Skip to main content

Advanced Enzyme Immobilization Technologies: An Eco-friendly Support, a Polymer-Stabilizing Immobilization Strategy, and an Improved Cofactor Co-immobilization Technique

  • Protocol
  • First Online:
Enzyme Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2397))

Abstract

There is a wide variety of protocols for enzyme immobilization, allowing for the reuse of the enzyme, integration in flow bioreactors, and easy separation from the final product. However, none of them have reached a generalized implementation and new immobilization technologies are continuously being developed to improve the properties of the immobilized biocatalysts. In this chapter, we describe three advanced strategies looking at the key points of enzyme immobilization: the sustainability of the support, the recovered activity of the immobilized enzyme, and the reuse of the cofactors. Lignin is presented as a suitable and versatile support for enzyme immobilization, offering a more cost-effective and biodegradable strategy. A cationic polymer is used during the enzyme immobilization procedure to prevent the subunit dissociation of multimeric enzymes as well as to avoid excessive rigidification of the covalently immobilized enzyme. Finally, the reversible co-immobilization of cofactors has been improved by increasing the reactive groups of the support.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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. Sheldon RA, van Pelt S (2013) Enzyme immobilisation in biocatalysis: why, what and how. Chem Soc Rev 42:6223–6235. https://doi.org/10.1039/C3CS60075K

    Article  CAS  PubMed  Google Scholar 

  2. Guisan JM, López-Gallego F, Bolivar JM et al (2020) The science of enzyme immobilization. In: Methods in molecular biology. Humana Press, pp 1–26

    Google Scholar 

  3. Sheldon RA, Woodley JM (2018) Role of biocatalysis in sustainable chemistry. Chem Rev 118:801–838. https://doi.org/10.1021/acs.chemrev.7b00203

    Article  CAS  PubMed  Google Scholar 

  4. Zdarta J, Meyer A, Jesionowski T, Pinelo M (2018) A general overview of support materials for enzyme immobilization: characteristics, properties, practical utility. Catalysts 8:92. https://doi.org/10.3390/catal8020092

    Article  CAS  Google Scholar 

  5. Zdarta J, Klapiszewski Ł, Wysokowski M et al (2015) Chitin-lignin material as a novel matrix for enzyme immobilization. Mar Drugs 13:2424–2446. https://doi.org/10.3390/md13042424

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zdarta J, Klapiszewski L, Jedrzak A et al (2017) Lipase B from candida Antarctica immobilized on a silica-lignin matrix as a stable and reusable biocatalytic system. Catalysts 7:14. https://doi.org/10.3390/catal7010014

    Article  CAS  Google Scholar 

  7. Jędrzak A, Rębiś T, Klapiszewski Ł et al (2018) Carbon paste electrode based on functional GOx/silica-lignin system to prepare an amperometric glucose biosensor. Sensors Actuators B Chem 256:176–185. https://doi.org/10.1016/j.snb.2017.10.079

    Article  CAS  Google Scholar 

  8. Talebi Amiri M, Dick GR, Questell-Santiago YM, Luterbacher JS (2019) Fractionation of lignocellulosic biomass to produce uncondensed aldehyde-stabilized lignin. Nat Protoc 14:921–954. https://doi.org/10.1038/s41596-018-0121-7

    Article  CAS  PubMed  Google Scholar 

  9. Virgen-Ortíz JJ, Dos Santos JCSS, Berenguer-Murcia Á et al (2017) Polyethylenimine: a very useful ionic polymer in the design of immobilized enzyme biocatalysts. J Mater Chem B 5:7461–7490. https://doi.org/10.1039/C7TB01639E

    Article  PubMed  Google Scholar 

  10. Devine PN, Howard RM, Kumar R et al (2018) Extending the application of biocatalysis to meet the challenges of drug development. Nat Rev Chem 2:409–421. https://doi.org/10.1038/s41570-018-0055-1

    Article  Google Scholar 

  11. Fessner W-D (2015) Systems biocatalysis: development and engineering of cell-free “artificial metabolisms” for preparative multi-enzymatic synthesis. New Biotechnol 32:658–664. https://doi.org/10.1016/j.nbt.2014.11.007

    Article  CAS  Google Scholar 

  12. Beauchamp J, Vieille C (2015) Activity of select dehydrogenases with sepharose-immobilized N6-carboxymethyl-NAD. Bioengineered 6:106–110. https://doi.org/10.1080/21655979.2014.1004020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ji X, Wang P, Su Z et al (2014) Enabling multi-enzyme biocatalysis using coaxial-electrospun hollow nanofibers: redesign of artificial cells. J Mater Chem B 2:181–190. https://doi.org/10.1039/C3TB21232G

    Article  CAS  PubMed  Google Scholar 

  14. Fu J, Yang YR, Johnson-Buck A et al (2014) Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat Nanotechnol 9:531

    Article  CAS  Google Scholar 

  15. Velasco-Lozano S, Benítez-Mateos AI, López-Gallego F (2017) Co-immobilized phosphorylated cofactors and enzymes as self-sufficient heterogeneous biocatalysts for chemical processes. Angew Chem Int Ed 56:771–775. https://doi.org/10.1002/anie.201609758

    Article  CAS  Google Scholar 

  16. Roura Padrosa D, Benítez-Mateos AI, Calvey L, Paradisi F (2020) Cell-free biocatalytic syntheses of l -pipecolic acid: a dual strategy approach and process intensification in flow. Green Chem 22:5310–5316. https://doi.org/10.1039/d0gc01817a

    Article  CAS  Google Scholar 

  17. Heydari M, Ohshima T, Nunoura-Kominato N, Sakuraba H (2004) Highly stable L-lysine 6-Dehydrogenase from the Thermophile Geobacillus stearothermophilus isolated from a japanese hot spring: characterization, gene cloning and sequencing, and expression. Appl Environ Microbiol 70:937–942. https://doi.org/10.1128/AEM.70.2.937-942.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Cerioli L, Planchestainer M, Cassidy J et al (2015) Characterization of a novel amine transaminase from Halomonas elongata. J Mol Catal B Enzym 120:141–150. https://doi.org/10.1016/j.molcatb.2015.07.009

    Article  CAS  Google Scholar 

  19. Mateo C, Fernández-Lorente G, Cortés E et al (2001) One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports. Biotechnol Bioeng 76:269–276. https://doi.org/10.1002/bit.10019

    Article  CAS  PubMed  Google Scholar 

  20. Benítez-Mateos AI, Contente ML, Velasco-Lozano S et al (2018) Self-sufficient flow-biocatalysis by coimmobilization of pyridoxal 5′-phosphate and ω-transaminases onto porous carriers. ACS Sustain Chem Eng 6:13151–13159. https://doi.org/10.1021/acssuschemeng.8b02672

    Article  CAS  Google Scholar 

  21. Mateo C, Grazu V, Palomo JM et al (2007) Immobilization of enzymes on heterofunctional epoxy supports. Nat Protoc 2:1022

    Article  CAS  Google Scholar 

  22. McCluer RH (1963) Methods in carbohydrate chemistry. Volume 1, analysis and preparation of sugars. J Chem Educ 40:A394. https://doi.org/10.1021/ed040pa394

    Article  Google Scholar 

Download references

Acknowledgments

The authors acknowledge the University of Bern for the “Seal of Excellence Fund” (SELF) postdoctoral grant to support A.I.B.M.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Ana I. Benítez-Mateos or Francesca Paradisi .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Benítez-Mateos, A.I., Paradisi, F. (2022). Advanced Enzyme Immobilization Technologies: An Eco-friendly Support, a Polymer-Stabilizing Immobilization Strategy, and an Improved Cofactor Co-immobilization Technique. In: Magnani, F., Marabelli, C., Paradisi, F. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 2397. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1826-4_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1826-4_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1825-7

  • Online ISBN: 978-1-0716-1826-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics