Applied Biochemistry and Biotechnology

, Volume 173, Issue 7, pp 1849–1857 | Cite as

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

  • Jiayu Yang
  • Xinjie Hu
  • Huaiyuan Zhang
  • Haiqin Chen
  • M’balu. R. Kargbo
  • Jianxin Zhao
  • Yuanda Song
  • Yong Q. Chen
  • Hao Zhang
  • Wei Chen
Article
First Online:
Received:
Accepted:

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.

Keywords

Malic enzyme Mortierella alpina Characterization 
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Notes

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).

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jiayu Yang
    • 1
  • Xinjie Hu
    • 1
    • 2
    • 3
  • Huaiyuan Zhang
    • 1
  • Haiqin Chen
    • 1
    • 2
  • M’balu. R. Kargbo
    • 1
  • Jianxin Zhao
    • 1
    • 2
  • Yuanda Song
    • 1
  • Yong Q. Chen
    • 1
    • 2
  • Hao Zhang
    • 1
    • 2
  • Wei Chen
    • 1
    • 2
  1. 1.State Key Laboratory of Food Science and Technology, School of Food Science and TechnologyJiangnan UniversityWuxiPeople’s Republic of China
  2. 2.Synergistic Innovation Center for Food Safety and NutritionWuxiPeople’s Republic of China
  3. 3.College of Food ScienceSichuan Agricultural UniversityYaanPeople’s Republic of China

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