Skip to main content

Advertisement

SpringerLink
Log in

Operational and Thermal Stability Analysis of Thermomyces lanuginosus Lipase Covalently Immobilized onto Modified Chitosan Supports

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The aim of this paper was to evaluate different strategies of chitosan activation using cross-linking reagent like glycidol, epichlorohydrin, and glutaraldehyde for Thermomyces lanuginosus lipase (TLL) immobilization. Operational activity and stability by esterification of oleic acid with ethanol and thermal inactivation using these derivatives were investigated. Derivative obtained by sequentially activation with glycidol, ethylenediamine, and glutaraldehyde and subsequent TLL immobilization showed the best performance, with high hydrolytic activity value. Its stability was 15-fold higher than solubilized TLL in the evaluated inactivation conditions (60 °C, 25 mM sodium phosphate buffer pH 7). After 5 cycles of oleic acid esterification, only a few percentage of its conversion has reduced. On the other hand, glycidol-activated chitosan derivative showed very low hydrolytic activity value. Epichlorohydrin-activated chitosan derivative showed regular hydrolytic activity value. Both derivatives showed low immobilization yields. Operational stability of this last derivative was very low, where after the first cycle of oleic acid esterification, only 56% of its initial conversion was obtained.

Graphical Abstract

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

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Hasan, F., Shah, A. A., & Hameed, A. (2009). Methods for detection and characterization of lipases: a comprehensive review. Biotechnology Advances, 27, 782–798.

    Article  CAS  Google Scholar 

  2. Castilho L. R, D. M. G. Freire. s.l. : Enzimas em Biotecnologia. Produção, Aplicações e Mercado, Interciência, Rio de Janeiro, Brazil, Vols. 2008; 1; p. 369–385.

  3. Secundo, F., Carrea, G., Tarabiono, C., Gatti-Lafranconi, P., Brocca, S., Lotti, M., Jaeger, K. E., Puls, M., & Eggert, T. (2006). The lid is a structural and functional determinant of lipase activity and selectivity. Journal of Molecular Catalysis B: Enzymatic, 39, 166–170.

    Article  CAS  Google Scholar 

  4. Wu, X., Ge, J., Zhu, J., Zhang, Y., Yong, Y., & Liu, Z. (2015). A general method for synthesizing enzyme-polymer conjugates in reverse emulsions using Pluronic as reactive surfactant. Chem. comm., 51(47), 9674–9677.

    Article  CAS  Google Scholar 

  5. Zhu, J., Zhang, Y., Lu, D., Zare, R. N., Ge, J., & Liu, Z. (2013). Temperature-responsive enzyme–polymer nanoconjugates with enhanced catalytic activities in organic media. Chemical Communications, 49, 6090–6092.

    Article  CAS  Google Scholar 

  6. Hou, M., Wang, R., Wu, X., Zhang, Y., Ge, J., & Liu, Z. (2015). Synthesis of lutein esters by using a reusable lipase-pluronic conjugate as the catalyst. Catalysis Letters, 145(10), 1825–1829.

    Article  CAS  Google Scholar 

  7. Wu, X., & Ge, J. (2015). Enzymatic catalysis in melted polymer as green and reusable solvent. Catalysis Letter, 145, 1510–1513.

    Article  CAS  Google Scholar 

  8. Royon, D., Daz, M., Ellenrieder, G., & Locatelli, S. (2007). Enzymatic production of biodiesel from cotton seed oil using t-butanol as a solvent. Bioresourse Technol., 98, 648–653.

    Article  CAS  Google Scholar 

  9. Wu, X., Wang, R., Zhang, Y., Ge, J., & Liu, Z. (2014). Enantioselective ammonolysis of phenylglycine methyl ester with lipase–pluronic nanoconjugate in tertiary butanol. Catalysis Letters, 144(8), 1407–1410.

    Article  CAS  Google Scholar 

  10. Zhang, Y., Dai, Y., Hou, M., Li, T., Ge, J., & Liu, Z. (2013). Chemo-enzymatic synthesis of valrubicin using pluronic conjugated lipase with temperature responsiveness in organic media. RSC Advances, 3(45), 22963–22966.

    Article  CAS  Google Scholar 

  11. Ge, J., Lu, D., Wang, J., & Liu, Z. (2009). Lipase nanogel catalyzed transesterification in anhydrous dimethyl sulfoxide. Biomacromolecules, 10(6), 1612–1618.

    Article  CAS  Google Scholar 

  12. Garcia-Galan, C., Berenguer-Murcia, A., Fernandez-Lafuente, R., & Rodrigues, R. C. (2011). Potential of different enzyme immobilization strategies to improve enzyme performance. Adv. Synthesis and Catalysis, 353(16), 2885–2904.

    Article  CAS  Google Scholar 

  13. Hung, T. C., Giridhar, R., Chiou, S. H., & Wu, W. T. (2003). Binary immobilization of Candida rugosa lipase on chitosan. Journal of Molecular Catalysis B: Enzymatic, 26, 69–78.

    Article  CAS  Google Scholar 

  14. Kayser, H., Pienkoß, F., & Domínguez de María, P. (2014). Chitosan-catalyzed biodiesel synthesis: proof-of-concept and limitations. Fuel, 116, 267–272.

    Article  CAS  Google Scholar 

  15. Dutta, P. K., Dutta, J., & Tripathi, V. S. (2004). Chitin and chitosan: chemistry, properties and applications. Journal of Scientific and Industrial Research, 63, 20–31.

    CAS  Google Scholar 

  16. Younes, I., Hajji, S., Frachet, V., Rinaudo, M., Jellouli, K., & Nasri, M. (2014). Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan. International Journal of Biological Macromolecules, 69, 489–498.

    Article  CAS  Google Scholar 

  17. Silva, J. A., Macedo, G. P., Rodrigues, D. S., Giordano, R. L. C., & Gonçalves, L. R. B. (2012). Immobilization of Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using different support activation strategies. Biochemical Engineering J., 60, 16–24.

    Article  CAS  Google Scholar 

  18. de Bezerra, T. M. S., Bassan, J. C., de Santos, V. T., O., Ferraz, A., & Monti, R. (2015). Covalent immobilization of laccase in green coconut fiber and use in clarification of apple juice. Process Biochemistry, 50, 417–423.

  19. Nevell, T. P. (1963). In R. L. Whistler (Ed.), S.L. : Methods in carbohydrate chemistry (Vol. 3). NY: Academic Press.

    Google Scholar 

  20. Beppu, M. M., Arruda, E. J., Vieira, R. S., & Santos, N. N. (2004). Adsorption of Cu(II) on porous chitosan membranes functionalized with histidine. J Membrane Scice, 240, 227–235.

    Article  CAS  Google Scholar 

  21. dos Santos, J. C. S., Bonazza, H. L., de Matos, L. J. B. L., Carneiro, E. A., Barbosa, O., Fernandez-Lafuente, R., Gonçalves, L. R. B., & de Sant’ Ana, H. B. (2017). Immobilization of CALB on activated chitosan: application to enzymatic synthesis in supercritical and near-critical carbon dioxide. Biotechnology Reports, 14, 16–26.

    Article  Google Scholar 

  22. Adriano, W. S., Costa-Filho, E. H., Silva, J. A., Giordano, R. L. C., & Gonçalves, L. R. B. (2005). Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan. Brazilian J. Chem.l Eng., 22, 529–538.

    Article  CAS  Google Scholar 

  23. Manoel, E. A., dos Santos, J. C. S., Freire, D. M. G., Rueda, N., & Fernandez-Lafuente, R. (2015). Immobilization of lipases on hydrophobic supports involves the open form of the enzyme. Enzyme and Microbial Technology, 71, 53–57.

    Article  CAS  Google Scholar 

  24. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  25. Souza, T. C., Fonseca, T. S., Da Costa, J. A., Rocha, M. V. P., Mattos, M. C., Fernandez-Lafuente, R., Gonçalves, L. R. B., & Dos Santos, J. C. S. (2016). Cashew apple bagasse as a support for the immobilization of lipase B from Candida antarctica: application to the chemoenzymatic production of (R)-Indanol. J. of Molecular Catalysis B: Enzymatic, 130, 58–69.

    Article  Google Scholar 

  26. Sadana, A., & Henley, J. P. (1987). Analysis of enzyme deactivations by a series-type mechanism: influence of modification on the activity and stability of enzymes. Annals of the New York Academy of Sciences, 501, 73–79.

    Article  CAS  Google Scholar 

  27. Manzo, R. M., Sousa, M., Fenoglio, C. L., Gonçalves, L. R. B., & Mammarella, E. J. (2015). Chemical improvement of chitosan-modified beads for the immobilization of Enterococcus faecium DBFIQ E36 l-arabinose isomerase through multipoint covalent attachment approach. Journal of Industrial Microbiology & Biotechnology, 42, 1325–1340.

    Article  CAS  Google Scholar 

  28. Silva, J. A., Macedo, G. P., Rodrigues, D. S., Giordano, R. L. C., & Gonçalves, L. R. B. (2012). Immobilization of Candida antarctica lipase B by covalent attachment on. Biochemical Engineering Journal, 60, 16–24.

    Article  CAS  Google Scholar 

  29. Rodrigues, D. S., Mendes, A. A., Adriano, W. S., Gonçalves, L. R. B., & Giordano, R. L. C. (2008). Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods. Journal of Molecular Catalysis B: Enzymatic, 51, 100–109.

    Article  CAS  Google Scholar 

  30. Ribeiro, B. D., Castro, A. M., Coelho, M. A. Z., & Freire, D. M. G. (2011). Production and use of lipases in bioenergy: a review from the feedstocks to biodiesel production. Enzyme Research. doi:10.4061/2011/615803 (article ID 615803, 16 pp.)

  31. Babaki, M., Yousefi, M., Habibi, Z., Mohammadi, M., Yousefi, P., Mohammadi, J., & Brask, J. (2016). Enzymatic production of biodiesel using lipases immobilized on silica nanoparticles as highly reusable biocatalysts: effect of water, t-butanol and blue silica gel contents. Renewable Energy, 91, 196–206.

    Article  CAS  Google Scholar 

  32. Halling, P. J. (2004). Thermodynamic predictions for biocatalysis in nonconventional media: theory, tests, and recommendations for experimental design and analysis. Enzyme and Microbial Technology, 16, 178–206.

    Article  Google Scholar 

  33. Li, N.-W., Zong, M.-H., & Wu, H. (2009). Highly efficient transformation of waste oil to biodiesel by immobilized lipase from Penicillium expansum. Process Biochemistry, 44, 685–688.

    Article  CAS  Google Scholar 

  34. Oliveira, J. F. G., Lucena, I. L., Saboya, R. M. A., Rodrigues, M. L., Torres, A. E. B., Fernandes, F. A. N., Cavalcante, C. L., & Parente, E. J. (2010). Biodiesel production from waste coconut oil by esterification with ethanol: the effect of water removal by adsorption. Renewable Energy, 35, 2581–2584.

    Article  CAS  Google Scholar 

  35. Li, Z., Zhang, Y., Su, Y., Ouyang, P., Ge, J., & Liu, Z. (2014). Spatial co-localization of multi-enzymes by inorganic nanocrystal-protein complexes. Chem. Comm, 50, 12465–12468.

    Article  CAS  Google Scholar 

  36. Li, Z., Ding, Y., Li, S., Jiang, Y., Liu, Z., & Ge, J. (2016). Highly active, stable and self-antimicrobial enzyme catalysts prepared by biomimetic mineralization of copper hydroxysulfate. Nanoscale, 8, 17440–17445.

    Article  CAS  Google Scholar 

  37. Wu, X., Ge, J., Yang, C., Hou, M., & Liu, Z. (2015). Facile synthesis of multiple enzyme-contained metal-organic frameworks in biomolecule-friendly environment. Chem. Comm., 51, 13408–13411.

    Article  CAS  Google Scholar 

  38. Wu, X., Yang, C., Ge, J., & Liu, Z. (2015). Polydopamine tethered enzyme/metal-organic framework composites with high stability and reusability. Nanoscale, 7, 18883–18886.

    Article  CAS  Google Scholar 

  39. Wu, X., Hou, M., & Ge, J. (2015). Metal-organic frameworks and inorganic nanoflowers: a type of emerging inorganic crystal nanocarriers for enzyme immobilization. Catalysis Science & Technology, 5, 5077–5085.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was partially sponsored by funds of the projects CAI + D 2011 501 201101 00357 LI (Universidad Nacional del Litoral, Santa Fe, Argentina), Argentina–Brazil Bilateral Cooperation Program BR/12/06 MINCyT-CAPES 2012 (Buenos Aires, Argentina) and CONICET. The authors declare no competing financial interest. The authors would like to thank the financial support of the Brazilian Research Agencies CNPq, CAPES, FINEP, FUNCAP, and FAPESP.

Author information

Authors and Affiliations

  1. Instituto de Desarrollo Tecnológico para la Industria Química (INTEC), Universidad Nacional del Litoral (UNL), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Paraje “El Pozo”, RN 168, km 0, CP. 3000, Santa Fe, Argentina

    Horacio L. Bonazza, Ricardo M. Manzo & Enrique J. Mammarella

  2. Instituto de Engenharias e Desenvolvimento Sustentável, Universidade da Integração Internacional da Lusofonia Afro-Brasileira, Acarape, CE, CEP 62785-000, Brazil

    José C. S. dos Santos

Authors
  1. Horacio L. Bonazza
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ricardo M. Manzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José C. S. dos Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Enrique J. Mammarella
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Enrique J. Mammarella.

Additional information

Chemical compounds used in this article

Chitosan (PubChem CID: 71853)Epichlorohydrin (PubChem CID: 7835)Glycidol (PubChem CID: 11164)Ethylenediamine (PubChem CID: 3301)p-Nitrophenyl butyrate (PubChem CID: 75834)

Highlights

✓ Thermomyces lanuginosus lipase was immobilized on activated chitosan.

✓ The best activation strategy involved glycidol, ethylenediamine, and glutaraldehyde.

✓ The stability of the best derivative was 15-fold higher than solubilized TLL.

✓ More than 90% of oleic acid conversion was reached after 12 h.

✓ The best derivative lost only 18% activity after 5 cycles of oleic acid esterification.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bonazza, H.L., Manzo, R.M., dos Santos, J.C.S. et al. Operational and Thermal Stability Analysis of Thermomyces lanuginosus Lipase Covalently Immobilized onto Modified Chitosan Supports. Appl Biochem Biotechnol 184, 182–196 (2018). https://doi.org/10.1007/s12010-017-2546-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-017-2546-9

Keywords

Search

Navigation