Abstract
Alcohol dehydrogenase from halophilic archaeon Haloferax volcanii (HvADH2) was successfully covalently immobilized on metal-derivatized epoxy Sepabeads. The immobilization conditions were optimized by investigating several parameters that affect the halophilic enzyme–support interaction. The highest immobilization efficiency (100 %) and retention activity (60 %) were achieved after 48 h of incubation of the enzyme with Ni-epoxy Sepabeads support in 100 mM Tris–HCl buffer, pH 8, containing 3 M KCl at 5 °C. No significant stabilization was observed after blocking the unreacted epoxy groups with commonly used hydrophilic agents. A significant increase in the stability of the immobilized enzyme was achieved by blocking the unreacted epoxy groups with ethylamine. The immobilization process increased the enzyme stability, thermal activity, and organic solvents tolerance when compared to its soluble counterpart, indicating that the immobilization enhances the structural and conformational stability. One step purification–immobilization of this enzyme has been carried out on metal chelate-epoxy Sepabeads, as an efficient method to obtain immobilized biocatalyst directly from bacterial extracts.
Similar content being viewed by others
References
Honda, K., Ishige, T., Kataoka, M., & Shimizu, S. (2006). Microbial and enzymatic processes for the production of chiral compounds. In R. N. Patel (Ed.), Biocatalysis in the pharmaceutical and biotechnology industries (pp. 529–546). New York: Taylor and Francis.
Faber, K. (1997). Biotranformations in organic chemistry (pp. 10–16). Berlin: Springer.
Margolin, A. L. (1993). Enzymes in the synthesis of chiral drugs. Enzyme and Microbial Technology, 15, 266–280.
Yang, Z. H., Zeng, R., Chang, X., Li, X. K., & Wang, G. H. (2008). Toxicity of aromatic ketone to yeast cell and improvement of the asymmetric reduction of aromatic ketone catalyzed by yeast cell with the introduction of resin adsorption. Food Technology and Biotechnology, 46, 322–327.
Goldberg, K., Schroer, K., Lütz, S., & Liese, A. (2007). Biocatalytic ketone reduction-a powerful tool for the production of chiral alcohols-part I: Processes with isolated enzymes. Applied Microbiology and Biotechnology, 76, 237–248.
Hanefeld, U., Gardossi, L., & Magner, E. (2009). Understanding enzyme immobilisation. Chemical Society Reviews, 38, 453–468.
Sheldon, R. A. (2007). Enzyme immobilization: The quest for optimum performance. Advanced Synthesis and Catalysis, 349, 1289–1307.
Garcia-Galan, C., Berenguer-Murcia, A., Fernandez-Lafuente, R., & Rodrigues, R. C. (2011). Potential of different enzyme immobilization strategies to improve enzyme performance. Advanced Synthesis and Catalysis, 353, 2885–2904.
Mateo, C., Abian, O., Fernández-Lorente, G., Pessela, B. C., Grazu, V., Guisan, J. M., et al. (2006). Immobilization-stabilization of enzymes by multipoint covalent attachment on supports activated with epoxy groups. In G. F. Bickerstaff (Ed.), Immobilization of enzymes and cells. Methods in biotechnology (Vol. 22, pp. 47–55). Totowa: Humana.
Mateo, C., Abian, C., Fernández- Lorente, G., Predoche, J., Fernández- Lafuente, R., & Guisan, J. M. (2002). Sepabeads: A novel epoxy-support for stabilization of industrial enzymes via very intense multipoint covalent attachment. Biotechnology Progress, 18, 629–634.
Katchalski-Katzir, E., & Kraemer, D. M. (2000). Eupergit® C, a carrier for immobilization of enzymes of industrial potential. Journal of Molecular Catalysis B, 10, 157–176.
Mateo, C., Abian, O., Fernandez-Lafuente, R., & Guisan, J. M. (2000). Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment. Enzyme and Microbial Technology, 26, 509–515.
Adams, J. P., Collis, A. J., Henderson, R. K., & Sutton, P. W. (2010). Biotransformations in small-molecule pharmaceutical development. In J. Whittall & P. W. Sutton (Eds.), Practical methods for biocatalysis and biotransformations (pp. 1–82). Chichester: Wiley.
Mateo, C., Fernández-Lorente, G., Cortés, E., Garcia, J. L., Fernández-Lafuente, R., & Guisan, J. M. (2001). One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports. Biotechnology and Bioengineering, 76, 269–276.
Pessela, B. C., Mateo, C., Carrascosa, A. V., Vian, A., García, J. L., Rivas, G., et al. (2003). One-step purification, covalent immobilization, and additional stabilization of a thermophilic poly-His-tagged β-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads. Biomacromolecules, 4, 107–113.
Timpson, L. M., Liliensiek, A. K., Alsafadi, D., Cassidy, J., Sharkey, M. A., Liddell, S., et al. (2013). A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii. Applied Microbiology and Biotechnology, 97, 195–203.
Alsafadi, D., & Paradisi, F. (2013). Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii. Extremophiles, 17, 115–122.
Koch-Schmidt, A. C., Mosbach, K., & Werber, M. M. (1979). A comparative study on the stability of immobilized halophilic and non-halophilic malate dehydrogenase at various ionic strengths. European Journal of Biochemistry, 100, 213–218.
Patel, S., Bagai, R., & Madamwar, D. (1999). Stabilization of a halophilic α-amylase by calcium alginate immobilization. Biocatalysis and Biotransformation, 14, 147–155.
D’Souza, S. E., Altekar, W., & D’Souza, S. F. (1997). Immobilization of Haloferax mediterranei aldolase by cross-linking in a proteinic matrix: Stability and halophilic characteristics. World Journal of Microbiology and Biotechnology, 13, 561–564.
Quaglia, D., Pori, M., Galletti, P., Emer, E., Paradisi, F., & Giacomini, D. (2013). His-tagged horse liver alcohol dehydrogenase: Immobilization and application in the bio-based enantioselective synthesis of (S)-arylpropanols. Process Biochemistry, 48, 810–818.
Kastritis, P. L., Papandreou, N. C., & Hamodrakas, S. J. (2007). Haloadaptation: Insights from comparative modeling studies of halophilic archaeal DHFRs. International Journal of Biological Macromolecules, 41, 447–453.
Roig, M. G., Bello, J. F., Moreno de Vega, M. A., Cachaza, J. M., & Kennedy, J. F. (1990). Liver alcohol dehydrogenase immobilized on polyvinylidene difluoride. Journal of Chemical Technology and Biotechnology, 49, 99–113.
Goldberg, K., Krueger, A., Meinhardt, T., Kroutil, W., Mautner, B., & Liese, A. (2008). Novel immobilization routes for the covalent binding of an alcohol dehydrogenase from Rhodococcus ruber DSM 4454. Tetrahedron Asymmetry, 19, 1171–1173.
Soni, S., Desai, J. D., & Devi, S. (2001). Immobilization of yeast alcohol dehydrogenase by entrapment and covalent binding to polymeric supports. Journal of Applied Polymer Science, 82, 1299–1305.
Bolivar, J. M., Wilson, L., Ferrarotti, S. A., Guisan, J. M., Fernandez-Lafuente, R., & Mateo, C. (2006). Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose. Journal of Biotechnology, 125, 85–94.
Hildebrand, F., & Lütz, S. (2006). Immobilisation of alcohol dehydrogenase from Lactobacillus brevis and its application in a plug-flow reactor. Tetrahedron Asymmetry, 17, 3219–3225.
Timpson, L., Alsafadi, D., Mac Donnchadha, C., Liddell, S., Sharkey, M., & Paradisi, F. (2012). Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea. Extremophiles, 16, 57–66.
Martins, S., Karmali, A., Andrade, J., & Serralheiro, M. L. (2006). Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa. Molecular Biotechnology, 33, 103–114.
Acknowledgments
This work was supported by funding provided by the Islamic Development Bank (IDB) and by Science Foundation Ireland (SFI). The company Resindion S. R. L. (Milano, Italy) kindly donated the epoxy sepabeads.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Alsafadi, D., Paradisi, F. Covalent Immobilization of Alcohol Dehydrogenase (ADH2) from Haloferax volcanii: How to Maximize Activity and Optimize Performance of Halophilic Enzymes. Mol Biotechnol 56, 240–247 (2014). https://doi.org/10.1007/s12033-013-9701-5
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-013-9701-5