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The BhbA Enzyme Assay
还原性脱卤素酶(BhbA)试验   

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Abstract

Reductive dehalogenation has been found primarily in anaerobic communities and is originally thought to rarely occur in aerobes. A reductive dehalogenase (BhbA) was characterized from an aerobic strain of Comamonas sp. 7D-2, which was isolated from a bromoxynil octanoate-contaminated soil sample collected in Jiangsu, China. BhbA catalyzes the reductive dehalogenation of bromoxynil and its derivative 3,5-dibromo-4-hydroxybenzoate under aerobic conditions. BhbA is membrane-associated and found to have the key features of anaerobic respiratory reductive dehalogenases. This protocol describes the method for enzyme analysis of the aerobic reductive dehalogenase (BhbA) in the membrane fraction.

Materials and Reagents

  1. Comamonas sp. 7D-2
  2. LB medium
  3. Substrate, 3,5-dibromo-4-hydroxybenzoate (DBHB) or 3-bromo-4-hydroxybenzoate (BHB) (Sigma-Aldrich)
  4. Electron donor, NADPH or NADH (Sangon Biotech, catalog numbers: Y4433000-100 mg and NB0642-1 g , respectively)
  5. Reaction inhibitor, sodium dithionite (Na2S2O4) (Sigma-Aldrich, catalog number: 7775-14-6 )
  6. Protein Quantification Kit (Sangon Biotech, catalog number: BE530-100 ml )
  7. Phosphate buffered saline (PBS) (Sambrook and Russell, 2001) (see Recipes)
  8. Mobile phase of HPLC (see Recipes)

Equipment

  1. 7 ml centrifuge tube
  2. Membrane filtration (pore size, 0.22 μm)
  3. Centrifuge
  4. HPLC (600 controller, Rheodyne 7725i manual injector and 2487 Dual λ Absorbance Detector) (Waters)
  5. Ultrasonic instrument
  6. Fast protein liquid chromatography

Procedure

  1. Comamonas sp. 7D-2 was inoculated in 100 ml LB medium (supplemented with 0.25 mM DBHB) and cultured at 30 °C for 14 h.
  2. Late exponential-phase cultures (approximately OD600=1.0) of strain 7D-2 were pelleted by centrifugation at 15,000 x g for 5 min, washed two times with PBS (pH 7.4), resuspended in PBS (containing 2 mM dithiothreitol) and then disrupted by sonication at 4 °C. BhbA was partially purified by fast protein liquid chromatography (Amersham Biosciences) at 4 °C (Chen et al., 2013; van de Pas et al., 1999).
  3. Protein concentrations were determined by the Bradford method (Bradford, 1976) with Protein Quantification Kit.
  4. A standard enzyme assay was performed under aerobic conditions in a 7 ml centrifuge tube containing 3.0 ml PBS, 0.1 mM DBHB (BHB) and 0.2 mM NADPH (NADH). The reaction was initiated by the addition of 30 μl of enzyme preparation and incubated at 30 °C for 60 min. Negative control without added enzyme was performed.
  5. The reaction was stopped by the addition of 15 μl sodium dithionite (1 M).
  6. The mixture was centrifuged at 16,000 x g for 10 min and filtered by membrane filtration; then, the DBHB concentration was analyzed using HPLC (Chen et al., 2013).
  7. One unit of BhbA activity was defined as the amount of BhbA that catalyzes the reduction of 1 nmol of DBHB per minute. Specific activity was expressed in units per milligram of protein. All assays were performed independently three times, and the means and standard deviations were calculated.

Recipes

  1. Phosphate buffered saline (PBS)
    8.0 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4 per liter of water
    pH 7.4
  2. Mobile phase of HPLC
    Acetonitrile: water: acetic acid (50: 50: 0.5/v:v:v)

Acknowledgments

This work was supported by grants from the Chinese National Science Foundation for Excellent Young Scholars (31222003), the Chinese National Natural Science Foundation (31070100), the Program for New Century Excellent Talents in University (NCET-12-0892), the Outstanding Youth Foundation of Jiangsu Province and the National Science and Technology Support Plan (2013AA102804). S. Zinder’s research on haloaromatic degradation was supported by funding by the DuPont Corporation.

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Chen, K., Huang, L., Xu, C., Liu, X., He, J., Zinder, S. H., Li, S. and Jiang, J. (2013). Molecular characterization of the enzymes involved in the degradation of a brominated aromatic herbicide. Mol Microbiol 89(6): 1121-1139.
  3. Sambrook, J., Russell, D. W. and Russell, D. W. (2001). Molecular cloning: a laboratory manual (3-volume set). Cold Spring Harbor Laboratory Press.
  4. van de Pas, B. A., Smidt, H., Hagen, W. R., van der Oost, J., Schraa, G., Stams, A. J. and de Vos, W. M. (1999). Purification and molecular characterization ofortho-chlorophenol reductive dehalogenase, a key rnzyme of halorespiration in Desulfitobacterium dehalogenans. J Biol Chem 274(29): 20287-20292.

简介

还原脱卤主要在厌氧社区中发现,并且最初被认为很少发生在aerobes中。 还原脱卤素酶(BhbA)的特征在于Comamonas的需氧菌株。 7D-2,其从中国江苏收集的溴苯腈辛酸盐污染的土壤样品中分离。 BhbA在需氧条件下催化溴苯腈及其衍生物3,5-二溴-4-羟基苯甲酸的还原脱卤。 BhbA是膜相关的并且发现具有厌氧呼吸还原脱卤素酶的关键特征。 该方案描述了膜部分中有氧还原脱卤酶(BhbA)的酶分析方法。

材料和试剂

  1. 7D-2
  2. LB培养基
  3. 底物,3,5-二溴-4-羟基苯甲酸酯(DBHB)或3-溴-4-羟基苯甲酸酯(BHB)(Sigma-Aldrich)
  4. 电子给体,NADPH或NADH(Sangon Biotech,目录号:分别为Y4433000-100mg和NB0642-1g)
  5. 反应抑制剂连二亚硫酸钠(Na 2 S 2 O 4 sub)(Sigma-Aldrich,目录号:7775-14-6)
  6. 蛋白定量试剂盒(Sangon Biotech,目录号:BE530-100ml)
  7. 磷酸盐缓冲盐水(PBS)(Sambrook和Russell,2001)(参见Recipes)
  8. HPLC的流动相(参见配方)

设备

  1. 7ml离心管
  2. 膜过滤(孔径,0.22μm)
  3. 离心机
  4. HPLC(600控制器,Rheodyne 7725i手动进样器和2487双λ吸光度检测器)(Waters)
  5. 超声仪器
  6. 快速蛋白质液相色谱法

程序

  1. 7D-2接种在100ml LB培养基(补充有0.25mM DBHB)中,并在30℃下培养14小时。
  2. 通过在15,000×g离心5分钟使菌株7D-2的晚指数期培养物(大约OD 600 = 1.0)沉淀,用PBS(pH7.4)洗涤两次),重悬于PBS(含有2mM二硫苏糖醇)中,然后在4℃下通过超声破碎。在4℃下通过快速蛋白质液相色谱(Amersham Biosciences)部分纯化BhbA(Chen等人,2013; van de Pas等人,1999)。 br />
  3. 蛋白质浓度通过Bradford方法(Bradford,1976)用蛋白质定量试剂盒测定
  4. 在需氧条件下在含有3.0ml PBS,0.1mM DBHB(BHB)和0.2mM NADPH(NADH)的7ml离心管中进行标准酶测定。通过加入30μl酶制剂引发反应,并在30℃温育60分钟。进行不加酶的阴性对照
  5. 通过加入15μl连二亚硫酸钠(1M)终止反应。
  6. 将混合物在16,000×g离心10分钟,通过膜过滤进行过滤;然后,使用HPLC分析DBHB浓度(Chen等人,2013)。
  7. 一个单位的BhbA活性定义为每分钟催化1nmol DBHB还原的BhbA的量。 比活性以每毫克蛋白质的单位表示。 所有测定独立进行三次,计算平均值和标准偏差

食谱

  1. 磷酸盐缓冲盐水(PBS)
    8.0克NaCl 0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2·PO 4 4 /升水
    pH 7.4
  2. HPLC的流动相
    乙腈:水:乙酸(50:50:0.5/v:v:v)

致谢

这项工作得到中国优秀青年学者科学基金会(31222003),中国国家自然科学基金(31070100),大学新世纪优秀人才计划(NCET-12-0892),杰出青年 江苏省基金会和国家科技支撑计划(2013AA102804)。 S.Zinder对卤代芳烃降解的研究得到了杜邦公司的资助。

参考文献

  1. Bradford,M.M。(1976)。 利用蛋白质染料结合原理的快速灵敏的微克量蛋白定量方法 。 Anal Biochem 72:248-254。
  2. Chen,K.,Huang,L.,Xu,C.,Liu,X.,He,J.,Zinder,S. H.,Li,S.and Jiang,J.(2013)。 涉及溴化芳香族除草剂降解的酶的分子表征 Mol Microbiol 89(6):1121-1139。
  3. Sambrook,J.,Russell,D.W。和Russell,D.W。(2001)。 分子克隆:实验室手册(3卷集)冷泉港L
  4. van de Pas,B.A.,Smidt,H.,Hagen,W.R.,van der Oost,J.,Schraa,G.,Stams,A.J.and de Vos,W.M。(1999)。 邻 - 氯苯酚还原脱卤素酶的纯化和分子表征, (Deulfitobacterium dehalogenans)中的呼吸的关键酶。 J Biol Chem 274(29):20287-20292。
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引用:Chen, K. and Jiang, J. (2014). The BhbA Enzyme Assay. Bio-protocol 4(8): e1111. DOI: 10.21769/BioProtoc.1111.
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