Purification, Characterization, and Overexpression of Psychrophilic and Thermolabile Malate Dehydrogenase of a Novel Antarctic Psychrotolerant, Flavobacterium frigidimaris KUC-1
Purification, Characterization, and Overexpression of Psychrophilic and Thermolabile Malate Dehydrogenase of a Novel Antarctic Psychrotolerant, Flavobacterium frigidimaris KUC-1
复制标题
DOI：
10.1271/bbb.69.2146
复制
发表时间：
2005-01
期刊：
影响因子：
--
通讯作者：
T. Oikawa;N. Yamamoto;K. Shimoke;S. Uesato;T. Ikeuchi;Toru Fujioka
中科院分区：
文献类型：
--
作者：
T. Oikawa;N. Yamamoto;K. Shimoke;S. Uesato;T. Ikeuchi;Toru Fujioka
我们纯化的嗜冷和不耐热的苹果酸脱氢酶的同质性从一种新的耐冷，Flavobacterium frigidimaris KUC-1，分离自南极海水。该酶为同源四聚体，分子量约为123 k，亚基分子量约为32 k。该酶以NAD（P）+为辅酶，特异性地催化L-苹果酸的氧化和N-乙酸的还原。反应通过有序的双-双机理进行。该酶对热处理非常敏感，在40 °C下的半衰期估计为3.0 min。L-苹果酸和NAD+在30 °C下的kcat/Km（μM−1·s−1）值分别为289和2，790。该酶表现出前R立体专一性的氢转移在C4位置的烟酰胺部分的辅酶。该酶含有311个氨基酸残基，脯氨酸和精氨酸残基的数量远低于其他苹果酸脱氢酶。
We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123 k and that of the subunit was about 32 k. The enzyme required NAD(P)+ as a coenzyme and catalyzed the oxidation of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi–bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40 °C was estimated to be 3.0 min. The kcat/Km (μM−1·s−1) values for L-malate and NAD+ at 30 °C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.