School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, 2052, Australia.
Department of Biotechnology, Mannheim University of Applied Sciences, 68163, Mannheim, Germany.
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) × 10 units mg protein . 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.
我们在此报告了一种来自严格厌氧菌 Dehalobacter sp. strain UNSWDHB 的膜部分的氯仿（CF）还原酶 TmrA 的纯化，与粗裂解物相比，其相对纯度提高了约 23 倍，达到明显的均一性。通过超速离心获得的膜部分在甘油存在下用 Triton X-100 溶解，然后通过阴离子交换层析进行纯化。通过 SDS-PAGE 和 MALDI-TOF/TOF 测定，纯化的 TmrA 的分子量为 44.5 kDa。在体外，用还原甲基紫精作为电子供体，纯化的脱卤酶将 CF 还原脱氯为二氯甲烷，比活为（1.27±0.04）×10 单位 mg 蛋白。最佳温度和 pH 值分别为 45°C 和 7.2。紫外可见光谱分析表明存在一个类咕啉和两个 [4Fe-4S] 簇，这是根据氨基酸序列预测的。这是首次报道 CF 还原脱卤酶的生产、纯化和生化特性。
