固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌（Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓ）Ａ１５０１菌体经超声破碎后，无细胞粗提液以ＰＥＧ－６０００分级沉淀，丙酮沉淀，再经蓝琼脂糖（ＢｌｕｅＳｅｐｈａｒｏｓｅＣＬ－６８）亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ｋＤ和６４５ｋＤ，亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值，在以Ｇｌｕ为氮源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ），５０ｍｍｏｌ／Ｌ（ＡＴＰ）和４５ｍｍｏｌ／Ｌ（ＮＨ~＋_４）；在以ＮＨ~＋_４为氮源的介质中培养时则分别为７０ｍｍｏｌ／Ｌ（Ｇｌｕ），４９ｍｍｏｌ／Ｌ（ＡＴＰ）和８０ｍｍｏｌ／Ｌ（ＮＨ~＋_４），表明ＮＨ~＋_４培养下形成高度腺苷化的ＧＳ对Ｇｌｕ及ＮＨ~＋_４的亲和力有所下降。

Purification and Characterization of Glutamine Synthetase from Alcaligenes faecalis

Using Southern blotting and glnA gene probe from Azospirillum brasilense Sp 7,the glnA gene encoding glutamine synthetase of Alcaligenes faecalis strain A1501 was found.It is located on chromosome of this bacterium. Glutamine synthetase (GS) was isolated and purified from both NH￣+_4-growm and glutamate (Glu)-grown cells of A. faecalis A1501 through PEG fraction (15%-25%), acetone precipitation and dye-ligand chromatography.Af GS was purified to electrophoresis homogeneity with rather high activity. The molecular weight of its subunit determined by SDS-PAGE was 55 kD and that of whole enzyme was 645 kD determined by gradient PAGE, indicating the A. faecalis GS consists of 12 subunits and all subunits are the same. The Af GS subunit contained 456 amino acid residues. The acidic amino acid residues (12%) are less than the basic ones (16%). The apparent Km values of Af GS from Glu-grown and NH￣+_4-grown cells for glutamate, ATP and ammonia are 20, 50,45 and 70, 49, 80, respectively. This result shows that Af GS from NH￣+_4-grown cells is highly adenylylated and in an inactive state.