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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.
Microbial Biotechnology
|June 21, 2017
Summary
Researchers purified TmrA, a novel chloroform (CF)-reducing enzyme from Dehalobacter sp. strain UNSWDHB. This enzyme is the first characterized CF reductive dehalogenase, offering insights into chlorinated compound metabolism.
Area of Science:
- Microbiology
- Environmental Science
- Biochemistry
Background:
- Chlorinated compounds like chloroform (CF) pose environmental risks.
- Microbial degradation is a key strategy for remediating CF contamination.
- Understanding the enzymes involved in CF reduction is crucial for bioremediation efforts.
Purpose of the Study:
- To isolate and purify the chloroform (CF)-reducing enzyme, TmrA, from Dehalobacter sp. strain UNSWDHB.
- To characterize the biochemical properties of the purified TmrA enzyme.
- To provide the first report on the production, purification, and biochemical characterization of a CF reductive dehalogenase.
Main Methods:
- Enzyme purification from the membrane fraction of Dehalobacter sp. strain UNSWDHB using ultracentrifugation, Triton X-100 solubilization, and anion exchange chromatography.
- Determination of molecular mass using SDS-PAGE and MALDI-TOF/TOF.
- In vitro activity assays with reduced methyl viologen as the electron donor.
- Optimization of temperature and pH for enzyme activity.
- UV-visible spectrometric analysis to identify cofactors.
Main Results:
- TmrA was purified to apparent homogeneity with a 23-fold increase in purity.
- The purified enzyme has a molecular mass of 44.5 kDa.
- TmrA reductively dechlorinated CF to dichloromethane with a specific activity of (1.27 ± 0.04) × 10³ units mg protein⁻¹.
- Optimal activity was observed at 45°C and pH 7.2.
- Spectrometric analysis indicated the presence of a corrinoid and two [4Fe-4S] clusters.
Conclusions:
- TmrA is a novel chloroform (CF)-reducing enzyme from Dehalobacter sp. strain UNSWDHB.
- This study presents the first biochemical characterization of a CF reductive dehalogenase.
- The findings contribute to understanding microbial pathways for chlorinated compound degradation and potential bioremediation strategies.
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