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Research for Improvement in Efficiency of Energy Metabolism of Soybean Rhizobium.

提高大豆根瘤菌能量代谢效率的研究。

基本信息

批准号：
02660068
负责人：
TAJIMA Shigeyuki
金额：
$ 1.28万
依托单位：
Kagawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660068/
关键词：
nitrogen fixation nodules malic enzyme Bradyrhizobiom gene cloning emergy metabolism nod-35 soybean nitrogen fixqtion Bradyrhizobium japonicum energy metabolism enzyme purification Rhizobium japonicum

项目摘要

Role of malic enzyme in Bradyrhizobium japonicum energy metabolism were surveyed, and the data suggested the enzyme was a key enzyme to improve the energy metabolism.An NADP-malic enzyme, a possible key enzyme for the energy metabolism of Bradyrhizobium japonicum A1017, was purified to homogeneity by ammonium sulfate precipitation, DEAE-cellulose column chromatography, preparative isoelectric-focusing, Sephacryl S-200 column, 2', 5'-ADP-Sepharose 4B column and NAD-agarose column chromatographies.The purified enzyme showed its optimun pH at 7.4, and the pI value was 6.4. The reaction was dependent on NADP, with the Km values of 4.9 muM and 61 muM against NADP and malate respectively. The molecular weight of the enzyme subunit was calculated to be 59, 000 on SDS-PAGE, and the result of Sephacryl S-200 gel chromatography suggested that the native enzyme was present as dimer. Among metabolites present in the nodule cell extract, malonate, succinate, oxalacetate, ATP, ADP and NADPH in the enzyme reaction system showed strong regulatory effect on the activity under physiological concentrations.From the purified enzyme, the N-terminal amino acid sequence was analysed. From the data 4 DNA probes were synthesized. A genomic DNA library was also prepared after selecting ca. 2 kbp DNA fragment by sucrose density gradient centrifugation. Following ligating and packaging of the DNA to lamda phage, the plaque hybridization was performed and selected the positive clone.

苹果酸酶是大豆慢生根瘤菌（Bradyrhizobium japonicum）A1017能量代谢的关键酶，通过硫酸铵沉淀、DEAE-cellulose柱层析、等电聚焦、Sephacryl S-200柱层析、2 ′-葡聚糖凝胶柱层析、等电聚焦等方法，对大豆慢生根瘤菌A1017的NADP-苹果酸酶进行了纯化。经5 ′-ADP-Sepharose 4 B柱层析和NAD-琼脂糖柱层析，纯化的酶最适pH为7.4，pI值为6.4。该反应依赖于NADP，对NADP和苹果酸的Km值分别为4.9 μ M和61 μ M。SDS-PAGE测得酶亚基的分子量为59，000，Sephacryl S-200凝胶层析结果表明天然酶以二聚体形式存在。在根瘤细胞提取物中存在的代谢产物中，生理浓度下酶反应体系中的丙二酸、琥珀酸、乙酸、ATP、ADP和NADPH对酶活性有较强的调节作用。根据这些数据合成了4个DNA探针。选择ca. 2 kbp的DNA片段。将DNA连接并包装至λ噬菌体后，进行噬斑杂交并选择阳性克隆。