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

A Bacterial Chloroform Reductive Dehalogenase: Purification and Biochemical Characterization

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A Bacterial Chloroform Reductive Dehalogenase: Purification and Biochemical Characterization

Bat-Erdene Jugder et al. Microb Biotechnol. .
Free PMC article

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.

Figures

Figure 1
Figure 1
The purified TmrA. A. SDSPAGE with the purified chloroform reductive dehalogenase, TmrA, of Dehalobacter UNSWDHB in lane 4. Molecular size markers (SeeBlue Plus2 Prestained Standard; Thermo Fisher Scientific) are shown in lane 1 and 5. The crude cell lysate and solubilized membrane fraction are in lane 2 and 3. The arrow indicates the purified protein band. The gel was stained with GelCode Blue Stain Reagent (Thermo Fisher Scientific). B. MALDITOF spectra of the TmrA at an intact mass of 44 511 Da.
Figure 2
Figure 2
Deduced amino acid sequence of TmrA from strain UNSWDHB. The black and grey boxes indicate the TAT signal peptide and a [4Fe‐4S] double cluster binding domain (NCBI: pfam13484), respectively. Underlined sequences are peptides that were detected by mass spectrometry with MASCOT (coverage 77.3%, score of 2223). A slash indicates the predicted location of the cleavage of the peptide signal predicted by PREDTAT.
Figure 3
Figure 3
The effect of pH and temperature on the activity of the purified TmrA. A. The anaerobic reaction mixtures containing the purified TmrA (4 μg), 2 mM titanium (III) citrate and 2 mM methyl viologen in 100 mM Tris–HCl buffer at pH values of 5.5, 6.2, 6.4, 6.72, 7.0, 7.2, 7.5, 7.75, 7.98 and 8.3 were incubated at 30°C. B. The mixtures were incubated at temperatures between 25 and 70°C, with 5°C increments, at pH 7.5. The average values of triplicate assays are presented.
Figure 4
Figure 4
CF dechlorinating kinetics of purified TmrA. Each point represents the initial rate of dechlorination determined by the concentration of DCM formed after 2 h incubation. Curves are nonlinear regression fitted to the Substrate Inhibition model (solid line) and the Michaelis–Menten model (dashed line), resulting from duplicates using GraphPad Prism v. 6.07. Error bars represent standard deviation.
Figure 5
Figure 5
UV‐visible absorption spectra of the purified TmrA. The purified enzyme (0.05 mg ml−1) exhibits features consistent with inclusion of both [4Fe‐4S] clusters (∼420 nm) and cob(II)alamin (310 nm and 450–475 nm) as cofactors.

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