Skip to main content

Design of Smart Biocatalysts

Immobilization of Enzymes on Smart Polymers

  • Protocol
Immobilization of Enzymes and Cells

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

Abstract

Smart polymers are water-soluble polymers that can be precipitated by an appropriate stimulus such as change of pH, ionic strength, temperature, or addition of a chemical species. Such polymers occur naturally (e.g., alginate, chitosan) but can also be synthesized chemically (e.g., methyl methacrylate polymers available commercially as Eudragitâ„¢). Linking of an enzyme to these polymers by noncovalent or covalent methods gives a biocatalyst that can be used as a homogeneous catalyst but can be recovered for possible re-use (after the reaction) by applying appropriate stimulus. The illustrative protocol shows that xylanase could be adsorbed on Eudragit L-100 and this reversibly soluble-insoluble biocatalyst could be used for hydrolysis of xylan. Interestingly, the adsorption removed cellulase impurity. This is useful for paper pulp bleaching because xylanase should be free of cellulase activity. The soluble Eudragit-xylanase conjugate could be studied by circular dichroism spectroscopy to examine conformational changes in enzymes on immobilization on Eudragit L-100.

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

Access this chapter

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

References

  1. Taniguchi M., Tanahashi S., and Fujii M. (1990) Properties and repeated use of a reversibly soluble-insoluble yeast lytic enzyme. Appl. Microbiol. Biotechnol. 33, 629–632.

    Article  CAS  Google Scholar 

  2. Fujii M. and Taniguchi M. (1991) Application of reversibly soluble polymers in bioprocessing. Trends Biotechnol. 9, 191–196.

    Article  CAS  Google Scholar 

  3. Roy I., Sharma S., and Gupta M. N. (2003) Smart biocatalysts: design and applications. Adv. Biochem. Eng. Biotechnol. 86, 159–189.

    Google Scholar 

  4. Roy I., and Gupta M. N. (2004) Repeated enzymatic hydrolysis of polygalacturonic acid, chitosan and chitin using a novel reversibly-soluble pectinase with the aid of К-carrageenan. Biocatal. Biotransformation (in press)

    Google Scholar 

  5. Hoffman A.S. (2000) Bioconjugation of intelligent polymers and recognition proteins for use in diagnostics and affinity separations. Clin. Chem. 46, 1478–1486.

    CAS  Google Scholar 

  6. Roy I. and Gupta M. N. (2003) Smart polymeric materials: emerging biochemical applications. Chem. Biol. 10, 1161–1171.

    Article  CAS  Google Scholar 

  7. Tyagi R., Roy I., Agarwal R. and Gupta M.N. (1998) Carbodiimide coupling of enzymes to the reversibly soluble-insoluble polymer Eudragit S-100. Biotechnol. Appl. Biochem. 28, 201–206.

    CAS  Google Scholar 

  8. Carlsson J., Janson J.-C., and Sparrman M. (1989) Affinity chromatography. In: Protein Purification: Principles, High Resolution Methods and Applications, (Janson J. C. and Ryden L. eds.), Wiley-VCH New York, 275–329.

    Google Scholar 

  9. Roy I., Sardar M., and Gupta M. N. (2003) Evaluation of a smart bioconjugate of pectinase for chitin hydrolysis. Biochem. Eng. J. 16, 329–335.

    Article  CAS  Google Scholar 

  10. Sardar M., Roy I. and Gupta M. N. (2003) A smart bioconjugate of alginate and pectinase with unusual biological activity towards chitosan. Biotechnol. Prog. 19, 1654–1658.

    Article  CAS  Google Scholar 

  11. Ding Z. L., Chen G. H., and Hoffman A. S. (1996) Synthesis and purification of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin. Bioconjug. Chem. 7, 121–126.

    Article  CAS  Google Scholar 

  12. Ding Z. L., Chen G. H., and Hoffman A. S. (1998) Unusual properties of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin. J. Biomed. Mater. Res. 39, 498–505.

    Article  CAS  Google Scholar 

  13. Morris J. E., Hoffman A. S., and Fisher R. R. (1993) Affinity precipitation of proteins by polyligands. Biotechnol. Bioeng. 41, 991–997.

    Article  CAS  Google Scholar 

  14. Shimoboji T., Ding Z., Stayton P. S., and Hoffman A. S. (2001) Mechanistic investigation of smart polymer-protein conjugates. Bioconjug. Chem. 12, 314–319.

    Article  CAS  Google Scholar 

  15. Ito Y., Sugimura N., Kown O. H., and Imanishi Y. (1999) Enzyme modification by polymers with solubilities that change in response to photoirradiation in organic media. Nature Biotechnol. 17, 73–75.

    Article  CAS  Google Scholar 

  16. Shimoboji S., Larenas E., Fowler T., Kulkarni S., Hoffman A. S., and Stayton P.S. (2002) Photoresponsive polymer-enzyme switches. Proc. Natl. Acad. Sci. USA 99, 16,592–16,596.

    Article  CAS  Google Scholar 

  17. Sardar M., Roy I., and Gupta M. N. (2000) Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer Eudragit™ L-100. Enzyme Microb. Technol. 27, 672–679.

    Article  CAS  Google Scholar 

  18. Miller G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428.

    Article  CAS  Google Scholar 

  19. Bailey M. J., Biely P., and Poutanen K. (1992) Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257–270.

    Article  CAS  Google Scholar 

  20. Guoqiang D., Batra R., Kaul R., Gupta M. N., and Mattiasson B. (1995) Alternative modes of precipitation of Eudragit S-100: A potential ligand carrier for affinity precipitation of proteins. Bioseparation 5, 339–350.

    Google Scholar 

  21. Fujimura M., Mori T., and Tosa T. (1987) Preparation and properties of solubleinsoluble immobilized proteases. Biotechnol. Bioeng. 29, 747–752.

    Article  CAS  Google Scholar 

  22. Dominguez E., Nilsson M., and Hahn-Hagerdal B. (1988) Carbodiimide coupling of β-galactosidase from Aspergillus oryzae to alginate. Enzyme Microb. Technol. 10, 606–610.

    Article  CAS  Google Scholar 

  23. Vazquez-Duhalt R., Tinoco R., D’Antonio P., Topoleski L. D. T., and Payne G. F. (2001) Enzyme conjugation to the polysaccharide chitosan: smart biocatalysts and biocatalytic hydrogels. Bioconjug. Chem. 12, 301–306.

    Article  CAS  Google Scholar 

  24. Willner I. and Rubin S. (1993) Reversible photoregulation of the activities of proteins. Reactive Polym. 21, 177–181.

    Article  CAS  Google Scholar 

  25. Okumura K., Ikura K., Yoshikawa M., Sasaki R., and Chiba H. (1984) Preparation of soluble-insoluble interconvertible enzymes: Enzyme polymerised as1-casein conjugates. Agric. Biol. Chem. 48, 2435–2440.

    CAS  Google Scholar 

  26. Mondal K., Roy I., and Gupta M. N. (2003) κ-Carrageenan as a carrier in affinity precipitation of yeast alcohol dehydrogenase. Protein Expr. Purif. 32, 151–160.

    Article  CAS  Google Scholar 

  27. Chen J. P. and Chang K. C. (1994) Immobilization of chitinase on a reversibly soluble-insoluble polymer for chitin hydrolysis. J. Chem. Technol. Biotechnol. 60, 133–140.

    Article  CAS  Google Scholar 

  28. Ito Y., Kotoura M., Chung D. J., and Imanishi Y. (1993) Trypsin modification by vinyl polymers with variable solubilities in response to external signals. Bioconjug. Chem. 4, 358–361.

    Article  CAS  Google Scholar 

  29. Nath N.I., and Chilkoti A. (2003) Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide. Anal. Chem. 75, 709–715.

    Article  CAS  Google Scholar 

  30. Brahim S., Narinesingh D., and Guiseppi-Elie A. (2002) Bio-smart hydrogels: co-joined molecular recognition and signal transduction in biosensor fabrication and drug delivery. Biosens. Bioelectron. 17, 973–981.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Roy, I., Gupta, M.N. (2006). Design of Smart Biocatalysts. 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_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-053-9_8

  • 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