Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1
Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1
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DOI：
10.1128/jb.177.17.5135-5139.1995
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发表时间：
1995-09
影响因子：
3.2
通讯作者：
Shuisong Ni;James K. Fredrickson;L. Xun
中科院分区：
文献类型：
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作者：
Shuisong Ni;James K. Fredrickson;L. Xun
虽然厌氧微生物的还原脱卤为卤烃的降解提供了巨大的潜力，但对所涉及的生化机制知之甚少。先前已经证明，脱硫单胞菌DCB-1的3-氯苯甲酸脱卤作用中涉及的脱卤酶活性存在于细胞提取物的膜部分中。本文报道了从D. tiedjei DCB-1.通过用还原甲基紫精作为还原剂将3-氯苯甲酸酯转化为苯甲酸酯来监测脱卤酶活性。通过超离心获得细胞提取物的膜部分，并且在甘油存在下用洗涤剂CHAPS（3-[（3-胆酰胺丙基）-二甲基-铵基]-1-丙磺酸盐）或Triton X-100溶解膜蛋白。溶解的脱卤酶纯化硫酸铵分级和阴离子交换，羟基磷灰石，疏水相互作用色谱的组合。该方法产生约7%的总脱卤酶活性，比活性增加120倍。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明，纯化的脱卤酶由两个亚基组成，分子量分别为64，000和37，000。该酶在38 ℃下在10 mM磷酸钾缓冲液（pH 7.2）中以其最高比活性将3-氯苯甲酸盐转化为苯甲酸盐。这种酶是黄色的，可能是血红素蛋白。该酶在408 nm处有一吸收峰。连二亚硫酸盐还原的酶在416、522和550 nm处显示吸收峰。连二亚硫酸盐还原的酶能够与一氧化碳络合。血红素发色团的性质目前尚不清楚。
Although reductive dehalogenation by anaerobic microorganisms offers great potential for the degradation of halocarbons, little is known about the biochemical mechanisms involved. It has previously been demonstrated that the dehalogenase activity involved in 3-chlorobenzoate dehalogenation by Desulfomonile tiedjei DCB-1 is present in the membrane fraction of the cell extracts. We report herein the purification of a 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of D. tiedjei DCB-1. The dehalogenase activity was monitored by the conversion of 3-chlorobenzoate to benzoate with reduced methyl viologen as a reducing agent. The membrane fraction of the cell extracts was obtained by ultracentrifugation, and the membrane proteins were solubilized with either the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) or Triton X-100 in the presence of glycerol. The solubilized dehalogenase was purified by ammonium sulfate fractionation and a combination of anion exchange, hydroxyapatite, and hydrophobic interaction chromatographies. This procedure yielded about 7% of the total dehalogenase activity with a 120-fold increase in specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified dehalogenase consisted of two subunits with molecular weights of 64,000 and 37,000. The enzyme converted 3-chlorobenzoate to benzoate at its highest specific activity in 10 mM potassium phosphate buffer (pH 7.2) at 38 degrees C. The enzyme was yellow and probably a heme protein. The enzyme had an adsorbance peak at 408 nm. The dithionite-reduced enzyme displayed absorbance peaks at 416, 522, and 550 nm. The dithionite-reduced enzyme was able to complex with carbon monoxide. The nature of the heme chromophore is currently unknown.