Purification and characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum multivorans
Purification and characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum multivorans
复制标题
DOI：
10.1074/jbc.271.28.16515
复制
发表时间：
1996-07-12
影响因子：
4.8
通讯作者：
Diekert, G
中科院分区：
文献类型：
--
作者：
Neumann, A;Wohlfarth, G;Diekert, G
从利用四氯乙烯的厌氧菌多食性脱卤螺杆菌中分离出的四氯乙烯还原脱卤酶纯化约100倍，达到表观均一。纯化的脱卤酶催化四氯乙烯还原脱氯为三氯乙烯，三氯乙烯还原脱氯为顺-1，2-二氯乙烯，还原甲基紫精为电子供体，比活度为2.6微卡/毫克。四氯乙烯和三氯乙烯的表观K-m值分别为0.20和0.24 mm。凝胶过滤测得该天然酶的表观分子质量为58 kDa。十二烷基硫酸钠凝胶电泳法显示一条相对分子质量为57 kDa的单一蛋白带。1摩尔脱卤酶中含有1.0摩尔皮质激素、9.8摩尔铁和8.0摩尔不稳定酸硫。最适pH为8.0左右。该酶的最适温度为42℃，对氧稍敏感，50℃以上不耐热。氯代甲烷对PCE脱卤酶活性有严重抑制作用。
Tetrachloroethene reductive dehalogenase from the tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, was purified approximately 100-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive dechlorination of tetrachloroethene (PCE) to trichloroethene and of trichloroethene to cis-1,2-dichloroethene with reduced methyl viologen as the electron donor at a specific activity of 2.6 microkatal/mg. The apparent K-m values for tetrachloroethene and trichloroethene were 0.20 and 0.24 mM, respectively. The apparent molecular mass of the native enzyme was determined by gel filtration to be 58 kDa. Sodium dodecyl sulfate-gel electrophoresis revealed a single protein band with a molecular mass of 57 kDa. One mol of dehalogenase contained 1.0 mol of corrinoid, 9.8 mol of iron, and 8.0 mol of acid-labile sulfur. The pH optimum was about 8.0. The enzyme had a temperature optimum of 42 degrees C. It was slightly oxygen-sensitive and was ther molabile above 50 degrees C. The dechlorination of PCE was stimulated by ammonium ions. Chlorinated methanes severely inhibited PCE dehalogenase activity.