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Cloning, Purification and Characterization of NADP+-dependent malic enzyme from the oleaginous fungus Mortierella alpina
Jiayu Yang 1 #,Xinjie Hu 1,M\'balu.R.Kargbo 2,Haiqin Chen 2,Jianxin Zhao 1,Yuanda Song 2 *,Yong Q. Chen 1,Hao Zhang 2,Wei Chen 1
1.State Key Laboratory of Food Science and Technology, Jiangnan University, JiangSu WuXi, 214122
2.School of Food Science and technology, Jiangnan University, JiangSu WuXi, 214122
*Correspondence author
#Submitted by
Subject:
Funding: the National High Technology Research and Development Program of China (No.No. 2012AA022105C), the National Natural Science Foundation of China (No.No. 31271812), the National Science Fund for Distinguished Young Scholars (No.No. 31125021)
Opened online:19 March 2013
Accepted by: none
Citation: Jiayu Yang,Xinjie Hu,M\'balu.R.Kargbo.Cloning, Purification and Characterization of NADP+-dependent malic enzyme from the oleaginous fungus Mortierella alpina[OL]. [19 March 2013] http://en.paper.edu.cn/en_releasepaper/content/4529176
 
 
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. NADP+-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this work the malic enzyme-encoding cDNA(mce2) from oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified by affinity chromatography using Ni-NTA Column. The purified enzyme used NADP+ as cofactor. Km value for L-malate and NADP+ were 2.19±0.01 mM and 0.38±0.02 mM respectively, while Vmax values were 147±2U/mg and 302±14U/mg respectively, at optimal condition of pH 7.5 and 33℃. MaME appeared to be activated by Mn2+, Mg2+, Co2+, Ni2+ and low concentration of Zn2+, but not by Ca2+, Cu2+ and high concentration of Zn2+. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate and their Ki values were 0.08mM and 0.6mM respectively.
Keywords:Malic enzyme; Mortierella alpina; Characterization
 
 
 

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