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Published on: October 15, 2015
A bacterial chloroform reductive dehalogenase: purification and biochemical characterization
Bat-Erdene Jugder1, Susanne Bohl1,2, Helene Lebhar1
1School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, 2052, Australia.
Abstract:
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.
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