Skip to main content
SpringerLink
Log in

Expression, Purification, and Characterization of NADP+-Dependent Malic Enzyme from the Oleaginous Fungus Mortierella Alpina

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)+ to NAD(P)H. The NADP+-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP+ as the cofactor. The K m values for l-malate and NADP+ were 2.19 ± 0.01 and 0.38 ± 0.02 mM, respectively, while the V max values were 147 ± 2 and 302 ± 14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn2+, Mg2+, Co2+, Ni2+, and low concentrations of Zn2+ rather than Ca2+, Cu2+, or high concentrations of Zn2+. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their K i values were 0.08 and 0.6 mM, respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Sakuradani, E., & Shimizu, S. (2009). Journal of Biotechnology, 144, 31–36.

    Article  CAS  Google Scholar 

  2. Eroshin, V. K., Satroutdinov, A. D., Dedyukhina, E. G., & Chistyakova, T. I. (2000). Process Biochemistry, 35, 1171–1175.

    Article  CAS  Google Scholar 

  3. Botham, P. A., & Ratledge, C. (1979). Journal of General Microbiology, 114, 361–375.

    Article  CAS  Google Scholar 

  4. Evans, C. T., & Ratledge, C. (1985). Canadian Journal of Microbiology, 31, 1000–1005.

    Article  CAS  Google Scholar 

  5. Ratledge, C., & Wynn, J. P. (2002). Advances in Applied Microbiology, 51, 1–51.

    Article  CAS  Google Scholar 

  6. Frenkel, R. (1975). Current Topics in Cellular Regulation, 9, 157–181.

    Article  CAS  Google Scholar 

  7. Evans, C. T., & Ratledge, C. (1983). Lipids, 18, 630–635.

    Article  CAS  Google Scholar 

  8. Whitworth, D. A., & Ratledge, C. (1975). Journal of General Microbiology, 88, 275–288.

    Article  CAS  Google Scholar 

  9. Wynn, J. P., Bin, A. H. A., & Ratledge, C. (1999). Microbiology, 145, 1911–1917.

    Article  CAS  Google Scholar 

  10. Wynn, J. P., Kendrick, A., & Ratledge, C. (1997). Lipids, 32, 605–610.

    Article  CAS  Google Scholar 

  11. Zhang, Y., & Ratledge, C. (2008). Mycological Research, 112, 725–730.

    Article  CAS  Google Scholar 

  12. Song, Y., Wynn, J. P., Li, Y., Grantham, D., & Ratledge, C. (2001). Microbiology, 147, 1507–1515.

    CAS  Google Scholar 

  13. Zhang, Y., Adams, I. P., & Ratledge, C. (2007). Microbiology, 153, 2013–2025.

    Article  CAS  Google Scholar 

  14. Rodríguez-Frómeta, R. A., Gutiérrez, A., Torres-Martí, S., & Garre, V. (2013). Applied Microbiology and Biotechnology, 97, 3063–3072.

    Article  Google Scholar 

  15. Li, Z., Sun, H., Mo, X., Li, X., Xu, B., & Tian, P. (2013). Applied Microbiology and Biotechnology, 97, 4927–4936.

    Article  CAS  Google Scholar 

  16. Zhu, Z., Zhang, S., Liu, H., Shen, H., Lin, X., Yang, F., et al. (2012). Nature Communications, 3, 1112.

    Article  Google Scholar 

  17. Ambrook, J., Fritsch, E. F., & Maniatis, T. (2002). Molecular cloning: a laboratory manual. New York: CSHL Press.

    Google Scholar 

  18. Hsu, R. Y., & Lardy, H. A. (1969). Methods in Enzymology, 37, 230–235.

    Article  Google Scholar 

  19. Lamed, R., & Zeikus, J. G. (1981). Biochimica et Biophysica Acta, 660, 251–255.

    Article  CAS  Google Scholar 

  20. Garrido-Pertierra, A., Martinez-Marcos, C., Martin-Fernandez, M., & Ruiz-Amil, M. (1983). Biochimie, 65, 629–635.

    Article  CAS  Google Scholar 

  21. Bartolucci, S., Rella, R., Guagliardi, A., Raia, C. A., Gambacorta, A., De Rosa, M., et al. (1987). Journal of Biological Chemistry, 262, 7725–7731.

    CAS  Google Scholar 

  22. Drincovich, M. F., Iglesias, A. A., & Andreo, C. S. (1991). Plant Physiology, 81, 462–466.

    Article  CAS  Google Scholar 

  23. Brown, D. A., & Cook, R. A. (1981). Biochemistry, 20, 2503–2512.

    Article  CAS  Google Scholar 

  24. Yang, Z., Floyd, D. L., Loeber, G., & Tong, L. (2000). Nature Structural & Molecular Biology, 7, 251–257.

    CAS  Google Scholar 

  25. Jernejc, K., & Legisa, M. (2002). FEMS Microbiology Letters, 217, 185–190.

    Article  CAS  Google Scholar 

  26. Hung, H. C., Chang, G. G., Yang, Z., & Tong, L. (2000). Biochemistry, 39, 14095–14102.

    Article  CAS  Google Scholar 

  27. Driscollt, B. T., & Finan, T. M. (1997). Microbiology, 143, 489–498.

    Article  Google Scholar 

  28. Chang, G. G., & Tong, L. (2003). Biochemistry, 42, 12721–12733.

    Article  CAS  Google Scholar 

  29. Pon, J., Napoli, E., Luckhart, S., & Giulivi, C. (2011). Malaria Journal, 10, 318.

    Article  CAS  Google Scholar 

  30. Sato, I., Yoshikawa, J., Furusawa, A., Chiku, K., Amachi, S., & Fujii, T. (2010). Bioscience, Biotechnology, and Biochemistry, 74, 75–81.

    Article  CAS  Google Scholar 

  31. Bologna, F. P., Andreo, C. S., & Drincovich, M. F. (2007). Journal of Bacteriology, 189, 5937–5946.

    Article  CAS  Google Scholar 

  32. Su, K. L., Chang, K. Y., & Hung, H. C. (2009). Bioorganic & Medicinal Chemistry, 17, 5414–5419.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 31271812, 21276108, and 31171636), the National High Technology Research and Development Program of China (2012AA022105C and 2011AA100905), the National Science Fund for Distinguished Young Scholars (31125021), the National Basic Research Program 973 of China (2012CB720802), and the Fundamental Research Funds for the Central Universities (No. JUSRP51320B).

Author information

Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu Province, People’s Republic of China

    Jiayu Yang, Xinjie Hu, Huaiyuan Zhang, Haiqin Chen, M’balu. R. Kargbo, Jianxin Zhao, Yuanda Song, Yong Q. Chen, Hao Zhang & Wei Chen

  2. Synergistic Innovation Center for Food Safety and Nutrition, Wuxi, 214122, Jiangsu Province, People’s Republic of China

    Xinjie Hu, Haiqin Chen, Jianxin Zhao, Yong Q. Chen, Hao Zhang & Wei Chen

  3. College of Food Science, Sichuan Agricultural University, Yaan, 625014, Sichuan Province, People’s Republic of China

    Xinjie Hu

Authors
  1. Jiayu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xinjie Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huaiyuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Haiqin Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M’balu. R. Kargbo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jianxin Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yuanda Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yong Q. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yuanda Song or Wei Chen.

Additional information

Jiayu Yang and Xinjie Hu contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, J., Hu, X., Zhang, H. et al. Expression, Purification, and Characterization of NADP+-Dependent Malic Enzyme from the Oleaginous Fungus Mortierella Alpina . Appl Biochem Biotechnol 173, 1849–1857 (2014). https://doi.org/10.1007/s12010-014-0971-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-014-0971-6

Keywords

Search

Navigation