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Single-Step Purification and Characterization of Recombinant Aspartase of Aeromonas media NFB-5

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Abstract

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b(+) expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K2HPO4 and 0.3% KH2PO4. Recombinant His tagged aspartase was purified using Ni–NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K m and V max values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5–9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg2+, Mn2+, and Ca2+ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

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References

  1. Papierz, M., Grazyna, G., Sobierajski, B., & Chmiel, A. (2007). Polish Journal of Microbiology, 56, 71–76.

    CAS  Google Scholar 

  2. Quastel, J. H., & Woolf, B. (1926). Journal of Biochemistry, 20, 545–555.

    CAS  Google Scholar 

  3. Kisumi, M., Ashikaga, Y., & Chibata, I. (1960). Bulletin of Agricultural Chemistry Japan, 24, 296–305.

    Article  CAS  Google Scholar 

  4. Takagi, J. S., Ida, N., Tokushige, M., Sakamoto, H., & Shimura, Y. (1985). Nucleic Acids Research, 13, 2063–2074.

    Article  CAS  Google Scholar 

  5. Kazuoka, T., Masuda, Y., Oikawa, T., & Soda, K. (2003). Journal of Biochemistry, 133, 51–56.

    Article  CAS  Google Scholar 

  6. Takagi, J. S., Tokushige, M., & Shimura, Y. (1986). Journal of Biochemistry, 100, 697–705.

    CAS  Google Scholar 

  7. Kawata, Y., Tamura, K., Kawamura, M., Ikei, K., Mizobata, T., Nagai, J., et al. (2000). European Journal of Biochemistry, 267, 1847–1857.

    Article  CAS  Google Scholar 

  8. Sun, D. X., & Setlow, P. (1991). Journal of Bacteriology, 173, 3831–3845.

    CAS  Google Scholar 

  9. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  10. Tokushige, M. (1985). Methods in Enzymology, 113, 618–627.

    Article  CAS  Google Scholar 

  11. Laemmli, U. K. (1970). Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  12. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., et al. (2005). In J. M. Walker (Ed.), The Proteomics Protocols Handbook (pp. 571–607). USA: Humana Press.

    Chapter  Google Scholar 

  13. Pal, A., & Khanum, F. (2010). Journal of Biochemistry and Technology, 2, 203–209.

    CAS  Google Scholar 

  14. Jayasekera, M. M., Shi, W., Farber, G. K., & Viola, R. E. (1997). Biochemistry, 36, 9145–9150.

    Article  CAS  Google Scholar 

  15. Shi, W., Dunbar, J., Jayasekera, M. M., Viola, R. E., & Farber, G. K. (1997). Biochemistry, 36, 9136–9144.

    Article  CAS  Google Scholar 

  16. Asano, Y., Kira, I., & Yokozeki, K. (2005). Biomolecular Engineering, 22, 95–101.

    Article  CAS  Google Scholar 

  17. Veetil, V. P., Raj, H., Quax, W. J., Janssen, D. B., & Poelarends, G. J. (2009). FEBS Journal, 276, 2994–3007.

    Article  CAS  Google Scholar 

  18. Komatsubara, S., Taniguchi, T., & Kisumi, M. (1986). Journal of Biotechnology, 3, 281–291.

    Article  CAS  Google Scholar 

  19. Williams, V. R., & Lartigue, D. J. (1967). Journal of Biological Chemistry, 242, 2973–2978.

    CAS  Google Scholar 

  20. Suzuki, S., Yamagushi, J., & Togushige, M. (1973). Biochimica et Biophysica Acta, 321, 369–381.

    Article  CAS  Google Scholar 

  21. Zhang, H. Y., Zhang, J., Lin, L., Du, W. Y., & Lu, J. (1993). Biochemical and Biophysical Research Communications, 192, 15–21.

    Article  CAS  Google Scholar 

  22. Marangoni, A. G. (2003). Enzyme Kinetics: A Modern Approach. Hoboken: Wiley.

    Google Scholar 

Download references

Acknowledgments

Authors are thankful to the Head, Department of Biotechnology, for providing necessary laboratory facilities. The financial assistance received for this work in the form of a Major Research Project No. CSIR 37(1339)/08/EMR-II from Council of Scientific and Industrial Research (CSIR), New Delhi, Govt. of India, is also duly acknowledged.

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  1. Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala, 147 002, Punjab, India

    Ram Sarup Singh & Mukesh Yadav

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  1. Ram Sarup Singh
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  2. Mukesh Yadav
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Correspondence to Ram Sarup Singh.

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Singh, R.S., Yadav, M. Single-Step Purification and Characterization of Recombinant Aspartase of Aeromonas media NFB-5. Appl Biochem Biotechnol 167, 991–1001 (2012). https://doi.org/10.1007/s12010-012-9589-8

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  • DOI: https://doi.org/10.1007/s12010-012-9589-8

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