Enzyme Stabilization via Cross-Linked Enzyme Aggregates

  • Munishwar N. GuptaEmail author
  • Smita Raghava
Part of the Methods in Molecular Biology book series (MIMB, volume 679)


Extensive cross-linking of a precipitate of a protein by a cross-linking reagent (glutaraldehyde has been most commonly used) creates an insoluble enzyme preparation called cross-linked enzyme aggregates (CLEAs). CLEAs show high stability and performance in both conventional aqueous media as well as nonaqueous media. These are also stable at fairly high temperatures. CLEAs having more than one kind of enzyme activity can be prepared and such CLEAs are called combi-CLEAs or multipurpose CLEAs. Extent of cross-linking often influences their morphology, stability, activity, and enantioselectivity.

Key words

Cross-linking agents Glutaraldehyde Cross-linked enzyme aggregates Immobilization Thermal stability Enantioselectivity 



This work was supported by funds obtained from Department of Science and Technology and Department of Biotechnology, both Government of India organizations. Finally, we thank our research group members (past and present); Dr. Kalyani Mondal, Dr. Shweta Shah, Abir Majumder, and Sohel Dalal, whose work has been described/quoted in this chapter.

Prof. Finn Wold, while at University of Minnesota, St. Paul, USA, introduced bifunctional reagents (more frequently called cross-linking reagents) to protein chemistry. Consequently several subsequent developments including CLEA design were possible. Prof. Wold was one of the early mentors of one of the authors (Munishwar N. Gupta). This chapter is dedicated to the memory of Prof. Finn Wold who was a great scientist and a great human being.


  1. 1.
    Wold, F. (1967) Bifunctional reagents. Methods Enzymol. 11, 617–640.CrossRefGoogle Scholar
  2. 2.
    Broun, G. B. (1976) Chemically aggregated enzymes. Methods Enzymol. 44, 263–280.PubMedCrossRefGoogle Scholar
  3. 3.
    Gupta, M. N. (1993) Applications of crosslinking techniques to enzyme/protein stabilization and bioconjugate preparation. In Biocatalyst Design for Stability and Specificity (Himmel, M. E., and Georgiou, G., Eds.) pp 307–324, ACS Symposium Series American Chemical Society, Washington, DC.CrossRefGoogle Scholar
  4. 4.
    Sheldon, R. A., Schoevaart, R., and van Langen, L. M. (2006) Cross-linked enzyme aggregates. In Immobilization of Enzymes and Cells (Guisan, J. M., Ed.) pp 43, Humana Press, New Jersey.Google Scholar
  5. 5.
    van Langen, L. M., Selassa, R. P., van Rantwijk, F., and Sheldon, R. A. (2005) Cross-linked aggregates of (R)-oxynitrilase: A stable, recyclable biocatalyst for enantioselective hydrocyanation. Org. Lett. 7, 327–329.PubMedCrossRefGoogle Scholar
  6. 6.
    Majumder, A. B., Mondal, K., Singh, T. P., and Gupta, M. N. (2008) Designing cross-linked lipase aggregates for optimum performance as biocatalysts. Biocatal. Biotransformation 26, 235–242.CrossRefGoogle Scholar
  7. 7.
    Dalal, S., Sharma, A., and Gupta, M. N. (2007) A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities. Chem. Cent. J. 1: 16.PubMedCrossRefGoogle Scholar
  8. 8.
    Shah, S., Sharma, A., and Gupta, M. N. (2006) Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder. Anal. Biochem. 351, 207–213.PubMedCrossRefGoogle Scholar
  9. 9.
    Sheldon, R. A. (2006) Immobilization of enzymes as cross-linked enzyme aggregates: A simple method for improving performance. In Biocatalysis in the Pharmaceutical and Biotechnology Industries (Patel, R. N., Ed.) pp 350–362, CRC Press, New York.Google Scholar
  10. 10.
    Illanes, A., Wilson, L., Caballero, E., Fernández-Lafuente, R., and Guisan, J. M. (2006) Cross-linked penicillin acylase aggregates for synthesis of β-lactam antibiotics in organic medium. Appl. Biochem. Biotechnol. 133, 189–202.PubMedCrossRefGoogle Scholar
  11. 11.
    Ruiz Toral, A., de los Rios, A. P., Hernandez, F. J., Janssen, M. H. A., Schoevaart, R., van Rantwijk, F., and Sheldon, R. A. (2007) Cross-linked Candida antarctica lipase B is active in denaturing ionic liquids. Enzyme Microb. Technol. 40, 1095–1099.CrossRefGoogle Scholar
  12. 12.
    Shah, S., and Gupta, M. N. (2007) Kinetic resolution of (±)–1-phenylethanol in [Bmim][PF6] using high activity preparations of lipases. Bioorg. Med. Chem. Lett. 17, 921–924.PubMedCrossRefGoogle Scholar
  13. 13.
    Hobbs, H. R., Kondor, B., Stephenson, P., Sheldon, R. A., Thomas, N. R., and Poliakoff, M. (2006) Continuous kinetic resolution catalysed by cross-linked enzyme aggregates, ‘CLEAs’, in supercritical CO2. Green Chem. 8, 816–821.CrossRefGoogle Scholar
  14. 14.
    Mateo, B., Chmura, A., Rustler, S., van Rantwijk, F., Stolz, A., and Sheldon, R. A. (2006) Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase–nitrilase bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activity. Tetrahedron Asymm. 17, 320–323.CrossRefGoogle Scholar
  15. 15.
    St. Clair, N. L., and Navia, M. A. (1992) Cross-linked enzyme crystal as robust biocatalysts, J. Am. Chem. Soc. 114, 7314–7316.CrossRefGoogle Scholar
  16. 16.
    Mondal, K., Ramesh, N. G., Roy, I., and Gupta, M. N. (2006) Enhancing the synthetic utility of aldolase antibody 38C2. Bioorg. Med. Chem. Lett. 16, 807–810.PubMedCrossRefGoogle Scholar
  17. 17.
    Wilson, L., Illanes, A., Abian, O., Pessela, B. C. C., Fernandez-Lafuente, R., and Guisán, J. M. (2004) Co-aggregation of penicillin G acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media. Biomacromolecules 5, 852–857.PubMedCrossRefGoogle Scholar
  18. 18.
    Kim, M. I., Kim, J., Lee, J., Jia, H., Na, H. B., Youn, J. K., Kwak, J. H., Dohnalkova, A., Grate, J. W., Wang, P., Hyeon, T., Park, H. G., Chang, H. N. (2007) Cross-linked enzyme aggregates in hierarchically-ordered mesoporous silica: A simple and effective method for enzyme stabilization. Biotechnol. Bioeng. 96, 210–218.PubMedCrossRefGoogle Scholar
  19. 19.
    Hilal, N., Nigmatullin, R., and Alpatova, A. (2004) Immobilization of cross-linked lipase aggregates within microporous polymeric membranes. J. Memb. Sci. 238, 131–141.CrossRefGoogle Scholar
  20. 20.
    Mateo, C., Palomo, J. M., van Langen, L. M., Rantwijik, F. V., and Sheldon, R. A. (2004) A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates. Biotechnol. Bioeng. 86, 273–276.PubMedCrossRefGoogle Scholar
  21. 21.
    Bell, G., Halling, P. J., Moore, B. D., Partridge, J., and Rees, D. G. (1995) Biocatalyst behavior in low-water systems. Trends Biotechnol. 13, 468–473.CrossRefGoogle Scholar
  22. 22.
    Dalal, S., Kapoor, M., and Gupta, M. N. (2007) Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions. J. Mol. Catal. B: Enzym. 44, 128132.CrossRefGoogle Scholar
  23. 23.
    Schoevaart R., Wolbers, M. W., Golubovic, M., Ottens, M., Kieboom, A. P. G., Rantwijik, F. V., van der Wielen, L. A. M., and Sheldon, R. A. (2004) Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs). Biotechnol. Bioeng. 20, 754–762.CrossRefGoogle Scholar
  24. 24.
    Tyagi, R., Batra, R., and Gupta, M. N. (1999) Amorphous enzyme aggregates: stability towards heat and aqueous-organic cosolvent mixtures. Enzyme Microb. Technol. 24, 348–353.CrossRefGoogle Scholar
  25. 25.
    López-Gallego, F., Betancor, L., Hidalgo, A., Alonso, N., Fernandez-Láfuente, R., and Guisán, J. M. (2005) Co-aggregation of enzymes and polyethyleneimine: a simple method to prepare stable and immobilized derivatives of glutaryl acylase. Biomacromolecules 6, 1639–1842.CrossRefGoogle Scholar
  26. 26.
    Vaidya, A., and Fischer, L. (2006) Stabilization of new imprint property of glucose oxidase in pure aqueous medium by cross-linked-imprinting approach. In Immobilization of enzymes and cells (Guisan, J. M., Ed.) pp 175–183, Humana Press, New Jersey.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Chemistry DepartmentIndian Institute of Technology DelhiHauz KhasIndia

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