Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.
Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.
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乳杆菌二醇脱水酶的纯化、表征和亚基鉴定。
DOI：
10.1046/j.1432-1033.2002.03288.x
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
J. Laplace
中科院分区：
文献类型：
--
作者：
N. Sauvageot;V. Pichereau;Loïc Louarme;A. Hartke;Y. Auffray;J. Laplace
已克隆了编码Collinoides乳杆菌二醇脱氢酶的三个基因pduCDE，用于在pQE 30载体中过表达。虽然蛋白质的三个亚基被高度诱导，但在细胞提取物中未检测到活性。因此，通过硫酸铵沉淀和凝胶过滤色谱法将酶纯化至接近均一。在显示二醇脱氢酶活性的组分中，SDS/PAGE后存在三个主要条带，分子量分别为63、28和22 kDa。通过质谱鉴定，它们分别对应于由pduC、pduD和pduE基因编码的淀粉酶的大、中和小亚基。根据从pduC、D、E基因（分别为61、24.7和19.1 kDa）和α 2 β 2 γ 2组成推导的计算分子量，天然复合物的分子量估计为207 kDa。三种主要底物的Km分别为1.6 mm（1，2-丙二醇）、5.5 mm（1，2-乙烷）和8.3 mm（甘油）。该酶需要腺苷钴胺素辅酶的催化活性，辅助因子的Km为8 μ m。甘油和维生素B12均可使酶失活。该酶的最适反应条件为pH8.75和37 ℃。活性受到钠离子和钙离子的抑制，镁离子的抑制程度较低。第四条59 kDa的条带与二醇脱氢酶共纯化，并被鉴定为丙醛脱氢酶，另一种参与1，2-丙二醇代谢途径的蛋白质。
The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.
二醇脱水酶：N-末端氨基酸序列和亚基化学计量。
DOI：
10.1016/0006-291x(82)90863-4
发表时间：
1982
影响因子：
3.1
作者：
McGee,DE;Carroll,SS;Bond,MW;Richards,JH
通讯作者：
Richards,JH
丙二醇脱水酶（一种膜相关酶）的纯化和亚基表征。
影响因子：
2.9
作者：
McGee,DE;Richards,JH
通讯作者：
Richards,JH