Preparation and Characterization of Single-Enzyme Nanogels

  • Jun Ge
  • Ming Yan
  • Diannan Lu
  • Zhixia Liu
  • Zheng Liu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 743)

Abstract

Enzymes have been incorporated in nanostructures in order to provide robust catalysts for valuable reactions, particularly those performed under harsh and denaturing conditions. This chapter describes the encapsulation of enzymes in polyacrylamide nanogels by a two-step in situ polymerization process for preparing robust biocatalysts. The first step in this process is the generation of vinyl groups on the enzyme surface, while the second step involves in situ polymerization using acrylamide as the monomer. Encapsulation of the enzyme in the hydrophilic, porous, and flexible polyacrylamide gel of several nanometers thick would help to both give a significantly enhanced thermostability and prevent the removal of essential water by polar solvents. The hydrophilic flexible polymer shell also allows the protein structure to undergo necessary conformational transitions during the catalytic reaction and, at the same time, impose marginal mass transfer restrictions for the substrates entering across the polymer shell. The effectiveness of this method is demonstrated with horseradish peroxidase (HRP), carbonic anhydrase, and lipase. The impacts of such an encapsulation on the activity and stability of enzymes are also discussed.

Key words

Nanostructured biocatalyst polyacrylamide nanogel enzyme encapsulation enzyme stability enzymatic catalysis molecular simulation 

Notes

Acknowledgments

We gratefully acknowledge the support from the National High-tech R&D Program (863 Program; project number 2008AA05Z406) and National Natural Science Foundation (project number 20776076). The authors extend their thanks to Prof. Yunfeng Lu at Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, for his helps and suggestions on the research into nanostructures.

References

  1. 1.
    Kim, J., Grate, J. W., and Wang, P. (2008) Nanobiocatalysis and its potential applications. Trends Biotechnol. 26, 639–646.CrossRefGoogle Scholar
  2. 2.
    Kim, J., Grate, J. W., and Wang, P. (2006) Nanostructures for enzyme stabilization. Chem. Eng. Sci. 61, 1017–1026.CrossRefGoogle Scholar
  3. 3.
    Klibanov, A. M. (2001) Improving enzymes by using them in organic solvents. Nature 409, 241–246.CrossRefGoogle Scholar
  4. 4.
    Schmid, A., Dordick, J. S., Hauer, B., Kiener, A., Wubbolts, M., and Witholt, B. (2001) Industrial biocatalysis today and tomorrow. Nature 409, 258–268.CrossRefGoogle Scholar
  5. 5.
    Xiao, Y., Patolsky, F., Katz, E., Hainfeld, J. F., and Willner, I. (2003) “Plugging into enzymes”: Nanowiring of redox enzymes by a gold nanoparticle. Science 299, 1877–1881.CrossRefGoogle Scholar
  6. 6.
    Vriezema, D. M., Aragonès, M. C., Elemans, J. A. A. W., Cornelissen, J. J. L. M., Rowan, A. E., and Nolte, R. J. M. (2005) Self-assembled nanoreactors. Chem. Rev. 105, 1445–1489.CrossRefGoogle Scholar
  7. 7.
    Wu, L., and Payne, G. F. (2004) Biofabrication: Using biological materials and biocatalysts to construct nanostructured assemblies. Trends Biotechnol. 22, 593–599.CrossRefGoogle Scholar
  8. 8.
    Rosi, N. L., and Mirkin, C. A. (2005) Nanostructures in biodiagnostics. Chem. Rev. 105, 1547–1562.CrossRefGoogle Scholar
  9. 9.
    Stephanopoulos, N., Solis, E. O. P., and Stephanopoulos, G. (2005) Nanoscale process systems engineering: Toward molecular factories, synthetic cells, and adaptive devices. AIChE J. 51, 1858–1869.CrossRefGoogle Scholar
  10. 10.
    Kinbara, K., and Aida, T. (2005) Toward intelligent molecular machines: Directed motions of biological and artificial molecules and assemblies. Chem. Rev. 105, 1377–1400.CrossRefGoogle Scholar
  11. 11.
    Pohorille, A., and Deamer, D. (2002) Artificial cells: Prospects for biotechnology. Trends Biotechnol. 20, 123–128.CrossRefGoogle Scholar
  12. 12.
    Tanaka, M., and Sackmann, E. (2005) Polymer-supported membranes as models of the cell surface. Nature 437, 656–663.CrossRefGoogle Scholar
  13. 13.
    Zaks, A., and Klibanov, A. M. (1984) Enzymatic catalysis in organic media at 100°C. Science 224, 1249–1251.CrossRefGoogle Scholar
  14. 14.
    Garza-Ramos, G., Darszon, A., Tuena de Gomez-Puyou, M., and Gomez-Puyou, A. (1989) Catalysis and thermostability of mitochondrial F1-ATPase in toluene-phospholipid-low-water systems. Biochemistry 28, 3177–3182.CrossRefGoogle Scholar
  15. 15.
    Volkin, D. B., Staubli, A., Langer, R., and Klibanov, A. M. (1991) Enzyme thermoinactivation in anhydrous organic solvents. Biotechnol. Bioeng. 37, 843–853.CrossRefGoogle Scholar
  16. 16.
    Song, J. K., and Rhee, J. S. (2001) Enhancement of stability and activity of phospholipase A1 in organic solvents by directed evolution. Biochim. Biophys. Acta 1547, 370–378.CrossRefGoogle Scholar
  17. 17.
    Zhong, Z., Liu, J. L. C., Dinterman, L. M., Finkelman, M. A. J., Mueller, W. T., Rollence, M. L., Whitlow, M., and Wong, C. H. (1991) Engineering subtilisin for reaction in dimethylformamide. J. Am. Chem. Soc. 113, 683–684.CrossRefGoogle Scholar
  18. 18.
    Knubovets, T., Osterhout, J. J., and Klibanov, A. M. (1999) Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies. Biotechnol. Bioeng. 63, 242–248.CrossRefGoogle Scholar
  19. 19.
    Yan, M., Ge, J., Liu, Z., and Ouyang, P. (2006) Encapsulation of single enzyme in nanogel with enhanced biocatalytic activity and stability. J. Am. Chem. Soc. 128, 11008–11009.CrossRefGoogle Scholar
  20. 20.
    Ge, J., Lu, D., Wang, J., and Liu, Z. (2009) Lipase nanogel catalyzed transesterification in anhydrous dimethyl sulfoxide. Biomacromolecules 10, 1612–1618.CrossRefGoogle Scholar
  21. 21.
    Kim, J., and Grate, J. W. (2003) Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Lett. 3, 1219–1222.CrossRefGoogle Scholar
  22. 22.
    Yan, M., Liu, Z., Lu, D., and Liu, Z. (2007) Fabrication of single carbonic anhydrase nanogel against denaturation and aggregation at high temperature. Biomacromolecules 8, 560–565.CrossRefGoogle Scholar
  23. 23.
    Ge, J., Lu, D. N., Wang, J., and Liu, Z. (2008) Molecular fundamentals of enzyme nanogels. J. Phys. Chem. B 112, 14319–14324.CrossRefGoogle Scholar
  24. 24.
    Davis, J. C., and Averill, B. A. (1981) Isolation from bovine spleen of a green heme protein with properties of myeloperoxidase. J. Biol. Chem. 256, 5992–5996.Google Scholar
  25. 25.
    Pocker, Y., and Stone, J. T. (1965) The catalytic versatility of erythrocyte carbonic anhydrase. The enzyme-catalyzed hydrolysis of p-nitrophenyl acetate. J. Am. Chem. Soc. 87, 5497–5498.CrossRefGoogle Scholar
  26. 26.
    Pocker, Y., and Stone, J. T. (1967) The catalytic versatility of erythrocyte carbonic anhydrase. III. Kinetic studies of the enzyme-catalyzed hydrolysis of p-nitrophenyl acetate. Biochemistry 6, 668–678.CrossRefGoogle Scholar
  27. 27.
    López, N., Pernas, M. A., Pastrana, L. M., Sánchez, A., Valero, F., and Rúa, M. L. (2004) Reactivity of pure Candida rugosa lipase isoenzymes (Lip1, Lip2, and Lip3) in aqueous and organic media. Influence of the isoenzymatic profile on the lipase performance in organic media. Biotechnol. Prog. 20, 65–73.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jun Ge
    • 1
  • Ming Yan
    • 1
  • Diannan Lu
    • 1
  • Zhixia Liu
    • 1
  • Zheng Liu
    • 1
  1. 1.Department of Chemical EngineeringTsinghua UniversityBeijingChina

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