Studies on biological dehalogenation of chlorinated organic compounds

一株严格厌氧细菌Desulfitobacterium sp.Y51通过三氯乙烯（TCE）高效地将作为最终电子受体的四氯乙烯（PCE）转化为顺式-1，2-二氯乙烯（cis-DCE）。这种反应与能量守恒有关，称为脱卤呼吸或卤呼吸。获得了以下结果：1.纯化了一种PCE脱卤酶（PceA），并制备了抗PceA的多克隆抗体。结果表明，PceA定位于细胞周质，与细胞膜结合。2.添加PCE或TCE可高度诱导PceA的产生。另一方面，cis-DCE在转录水平上抑制PceA的产生。3.克隆了pceAB基因簇的上下游区域的新基因。在pceAB的上游发现了推定的调控基因pceR。PceR蛋白与PCE和/或TCE结合，可刺激pceAB基因簇的转录。4.商品化的cis-DCE含有大量的氯仿。用1μM氯仿培养细胞，可使约6.6kb的DNA（包括pceAB基因）高度缺失（80%以上）。野生型菌株的生长受到很大抑制，而缺失突变株的生长没有受到明显抑制。5.获得了一个能在10 d内将PCE完全脱氯为乙烯的微生物菌群，其中存在能将各种氯乙烯完全脱氯的Dehalococcoides菌株。

A strictly anaerobic bacterium Desulfitobacterium sp.Y51 converts tetrachloroethene (PCE) as a final electron acceptor to cis-1,2-dichloroethene (cis-DCE,) via trichloroethene (TCE) very efficiently. This reaction is associated with energy conservation and called as dehalorespiration or halorespiration. We obtained the following results.1.We purified a PCE-dehalogenase (PceA encoded by pceA), and raised polychlonal antibody against purified PceA. Using the antibody we found that PceA is localized at periplasm, associating the cell membrane.2.The production of PceA was highly inducible by addition of PCE or TCE. cis-DCE, on the other hand, inhibited the PceA production at the transcriptional level. The enzyme reaction was also inhibited by cis-DCE.3.We cloned additional genes upstream and downstream regions of pceAB gene cluster. The putatative regulatory pceR gene was found upstream of pceAB. PceR protein was bound with PCE and/or TCE and considered to stimulate the transcription of pceAB gene cluster.4.The commercial cis-DCE contained high amount of chloroform. About 6.6-kb DNA including pceAB genes were highly deleted (more than 80%) when the cells were grown with 1μM chloroform. The cell growth of wild type strain was greatly inhibited, but the growth of deletion mutant was not.5.We obtained a microbial consortium that dechlorinate PCE to ethylene completely in 10 days, in which Dehalococcoides strains, known as a completed dechlorination of various chlroethenes, were present.