Skip to main content
SpringerLink
Log in

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

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

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.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • 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 13:749–755

    Article  CAS  Google Scholar 

  • 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 287:19674–19686

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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 26:397–405

    CAS  PubMed  Google Scholar 

  • 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 4:1004–1009

    Article  CAS  Google Scholar 

  • 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 25:130–134

    Article  CAS  Google Scholar 

  • 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 6:981–986

    Article  CAS  Google Scholar 

  • Cao L (2005a) Carrier-bound immobilized enzymes: principles, applications and design. Wiley-VCH, Weinheim

    Book  Google Scholar 

  • Cao L (2005b) Immobilised enzymes: science or art? Curr Opin Chem Biol 9:217–226

    Article  CAS  PubMed  Google Scholar 

  • Ge J, Lu D, Liu Z, Liu Z (2009) Recent advances in nanostructured biocatalysts. Biochem Eng J 44:53–59

    Article  CAS  Google Scholar 

  • 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 105:26–32

    Article  CAS  Google Scholar 

  • 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 35:19–27

    Article  CAS  Google Scholar 

  • 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 35:591–598

    Article  PubMed  Google Scholar 

  • 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 93:73–83

    Article  CAS  Google Scholar 

  • 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 86:4447–4454

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sheldon RA (2007) Enzyme immobilization: the quest for optimum performance. Adv Synth Catal 349:1289–1307

    Article  CAS  Google Scholar 

  • Skowronek M, Fiedurek J (2006) Purification and properties of extracellular endoinulinase from Aspergillus niger 20 OSM. Food Technol Biotechnol 44:53–58

    CAS  Google Scholar 

  • 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 36:1661–1667

    Article  CAS  PubMed  Google Scholar 

  • 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 23:5275–5282

    Article  CAS  PubMed  Google Scholar 

  • 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 36:1999–2010

    Article  CAS  PubMed  Google Scholar 

  • 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 77:492–501

    Article  CAS  PubMed  Google Scholar 

  • 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 32:314–326

    Article  Google Scholar 

  • 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 64:29–37

    Article  CAS  Google Scholar 

  • Worsfold PJ (1995) Classification and chemical characteristics of immobilized enzymes—technical report. Pure Appl Chem 67:597–600

    Article  CAS  Google Scholar 

  • 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 89:1103–1109

    Article  CAS  Google Scholar 

  • 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 36:63–68

    Article  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Instituto de Catálisis y Petroleoquímica, CSIC, Cantoblanco, Marie Curie 2, 28049, Madrid, Spain

    Lucia Fernandez-Arrojo, Paloma Santos-Moriano, Barbara Rodriguez-Colinas, Antonio O. Ballesteros & Francisco J. Plou

Authors
  1. Lucia Fernandez-Arrojo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paloma Santos-Moriano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Barbara Rodriguez-Colinas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Antonio O. Ballesteros
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Francisco J. Plou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francisco J. Plou.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fernandez-Arrojo, L., Santos-Moriano, P., Rodriguez-Colinas, B. et al. Micro-scale procedure for enzyme immobilization screening and operational stability assays. Biotechnol Lett 37, 1593–1600 (2015). https://doi.org/10.1007/s10529-015-1835-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-015-1835-z

Keywords

Search

Navigation