Skip to main content
SpringerLink
Log in

Immobilization of Enzymes as Cross-Linked Enzyme Aggregates: General Strategy to Obtain Robust Biocatalysts

  • Protocol
  • First Online:
Immobilization of Enzymes and Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2100))

Abstract

Among carrier-free immobilization techniques, cross-linked enzyme aggregates (CLEA) have been extensively described for a great number of diverse enzymes. During the last two decades, numerous efforts have been devoted to identify and understand the main variables involved in CLEA’s preparation process leading to robust immobilized biocatalysts. Since every enzyme immobilized as CLEA requires specific conditions and protocols, herein we provide a general preparation strategy where main parameters are highlighted and correlated with a possible desired improved enzyme feature.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 249.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 329.99
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. Dewan SS (2017) Global markets for enzymes in industrial applications. A BBC research report, Wesley, MD

    Google Scholar 

  2. Robinson PK (2015) Enzymes: principles and biotechnological applications. Essays Biochem 59:1–41

    Article  PubMed  PubMed Central  Google Scholar 

  3. Stepankova V, Bidmanova S, Koudelakova T, Prokop Z, Chaloupkova R, Damborsky J (2013) Strategies for stabilization of enzymes in organic solvents. ACS Catal 3(12):2823–2836

    Article  CAS  Google Scholar 

  4. Bommarius AS, Paye MF (2013) Stabilizing biocatalysts. Chem Soc Rev 42(15):6534–6565

    Article  CAS  PubMed  Google Scholar 

  5. Iyer PV, Ananthanarayan L (2008) Enzyme stability and stabilization—aqueous and non-aqueous environment. Process Biochem 43(10):1019–1032

    Article  CAS  Google Scholar 

  6. Cao L, Van Rantwijk F, Sheldon RA (2000) Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase. Org Lett 2(10):1361–1364

    Article  CAS  PubMed  Google Scholar 

  7. Talekar S, Joshi A, Joshi G, Kamat P, Haripurkar R, Kambale S (2013) Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs). RSC Adv 3(31):12485–12511

    Article  CAS  Google Scholar 

  8. Cui JD, Jia SR (2015) Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges. Crit Rev Biotechnol 35(1):15–28

    Article  CAS  PubMed  Google Scholar 

  9. Sheldon RA (2011) Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs). Appl Microbiol Biotechnol 92(3):467–477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Velasco-Lozano S, López-Gallego F, Mateos Diaz JC, Favela-Torres E (2015) Cross-linked enzyme aggregates (CLEA) in enzyme improvement—a review. Biocatalysis 1:166–177

    Google Scholar 

  11. Cruz J, Barbosa O, Rodrigues RC, Fernandez-Lafuente R, Torres R, Ortiz C (2012) Optimized preparation of CALB-CLEAs by response surface methodology: the necessity to employ a feeder to have an effective crosslinking. J Mol Catal B Enzym 80:7–14

    Article  CAS  Google Scholar 

  12. Zerva A, Antonopoulou I, Enman J, Iancu L, Jütten P, Rova U, Christakopoulos P (2018) Optimization of transesterification reactions with clea-immobilized feruloyl esterases from Thermothelomyces thermophila and Talaromyces wortmannii. Molecules 23(9):2403

    Article  CAS  PubMed Central  Google Scholar 

  13. Rajendhran J, Gunasekaran P (2007) Application of cross-linked enzyme aggregates of Bacillus badius penicillin G acylase for the production of 6-aminopenicillanic acid. Lett Appl Microbiol 44(1):43–49

    Article  CAS  PubMed  Google Scholar 

  14. Ba S, Haroune L, Cruz-Morató C, Jacquet C, Touahar IE, Bellenger JP, Legault CY, Jones JP, Cabana H (2014) Synthesis and characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters. Sci Total Environ 487(1):748–755

    Article  CAS  PubMed  Google Scholar 

  15. Talekar S, Desai S, Pillai M, Nagavekar N, Ambarkar S, Surnis S, Ladole M, Nadar S, Mulla M (2013) Carrier free co-immobilization of glucoamylase and pullulanase as combi-cross linked enzyme aggregates (combi-CLEAs). RSC Adv 3(7):2265–2271. https://doi.org/10.1039/c2ra22657j

    Article  CAS  Google Scholar 

  16. Dalal S, Kapoor M, Gupta MN (2007) Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions. J Mol Catal B Enzym 44(3):128–132

    Article  CAS  Google Scholar 

  17. Nguyen LT, Yang KL (2017) Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions. Enzym Microb Technol 100:52–59

    Article  CAS  Google Scholar 

  18. Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom APG, Van Rantwijk F, Van Der Wielen LAM, Sheldon RA (2004) Preparation, optimization, and structures, of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 87(6):754–762

    Article  CAS  PubMed  Google Scholar 

  19. Talekar S, Nadar S, Joshi A, Joshi G (2014) Pectin cross-linked enzyme aggregates (pectin-CLEAs) of glucoamylase. RSC Adv 4(103):59444–59453

    Article  CAS  Google Scholar 

  20. Zerva A, Antonopoulou I, Enman J, Iancu L, Rova U, Christakopoulos P (2018) Cross-linked enzyme aggregates of feruloyl esterase preparations from Thermothelomyces thermophila and Talaromyces wortmannii. Catalysts 8(5):208

    Article  CAS  Google Scholar 

  21. Velasco-Lozano S, López-Gallego F, Vázquez-Duhalt R, Mateos-Díaz JC, Guisán JM, Favela-Torres E (2014) Carrier-free immobilization of lipase from candida rugosa with polyethyleneimines by carboxyl-activated cross-linking. Biomacromolecules 15(5):1896–1903

    Article  CAS  PubMed  Google Scholar 

  22. Wilson L, Illanes A, Soler L, Henríquez MJ (2009) Effect of the degree of cross-linking on the properties of different CLEAs of penicillin acylase. Process Biochem 44(3):322–326

    Article  CAS  Google Scholar 

  23. Majumder AB, Mondal K, Singh TP, Gupta MN (2008) Designing cross-linked lipase aggregates for optimum performance as biocatalysts. Biocatal Biotransformation 26(3):235–242

    Article  CAS  Google Scholar 

  24. Kim MH, Park S, Kim YH, Won K, Lee SH (2013) Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates. J Mol Catal B Enzym 97:209–214

    Article  CAS  Google Scholar 

  25. Velasco-Lozano S, López-Gallego F, Rocha-Martin J, Guisán JM, Favela-Torres E (2016) Improving enantioselectivity of lipase from Candida rugosa by carrier-bound and carrier-free immobilization. J Mol Catal B Enzym 130:32–39

    Article  CAS  Google Scholar 

  26. Wang A, Zhang F, Chen F, Wang M, Li H, Zeng Z, Xie T, Chen Z (2011) A facile technique to prepare cross-linked enzyme aggregates using p-benzoquinone as cross-linking agent. Korean J Chem Eng 28(4):1090–1095

    Article  CAS  Google Scholar 

  27. Valdés EC, Soto LW, Arcaya GA (2011) Influence of the pH of glutaraldehyde and the use of dextran aldehyde on the preparation of cross-linked enzyme aggregates (CLEAs) of lipase from Burkholderia cepacia. Electron J Biotechnol 14(3). https://doi.org/10.2225/vol14-issue3-fulltext-1

  28. Yu HW, Chen H, Wang X, Yang YY, Ching CB (2006) Cross-linked enzyme aggregates (CLEAs) with controlled particles: application to Candida rugosa lipase. J Mol Catal B Enzym 43(1–4):124–127

    Article  CAS  Google Scholar 

  29. Kumar S, Mohan U, Kamble AL, Pawar S, Banerjee UC (2010) Cross-linked enzyme aggregates of recombinant Pseudomonas putida nitrilase for enantioselective nitrile hydrolysis. Bioresour Technol 101(17):6856–6858

    Article  CAS  PubMed  Google Scholar 

  30. Dinh TH, Jang NY, McDonald KA, Won K (2015) Cross-linked aggregation of glutamate decarboxylase to extend its activity range toward alkaline pH. J Chem Technol Biotechnol 90:2100–2105

    Article  CAS  Google Scholar 

  31. Mateo C, Palomo JM, Van Langen LM, Van Rantwijk F, Sheldon RA (2004) A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates. Biotechnol Bioeng 86(3):273–276

    Article  CAS  PubMed  Google Scholar 

  32. Zhen Q, Wang M, Qi W, Su R, He Z (2013) Preparation of β-mannanase CLEAs using macromolecular cross-linkers. Catal Sci Technol 3(8):1937–1941

    Article  CAS  Google Scholar 

  33. Šulek F, Fernández DP, Knez Ž, Habulin M, Sheldon RA (2011) Immobilization of horseradish peroxidase as crosslinked enzyme aggregates (CLEAs). Process Biochem 46(3):765–769

    Article  CAS  Google Scholar 

  34. Arsenault A, Cabana H, Jones JP (2011) Laccase-based CLEAs: Chitosan as a novel cross-linking agent. Enzyme Res 2011:376015, 10 pages

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yang Xe ZP, Ni Y, Sun Z (2012) Highly efficient biosynthesis of sucrose-6-acetate with cross-linked aggregates of Lipozyme TL 100 L. J Biotechnol 161(1):27–33

    Article  CAS  Google Scholar 

  36. Cui JD, Sun LM, Li LL (2013) A simple technique of preparing stable cleas of phenylalanine ammonia lyase using co-aggregation with starch and bovine serum albumin. Appl Biochem Biotechnol 170(8):1827–1837

    Article  CAS  PubMed  Google Scholar 

  37. Miletic N, Loos K (2009) Over-stabilization of chemically modified and cross-linked Candida antarctica lipase B using various epoxides and diepoxides. Aust J Chem 62(8):799–805

    Article  CAS  Google Scholar 

  38. Shah S, Sharma A, Gupta MN (2006) Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder. Anal Biochem 351(2):207–213

    Article  CAS  PubMed  Google Scholar 

  39. Yamaguchi H, Miyazaki M, Asanomi Y, Maeda H (2011) Poly-lysine supported cross-linked enzyme aggregates with efficient enzymatic activity and high operational stability. Catal Sci Technol 1(7):1256–1261

    Article  CAS  Google Scholar 

  40. Wilson L, Fernández-Lorente G, Fernández-Lafuente R, Illanes A, Guisán JM, Palomo JM (2006) CLEAs of lipases and poly-ionic polymers: a simple way of preparing stable biocatalysts with improved properties. Enzym Microb Technol 39(4):750–755

    Article  CAS  Google Scholar 

  41. Tirunagari H, Basetty S, Rode HB, Fadnavis NW (2018) Crosslinked enzyme aggregates (CLEA) of phytase with soymilk proteins. J Biotechnol 282:67–69

    Article  CAS  PubMed  Google Scholar 

  42. Goetze D, Foletto EF, da Silva HB, Silveira VCC, Dal Magro L, Rodrigues RC (2017) Effect of feather meal as proteic feeder on combi-CLEAs preparation for grape juice clarification. Process Biochem 62:122–127

    Article  CAS  Google Scholar 

  43. Vaidya BK, Kuwar SS, Golegaonkar SB, Nene SN (2012) Preparation of cross-linked enzyme aggregates of l-aminoacylase via co-aggregation with polyethyleneimine. J Mol Catal B Enzym 74(3–4):184–191

    Article  CAS  Google Scholar 

  44. Li X, Yu Z, Bian Z, Xu J, Zhang L, Qiao M (2018) Physiochemical characterization of α-amylase as crosslinked enzyme aggregates. Catalysts 8(8):299

    Article  CAS  Google Scholar 

  45. Cabana H, Jones JP, Agathos SN (2007) Preparation and characterization of cross-linked laccase aggregates and their application to the elimination of endocrine disrupting chemicals. J Biotechnol 132(1):23–31

    Article  CAS  PubMed  Google Scholar 

  46. Li XD, Wu J, Jia DC, Wan YH, Yang N, Qiao M (2016) Preparation of cross-linked glucoamylase aggregates immobilization by using dextrin and xanthan gum as protecting agents. Catalysts 6(6):77

    Article  CAS  Google Scholar 

  47. Pan J, Kong XD, Li CX, Ye Q, Xu JH, Imanaka T (2011) Crosslinking of enzyme coaggregate with polyethyleneimine: a simple and promising method for preparing stable biocatalyst of Serratia marcescens lipase. J Mol Catal B Enzym 68(3–4):256–261

    Article  CAS  Google Scholar 

  48. Gupta P, Dutt K, Misra S, Raghuwanshi S, Saxena RK (2009) Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde. Bioresour Technol 100(18):4074–4076

    Article  CAS  PubMed  Google Scholar 

  49. López-Serrano P, Cao L, Van Rantwijk F, Sheldon RA (2002) Cross-linked enzyme aggregates with enhanced activity: application to lipases. Biotechnol Lett 24(16):1379–1383

    Article  Google Scholar 

  50. Guimarães JR, de Lima Camargo Giordano R, Fernandez-Lafuente R, Tardioli PW (2018) Evaluation of strategies to produce highly porous cross-linked aggregates of porcine pancreas lipase with magnetic properties. Molecules 23(11):2993

    Article  CAS  PubMed Central  Google Scholar 

  51. Wang M, Jia C, Qi W, Yu Q, Peng X, Su R, He Z (2011) Porous-CLEAs of papain: application to enzymatic hydrolysis of macromolecules. Bioresour Technol 102(3):3541–3545

    Article  CAS  PubMed  Google Scholar 

  52. Rodrigues RC, Ortiz C, Berenguer-Murcia A, Torres R, Fernández-Lafuente R (2013) Modifying enzyme activity and selectivity by immobilization. Chem Soc Rev 42(15):6290–6307

    Article  CAS  PubMed  Google Scholar 

  53. Fishman A, Cogan U (2003) Bio-imprinting of lipases with fatty acids. J Mol Catal B Enzym 22(3–4):193–202

    Article  CAS  Google Scholar 

  54. Cui JD, Liu RL, Li LL, Cui JD (2015) Imprinted cross-linked enzyme aggregate (iCLEA) of phenylalanine ammonia lyase: a new stable biocatalyst. In: Zhang TC, Nakajima M (eds) Advances in applied biotechnology. Lecture notes in electrical engineering, vol 332. Springer, Berlin, Heidelberg

    Google Scholar 

  55. Sampath C, Belur PD, Iyyasami R (2018) Enhancement of n-3 polyunsaturated fatty acid glycerides in Sardine oil by a bioimprinted cross-linked Candida rugosa lipase. Enzym Microb Technol 110:20–29

    Article  CAS  Google Scholar 

  56. De Winter K, Soetaert W, Desmet T (2012) An imprinted cross-linked enzyme aggregate (iCLEA) of sucrose phosphorylase: combining improved stability with altered specificity. Int J Mol Sci 13(9):11333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Amaral-Fonseca M, Kopp W, Giordano RLC, Fernández-Lafuente R, Tardioli PW (2018) Preparation of magnetic cross-linked amyloglucosidase aggregates: solving some activity problems. Catalysts 8(11):496

    Article  CAS  Google Scholar 

  58. Kopp W, Da Costa TP, Pereira SC, Jafelicci M Jr, Giordano RC, Marques RFC, Araújo-Moreira FM, Giordano RLC (2014) Easily handling penicillin G acylase magnetic cross-linked enzymes aggregates: catalytic and morphological studies. Process Biochem 49(1):38–46

    Article  CAS  Google Scholar 

  59. Lee J, Na HB, Kim BC, Lee JH, Lee B, Kwak JH, Hwang Y, Park JG, Gu MB, Kim J, Joo J, Shin CH, Grate JW, Hyeon T, Kim J (2009) Magnetically-separable and highly-stable enzyme system based on crosslinked enzyme aggregates shipped in magnetite-coated mesoporous silica. J Mater Chem 19(42):7864–7870

    Article  CAS  Google Scholar 

  60. Matijošyte I, Arends IWCE, de Vries S, Sheldon RA (2010) Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases. J Mol Catal B Enzym 62(2):142–148

    Article  CAS  Google Scholar 

  61. Wine Y, Cohen-Hadar N, Freeman A, Frolow F (2007) Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis. Biotechnol Bioeng 98(3):711–718

    Article  CAS  PubMed  Google Scholar 

  62. Okuda K, Urabe I, Yamada Y, Okada H (1991) Reaction of glutaraldehyde with amino and thiol compounds. J Ferment Bioeng 71(2):100–105

    Article  CAS  Google Scholar 

  63. Guauque Torres MP, Foresti ML, Ferreira ML (2013) Cross-linked enzyme aggregates (CLEAs) of selected lipases: a procedure for the proper calculation of their recovered activity. AMB Express 3(1):1–11

    Article  CAS  Google Scholar 

Download references

Acknowledgments

S. Velasco acknowledges the Mexican Council of Science and Technology (CONACyT) for the received postdoctoral fellowship, as well as the scientific illustrator Daniela Velasco (https://danielavelasco.myportfolio.com/scientific-illustration), for illustrating the Fig. 1.

Author information

Authors and Affiliations

  1. Catálisis Heterogénea en Síntesis Orgánicas Selectivas (ISQCH-CSIC), Universidad de Zaragoza, Zaragoza, Spain

    Susana Velasco-Lozano

Authors
  1. Susana Velasco-Lozano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Susana Velasco-Lozano .

Editor information

Editors and Affiliations

  1. CSIC Campus Cantoblanco, Instituto de Catalisis, Madrid, Spain

    Jose M. Guisan

  2. Institute of Biotech and Biochemical Engineering, Graz University of Technology, Graz, Austria

    Juan M. Bolivar

  3. CIC biomaGUNE, Donostia-San Sebastian, Spain

    Fernando López-Gallego

  4. Campus UAM-CSIC, Institute of Catatalysis, Madrid, Spain

    Javier Rocha-Martín

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Velasco-Lozano, S. (2020). Immobilization of Enzymes as Cross-Linked Enzyme Aggregates: General Strategy to Obtain Robust Biocatalysts. In: Guisan, J., Bolivar, J., López-Gallego, F., Rocha-Martín, J. (eds) Immobilization of Enzymes and Cells. Methods in Molecular Biology, vol 2100. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0215-7_23

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0215-7_23

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0214-0

  • Online ISBN: 978-1-0716-0215-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation