Neumann A, Wohlfarth G, Diekert G
Department of Microbiology, University of Stuttgart, Allmandring 31, D-70550 Stuttgart, Federal Republic of Germany.
J Biol Chem. 1996 Jul 12;271(28):16515-9. doi: 10.1074/jbc.271.28.16515.
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 Km 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 thermolabile above 50 degrees C. The dechlorination of PCE was stimulated by ammonium ions. Chlorinated methanes severely inhibited PCE dehalogenase activity.
从利用四氯乙烯的厌氧微生物——多噬脱卤螺旋菌中提取的四氯乙烯还原脱卤酶，经纯化后纯度提高了约100倍，达到了表观均一性。纯化后的脱卤酶以还原型甲基紫精作为电子供体，催化四氯乙烯（PCE）还原脱氯生成三氯乙烯，以及三氯乙烯还原脱氯生成顺式1,2 - 二氯乙烯，比活性为2.6微 katal/毫克。四氯乙烯和三氯乙烯的表观Km值分别为0.20和0.24毫摩尔。通过凝胶过滤法测定天然酶的表观分子量为58 kDa。十二烷基硫酸钠 - 凝胶电泳显示有一条分子量为57 kDa的单一蛋白带。1摩尔脱卤酶含有1.0摩尔类咕啉、9.8摩尔铁和8.0摩尔酸不稳定硫。最适pH约为8.0。该酶的最适温度为42℃。它对氧气略有敏感，在50℃以上热不稳定。铵离子可刺激PCE的脱氯反应。氯代甲烷严重抑制PCE脱卤酶的活性。
