Skip to main content
SpringerLink
Log in

Practical Protocols for Lipase Immobilization Via Sol-Gel Techniques

  • Protocol
Immobilization of Enzymes and Cells

Part of the book series: Methods in Biotechnologyâ„¢ ((MIBT,volume 22))

Abstract

Lipases can be efficiently entrapped in the pores of hydrophobic silicates by a simple and cheap sol-gel process in which a mixture of an alkylsilane [RSi(OCH3)3 and Si(OCH3)4] is hydrolyzed under basic conditions in the presence of the enzyme. Additives such as isopropanol, polyvinyl alcohol, cyclodextrins, or surfactants enhance the efficiency of this type of lipase immobilization. The main area of application of these heterogeneous biocatalysts concerns esterification or transesterification in organic solvents, ionic liquids, or supercritical carbon dioxide. Rate enhancements (relative to the traditional use of lipase powders) of several orders of magnitude have been observed, in addition to higher thermal stability. The lipase immobilizates are particularly useful in the kinetic resolution of chiral esters, enantioselectivity often being higher than what is observed when using the commercial forms of these lipases (powder or classical immobilizates). Thus, because of the low price of sol-gel entrapment, the excellent performance of the lipase immobilizates, and the ready recyclability, this method is industrially viable.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Faber K. (1997) Biotransformations in Organic Chemistry, 3rd edition, Springer Berlin.

    Google Scholar 

  2. Drauz K. and Waldmann H. (2002) Enzyme Catalysis in Organic Synthesis: A Comprehensive Handbook, Vol. I–III, VCH Weinheim.

    Book  Google Scholar 

  3. Klibanov A. M. (2001) Improving enzymes by using them in organic solvents. Nature 409, 241–246.

    Article  CAS  Google Scholar 

  4. Schmid R. D. and Verger R. (1998) Lipases: interfacial enzymes with attractive applications. Angew. Chem. 110, 1694–1720; Angew. Chem. Int. Ed. 37, 1608–1633.

    Article  Google Scholar 

  5. Villeneuve P., Muderhwa J. M., Graille J., and Haas M. J. (2000) Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches. J. Mol. Catal. B: Enzym. 9, 113–148.

    Article  CAS  Google Scholar 

  6. Reetz M. T. (2002) Lipases as practical biocatalysts. Curr. Opin. Chem. Biol. 6, 145–150.

    Article  CAS  Google Scholar 

  7. Brzozowski A. M., Derewenda U., Derewenda Z. S., et al. (1991) A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature (London) 351, 491–494.

    Article  CAS  Google Scholar 

  8. Van Tilbeurgh H., Egloff M.-P., Martinez C., Rugani N., Verger R. and Cambillau C. (1993) Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography. Nature (London) 362, 814–820.

    Article  Google Scholar 

  9. Avnir D., Braun S., Lev O., and Ottolenghi M. (1994) Enzymes and other proteins entrapped in sol-gel materials. Chem. Mater. 6, 1605–1614.

    Article  CAS  Google Scholar 

  10. Johnson P. and Whateley T. L. (1971) Use of polymerizing silica gel systems for immobilization of trypsin. J. Colloid Interface Sci. 37, 557–563.

    Article  CAS  Google Scholar 

  11. Glad M., Norrlöw O., Sellergren B., Siegbahn N., and Mosbach K. (1985) Use of silane monomers for molecular imprinting and enzyme entrapment in polysiloxane-coated porous silica. J. Chromatogr. 347, 11–23.

    Article  CAS  Google Scholar 

  12. Avnir D. (1995) Organic chemistry within ceramic matrixes: Doped sol-gel materials. Acc. Chem. Res. 28, 328–334.

    Article  CAS  Google Scholar 

  13. Livage J. (1996) Bioactivity in sol-gel glasses. C. R. Acad. Sci., Ser. IIb: Mec., Phys. Chim., Astron. 322, 417–427.

    CAS  Google Scholar 

  14. Gill I. (2001) Bio-doped nanocomposite polymers: sol-gel bioencapsulates. Chem. Mater. 13, 3404–3421.

    Article  CAS  Google Scholar 

  15. Hench L. L. and West J. K. (1990) The sol-del process. Chem. Rev. 90, 33–72.

    Article  CAS  Google Scholar 

  16. Brinker C. J. and Scherer G. W. (1990) Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing, Academic Press Boston.

    Google Scholar 

  17. Reetz M. T., Zonta A., and Simpelkamp J. (1995) Efficient heterogeneous biocatalysts by entrapment of lipases in hydrophobic sol-gel materials. Angew. Chem. 107, 373–376; Angew. Chem., Int. Ed. Engl. 34, 301–303.

    Article  Google Scholar 

  18. Reetz M. T., Zonta A., and Simpelkamp J. (1996) Efficient immoblization of lipases by entrapment in hydrophobic sol-gel materials. Biotechnol. Bioeng. 49, 527–534.

    Article  CAS  Google Scholar 

  19. Reetz M. T., Zonta A., Simpelkamp J., and Könen W. (1996) In situ fixation of lipase-containing hydrophobic sol-gel materials on sintered glass—highly efficient heterogeneous biocatalysts. Chem. Commun. (Cambridge, UK), 1397–1398.

    Google Scholar 

  20. Reetz M. T., Zonta A., Simpelkamp J., Rufinska A., and Tesche B. (1996) Characterization of hydrophobic sol-gel materials containing entrapped lipases. J. Sol-Gel Sci. Technol. 7, 35–43.

    Article  CAS  Google Scholar 

  21. Reetz M. T. (1997) Entrapment of biocatalysts in hydrophobic sol-gel materials for use in organic chemistry. Adv. Mater. (Weinheim, Ger.) 9 943–954.

    Article  CAS  Google Scholar 

  22. Reetz M. T., Wenkel R., and Avnir D. (2000) Entrapment of lipases in hydrophobic sol-gel-materials: efficient heterogeneous biocatalysts in aqueous medium. Synthesis 781–783.

    Google Scholar 

  23. Reetz M. T., Zonta A., Vijayakrishnan V., and Schimossek K. (1998) Entrapment of lipases in hydrophobic magnetite-containing sol-gel materials: magnetic separation of heterogeneous biocatalysts. J. Mol. Catal. A: Chem. 134, 251–258.

    Article  CAS  Google Scholar 

  24. Pierre M., Buisson P., Fache F., and Pierre A. (2000) Influence of the drying technique of silica gels on the enzymatic activity of encapsulated lipase. Biocatal. Biotransform. 18, 237–251.

    Article  CAS  Google Scholar 

  25. Buisson P., Hernandez C., Pierre M., and Pierre A. C. (2001) Encapsulation oflipases in aerogels. J. Non-Cryst. Solids 285, 295–302.

    Article  CAS  Google Scholar 

  26. Reetz M. T., Tielmann P., Wiesenhöfer W., Könen W., and Zonta A. (2003) Second generation sol-gel encapsulated lipases: Robust heterogeneous biocatalysts. Adv. Synth. Catal. 345, 717–728.

    Article  CAS  Google Scholar 

  27. Reinhoudt D. N., Eendebak A. M., Nijenhuis W. F., Verboom W., Kloosterman M., and Schoemaker H. E. (1989) The effect of crown ethers on enzyme-catalyzed reactions in organic solvents. J. Chem. Soc. Chem. Commun. 399–400.

    Google Scholar 

  28. Engbersen J. F. J., Broos J., Verboom W., and Reinhoudt D. N. (1996) Effects of crown ethers and small amounts of cosolvent on the activity and enantioselectivity of a-chymotrypsin in organic solvents. Pure Appl. Chem. 68, 2171–2178.

    Article  CAS  Google Scholar 

  29. van Unen D.-J., Engbersen J. F. J., and Reinhoudt D. N. (2002) Why do crown ethers activate enzymes in organic solvents? Biotechnol. Bioeng. 77, 248–255.

    Article  Google Scholar 

  30. Griebenow K., Laureano Y. D., Santos A. M., et al. (1999) Improved enzyme activity and enantioselectivity in organic solvents by methyl-β-cyclodextrin. J. Am. Chem. Soc. 121, 8157–8163.

    Article  CAS  Google Scholar 

  31. Santos A. M., Clemente I. M., Barletta G., and Griebenow K. (1999) Activation of serine protease subtilisin Carlsberg in organic solvents: combined effect of methl-β-cyclodextrin and water. Biotechnol. Lett. 21, 1113–1118.

    Article  CAS  Google Scholar 

  32. Khmelnitsky Y. L., Welch S. H., Clark D. S., and Dordick J. S. (1994) Salts dramatically enhance activity of enzymes suspended in organic solvents. J. Am. Chem. Soc. 116, 2647–2648.

    Article  CAS  Google Scholar 

  33. Altreuter D. H., Dordick J. S., and Clark D. S. (2002) Nonaqueous biocatalytic synthesis of new cyclotoxic doxorubicin derivatives: exploiting unexpected differences in the regioselectivity of salt-activated and solubilized subtilisin. J. Am. Chem. Soc. 124, 1871–1876.

    Article  CAS  Google Scholar 

  34. Liu Y.-Y., Xu J.-H., and Hu Y. (2000) Enhancing effect of Tween-80 on lipase performance in enantioselective hydrolysis of ketoprofen ester. J. Mol. Catal. B: Enzym. 10, 523–529.

    Article  CAS  Google Scholar 

  35. Colton I. J., Ahmed S. N., and Kazlauskas R. J. (1995) A 2-propanol treatment increases the enantioselectivity of Candida rugosa lipase toward esters of chiral carboxylic acids. J. Org. Chem. 60, 212–21

    Article  CAS  Google Scholar 

  36. Zhu K., Jutila A., Tuominen E. K. J., and Kinnunen P. K. J. (2001) Effects of ipropanol on the structural dynamics of Thermomyces lanuginosa lipase revealed by tryptophan fluorescence. Protein Sci. bd10}, 339–351.

    Article  Google Scholar 

  37. Cipiciani A. and Bellezza F. (2002) Primary allenic alcohols of high optical purity via lipase catalyzed resolution. J. Mol. Catal. B: Enzym. 17, 261–266.

    Article  CAS  Google Scholar 

  38. Khalaf N., Govardhan C. P., Lalonde J. J., Persichetti R. A., Wang Y.-F., and Margolin A. L. (1996) Cross-linked enzyme crystals as high active catalysts in organic solvents. J. Am. Chem. Soc. 118, 5494–5495.

    Article  CAS  Google Scholar 

  39. Badjic J. D., Kadnikova E. N., and Kostic N. M. (2001) Enantioselective aminolysis of an a-chloroester catalyzed by Candida cylindracea lipase encapsulated in sol-gel silica glass. Org. Lett. 3, 2025–2028.

    Article  CAS  Google Scholar 

  40. Furukawa S.-Y. and Kawakami K. (1998) Characterization of Candida rugosa lipase entrapped into organically modified silicates in esterification of menthol with butyric acid. J. Ferment. Bioeng. 85, 240–242.

    Article  CAS  Google Scholar 

  41. Pfau R. and Kunz H. (1999) Selectively deprotectable carbohydrates based on regioselective enzymatic reactions. Synlett 1817–1819.

    Google Scholar 

  42. Gill I., Pastor E., and Ballesteros A. (1999) Lipase-silicone biocomposites: Efficient and versatile immobilized biocatalysts. J. Am. Chem. Soc. 121, 9487–9496.

    Article  CAS  Google Scholar 

  43. Ragheb A., Brook M. A., and Hrynyk M. (2003) Highly activated, silicone entrapped, lipase. Chem. Commun. (Cambridge, UK) Issue 18, 2314–2315.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max-Planck-Institut für Kohlenforschung, Mülheim/Ruhr, Germany

    Manfred T. Reetz

Authors
  1. Manfred T. Reetz
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

    Jose M. Guisan

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Reetz, M.T. (2006). Practical Protocols for Lipase Immobilization Via Sol-Gel Techniques. In: Guisan, J.M. (eds) Immobilization of Enzymes and Cells. Methods in Biotechnologyâ„¢, vol 22. Humana Press. https://doi.org/10.1007/978-1-59745-053-9_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-053-9_6

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-290-2

  • Online ISBN: 978-1-59745-053-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation