Abstract
The kinetic behavior of the enzyme laccase in solution and immobilized onto carbon platforms using poly(amido amine) (PAMAM) dendrimers has been investigated. The results with the immobilized enzymes have demonstrated that almost ten times more enzyme on the carbon support is required for satisfactory kinetic rates to be achieved. Furthermore, the study as a function of the substrate concentration revealed that the kinetic behavior of the enzyme in solution fits the Michaelis–Menten model. However, when the enzyme is immobilized onto the carbon surface, the catalyzed reaction follows a particular kinetic behavior with apparent positive cooperativity. The highest activity with laccase (in solution or immobilized) is achieved around pH 4.5, and the substrate conversion rate clearly diminishes with rising pH. The optimum temperature lies around 60 °C. The enzyme displays good catalytic activity in a wide range of pH and temperature values. The stability tests evidenced that there is no appreciable reduction in the enzymatic activity after immobilization within the first 30 days. Taking into account both the kinetic and stability tests, one can infer that the use of PAMAM dendrimers seems to be a very attractive approach for the immobilization of enzymes, as well as a feasible and useful methodology for the anchoring of enzymes with potential application in many biotechnological areas.
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Gianfreda, L., Xu, F., & Bollag, J. (1999). Bioremediation Journal, 3, 1–25.
Ducros, V., Brzozowski, A. M., Wilson, K. S., Brown, S. H., Ostergaard, P., Schneider, P., Yaver, D. S., Pedersen, A. H., & Davies, G. J. (1998). Nature Structural and Molecular Biology, 5, 310–316.
Palmore, G. T. R., & Kim, H.-H. (1999). Journal of Electroanalytical Chemistry, 464, 110–117.
Zheng, W., Zhou, H. M., Zheng, Y. F., & Wang, N. (2008). Chemical Physics Letters, 457, 381–385.
Kavanagh, P., Jenkins, P., & Leech, D. (2008). Electrochemistry Communications, 10, 970–972.
Szamocki, R., Flexer, V., Levin, L., Forchiasin, F., & Calvo, E. J. (2009). Electrochimica Acta, 54, 1970–1977.
Piontek, K., Antorini, M., & Choinowski, T. (2002). Journal of Biological Chemistry, 277, 37663–37669.
Harvey, B. M., & Walker, J. R. K. (1999). Biochemistry, Molecular Biology and Biophysics, 3, 45–51.
Claus, H. (2004). Micron, 35, 93–96.
Kumar, S. V., Phale, P. S., Durani, S., & Wangikar, P. P. (2003). Biotechnology and Bioengineering, 83, 386–394.
Aquino Neto, S., Forti, J. C., & De Andrade, A. R. (2010). Electrocatalysis, 1, 87–94.
Durán, N., Rosa, M. A., Annibale, A. D’., & Gianfreda, L. (2002). Enzyme and Microbial Technology, 31, 907–931.
Aquino Neto, S., Forti, J. C., Zucolotto, V., Ciancaglini, P., & De Andrade, A. R. (2011). Process Biochemistry, 46, 2347–2352.
Aquino Neto, S., Forti, J. C., Zucolotto, V., Ciancaglini, P., & De Andrade, A. R. (2011). Biosensors and Bioelectronics, 26, 2922–2926.
Forti, J. C., Aquino Neto, S., Zucolotto, V., Ciancaglini, P., & De Andrade, A. R. (2011). Biosensors and Bioelectronics, 26, 2675–2679.
Zucolotto, V., Daghastanli, K. R. P., Hayasaka, C. O., Riul, A. R., Jr., Ciancaglini, P., & Oliveira, O. N., Jr. (2007). Analytical Chemistry, 79, 2163–2167.
Zucolotto, V., Pinto, P. A. A., Tumolo, T., Moraes, M. L., Baptista, M. S., Riul, A., Jr., Araújo, A. P. U., & Oliveira, O. N., Jr. (2007). Biosensors and Bioelectronics, 40, 466–470.
Perinotto, A. C., Caseli, L., Hayasaka, C. O., Riul, A., Jr., Oliveira, O. N., Jr., & Zucolotto, V. (2008). Thin Solid Films, 516, 9002–9005.
Lineweaver, H., & Burk, D. (1934). Journal of the American Chemical Society, 56, 658–666.
Leone, F. A., Baranauskas, J. A., & Ciancaglini, P. (1995). Biochemical Education, 23, 35–37.
Wu, J., Taylor, K. E., Bewtra, J. K., & Biswas, N. (1998). Enzyme and Microbial Technology, 22, 315–322.
Caza, N., Taylor, K. E., Bewtra, J. K., & Biswas. (1999). Water Research, 33, 3012–3018.
Nakamoto, S., & Machida, N. (1992). Water Research, 26, 49–54.
Kinsley, C., & Nicell, J. A. (2000). Bioresource Technology, 73, 139–146.
Nicell, J. A., Saadi, K. W., & Buchanan, I. D. (1995). Bioresource Technology, 54, 5–16.
Wu, Y., Taylor, K. E., Biswas, N., & Bewtra, J. K. (1997). Water Research, 31, 2699–2704.
Quan, D., Kim, Y., & Shin, W. (2004). Journal of Electroanalytical Chemistry, 561, 181–189.
Martinek, K., et al. (1998). Biokhimiya, 53, 1013–1016.
Moore, C. M., Akers, N. L., Hill, A. D., Johnson, Z. C., & Minteer, S. D. (2004). Biomacromolecules, 5, 1241–1247.
Pointing, S. B., Jones, E. B. G., & Vrijomed, L. L. P. (2000). Mycologia, 92, 139–144.
Nagai, M., Kawata, M., Watanabe, H., Ogawa, M., Saito, K., Takesawa, T., Kand, K., & Sato, T. (2003). Microbiology, 149, 2455–2462.
Jaouani, A., Guillén, F., Penninckx, M., Martinéz, A. T., & Martinéz, M. J. (2005). Enzyme and Microbial Technology, 36, 478–486.
Brunel, L., Denele, J., Servat, K., Kokoh, K. B., Jolivat, C., Innocent, C., Cretin, M., Rolland, M., & Tingry, S. (2007). Electrochemistry Communications, 9, 331–336.
Rincón, R. A., Lau, C., Luckarift, H. R., Garcia, K. E., Adkins, E., Johnson, G. R., & Atanassov, P. (2011). Biosensors and Bioelectronics, 27, 132–136.
Scherer, M., & Fischer, R. (1998). Microbiology, 170, 78–84.
Souza, C. G. M., & Peralta, R. M. (2003). Journal of Basic Microbiology, 43, 278–286.
Xiao, Y. Z., Tu, X. M., Wang, J., Zhang, M., Cheng, Q., Zeng, W. Y., & Shi, Y. Y. (2003). Applied Microbiology and Biotechnology, 60, 700–707.
Record, E., Punt, P. J., Chamkha, M., Labat, M., & Van Den Hondel, C. A. M. J. J. (2002). European Journal of Biochemistry, 269, 602–609.
Baldrian, P., & Gabriel, J. (2002). FEMS Microbiology Letters, 206, 69–74.
Prévoteau, A., & Faure, C. (2011). Biochimie. doi:10.1016/j.biochi.2011.10.010.
Qiu, H., Xu, C., Huang, X., Ding, Y., Qu, Y., & Gao, P. (2008). Journal of Physical Chemistry, 112, 14781–14785.
Hu, X., Zhao, X., & Hwang, H. M. (2007). Chemosphere, 66, 1618–1626.
Jiang, D. S., Long, S. Y., Huang, J., Xiao, H.-Y., & Zhou, J.-Y. (2005). Biochemical Engineering Journal, 25, 15–23.
Wang, P., Sergeeva, M. V., Lim, L., & Dordick, J. S. (1997). Nature Biotechnology, 15, 789.
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Financial support from FAPESP, CAPES, and CNPq is gratefully acknowledged.
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Cardoso, F.P., Aquino Neto, S., Ciancaglini, P. et al. The Use of PAMAM Dendrimers as a Platform for Laccase Immobilization: Kinetic Characterization of the Enzyme. Appl Biochem Biotechnol 167, 1854–1864 (2012). https://doi.org/10.1007/s12010-012-9740-6
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DOI: https://doi.org/10.1007/s12010-012-9740-6