Preparation of a Lipid-Coated Enzyme and Activity for Reverse Hydrolysis Reactions in Homogeneous Organic Media

  • Toshiaki Mori
  • Yoshio Okahata
Part of the Methods in Biotechnology book series (MIBT, volume 15)


In the last more than two decades, a considerable number of studies have been made on the conduct of enzyme reactions in organic solvents as non-aqueous media (1-5). Merits of employing hydrolytic enzymes in organic solvents are to increase the solubility of lipophilic substrates and to cause reverse reactions such as esterification, transesterification, and transglycosylation by lipases, esterases, and glycosidases, respectively. There have been several approaches to use enzymes as a synthetic catalyst in organic solvents (6-8). In addition to the water-in-oil emulsion and the reversed micellar system containing a small amount of water (9-11), there are two previous reports (12,13) of the use of both hydrophobic and hydrophilic organic solvents as a reaction medium for lipase: (1) Klibanov and coworkers reported the direct dispersion of powdered lipase in organic solvents, to produce an ester exchange catalyst for heterogeneous solutions (2,8,14-16) and (2) Inada and coworkers prepared a poly(ethylene glycol) (PEG)-grafted lipase that is soluble or swelled in hydrophobic organic solvents and catalyzes simple ester syntheses from aliphatic alcohols and acids (17-19).


Lauric Acid Organic Medium Aliphatic Alcohol Ester Synthesis Catalytic Antibody 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Whitesides, G. M. and Wong, C.-H. (1985) Enzymes as catalysts in synthetic organic chemistry. Angew. Chem. Int. Ed. Engl. 24, 617–638.CrossRefGoogle Scholar
  2. 2.
    Klibanov, A. M. (1983) Immobilized enzymes and cells as practical catalysts. Science 219, 722–729.CrossRefGoogle Scholar
  3. 3.
    Klibanov, A. M. (1986) Enzymes that work in organic solvents. CHEMTECH 16, 354–359.Google Scholar
  4. 4.
    Klibanov, A. M. (1990) Asymmetric transformations catalyzed by enzymes in organic solvents. Acc. Chem. Res. 23, 114–120.CrossRefGoogle Scholar
  5. 5.
    Jones, J. B. (1986) Enzymes in organic synthesis. Tetrahedron 42, 3351–3403.CrossRefGoogle Scholar
  6. 6.
    Chen, C.-S. and Sih, C. J. (1989) General aspects and optimization of enantioselective biocatalysis in organic solvents: the use of lipases. Angew. Chem. Int. Ed. Engl. 28, 695–707.CrossRefGoogle Scholar
  7. 7.
    Yokozeki, K., Yamanaka, S., Takinami, K., Hirose, Y., Tanaka, A., Sonomoto, K., and Fukui, S. (1982) Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent. Eur. J. Appl. Microbiol. Biotechnol. 14, 1–5.CrossRefGoogle Scholar
  8. 8.
    Zaks, A. and Klibanov, A. M. (1984) Enzymatic catalysis in organic media at 100°C. Science 224, 1249–1251.CrossRefGoogle Scholar
  9. 9.
    Luisi, P. L. (1985) Enzymes hosted in reverse micelles in hydrocarbon solution. Angew. Chem. Int. Ed. Engl. 24, 439–450.CrossRefGoogle Scholar
  10. 10.
    Martinek, K., Levashov, A. V., Klyachko, N., Khmelnitski, Y. L., and Berezin, I. V. (1986) Micellar enzymology. Eur. J. Biochem. 155, 453–468.CrossRefGoogle Scholar
  11. 11.
    Hayes, D. G. and Gulari, E. (1990) Esterification reactions of lipase in reverse micelles. Biotechnol. Bioeng. 35, 793–801.CrossRefGoogle Scholar
  12. 12.
    Dastoli, F. R., Musto, N. A., and Price, S. (1966) Reactivity of active sites of chymotrypsin suspended in an organic medium. Arch. Biochem. Biophys. 115, 44–47.CrossRefGoogle Scholar
  13. 13.
    Dastoli, F. R. and Price, S. (1967) Catalysis by xanthine oxidase suspended in organic media. Arch. Biochem. Biophys. 118, 163–165.CrossRefGoogle Scholar
  14. 14.
    Klibanov, A. M. (1989) Enzymatic catalysis in anhydrous organic solvents. Trends Biochem. Sci. 14, 141–144.CrossRefGoogle Scholar
  15. 15.
    Russell, A. J. and Klibanov, A. M. (1988) Inhibitor-induced enzyme activation in organic solvents. J. Biol. Chem. 263, 11,624–11,626.Google Scholar
  16. 16.
    Kitaguchi, H., Fitzpatrick, P. A., Huber, J. E., and Klibanov, A. M. (1989) Enzymatic resolution of racemic amines: crucial role of the solvent. J. Am. Chem. Soc. 111, 3094,3095.Google Scholar
  17. 17.
    Takahashi, K., Ajima, A., Yoshimoto, T., Okada, M., Matsushima, A., Tamaura, Y., and Inada, Y. (1985) Chemical reactions by polyethylene glycol modified enzymes in chlorinated hydrocarbons. J. Org. Chem. 50, 3414,3415.Google Scholar
  18. 18.
    Inada, Y., Yoshimoto, T., Matsushima, A., and Saito, Y. (1986) Engineering physicochemical and biological properties of proteins by chemical modification. Trends Biotechnol. 4, 68–73.CrossRefGoogle Scholar
  19. 19.
    Matsushima, A., Kodera, Y., Takahashi, K., Saito, Y., and Inada, Y. (1986) Ester-exchange reaction between triglycerides with polyethylene glycol-modified lipase. Biotechnol. Lett. 8, 73–78.CrossRefGoogle Scholar
  20. 20.
    Okahata, Y. and Ijiro, K. (1988) A lipid-coated lipase as a new catalyst for triglyceride synthesis in organic solvents. J. Chem. Soc. Chem. Commun. 1392–1394.Google Scholar
  21. 21.
    Okahata, Y., Fujimoto, Y., and Ijiro, K. (1988) Lipase-lipid complex as aresolution catalyst of racemic alcohols in organic solvents. Tetrahedron Lett. 29, 5133,5134.CrossRefGoogle Scholar
  22. 22.
    Okahata, Y. and Ijiro, K. (1992) Preparation of a lipid-coated lipase and catalysis of glyceride ester synthesis in homogeneous organic solvents. Bull. Chem. Soc. Jpn. 65, 2411–2429.CrossRefGoogle Scholar
  23. 23.
    Okahata, Y., Fujimoto, Y., and Ijiro, K. (1995) A lipid-coated lipase as an enantioselective ester synthesis catalyst in homogeneous organic solvents. J. Org. Chem. 60, 2244–2250.CrossRefGoogle Scholar
  24. 24.
    Okahata, Y., Hatano, A., and Ijiro, K. (1995) Enhancing enantioselectivity of a lipid-coated lipase via imprinting methods for esterification in organic solvents. Tetrahedron Asymmetry 6, 1311–1322.CrossRefGoogle Scholar
  25. 25.
    Tsuzuki, W., Okahata, Y., Katayama, O., and Suzuki, T. (1991) Preparation of organic-solvent-soluble enzyme (lipase B) and characterization by gel permeation chromatography. J. Chem. Soc., Perkin Trans. 1, 1245–1247.CrossRefGoogle Scholar
  26. 26.
    Okahata, Y., Niikura, K., and Ijiro, K. (1995) A facile transphosphatidylation of phospholipids catalyzed by a lipid-coated phospholipase D in organic solvents. J. Chem. Soc. Perkin Trans. 1, 919–925.CrossRefGoogle Scholar
  27. 27.
    Okahata, Y., Yamaguchi, M., Tanaka, F., and Fujii, I. (1995) A lipid-coated catalytic antibody in water-miscible organic solvents. Tetrahedron 51, 7673–7680.CrossRefGoogle Scholar
  28. 28.
    Okahata, Y. and Mori, T. (1996) Effective transgalactosylation catalyzed by a lipid-coated β-D-galactosidase in organic solvents. J. Chem. Soc. Perkin Trans. 1, 2861–2866.CrossRefGoogle Scholar
  29. 29.
    Okahata, Y. and Mori, T. (1998) Transglycosylation catalyzed by a lipid-coated β-D-galactosidase in a two-phase aqueous-organic system. J. Mol. Catal. B: Enzymatic 5, 119–123.CrossRefGoogle Scholar
  30. 30.
    Mori, T., Fujita, S., and Okahata, Y. (1997) Transglycosylation in a two-phase aqueous-organic system with catalysis by a lipid-coated β-D-galactosidase. Carbohydr. Res. 298, 65–73.CrossRefGoogle Scholar
  31. 31.
    Mori, T., Fujita, S., and Okahata, Y. (1997) A facile transglycosylation catalyzed by a lipid-coated β-D-galactosidase in the water-organic two phases. Chem. Lett. 73.Google Scholar
  32. 32.
    Mori, T. and Okahata, Y. (1997) A variety of lipid-coated glycoside hydrolases as effective glycosyl transfer catalysts in homogeneous organic solvents, Tetrahedron Lett. 38, 1971–1974.CrossRefGoogle Scholar
  33. 33.
    Mori, T. and Okahata, Y. (1998) Effective biocatalytic transgalactosylation in a supercritical fluid using a lipid-coated enzyme. Chem. Commun. 2215,2216.Google Scholar
  34. 34.
    Mori, T., Kobayashi, A., and Okahata, Y. (1998) Biocatalytic esterification in supercritical carbon dioxide by using a lipid-coated lipase. Chem. Lett. 921,922.Google Scholar
  35. 35.
    Okahata, Y. and Mori, T. (1997) Lipid-coated enzymes as efficient catalysts in organic media. Trends Biotechnol. 15, 50–54.CrossRefGoogle Scholar
  36. 36.
    Okahata, Y. and Mori, T. (1997) Catalytic activity of lipid-coated enzymes in organic media. Proc. Jpn. Acad. 73, 210–214.CrossRefGoogle Scholar
  37. 37.
    Okahata, Y. and Mori, T. (1998) Biocatalytic reactions in organic media by using lipid-coated enzymes. J. Synth. Org. Chem. Jpn. 56, 931–939.Google Scholar
  38. 38.
    Okahata, Y., Tsuruta, T., Ijiro, K., and Ariga, K. (1988) Langmuir-Blodgett films of an enzyme-lipid complex for sensor membranes. Langmuir 4, 1373–1375.CrossRefGoogle Scholar
  39. 39.
    Okahata, Y., Tsuruta, T., Ijiro, K. and Ariga, K. (1989) Preparations of Langmuir-Blodgett films of enzyme-Lipid complex: a glucose sensor membrane. Thin Solid Films 180, 65–72.CrossRefGoogle Scholar
  40. 40.
    Lerner, R.A., Benkovic, S. J., and Schultz, P. G. (1991) At the crossroads of chemistry and immunology: catalytic antibodies. Science 252, 659–667.CrossRefGoogle Scholar
  41. 41.
    Benkovic, S. (1992) Catalytic antibody. J. Annu. Rev. Biochem. 61, 29–54.CrossRefGoogle Scholar
  42. 42.
    Hilvert, D. (1992) Antibody catalysis. Pure Appl.Chem. 64, 1103–1109.CrossRefGoogle Scholar
  43. 43.
    Stewart, J. D., Liotta, L. J., and Benkovic, S. J. (1993) Reaction mechanisms displayed by catalytic antibodies. Acc. Chem. Res. 26, 396–404.CrossRefGoogle Scholar
  44. 44.
    Hilvert, D. (1993) Antibody catalysis of carbon-carbon bond formation and cleavage. Acc. Chem. Res. 26, 552–558.CrossRefGoogle Scholar
  45. 45.
    Miyashita, H., Karaki, Y., Kikuchi, M., and Fujii, I. (1993) Prodrug activation via catalytic antibodies. Proc. Natl. Acad. Sci. USA 90, 5337–5340.CrossRefGoogle Scholar
  46. 46.
    Miyashita, H., Hara, T., Tanimura, R., Tanaka, F., Kikuchi, M., and Fujii, I. (1994) A common ancestry for multiple catalytic antibodies generated against a single transition-state analog. Proc. Natl. Acad. Sci. USA 91, 6045–6049.CrossRefGoogle Scholar
  47. 47.
    Usui, T., Kubota, S., and Ohi, H. (1993) A convenient synthesis of β-D-galactosyl disaccharide derivatives using the β-D-galactosidase from Bacillus circulans. Carbohydr. Res. 244, 315–323.CrossRefGoogle Scholar
  48. 48.
    Ooi, Y., Hashimoto, T., Mitsuo, N., and Satoh, T. (1985) Enzymic formation of β-alkyl glycosides by β-galactosidase from aspergillus oryzae and its application to the synthesis of chemistry unstable cardiac glycosides. Chem. Pharm. Bull. 33, 1808–1814.Google Scholar
  49. 49.
    Sauerbrei, B. and Thiem, J. (1992) Galactosylation and glucosylation by use of β-galactosidase. Tetrahedron Lett. 33, 201–204.CrossRefGoogle Scholar
  50. 50.
    Vulfson, E. N., Patel, R., Beecher, J. E., Andrews, A. T., and Law, B. A. (1990) Glycosidases in organic solvents: I. Alkyl-β-glucoside synthesis in a water-organic two-phase system. Enzyme Microb. Technol. 12, 950–959.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Toshiaki Mori
    • 1
  • Yoshio Okahata
    • 1
  1. 1.Department of Biomolecular EngineeringTokyo Institute of TechnologyYokohamaJapan

Personalised recommendations