Characterisation of microbial dehalogenation using compound specific stable isotope analysis

虽然有机卤化物的呼吸作用在过去的几十年里已经被研究，但仍然不清楚涉及哪些反应机制。虽然类似的途径和酶被认为是参与不同的有机卤化物呼吸，我们最近的研究比较了各种微生物菌株中的氯化乙烯的还原脱氯，表明在表观动力学碳同位素分馏的高度可变性。表观动力学同位素效应的变化引起的假设，脱卤反应的途径和机制略有不同，需要一个系统的研究比较酶的库存和分馏因素，以更好地了解脱卤过程。因此，本项目的主要目的是利用氯和碳同位素分馏模式和氢溶剂动力学效应表征还原脱卤的反应机理。在该提案中，Dehalococcoides spp.，具有不同的酶，能够脱卤各种各样的底物，将被用作模型系统之一，以研究与同位素分馏模式有关的还原脱卤酶的变异性。将使用所描述的Dehalococcoides sp.菌株195和CBDB 1，并与其他有机卤化物呼吸菌株如硫杆菌属、脱硫杆菌属或脱卤杆菌属以及我们的源自Bitterfeld地下水的高度富集的Dehaloccoides培养物BTF 08进行比较。同位素分馏模式使用多同位素分析，包括Cl和H同位素应该允许表征的反应机制，如以前所做的BTEX和MTBE在我们的研究小组。

Although organohalide respiration has been investigated over the last few decades, it is still unclear which reaction mechanisms are involved. Although similar pathways and enzymes are thought to be involved in different organohalide respireres, our recent research comparing the reductive dechlorination of chlorinated ethenes in a variety of microbial strains showed a high variability in apparent kinetic carbon isotope fractionation. The variability of apparent kinetic isotope effects gives rise to the hypothesis that pathways and mechanisms of dehalogenation reactions are slightly different and a systematic study comparing the enzymatic inventory and fractionation factors is needed for a better understanding of the dehalogenation process. Therefore, the main aim of this project is the characterization of the reaction mechanism of reductive dehalogenation using chlorine and carbon isotope fractionation patterns and hydrogen solvent kinetic effects. In this proposal, Dehalococcoides spp., possessing different enzymes capable of dehalogenating a wide variety of substrates, will be used as one of the model systems to investigate the variability of reductive dehalogenases related to the isotope fractionation pattern. The described Dehalococcoides sp. strains 195 and CBDB1 will be used and compared to other organohalide respiring strains such as Sulfurospirillum, Desulfitobacterium or Dehalobacter as well as to our highly enriched Dehalococcoides culture BTF08 derived from Bitterfeld groundwater. Isotope fractionation patterns using multi-isotope analysis including Cl and H isotopes should allow the characterisation of the reaction mechanism as previously done for BTEX and MTBE in our research group.