Improving Lipase Activity by Immobilization and Post-immobilization Strategies

  • Jose M. Palomo
  • Marco Filice
  • Oscar Romero
  • Jose M. Guisan
Part of the Methods in Molecular Biology book series (MIMB, volume 1051)


One important parameter for the application of lipase catalysts in chemical industries is the specific activity displayed towards natural or unnatural substrates. Different strategies to enhance the lipase activity have been described. The immobilization of lipases on hydrophobic supports by interfacial adsorption at low ionic strength permitted the hyper-activation of these enzymes by fixing the open conformation of the lipase on the hydrophobic support. Improvements of activity from 1.2- up to 20-fold with respect to the initial one have been observed for lipases from different sources.

A second strategy was based on the presence of additives, in particular surfactants or ionic liquids, with hydrophobic character to enhance the activity of lipases immobilized on macroporous supports up to eightfold and even more than 100-fold in some cases for soluble lipases.

Finally, a third strategy to improve the activity in immobilized lipases was based on a site-directed chemical modification of the protein by glycosylation on the enzyme N-terminal group or on a unique reactive cysteine of the enzyme by disulfide exchange using different tailor-made disulfide activated activated polymers.

Key words

Lipase Activation Immobilization Site-directed modification Additives Polymers 



This work has been sponsored by the Spanish Ministry of Science and Innovation (AGL-2009-07526) and the CSIC by Intramural project (200980I133). The authors are grateful to CSIC for the JAE-DOC contract of M.F. and to CONICYT and Programa Bicentenario Becas-Chile for financial support of O.R.


  1. 1.
    Patel RN (2006) Biocatalysis: synthesis of chiral intermediates for pharmaceuticals. Curr Org Chem 10:1289–1321CrossRefGoogle Scholar
  2. 2.
    Fukuda H, Hama S, Tamalampudi S, Noda H (2008) Whole-cell biocatalysts for biodiesel fuel production. Trends Biotechnol 26:668–673PubMedCrossRefGoogle Scholar
  3. 3.
    Verger R (1997) Interfacial activation of lipases: facts and artifacts. Trends Biotechnol 15:32–38CrossRefGoogle Scholar
  4. 4.
    Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Lawson DM, Turkenburg JP, Bjorkling F, Huge-Jensen B, Patkar SA, Thim L (1991) A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351:491–494PubMedCrossRefGoogle Scholar
  5. 5.
    Derewenda U, Brzozowski AM, Lawson DM, Derewenda ZS (1992) Catalysis at the interface: the anatomy of a conformational change in a triglyceride lipase. Biochemistry 31:1532–1541PubMedCrossRefGoogle Scholar
  6. 6.
    Lowrier A, Drtina GJJ, Klibanov AM (1996) On the issue of interfacial activation of lipase in nonaqueous media. Biotechnol Bioeng 50:1–5CrossRefGoogle Scholar
  7. 7.
    Sarda L, Desnuelle P (1958) Actions of pancreatic lipase on esters in emulsions. Biochim Biophys Acta 30:513–521PubMedCrossRefGoogle Scholar
  8. 8.
    Bastida A, Sabuquillo P, Armisen P, Fernández-Lafuente R, Huguet J, Guisán JM (1998) A single step purification, immobilization and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol Bioeng 58:486–493PubMedCrossRefGoogle Scholar
  9. 9.
    Palomo JM, Muñoz G, Fernández-Lorente G, Mateo C, Fernández-Lafuente R, Guisán JM (2002) Interfacial adsorption of lipases on very hydrophobic support (octadecyl-Sepabeads): immobilization, hyperactivation and stabilization of the open form of lipases. J Mol Catal B Enzym 19–20:279–286CrossRefGoogle Scholar
  10. 10.
    Fernández-Lorente G, Cabrera Z, Godoy C, Fernandez-Lafuente R, Palomo JM, Guisan JM (2008) Interfacially activated lipases against hydrophobic supports: effect of the support nature on the biocatalytic properties. Process Biochem 43:1061–1067CrossRefGoogle Scholar
  11. 11.
    Mogensen JE, Sehgal P, Otzen DE (2005) Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents. Biochemistry 44:1719–1730PubMedCrossRefGoogle Scholar
  12. 12.
    Fishman A, Cogan U (2003) Bio-imprinting of lipases with fatty acids. J Mol Catal B Enzym 22:193–202CrossRefGoogle Scholar
  13. 13.
    Lopez-Serrano P, Cao L, Van Rantwijk F, Sheldon RA (2002) Cross-linked enzyme aggregates with enhanced activity: application to lipases. Biotechnol Lett 24:1379–1383CrossRefGoogle Scholar
  14. 14.
    Filice M, Marciello M, Betancor L, Carrascosa AV, Guisan JM, Fernandez-Lorente G (2011) Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange. Biotechnol Prog 27(4):961–968PubMedCrossRefGoogle Scholar
  15. 15.
    Fernandez-Lorente G, Palomo JM, Mateo C, Munilla R, Ortiz C, Cabrera Z et al (2006) Glutaraldehyde crosslinking in the presence of detergents of lipases adsorbed on aminated supports: improving lipases performance. Biomacromolecules 7:2610–2615PubMedCrossRefGoogle Scholar
  16. 16.
    Palomo JM, Segura RL, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Glutaraldehyde modification of lipases adsorbed on aminated supports: a simple way to improve their behaviour as enantioselective biocatalyst. Enzyme Microb Technol 40:704–707CrossRefGoogle Scholar
  17. 17.
    Palomo JM, Fuentes M, Fernández-Lorente G, Mateo C, Guisan JM, Fernández-Lafuente R (2003) General trend of lipase to self-assemble giving bimolecular aggregates greatly modifies the enzyme functionality. Biomacromolecules 4:1–6PubMedCrossRefGoogle Scholar
  18. 18.
    Helenius A, Simons K (1975) Solubilization of membranes by detergents. Biochem Biophys Acta 415:29–79PubMedCrossRefGoogle Scholar
  19. 19.
    Filice M, Guisan JM, Palomo JM (2010) Effect of ionic liquids as additives in the catalytic properties of different immobilized preparations of Rhizomucor miehei lipase in the hydrolysis of peracetylated lactal. Green Chem 12:1365–1369CrossRefGoogle Scholar
  20. 20.
    Hackenberger CPR, Schwarzer D (2008) Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed 47:10030–10074CrossRefGoogle Scholar
  21. 21.
    Gutarra MLE, Romero O, Abian O, Torres FAG, Freire DMG, Castro AM, Guisan JM, Palomo JM (2011) Enzyme surface glycosylation in the solid phase: improved activity and selectivity of Candida antarctica. ChemCatChem 3:1902–1910CrossRefGoogle Scholar
  22. 22.
    Chalker JM, Bernardes GJL, Lin YA, Davis BG (2009) Chemical modification of proteins at cysteine: opportunities in chemistry and biology. Chem Asian J 4:630–640PubMedCrossRefGoogle Scholar
  23. 23.
    Godoy C, de las Rivas B, Filice M, Fernández-Lorente G, Guisan JM, Palomo JM (2010) Enhanced activity of an immobilized lipase promoted by site-directed chemical modification with polymers. Process Biochem 45:534–541CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Jose M. Palomo
    • 1
  • Marco Filice
    • 1
  • Oscar Romero
    • 1
  • Jose M. Guisan
    • 2
  1. 1.Institute of Catalysis, CSICMadridSpain
  2. 2.Instituto de Catalisis y Petroleoquimica, CSICMadridSpain

Personalised recommendations