产油真菌胞质中NADP+依赖型异柠檬酸脱氢酶(isocitrate dehydrogenase, IDH)能够为脂肪酸合成提供还原力,前期研究表明高山被孢霉的异柠檬酸脱氢酶4(Mortierella alpina isocitrate dehydrogenase4, MaIDH4)对脂质积累有积极的影响。为了深入研究MaIDH4的性质,该文将其在大肠杆菌和毕赤酵母系统中分别进行异源表达并纯化,均成功获得纯酶,并考察了不同表达系统对酶活性和产量的影响。首先克隆MaIDH4至表达载体pET28a(+)和pPink-HC-3CZHEK构建重组质粒,然后分别转化至Escherichia coli BL21(DE3)和PichiaPinkTM Strain 2进行诱导表达,最后采用镍柱纯化获得携带10×His标签的MaIDH4重组酶,检测纯酶的比活力分别是34.10 U/mg和43.00 U/mg,酶产量分别为10.28 U/L和159.12 U/L。通过纯酶比活力和酶产量分析表明相比于大肠杆菌表达系统,毕赤酵母宿主更适合MaIDH4的异源表达。
The cytosolic NADP-isocitrate dehydrogenase (IDH) of oleaginous fungi provides the reducing power for fatty acid synthesis. Previous studies have shown that IDH4 from Mortierella alpina (MaIDH4) has a positive effect on lipid accumulation. To investigate the properties of MaIDH4, it was heterologously expressed and purified in Escherichia coli and Pichia pastoris expression systems respectively. The pure enzyme was successfully obtained in both systems. The effects of different expression systems on enzyme activity and yield were also investigated. Firstly, MaIDH4 was cloned into the expression vectors pET28a(+) and pPink-HC-3CZHEK to construct recombinant plasmids, which were then transformed into E. coli BL21 (DE3) and PichiaPinkTM Strain 2 respectively for induction of expression. Finally, the MaIDH4 recombinase carrying a 10×His tag was obtained by purification with a nickel column. The specific activity of the pure enzymes were 34.10 and 43.00 U/mg, with enzyme yields of 10.28 and 159.12 U/L, respectively. The analysis of the specific activity and enzyme yield of the pure enzyme indicated that the host of Pichia pastoris was more suitable for the heterologous expression of MaIDH4 than the E. coli expression system.
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