title: Expression, purification, and characterization of NADP+-dependent malic enzyme from the oleaginous fungus Mortierella alpina.
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.
authors.affiliation: 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.
authors.forename: Jiayu
authors.initials: J
authors.lastname: Yang
authors.forename: Xinjie
authors.initials: X
authors.lastname: Hu
authors.forename: Huaiyuan
authors.initials: H
authors.lastname: Zhang
authors.forename: Haiqin
authors.initials: H
authors.lastname: Chen
authors.forename: M'balu R
authors.initials: MR
authors.lastname: Kargbo
authors.forename: Jianxin
authors.initials: J
authors.lastname: Zhao
authors.forename: Yuanda
authors.initials: Y
authors.lastname: Song
authors.forename: Yong Q
authors.initials: YQ
authors.lastname: Chen
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D002262:Carboxy-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008293:Malates / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D020094:Mortierella / Q000201:enzymology* / Q000235:genetics; D009249:NADP / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D008293:Malates; D008670:Metals; D011994:Recombinant Proteins; D009249:NADP; C030298:malic acid; D002262:Carboxy-Lyases