Characterization of a malic enzyme isoform V from Mucor circinelloides

Abstract:

[Objective] We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. [Methods] me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using Ni-NTA column and characterized subsequently. [Results] The optimum conditions were pH at 8.0 and temperature at 33 ℃. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K_m for L-malate and NADP⁺ were 0.74960±0.06120 mmol/L and 0.22070±0.01810 mmol/L, the V_max for L-malate and NADP⁺ were 72.820±1.077 U/mg and 86.110±1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn²⁺, Mg²⁺, Co²⁺, Ni²⁺ could activate BLME1, whereas Ca²⁺, Cu²⁺ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. [Conclusion] A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.