Abstract
Periplasmic phytase, appA from E. coli has been noticed as a superior feed and food additive owing to its high specific activity, acidic pH optimum and resistance to gastric proteases. E. coli phytase was expressed as a fusion protein with maltose-binding protein, affinity-purified to homogeneity and, subsequently, immobilized in one step using a cost-effective matrix prepared from starch agar bead. Immobilized enzyme revealed an activity optimum at pH 6, while that of free enzyme was observed at pH 4. Both the immobilized and free enzyme showed a temperature optimum at 60 °C. Cleavage of 87 kDa fusion protein using factor Xa released 45 kDa appA. Hydrolysis of soy milk using immobilized enzyme led to 10% increase in release of inorganic phosphate at 50 °C relative to free fusion protein. This study suggests the usability of MBP as an immobilizing linker to other food enzymes for economical use in industry.
Similar content being viewed by others
References
Kumar, V., Sinha, A. K., Makkar, H. P. S., & Becke, K. (2010). Food Chemistry, 120, 945–959.
Afinah, S., Yazid, A. M., Anis, S. M. H., & Shuhaimi, M. (2010). International Food Research Journal, 17, 13–21.
Pandey, A., Szakacs, G., Soccol, C. R., Rodriguez-lenon, J. A., & Soccol, V. T. (2001). Bioresource Technology, 77, 203–214.
Ramachandran, S., Krishnan, R., Nampoothiri, K. M., Szakacs, G., & Pandey, A. (2005). Process Biochemistry, 40, 1749–1754.
Krishnan, R., Sumitra, R., Nampoothiri, K. M., Szakacs, G., & Pandey, A. (2006). Bioresource Technology, 97, 506–511.
Golovan, S. P., Wang, G., Zhang, J., & Forsberg, C. W. (2000). Canadian. Journal of Microbiology, 46, 59–71.
Rodriguez, E., Han, Y., & Lei, X. G. (1999). Biochemical and Biophysical Research Communications, 257, 117–123.
Igbasan, F. A., Simon, O., Miksch, G., & Manner, K. (2001). Archives of Animal Nutrition, 54, 117–126.
Kapust, R. B., & Waugh, D. S. (1999). Protein Science, 8, 1668–1674.
Smita, R., Samina, A., Sanchari, B., Raghavan, V., & Munishwar, N. G. (2008). Journal of Chromatography A, 1194, 90–95.
Han, M., Park, I. S., Kim, S. H., Kim, B. S., & Kim, S. H. (2009). Biotechnology Letters, 31, 1677–84.
Celem, E. B., & Onal, S. (2009). Artificial Cells, Blood Substitutes, and Biotechnology, 37, 195–202.
Eun-Ah, C., Eui-Joong, K., & Jae-Gu, P. (2011). Enzyme and Microbial Technology, 49, 66–71.
Greiner, R., & Konietzny, U. (1996). Journal of Biotechnology, 48, 153–159.
Sambrook, E., Fritsch, F., & Maniatis, T. (2001). Molecular Cloning: A Laboratory Manuel (3rd ed.). New York, USA: Cold Spring Harbor Laboratory.
Heinonen, J. K., & Lahti, R. J. (1981). Analytical Biochemistry, 113, 313–317.
Ullah, A. H. J., & Cummins, B. J. (1987). Preparative Biochemistry, 17, 397–422.
Berkmen, M., Dana, B., & Jon, B. (2005). The Journal of Biological Chemistry, 280, 11387–11394.
Seonho, L., Taewan, K., Chad, H. S., & Xin, G. L. (2005). Biotechnology Letters, 27, 327–334.
Stahl, C. H., Wilson, D. B., & Lei, X. G. (2003). Biotechnology Letters, 25, 827–831.
Miksch, G., Kleist, S., Friehs, K., & Flaschel, E. (2002). Applied Microbiology and Biotechnology, 59, 685–694.
Aarfman, N., Worrell, V., & Ingram, L. (1992). Journal of Bacteriology, 172, 7370–7378.
Srinivasan, U., & Jeffrey, A. B. (1998). Journal of Biotechnology, 62, 163–167.
Stephen, N. H., Ryan, C., Angela, K., Gregory, J. C., Alberto, J. N., & Wesley, C. V. V. (2011). Acta Crystallographica, 67, 1006–1009.
Yoshiyuki, I., Syuji, S., Yasushi, I., Eiichi, T., Yoshikazu, S., Koichi, O., Hiroaki, I.-O., Yasuhiko, K., & Akihiko, K. (1998). Journal of Biochemistry, 124, 842–847.
Ogiwara, K., Nagaoka, M., Cho, C. S., & Akaike, T. (2005). Biotechnology Letters, 27, 1633–1637.
Kaur, P., & Satyanarayana, T. (2010). Journal of Applied Microbiology, 108, 2041–2049.
Acknowledgements
The authors gratefully acknowledge the Department of Biotechnology, New Delhi for the financial support.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ushasree, M.V., Gunasekaran, P. & Pandey, A. Single-step Purification and Immobilization of MBP–phytase Fusion on Starch Agar Beads: Application in Dephytination of Soy Milk. Appl Biochem Biotechnol 167, 981–990 (2012). https://doi.org/10.1007/s12010-012-9598-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-012-9598-7