A Bacterial Chloroform Reductive Dehalogenase: Purification and Biochemical Characterization

Microb Biotechnol. 2017 Nov;10(6):1640-1648. doi: 10.1111/1751-7915.12745. Epub 2017 Jun 20.


We report herein the purification of a chloroform (CF)-reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23-fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X-100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS-PAGE and MALDI-TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 103 units mg protein-1 . The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV-visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe-4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification*
  • Bacterial Proteins / metabolism
  • Chloroform / metabolism*
  • Chromatography, Ion Exchange
  • Clostridiales / chemistry
  • Clostridiales / enzymology*
  • Clostridiales / genetics
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Weight
  • Oxidoreductases / chemistry*
  • Oxidoreductases / genetics
  • Oxidoreductases / isolation & purification*
  • Oxidoreductases / metabolism
  • Substrate Specificity


  • Bacterial Proteins
  • Chloroform
  • Oxidoreductases