解脂耶氏酵母异柠檬酸脱氢酶基因的克隆与表达.doc

大肠杆菌苹果酸脱氢酶的原核表达、纯化与酶活力测定 摘 要：苹果酸脱氢酶(malate dehydrogenase, MDH)普遍存在动物、植物、细 菌等多种生物体中，负责催化苹果酸与草酰乙酸之间的可逆转换，在细胞的多种生理活动中发挥重要作用，如C4循环、光合作用、TCA循环。实验室保存的DH5αpET28b-mdh重组质粒，并对其进行双酶切鉴定。将重组质粒pET8b-mdh转化入Rosetta(DE3)感受态细胞中，通过卡那霉素和氯霉素抗性进行筛选。挑取单菌落培养600≈0.6时，用0.1 mM的IPTG诱导表达MDH蛋白Expression and Purification of Malate Dehydrogenase from Escherichia coli and its Activity Determination Abstract: Malate Dehydrogenase is ubiquitous in animals, plants, and bacterias, which catalyzed the interconversion of oxaloacetate and malate linked to the oxidation/reduction of dinucleotide coenzymes, which play a key role in many metabolic pathways such as TCA cycle, photosynthesis, C4 cycle and so on. In this study, we extracted pET28b-mdh recombinant plasmid from DH5α, which was preserved in our laboratory, and examined the recombinant plasmid correctness through double enzymes digestion reaction. Then we transformed the recombinant plasmid into E.coli Rosetta(DE3) and screened the positive recombinant strain thought kalamycin and chloromycetin resistance. And then we cultured the individual colony until OD600≈0.6, then added IPTG to the finally concentration 0.1mM to induce the expression of MDH. We collected and purified the MDH and then analyzed MDH enzyme activity. The result showed that MDH can be highly induced to express in E.coli and its activity to reduce oxaloacetate is 194.75 U/mg. Keywords: malate dehydrogenase; prokaryotic expression; activity assay 1 前 言 1.1malate dehydrogenase, MDH; EC 1.1.1.37)普遍存在于动物、植物和细菌等各种生物体中，是生物体糖代谢的关键酶之一。根据不同的辅酶特异性，MDHs分为NAD-依赖性的MDHs和NADP-依赖性MDHs. 细菌中通常只含有NAD-MDHs，存在于细胞膜上。在真核细胞中，NAD-MDHs分布于细胞质和线粒体中，NADP-MDHs位于叶绿体。 NAD-MDHs一般不受代谢调节， NADP-MDHs间接受光调节[1]。MDHs为同源二聚体或四聚体，目前发现的真核生物MDHs都是二聚体，如猪心cyMDH和大米的cyMDH。大肠杆菌的MDH都是二聚体，但芽孢杆菌类类多为四聚体，如枯草杆菌和嗜热脂肪芽孢杆菌的MDH。NAD-MDHs亚基分子量在32~37kDa之间，NADP-MDHs亚基分子量约42kDa[2]。MDH能催化苹果酸和草酰乙酸之间的转化： malate + NAD+ oxaloacetate + NADH + H+ 该反应在三羧酸循环、光合作用和C4循环等途径中都有重要作用。 1.2 苹果酸脱氢酶的研究意义 ]可以螯合土壤中的铝离子，降低铝对植物体的毒害作用[5]。 这些有机酸中，苹果酸是植物体