Applied Biochemistry and Biotechnology

, Volume 188, Issue 2, pp 310–325 | Cite as

One-Step Immobilization and Stabilization of a Recombinant Enterococcus faecium DBFIQ E36 l-Arabinose Isomerase for d-Tagatose Synthesis

  • Marylane de Sousa
  • Vânia M. M. Melo
  • Denise C. Hissa
  • Ricardo M. Manzo
  • Enrique J. Mammarella
  • André Saraiva Leão Marcelo Antunes
  • José L. García
  • Benevides C. PesselaEmail author
  • Luciana R. B. GonçalvesEmail author


A recombinant l-arabinose isomerase from Enterococcus faecium DBFIQ E36 was immobilized onto multifunctional epoxide supports by chemical adsorption and onto a chelate-activated support via polyhistidine-tag, located on the N-terminal (N-His-L-AI) or on the C-terminal (C-His-L-AI) sequence, followed by covalent bonding between the enzyme and the support. The results were compared to reversible L-AI immobilization by adsorption onto charged agarose supports with improved stability. All the derivatives presented immobilization yields of above 75%. The ionic interaction established between agarose gels containing monoaminoethyl-N-aminoethyl structures (MANAE) and the enzyme was the most suitable strategy for L-AI immobilization in comparison to the chelate-activated agarose. In addition, the immobilized biocatalysts by ionic interaction in MANAE showed to be the most stable, retaining up to 100% of enzyme activity for 60 min at 60 °C and with Km values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively.


l-Arabinose isomerase Chelate-agarose d-Tagatose Enterococcus faecium Immobilization Enzyme activity 


Funding Information

This study received financial support from the Brazilian Research Agencies CAPES, CNPq, and FUNCAP. This work was partially sponsored by funds from the project Argentina–Brazil Bilateral Cooperation Program BR/12/06 MINCyT-CAPES 2012 (Buenos Aires, Argentina).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that there is no conflict of interest.


  1. 1.
    Hong, Y. H., Lee, D. W., Lee, S. J., Choe, E. A., Kim, S. B., Lee, Y. H., Cheigh, C. I., & Pyun, Y. R. (2007). Production of d-tagatose at high temperatures using immobilized escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana. Biotechnology Letters, 29(4), 569–574.Google Scholar
  2. 2.
    Levin, G. V., Zehner, L. R., Saunders, J. P., & Beadle, J. R. (1995). Sugar substitutes: their energy values, bulk characteristics, and potential health benefits. The American Journal of Clinical Nutrition, 62(5), 1161S–1168S.Google Scholar
  3. 3.
    Livesey, G., & Brown, J. C. (1996). D-Tagatose is a bulk sweetener with zero energy determined in rats. The Journal of Nutrition, 126(6), 1601–1609.Google Scholar
  4. 4.
    Beadle, J. R., Saunder, J. P., & Wajada, T. J. (1992). Process for manufacturing tagatose. US Patent 5,078,796.Google Scholar
  5. 5.
    Oh, D. K. (2007). Tagatose: properties, applications, and biotechnological processes. Applied Microbiology and Biotechnology, 76(1), 1–8.Google Scholar
  6. 6.
    Illanes, A., Guerrero, C., Vera, C. Wilson, L., Conejeros, R., Scott, F. (2016). Lactose-derived prebiotics: a process perspective. 1st Ed., San Diego: Academic Press.
  7. 7.
    Oh, D. K., Kim, H. J., Ryu, S. A., Rho, H. J., & Ki, P. (2001). Development of an immobilization method of L-arabinose isomerase for industrial production of tagatose. Biotechnology Letters, 23(22), 1859–1862.Google Scholar
  8. 8.
    Kim, H. J., Ryu, S. A., Kim, P., & Oh, D. K. (2003). A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor. Biotechnology Progress, 19(2), 400–404.Google Scholar
  9. 9.
    Ryu, S. A., Kim, C. S., Kim, H. J., Baek, D. H., & Oh, D. K. (2003). Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor. Biotechnology Progress, 19(6), 1643–1647.Google Scholar
  10. 10.
    Jung, E. S., Kim, H. J., & Oh, D. K. (2005). Tagatose production by immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus L-arabinose isomerase mutant in a packed-bed bioreactor. Biotechnology Progress, 21(4), 1335–1340.Google Scholar
  11. 11.
    Rhimi, M., Messaoud, E. B., Borgi, M. A., khadra, K. B., & Bejar, S. (2007). Co-expression of l-arabinose isomerase and d-glucose isomerase in E. coli and development of an efficient process producing simultaneously d-tagatose and d-fructose. Enzyme and Microbial Technology, 40(6), 1531–1537.Google Scholar
  12. 12.
    Pessela, B. C. C., Vian, A., Mateo, C., Lafuente, R., García, J. L., Guisan, J. M., & Carrascosa, A. V. (2003). Overproduction of Thermus sp. strain T2 B-galactosidase in Escherichia coli and preparation by using tailor-made metal chelate supports. Applied and Environmental Microbiology, 69(4), 1967–1972.Google Scholar
  13. 13.
    Guisán, J. M. (1988). Aldehyde-agarose gels as activated supports for immobilization-stabilization of enzyme. Enzyme and Microbial Technology, 10(6), 375–382.Google Scholar
  14. 14.
    Mateo, C., Grazú, V., Palomo, J. M., López-Gallego, F., Fernández-Lafuente, R., & Guisán, J. M. (2007). Immobilization of enzymes on heterofunctional epoxy supports. Nature, 2, 1022–1033.Google Scholar
  15. 15.
    Fernández-Lorente, G., Lopez-Gallego, F., Bolivar, J. M., Rocha-Martin, J., Perez, S. M., & Guisán, J. M. (2015). Immobilization of proteins on highly activated glyoxyl supports: dramatic increase of the enzyme stability via multipoint immobilization on pre-existing carriers. Current Organic Chemistry, 19(17), 1719–1731.Google Scholar
  16. 16.
    Bolívar, J. M., Rocha-Martin, J., Godoy, C., Rodrigues, R. C., & Guisán, J. M. (2010). Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus. Process Biochemistry, 45(1), 107–113.Google Scholar
  17. 17.
    Mateo, C., Bolivar, J. M., Godoy, C. A., Rocha-Martin, J., Pessela, B. C., Curiel, J. A., Munoz, R., Guisan, J. M., & Fernandez-Lorete, G. (2010). Improvement of enzyme properties with a two-step immobilization process on novel heterofunctional supports. Biomacromolecules, 11(11), 3112–3117.Google Scholar
  18. 18.
    Manzo, R. M., de 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(10), 1325–1340.Google Scholar
  19. 19.
    Armisén, P., Mateo, C., Cortés, E., Barredo, J. L., Salto, F., Diez, B., Rodés, L., García, J. L., Fernández-Lafuente, R., & Guisan, J. M. (1999). Selective adsorption of poly-His tagged glutaryl acyiase on tailor-made metal chelate supports. Journal of Chromatography. A, 848(1-2), 61–70.Google Scholar
  20. 20.
    Hernaiz, M. J., & Crout, D. H. G. (2000). Immobilization-stabilization on Eupergit C of the‚ β-galatosidase from B. circulans and an β-galactosidase from A. oryzae. Enzyme and Microbial Technology, 27(1-2), 26–32.Google Scholar
  21. 21.
    Katchalski-Katzir, E., & Kraemer, D. (2000). Eupergit C, a carrier for immobilization of enzymes of industrial potential. Journal of Molecular Catalysis B: Enzymatic, 10(1-3), 157–176.Google Scholar
  22. 22.
    Mateo, C., Femández-Lorente, G., Benevides, C. C. P., Vían, A., Carrascosa, A. V., García, J. L., Fernández-Lafuente, R., & Guisan, J. M. (2001). Affinity chomatography of polyfíistidine tagged enzimes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. Journal of Chromatography A, 915(1-2), 97–106.Google Scholar
  23. 23.
    Mateo, C., Femández-Lorente, G., Abian, O., Fernández-Lafuente, R., & Guisan, J. M. (2000a). Multifunctionai Epoxi Suports. A new tool to improve the covalent immobiiization of proteins. The promotion of physical adsorptions of proteins on the supports before theírcovalent linkage. Biomacromoleculas, 1(4), 739–745.Google Scholar
  24. 24.
    Sousa, M., Manzo, R. M., García, J. L., Mammarella, E. J., Gonçalves, L. R. B., & Pessela, B. C. (2017). Engineering the L-arabinose isomerase from Enterococcus Faecium for D-tagatose synthesis. Molecules, 22(12), 2164.Google Scholar
  25. 25.
    Fernández-Lafuente, R., Rossell, C. M., Rodríguez, V., Santana, C., Soler, G., Bastida, A., & Guisan, J. M. (1993). Preparation of activated supports containing low pK amino groups. A new tool or protein immobilization via the carboxyl coupling method. Enzyme and Microbial Technology, 15(7), 546–550.Google Scholar
  26. 26.
    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(1-2), 248–254.Google Scholar
  27. 27.
    Dische, Z., & Borenfreund, E. (1951). A new spectrophotometric method for the detection and determination of keto sugars and trioses. The Journal of Biological Chemistry, 192, 583–587.Google Scholar
  28. 28.
    Hung, X. G., Tseng, W. C., Liu, S. M., Tzou, W. S., & Fang, T. Y. (2014). Characterization of a thermophilic L-arabinose isomerase from Thermoanaerobacterium saccharolyticum NTOU1. Biochemical Engineering Journal, 83, 121–128.Google Scholar
  29. 29.
    Guisan, J. M. (2006). Immobilization of enzymes and cells. Methods in biotechnology (Vol. 22). Totowa: Humana Press.Google Scholar
  30. 30.
    Laemmli, U. K. (1970). Cleavage of structural protein during the assembly of the heat of bacteriophage T4. Nature, 227(5259), 680–685.Google Scholar
  31. 31.
    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 covalente attachment on chitosan-based hydrogels using diferente support activation strategies. Biochemical Engineering Journal, 60, 16–24.Google Scholar
  32. 32.
    Illanes, A. (2008). Enzyme biocatalysis: principles and applications. Netherlands: Springer.Google Scholar
  33. 33.
    Cheng, L. F., Mu, W. M., Zhang, T., & Jiang, B. (2010). Na L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization. Applied Microbiology and Biotechnology, 86(4), 1089–1097.Google Scholar
  34. 34.
    Rhimi, M., Ilhammami, R., Bajic, G., Boudebbouze, S., Maguin, E., Haser, R., & Aghajari, N. (2010). The acid tolerant L-arabinose isomerase from the food grade Lactobacillus sakei 23 K is an attractive D-tagatose producer. Bioresource Technology, 101(23), 9171–9177.Google Scholar
  35. 35.
    Lee, S. J., Lee, D. W., Choe, E. A., Hong, Y. H., Kim, S. B., Kim, B. C., & Pyun, Y. R. (2005). Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum. Applied and Environmental Microbiology, 71(12), 7888–7896.Google Scholar
  36. 36.
    Mateo, C., Abian, O., Lafuente, R., & Guisan, J. M. (2000b). Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support–polyethylenimine composites. Biotechnology and Bioengineering, 68(1), 98–105.Google Scholar
  37. 37.
    Fernández-Lafuente, R., Rodríguez, V., Mateo, C., Penzol, G., Hernández-Justiz, O., Irazoqui, G., Villaríno, A., Ovsejevi, K., Francisco, B., & Guisan, J. M. (1999). Stabilization of multimeric enzymes via immobilization and post-immobilization techniques. Journal of Molecular Catalysis B: Enzymatic, 7(1-4), 181–189.Google Scholar
  38. 38.
    Cardoso, C. L., de Moraes, M. C., & Cass, Q. B. (2009). Imobilização de enzimas em suportes cromatográficos: uma ferramenta na busca por substâncias bioativas. Quimica Nova, 32(1), 175–187.Google Scholar
  39. 39.
    Pessela, B. C. C. (2002). Ingeniería de Biocatalizadores y Bioprocesos: β-Galactosidasa de Thermus sp., Cepa T2. PhD Thesis. Universidad Politécnica de Madrid, Espanha.Google Scholar
  40. 40.
    Pisan, F. M., Relia, C. A., Raya, C., Rozzo, R., Nucci, A., Gambacorta, M., Rosa, D., & Rossi, M. (1990). Thermostable p-galactosidase from archaebactrium Solfulobus solfatarius. Purification and properties. European Journal of Biochemistry, 187(2), 321–328.Google Scholar
  41. 41.
    Coolbear, T., Daniel, R. M., & Morgan, H. W. (1992). The enzymes from extreme thermophiles: bacterial source, thermostabilities and industrial relevance. Advances in Biochemical Engineering / Biotechnology, 45, 58–98.Google Scholar
  42. 42.
    da Silva, E. S., Gómez-Vallejo, V., & López-Gallego, F. (2015). Efficient nitrogen-13 radiochemistry catalyzed by a highly stable immobilized biocatalyst. Catalysis Science & Technology, 5(5), 2705–2713.Google Scholar
  43. 43.
    Silva, T. M., Pessela, B. C., Silva, J. C. R., Lima, M. S., Jorge, J. A., Guisán, J. M., & Polizeli, M. L. T. M. (2014). Immobilization and high stability of an extracellular β-glucosidase from Aspergillus japonicus by ionic interactions. Journal of Molecular Catalysis B: Enzymatic, 104, 95–100.Google Scholar
  44. 44.
    Mateo, C., Palomo, J. M., Fuentes, M., Betancor, L., Grazu, V., López-Gallego, F., Pessela, B. C. C., Hidalgo, A., Fernández-Lorente, G., Fernández-Lafuente, R., & Guisán, J. M. (2006). Glyoxyl agarose: a fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzyme and Microbial Technology, 39(2), 274–280.Google Scholar
  45. 45.
    Blanco, R. M., Calvete, J. J., & Guisan, J. M. (1989). Immobilization e stabilization of enzymes. Variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multi-point covalent attachment. Enzyme and Microbial Technology, 11(6), 353–359.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Marylane de Sousa
    • 1
  • Vânia M. M. Melo
    • 2
  • Denise C. Hissa
    • 2
  • Ricardo M. Manzo
    • 3
  • Enrique J. Mammarella
    • 3
  • André Saraiva Leão Marcelo Antunes
    • 4
  • José L. García
    • 5
  • Benevides C. Pessela
    • 6
    • 7
    Email author
  • Luciana R. B. Gonçalves
    • 1
    Email author
  1. 1.Department of Chemical EngineeringFederal University of CearáFortalezaBrazil
  2. 2.Department of BiologyFederal University of CearáFortalezaBrazil
  3. 3.Food and Biotechnology Engineering Group, Institute of Technological Development for the Chemical IndustryNational University of the Litoral (UNL), National Council of Scientific and Technical Research (CONICET)Santa FeArgentina
  4. 4.Department of Infectious DiseasesKing’s College LondonLondonUK
  5. 5.Center for Biological Research, CIBHigher Council for Scientific Research, CSICMadridSpain
  6. 6.Department of Food Biotechnology and Microbiology, Institute of Research in Food Sciences, CIALHigher Council for Scientific Research, CSICMadridSpain
  7. 7.Department of Engineering and TechnologyPolytechnic Institute of Sciences and TechnologyLuandaAngola

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