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Biotechnology Letters

, Volume 37, Issue 8, pp 1593–1600 | Cite as

Micro-scale procedure for enzyme immobilization screening and operational stability assays

  • Lucia Fernandez-Arrojo
  • Paloma Santos-Moriano
  • Barbara Rodriguez-Colinas
  • Antonio O. Ballesteros
  • Francisco J. PlouEmail author
Original Research Paper

Abstract

Objective

A simple and inexpensive methodology, based on the use of micro-centrifuge filter tubes, is proposed for establishing the best enzyme immobilization conditions.

Results

The immobilized biocatalyst is located inside the filter holder during the whole protocol, thus facilitating the incubations, filtrations and washings. This procedure minimizes the amount of enzyme and solid carrier needed, and allows exploring different immobilization parameters (pH, buffer concentration, enzyme/carrier ratio, incubation time, etc.) in a fast manner. The handling of immobilized enzymes using micro-centrifuge filter tubes can also be applied to assess the apparent activity of the biocatalysts, as well as their reuse in successive batch reaction cycles. The usefulness of the proposed methodology is shown by the determination of the optimum pH for the immobilization of an inulinase (Fructozyme L) on two anion-exchange polymethacrylate resins (Sepabeads EC-EA and Sepabeads EC-HA).

Conclusion

The micro-scale procedure described here will help to overcome the lack of guidelines that usually govern the selection of an immobilization method, thus favouring the development of stable and robust immobilized enzymes that can withstand harsh operating conditions in industry.

Keywords

Biotransformations Enzyme immobilization Enzyme carriers Immobilized biocatalysts Inulinase Operational stability 

Notes

Acknowledgments

This work was supported by a Grant from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysts. P. S-M. thanks the Spanish Ministry of Education for FPU Grant.

References

  1. Alcalde M, Plou FJ, de Segura AG, Remaud-Simeon M, Willemot RM, Monsan P, Ballesteros A (1999) Immobilization of native and dextran-free dextransucrases from Leuconostoc mesenteroides NRRL B-512F for the synthesis of glucooligosaccharides. Biotechnol Tech 13:749–755CrossRefGoogle Scholar
  2. Alvaro-Benito M, Sainz-Polo MA, Gonzalez-Perez D, Gonzalez B, Plou FJ, Fernandez-Lobato M, Sanz-Aparicio J (2012) Structural and kinetic insights reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, an enzyme that produces prebiotics. J Biol Chem 287:19674–19686PubMedCentralPubMedCrossRefGoogle Scholar
  3. Basso A, Spizzo P, Ferrario V, Knapic L, Savko N, Braiuca P, Ebert C, Ricca E, Calabro V, Gardossi L (2010) Endo- and exo-inulinases: enzyme-substrate interaction and rational immobilization. Biotechnol Prog 26:397–405PubMedGoogle Scholar
  4. Bautista-Barrufet A, Lopez-Gallego F, Rojas-Cervellera V, Rovira C, Pericas MA, Guisan JM, Gorostiza P (2014) Optical control of enzyme enantioselectivity in solid phase. ACS Catal 4:1004–1009CrossRefGoogle Scholar
  5. Berrio J, Plou FJ, Ballesteros A, Martin AT, Martinez MJ (2007) Immobilization of Pycnoporus coccineus laccase on Eupergit C: stabilization and treatment of oil mill wastewaters. Biocatal Biotransform 25:130–134CrossRefGoogle Scholar
  6. Bolivar JM, Schelch S, Mayr T, Nidetzky B (2014) Dissecting physical and biochemical factors of catalytic effectiveness in immobilized D-amino acid oxidase by real-time sensing of O2 availability inside porous carriers. ChemCatChem 6:981–986CrossRefGoogle Scholar
  7. Cao L (2005a) Carrier-bound immobilized enzymes: principles, applications and design. Wiley-VCH, WeinheimCrossRefGoogle Scholar
  8. Cao L (2005b) Immobilised enzymes: science or art? Curr Opin Chem Biol 9:217–226PubMedCrossRefGoogle Scholar
  9. Ge J, Lu D, Liu Z, Liu Z (2009) Recent advances in nanostructured biocatalysts. Biochem Eng J 44:53–59CrossRefGoogle Scholar
  10. George R, Sugunan S (2014) Kinetics of adsorption of lipase onto different mesoporous materials: evaluation of Avrami model and leaching studies. J Mol Catal B Enzym 105:26–32CrossRefGoogle Scholar
  11. Ghazi I, Gómez de Segura A, Fernández-Arrojo L, Alcalde M, Yates M, Rojas-Cervantes ML, Plou FJ, Ballesteros A (2005) Immobilisation of fructosyltransferase from Aspergillus aculeatus on epoxy-activated Sepabeads EC for the synthesis of fructo-oligosaccarides. J Mol Catal B Enzym 35:19–27CrossRefGoogle Scholar
  12. Gonçalves HB, Jorge JA, Pessela BC, Lorente GF, Guisan JM, Guimaräes LHS (2013) Characterization of a tannase from Emericela nidulans immobilized on ionic and covalent supports for propyl gallate synthesis. Biotechnol Lett 35:591–598PubMedCrossRefGoogle Scholar
  13. Izrael-Zivkovic LT, Zivkovic LS, Babic BM, Kokunesoski MJ, Jokic BM, Karadzic IM (2015) Immobilization of Candida rugosa lipase by adsorption onto biosafe meso/macroporous silica and zirconia. Biochem Eng J 93:73–83CrossRefGoogle Scholar
  14. Oliver-Calixte NJ, Uba FI, Battle KN, Weerakoon-Ratnayake KM, Soper SA (2014) Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs. Anal Chem 86:4447–4454PubMedCentralPubMedCrossRefGoogle Scholar
  15. Sheldon RA (2007) Enzyme immobilization: the quest for optimum performance. Adv Synth Catal 349:1289–1307CrossRefGoogle Scholar
  16. Skowronek M, Fiedurek J (2006) Purification and properties of extracellular endoinulinase from Aspergillus niger 20 OSM. Food Technol Biotechnol 44:53–58Google Scholar
  17. Tobis J, Tiller JC (2014) Impact of the configuration of a chiral, activating carrier on the enantioselectivity of entrapped lipase from Candida rugosa in cyclohexane. Biotechnol Lett 36:1661–1667PubMedCrossRefGoogle Scholar
  18. Torres-Salas P, Del Monte-Martinez A, Cutiño-Avila B, Rodriguez-Colinas B, Alcalde M, Ballesteros AO, Plou FJ (2011) Immobilized biocatalysts: novel approaches and tools for binding enzymes to supports. Adv Mater 23:5275–5282PubMedCrossRefGoogle Scholar
  19. Vasquez C, Anderson D, Oyarzun M, Carvajal A, Palma C (2014) Method for the stabilization and immobilization of enzymatic extracts and its application to the decolorization of textile dyes. Biotechnol Lett 36:1999–2010PubMedCrossRefGoogle Scholar
  20. Volkov PV, Sinitsyna OA, Fedorova EA, Rojkova AM, Satrutdinov AD, Zorov IN, Okunev ON, Gusakov AV, Sinitsyn AP (2012) Isolation and properties of recombinant inulinases from Aspergillus sp. Biochemistry-Moscow 77:492–501PubMedCrossRefGoogle Scholar
  21. Wang F, Nie TT, Shao LL, Cui Z (2014) Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix. Biocatal Biotransform 32:314–326CrossRefGoogle Scholar
  22. Weber E, Sirim D, Schreiber T, Thomas B, Pleiss J, Hunger M, Gläser R, Urlacher VB (2010) Immobilization of P450 BM-3 monooxygenase on mesoporous molecular sieves with different pore diameters. J Mol Catal B Enzym 64:29–37CrossRefGoogle Scholar
  23. Worsfold PJ (1995) Classification and chemical characteristics of immobilized enzymes—technical report. Pure Appl Chem 67:597–600CrossRefGoogle Scholar
  24. Wu R, He B, Zhang B, Zhao G, Li J, Li X (2014) Preparation of immobilized pectinase on regenerated cellulose beads for removing anionic trash in whitewater from papermaking. J Chem Technol Biotechnol 89:1103–1109CrossRefGoogle Scholar
  25. Zhou GX, Chen GY, Yan BB (2014) Biodiesel production in a magnetically-stabilized, fluidized bed reactor with an immobilized lipase in magnetic chitosan microspheres. Biotechnol Lett 36:63–68PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Lucia Fernandez-Arrojo
    • 1
  • Paloma Santos-Moriano
    • 1
  • Barbara Rodriguez-Colinas
    • 1
  • Antonio O. Ballesteros
    • 1
  • Francisco J. Plou
    • 1
    Email author
  1. 1.Instituto de Catálisis y Petroleoquímica, CSICMadridSpain

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