• Journal of environmental and Sanitary engineering
• /
• v.23 no.2
• /
• pp.1-18
• /
• 2008

The tetrachloroethylene (PCE) dehalogenase of Clostridium bifermentans DPH-1 (a halorespiring organism) was purified, cloned, and sequenced. This enzyme is a homodimer with a molecular mass of ca. 70 kDa and exhibits dehalogenation of dichloroethylene isomers along with PCE and trichloroethylene (TCE). Broad range of substrate specificity for chlorinated aliphatic compounds (PCE, TCE, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropene, and 1,1,2-trichloroethane) for this enzyme was also observed. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. A partial sequence (81 bp) of the encoding gene for PCE dehalogenase was amplified and sequenced with degenerateprimers designed from the N-terminal sequence (27 amino acid residues). Southern analysis of C. bifermentans genomic DNA using the polymerase chain reaction product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase. So, C. bifermentans could play some important role in the initial breakdown of PCE and other chlorinated aliphatic compounds in sites contaminated with mixtures of halogenated substances.

• Journal of Environmental Health Sciences
• /
• v.26 no.2
• /
• pp.14-21
• /
• 2000

DEAE-Toyopearl 650S, Superdex pg-75, Poros HQ, Superdex H200의 각종 칼러크로마토그래피를 이용하여 C.bifermentans DPH-1균주로부터 테트라클로로에틸렌(PCE) 분해 효소를 정제했다. 이 PCE 분해효소 (PCE dehalogenase)는 PCE를 환원적 탈염소화 반응에 의해 시스디클로로에딜렌 (cis-1,2-dichloroethylene)에 전환 가능하여, 이 때의 Vmax 및 Km 치는 각각 73 nmol/h.mg protein, 12$\mu$M이었다. 본 PCE dehalogenase의 겔여과 분자량 Maker Kit를 이용한 분석결과(70kDa)는 SDS-PAGE에 나타난 분자량(35kDa)의 약 2배인 것으로 확인되었다. 따라서 본 효소는 분자량 70kDa의 이량체(Homo dimer)인 것으로 추정되었다. 본 효소의 최적온도 및 pH는 각각 35$^{\circ}C$ 및 8.0 이었다. 또한 본 효소는 PCE외의 트리클로로에틸렌, 디클로로에틸렌 이성체, 1,2-디클로로에템, 1,2-디클로로프로판, 1,1,2-트리클로로에탄에 대하여도 활성을 타나내었다. N-말단 아미노산 분석결과에서도 본 효소는 현재 알려진 PCE dehalogenase와 그 배열이 전혀 다른 것으로 나타나 각종 유기염소 화합물의 분해능을 보유한 신종의 PCE 분해효소인 것이 확인되었다.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.1
• /
• pp.120-129
• /
• 2016

An industrial complex in Wonju, contaminated with trichloroethene (TCE), was one of the most problematic sites in Korea. Despite repeated remedial trials for decades, chlorinated ethenes remained as sources of down-gradient groundwater contamination. Recent efforts were being made to remove the contaminants of the area, but knowledge of the indigenous microbial communities and their dechlorination abilities were unknown. Thus, the objectives of the present study were (i) to evaluate the dechlorination abilities of indigenous microbes at the contaminated site, (ii) to characterize which microbes and reductive dehalogenase genes were responsible for the dechlorination reactions, and (iii) to develop a PCE-to-ethene dechlorinating microbial consortium. An enrichment culture that dechlorinates PCE to ethene was obtained from Wonju stream, nearby a trichloroethene (TCE)-contaminated industrial complex. The community profiling revealed that known organohalide-respiring microbes, such as Geobacter, Desulfuromonas, and Dehalococcoides grew during the incubation with chlorinated ethenes. Although Chloroflexi populations (i.e., Longilinea and Bellilinea) were the most enriched in the sediment microcosms, those were not found in the transfer cultures. Based upon the results from pyrosequencing of 16S rRNA gene amplicons and qPCR using TaqMan chemistry, close relatives of Dehalococcoides mccartyi strains FL2 and GT seemed to be dominant and responsible for the complete detoxification of chlorinated ethenes in the transfer cultures. This study also demonstrated that the contaminated site harbors indigenous microbes that can convert PCE to ethene, and the developed consortium can be an important resource for future bioremediation efforts.