversed micelles. J. Am. Chem. Soc. 103:4239-4244(1981)
  23. Lowry, R.R. and Tinsley, I.J. Colorimetric analysis of free fatty acids by cupric sulfate, J. Am. Oil Chem. Soc. 53: 470-472(1976)
  24. Kruger, J.E., Lineback, D. and Stauffen, C.E. Enzymes and Their Role in Cereal Technology. American Association of Cereal Chemists, Inc., p. 241 (1987)
  25. Chang, P.S. Hydrolysis of phosphatidylcholine in aerosol-OT/isooctane reversed micelles by phospholipase $A_2$. Korean J. Food Sci. Technol. 29: 26-31 (1997)
  26. Chang, P.S., Rhee, J.S. and Kim, J.J. Continuous glycerolysis of olive oil by immobilization on liposome in reversed micelles. Biotechnol. Bioeng. 38: 1159-1165 (1991)
  27. Grandi, C., Smith, R.E., and Luisi, P.L. Micellar solubilization of biopolymers in organic solvents: Activity and conformation of lysozyme in isooctane reverse micelles. J. Biol. Chem. 256: 837-843(1981)
  28. Grosch, W. and Laskawy, G. Differences in the amount and range of volatile carbonyl compounds fonned by lipoxygenase isoen-zymes from soybeans. J. Agric. Food Chem. 23: 791-794 (1975)
  29. Shin, H.S. The Newest Food Chemistry, p. 445. Kwangmungak,Seoul(1995)