Lipase Activation and Stabilization in Room-Temperature Ionic Liquids

  • Joel L. KaarEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 679)


Widespread interest in the use of room temperature ionic liquids (RTILs) as solvents in anhydrous biocatalytic reactions has largely been met with underwhelming results. Enzymes are frequently inactivated in RTILs as a result of the influence of solvent on the enzyme’s microenvironment, be it through interacting with the enzyme or enzyme-bound water molecules. The purpose of this chapter is to present a rational approach to mediate RTIL–enzyme interactions, which is essential if we are to realize the advantages of RTILs over conventional solvents for biocatalysis in full. The underlying premise for this approach is the stabilization of enzyme structure via multipoint covalent immobilization within a polyurethane foam matrix. Additionally, the approach entails the use of salt hydrates to control the level of hydration of the immobilized enzyme, which is critical to the activation of enzymes in nonaqueous media. Although lipase is used as a model enzyme, this approach may be effective in activating and stabilizing virtually any enzyme in RTILs.

Key words

Nonaqueous biocatalysis Ionic liquids Enzyme stabilization Immobilization Salt hydrates Water activity Lipase Green chemistry 



This work was funded by SACHEM Inc. and by a research grant from the Environmental Protection Agency (R-82813101-0) to Alan J. Russell, to whom I am grateful for support, both financial and scientific. I am thankful to Jason A. Berberich for technical advice and helpful discussion on all aspects of this work. I also wish to thank Jason A. Berberich and Rick R. Koepsel for their critical reading of this manuscript.


  1. 1.
    Brennecke, J. F. and Maginn, E. J. (2001) Ionic liquids: innovative fluids for chemical processing, AIChE J 47, 2384–2389.CrossRefGoogle Scholar
  2. 2.
    Yang, Z. and Pan, W. (2005) Ionic liquids: green solvents for nonaqueous biocatalysis, Enzyme Microb Technol 37, 19–28.CrossRefGoogle Scholar
  3. 3.
    van Rantwijk, F. and Sheldon, R. A. (2007) Biocatalysis in ionic liquids, Chem Rev 107, 2757–2785.PubMedCrossRefGoogle Scholar
  4. 4.
    Roosen, C., Muller, P., and Greiner, L. (2008) Ionic liquids in biotechnology: applications and perspectives for biotransformations, Appl Microbiol Biotechnol 81, 607–614.PubMedCrossRefGoogle Scholar
  5. 5.
    Kaar, J. L., Jesionowski, A. M., Berberich, J. A., Moulton, R., and Russell, A. J. (2003) Impact of ionic liquid physical properties on lipase activity and stability, J Am Chem Soc 125, 4125–4131.PubMedCrossRefGoogle Scholar
  6. 6.
    Micaêlo, N. M. and Soares, C. M. (2008) Protein structure and dynamics in ionic liquids. Insights from molecular dynamics simulation studies, J Phys Chem B 112, 2566–2572.PubMedCrossRefGoogle Scholar
  7. 7.
    Halling, P. J. (1994) Thermodynamic predictions for biocatalysis in nonconventional media: theory, tests, and recommendations for experimental design and analysis, Enzyme Microb Technol 16, 178–206.PubMedCrossRefGoogle Scholar
  8. 8.
    Halling, P. J. (2004) What can we learn by studying enzymes in non-aqueous media? Philos Trans R Soc Lond B Biol Sci 359, 1287–1296; discussion 1296–1287, 1323–1288.PubMedCrossRefGoogle Scholar
  9. 9.
    Nakashima, K., Maruyama, T., Kamiya, N., and Goto, M. (2006) Homogeneous enzymatic reactions in ionic liquids with poly(ethylene glycol)-modified subtilisin, Org Biomol Chem 4, 3462–3467.PubMedCrossRefGoogle Scholar
  10. 10.
    Berberich, J. A., Kaar, J. L., and Russell, A. J. (2003) Use of salt hydrate pairs to control water activity for enzyme catalysis in ionic liquids, Biotechnol Prog 19, 1029–1032.PubMedCrossRefGoogle Scholar
  11. 11.
    Lejeune, K. E., Mesiano, A. J., Bower, S. B., Grimsley, J. K., Wild, J. R., and Russell, A. J. (1997) Dramatically stabilized phosphotriesterase-polymers for nerve agent degradation, Biotechnol Bioeng 54, 105–114.PubMedCrossRefGoogle Scholar
  12. 12.
    Gordon, R. K., Feaster, S. R., Russell, A. J., LeJeune, K. E., Maxwell, D. M., Lenz, D. E., Ross, M., and Doctor, B. P. (1999) Organophosphate skin decontamination using immobilized enzymes, Chem Biol Interact 119-120, 463–470.PubMedCrossRefGoogle Scholar
  13. 13.
    LeJeune, K. E., Swers, J. S., Hetro, A. D., Donahey, G. P., and Russell, A. J. (1999) Increasing the tolerance of organophosphorus hydrolase to bleach, Biotechnol Bioeng 64, 250–254.PubMedCrossRefGoogle Scholar
  14. 14.
    Gill, I. and Ballesteros, A. (2000) Bioencap­sulation within synthetic polymers (Part 2): non-sol-gel protein-polymer biocomposites, Trends Biotechnol 18, 469–479.PubMedCrossRefGoogle Scholar
  15. 15.
    Drevon, G. F., Hartleib, J., Scharff, E., Ruterjans, H., and Russell, A. J. (2001) Thermoinactivation of diisopropylfluorophosphatase-containing polyurethane polymers, Biomacromolecules 2, 664–671.PubMedCrossRefGoogle Scholar
  16. 16.
    Drevon, G. F., Danielmeier, K., Federspiel, W., Stolz, D. B., Wicks, D. A., Yu, P. C., and Russell, A. J. (2002) High-activity enzyme-polyurethane coatings, Biotechnol Bioeng 79, 785–794.PubMedCrossRefGoogle Scholar
  17. 17.
    Vasudevan, P. T., Lopez-Cortes, N., Caswell, H., Reyes-Duarte, D., Plou, F. J., Ballesteros, A., Como, K., and Thomson, T. (2004) A novel hydrophilic support, CoFoam, for enzyme immobilization, Biotechnol Lett 26, 473–477.PubMedCrossRefGoogle Scholar
  18. 18.
    Burrell, A. K., Del Sesto, R. E., Baker, S. N., McCleskey, T. M., and Baker, G. A. (2007) The large scale synthesis of pure imidazolium and pyrrolidinium ionic liquids, Green Chem 9, 449–454.CrossRefGoogle Scholar
  19. 19.
    Park, S. and Kazlauskas, R. J. (2001) Improved preparation and use of room-temperature ionic liquids in lipase-catalyzed enantio- and regioselective acylations, J Org Chem 66, 8395–8401.PubMedCrossRefGoogle Scholar
  20. 20.
    Lee, S. H., Ha, S. H., Lee, S. B., and Koo, Y. M. (2006) Adverse effect of chloride impurities on lipase-catalyzed transesterifications in ionic liquids, Biotechnol Lett 28, 1335–1339.PubMedCrossRefGoogle Scholar
  21. 21.
    Lejeune, K. E. and Russell, A. J. (1996) Covalent binding of a nerve agent hydrolyzing enzyme within polyurethane foams, Biotechnol Bioeng 51, 450–457.PubMedCrossRefGoogle Scholar
  22. 22.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72, 248–254.PubMedCrossRefGoogle Scholar
  23. 23.
    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.PubMedCrossRefGoogle Scholar
  24. 24.
    Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein, Protein Sci 4, 2411–2423.PubMedCrossRefGoogle Scholar
  25. 25.
    Halling, P. J. (1992) Salt hydrates for water activity control with biocatalysts in organic media, Biotechnol Tech 6, 271–276.CrossRefGoogle Scholar
  26. 26.
    Zacharis, E., Omar, I. C., Partridge, J., Robb, D. A., and Halling, P. J. (1997) Selection of salt hydrate pairs for use in water control in enzyme catalysis in organic solvents, Biotechnol Bioeng 55, 367–374.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Chemical Engineering, McGowan Institute for Regenerative MedicineUniversity of PittsburgPittsburgUSA

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