卷枝毛霉中苹果酸酶同工酶V的酶学性质

Characterization of a malic enzyme isoform V from Mucor circinelloides

摘要:

[目的] 探讨卷枝毛霉中苹果酸酶同工酶V的性质。[方法] 克隆卷枝毛霉中编码苹果酸酶同工酶V的me1基因并在大肠杆菌BL21(DE3)中表达,利用His标签纯化获得了高纯度的重组酶BLME1,并进行酶学性质分析。[结果] 该重组酶最适pH为8.0,最适温度为33 ℃,在此条件下酶活达到92.8 U/mg,对底物L-苹果酸和NADP⁺的米氏常数K_m值为0.74960±0.06129 mmol/L和0.22070± 0.01810 mmol/L,最大反应速度V_max分别为72.820±1.077 U/mg和86.110±1.665 U/mg。金属离子Mg²⁺、Mn²⁺、Co²⁺、Ni²⁺可以激活BLME1的活性,而Ca²⁺、Cu²⁺对BLME1活性则有抑制作用,中间代谢产物草酰乙酸和α-酮戊二酸也会抑制BLME1的活性,但琥珀酸却对BLME1有激活作用。[结论] 本实验调查了卷枝毛霉苹果酸酶同工酶V的最适反应温度和pH、动力学参数,以及各种金属离子和中间代谢产物对酶活力的影响,这为以后深入研究该苹果酸酶的功能提供了理论依据和参考。

关键词:卷枝毛霉;苹果酸酶;酶学性质;金属离子;中间代谢产物

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.

Key words:Mucor circinelloides;malic enzyme;enzymatic property;metal iron;metabolic intermediate