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

研究了苹果酸酶在缓生根瘤菌能量代谢中的作用，结果表明苹果酸酶是缓生根瘤菌能量代谢的关键酶。采用硫酸铵沉淀、deae -纤维素柱层析、制备等电聚焦、Sephacryl S-200柱、2′，5′-ADP-Sepharose 4B柱和nada -琼脂糖柱层析纯化了一种nadp -苹果酸酶，该酶可能是日本慢生根瘤菌A1017能量代谢的关键酶。纯化酶的最佳pH值为7.4，pI值为6.4。该反应依赖于NADP，对NADP和苹果酸盐的Km值分别为4.9 muM和61 muM。SDS-PAGE计算该酶亚基的分子量为59000，Sephacryl S-200凝胶层析结果表明该酶以二聚体的形式存在。在结节细胞提取物中代谢产物中，酶反应体系中的丙二酸盐、琥珀酸盐、草酸盐、ATP、ADP和NADPH在生理浓度下对活性表现出较强的调节作用。对纯化酶的n端氨基酸序列进行了分析。根据数据合成了4个DNA探针。通过蔗糖密度梯度离心选择约2 kbp的DNA片段，建立基因组DNA文库。将DNA与lamda噬菌体结扎和包装后，进行斑块杂交并选择阳性克隆。

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.