参见作者原研究论文

本实验方案简略版
Apr 2020

本文章节


 

In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract
耻垢分枝杆菌粗提物硝酸还原酶活性的体外测定   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate reductase (NaR) activity of NAS enzymes from Mycolicibacterium smegmatis crude extract using both physiological and non-physiological electron donors.

Keywords: Mycolicibacterium smegmatis (耻垢分枝杆菌), Assimilatory nitrate reductase (同化硝酸还原酶), In vitro nitrate reductase activity assay (体外硝酸还原酶活性测定), NADPH (NADPH), NADH (NADH), Methyl viologen (甲基紫精)

Background

In addition to being an important nitrogen source for plants, nitrate (NO3-) is universally used as an inorganic nitrogen source for microorganisms, particularly soil bacteria such as actinomycetes, as well as marine bacteria such as cyanobacteria, due to fierce competition for limited nutrients in the natural environment (Kuypers et al., 2018). In prokaryotes, nitrate is reduced to ammonia (NH3) through two sequential steps, with the first committed step catalyzed by NAS. Prokaryotic NASs are categorized based on the physiological electron donors utilized (Morozkina and Zvyagilskaya, 2007; Tan et al., 2020), i.e., the flavodoxin- or ferredoxin-dependent NAS and the NAD(P)H-dependent NAS; however, to date, there have been few reports detecting in vitro NaR activity of bacterial NAD(P)H-dependent NASs employing their physiological electron donors. Instead, methyl viologen (an artificial chemical reductant) is the most commonly used electron donor for in vitro NaR activity assays.


In this protocol, employing an optimized assay system, NAD(P)H-dependent NaR activity can be measured in M. smegmatis crude cell extract. Briefly, the enzyme samples prepared under anaerobic conditions can use both NADPH and NADH as electron donors for catalysis in vitro. Nitrite produced by nitrate reduction forms a colored azo compound with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride, which can be measured by its absorbance at 540 nm. This method is suitable for the determination of in vitro NaR activity of bacterial NAS enzymes.


Materials and Reagents

  1. Mycolicibacterium smegmatis mc2155

  2. Middlebrook 7H10 agar (BD Biosciences, catalog number: 262710)

  3. Middlebrook 7H9 broth (BD Biosciences, catalog number: 271310)

  4. Glycerol (Sigma-Aldrich, catalog number: G5516)

  5. Tween 80 (Sigma-Aldrich, catalog number: P1754)

  6. Ethanol (Sigma-Aldrich, catalog number: 51976)

  7. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626)

  8. Bradford protein assay kit (Beyotime, catalog number: P0006)

  9. Sodium nitrate (NaNO3) (Sigma-Aldrich, catalog number: S5506)

  10. Sodium nitrite (NaNO2) (Sigma-Aldrich, catalog number: S2252)

  11. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793)

  12. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S3139)

  13. Nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma-Aldrich, catalog number: N7505)

  14. Nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich, catalog number: 43420)

  15. Methyl viologen (Sigma-Aldrich, catalog number: 856177)

  16. Flavin mononucleotide (FMN) (Sigma-Aldrich, catalog number: F2253)

  17. Flavin adenine dinucleotide (FAD) (Sigma-Aldrich, catalog number: F6625)

  18. Sodium hydrosulfite (Na2S2O4) (Sigma-Aldrich, catalog number: 71699)

  19. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6014)

  20. Sulfanilamide (Sigma-Aldrich, catalog number: S9251)

  21. N-(1-naphthyl)ethylenediamine dihydrochloride (NED) (Sigma-Aldrich, catalog number: 222488)

  22. 7H10 agar (see Recipes)

  23. 7H9 medium (see Recipes)

  24. MPLN medium (see Recipes)

  25. 0.5 M sodium phosphate buffer, pH 7.5 (see Recipes)

  26. 0.1 M sodium phosphate buffer, pH 7.5 (see Recipes)

  27. 0.1 M PMSF (see Recipes)

  28. 60 mM Na2S2O4/NaHCO3 (see Recipes)

  29. 1% (w/v) sulfanilamide solution (see Recipes)

  30. 0.01% (w/v) NED solution (see Recipes)

Equipment

  1. Pipette kit (Gilson, catalog number: F167300)

  2. High-speed centrifuge (Beckman, catalog number: AVANTI JXN-26)

  3. Ultrasonic cell disruptor (Beyotime, catalog number: E0385)

  4. Ultrasonic bath equipped with a degas mode (Beyotime, catalog number: E0439)

  5. 250-ml and 2-L Erlenmeyer flasks

  6. Petri dish (90 mm diameter)

  7. 37°C incubator

  8. Incubator shaker

  9. Water bath

  10. Spectrophotometer

  11. Anaerobic chamber (glove box) filled with mixed gas (nitrogen, 95%; hydrogen, 5%)

    Note: Place the pipette kit, ultrasonic cell disruptor, and water bath inside the anaerobic chamber.

Software

  1. Microsoft Excel

Procedure

  1. Preparation of M. smegmatis culture

    1. Streak M. smegmatis frozen culture on a 7H10 agar plate and incubate aerobically at 37°C for 3 days until colonies are visible.

    2. Inoculate a single colony into 50 ml 7H9 medium in a 250-ml Erlenmeyer flask and incubate aerobically at 37°C for 16 h with shaking at 180 rpm (overnight culture).

    3. Collect cells from the overnight culture by centrifuging at 3,000 × g for 10 min. Wash the pellet twice with nitrogen-free MPLN medium and centrifuge as above. Resuspend the cells in 50 ml MPLN medium as the seeding culture.

    4. Re-inoculate 10 ml seeding culture into 1 L MPLN medium supplemented with 10 mM nitrate in a 2-L Erlenmeyer flask and incubate aerobically at 37°C for 30 h with shaking to late-log phase (OD600 0.8–1.0).


  2. Preparation of M. smegmatis crude extract

    1. Collect cells by centrifuging at 5,000 × g for 10 min at 4°C.

    2. Wash the cell pellet twice with 30 ml degassed 0.1 M sodium phosphate buffer (pH 7.5).

    3. Transfer the cell pellet into an anaerobic chamber and resuspend in 20 ml degassed 0.1 M sodium phosphate buffer (pH 7.5) supplemented with 10% v/v glycerol and 1 mM PMSF.

    4. Disrupt cells by sonication (pulse: 0.3 s, stop: 0.7 s) for 10 min on ice and transfer the lysate to a centrifuge tube inside the anaerobic chamber.

    5. Centrifuge at 16,000 × g for 1 h at 4°C outside the anaerobic chamber.

    6. Bring the centrifuge tube back into the anaerobic chamber and collect the supernatant (cytoplasmic fraction) as the crude enzyme extract.

      Note: Keep on ice during the assay and use within 24 h.

    7. Determine the total protein concentration of the cytoplasmic fraction using the Bradford method.


  3. Preparation of the nitrite standard curve

    1. Prepare a nitrite dilution series using sodium nitrite (0, 3, 30, 60, 120, 240, and 300 μM) in deionized water.

    2. Pipette 0.5 ml nitrite dilution series (Test sample) into 1.5-ml tubes. Use 0.5 ml 10 mM sodium nitrate as a control (Blank sample).

    3. Add 0.5 ml sulfanilamide solution and 0.5 ml NED solution to all tubes.

    4. Mix by swirling and incubate at 25°C for 10 min.

    5. Transfer 1 ml assay mixture to a cuvette.

    6. Measure the visible absorption at 540 nm using the spectrophotometer.

    7. Calculate the absorbance change using the following equation:
      ΔA540 nm (Sample) = A540 nm (Test) - A540 nm (Blank)

    8. Plot the standard curve with the nitrite concentration on the horizontal axis and the absorbance change on the vertical axis (Figure 1).



      Figure 1. Nitrite standard curve. Error bars indicate the average value of three biological replicates ± SD (n = 3). According to the standard curve, the regression equation is x (µM) = (y-0.0204)/0.0092, where x stands for nitrite concentration and y stands for ΔA540 nm (Sample).


  4. In vitro NaR activity assay

    1. Before preparing the reaction mixture for the enzyme assay, degas the solutions used in the assays and place in the anaerobic chamber before use.

    2. Pipette the following reagents for the nitrate reduction reaction (Tables 1 and 2) into a 1.5-ml tube inside the anaerobic chamber:


      Table 1. Assay mixtures for determining NAD(P)H-dependent NaR activity

      Assay sample
      Test Blank
      Deionized water 0.090 ml 0.100 ml
      Sodium phosphate buffer (0.5 M) 0.100 ml 0.100 ml
      NaNO3 (50 mM) 0.100 ml 0.100 ml
      NADPH (2 mM) or NADH (2 mM) 0.100 ml 0.100 ml
      FMN (0.25 mM) 0.050 ml 0.050 ml
      FAD (0.25 mM) 0.050 ml 0.050 ml
      Crude enzyme extract 0.010 ml -
      Step 1: Mix gently and incubate at 30°C for 10 min inside the anaerobic chamber
      1% (w/v) sulfanilamide solution 0.500 ml 0.500 ml
      0.01% (w/v) NED solution 0.500 ml 0.500 ml
      Step 2: Mix gently and incubate at 25°C in the dark for 10 min inside the anaerobic chamber


      Table 2. Assay mixtures for determining methyl viologen-dependent NaR activity

      Assay sample
      Test Blank
      Deionized water 0.090 ml 0.100 ml
      Sodium phosphate buffer (0.5 M) 0.100 ml 0.100 ml
      NaNO3 (50 mM) 0.100 ml 0.100 ml
      Methyl viologen (0.75 mM) 0.100 ml 0.100 ml
      Na2S2O4/NaHCO3 (60 mM) 0.100 ml 0.100 ml
      Crude enzyme extract 0.010 ml -

      Step 1: Mix gently and incubate at 30°C for 10 min inside the anaerobic chamber
      Step 2: Stop the reaction by vigorous stirring in the open air outside the anaerobic chamber until the blue color disappears

      1% (w/v) sulfanilamide solution 0.500 ml 0.500 ml
      0.01% (w/v) NED solution 0.500 ml 0.500 ml
      Step 3: Mix gently and incubate at 25°C in the dark for 10 min outside the anaerobic chamber

      Note: The assay mixture turns deep blue after adding Na2S2O4/NaHCO3, an indication of methyl viologen being reduced.


    3. Transfer 1 ml assay mixture to a cuvette.

    4. Measure the visible absorption at 540 nm for both the Test and Blank samples.

    5. Calculate the NaR specific activity using the following equation:


      NaR specific activity (nmol/min/mg) = Cnitrite DF/T/Cenzyme/Venzyme


      where,

      Cnitrite (µM) = [A540 nm (Test) - A540 nm (Blank)-0.0204]/0.0092, the concentration of nitrite formed in the assay

      DF: The dilution factor

      T (min): The reaction time for nitrate reduction in the assay

      Note: An incubation time of 10 min is sufficient for measuring the in vitro nitrate reductase activity.

      Cenzyme (mg/ml): The protein concentration of the crude enzyme extract

      Venzyme (ml): The volume of the crude enzyme extract used in the assay

Recipes

  1. 7H10 agar

    Dissolve 19 g 7H10 powder in 1 L deionized water

    Add 4 ml 50% (v/v) glycerol and 5 ml 10% (v/v) Tween 80

    Autoclave at 121°C for 15 min

  2. 7H9 medium

    Dissolve 4.7 g 7H9 powder in 1 L deionized water

    Add 4 ml 50% (v/v) glycerol and 5 ml 10% (v/v) Tween 80

    Autoclave at 121°C for 15 min

  3. Nitrogen-free MPLN medium (pH 6.6)

    36.7 mM KH2PO4

    7.7 mM sodium citrate

    5 mM MgSO4

    60 µM FeCl3

    20 µM NaMoO4

    3.4 µM CaCl2

    3.5 µM ZnSO4

    4 µM CuSO4

    2% (v/v) glycerol

    0.05% (v/v) Tween 80

    Autoclave at 121°C for 15 min.

  4. 0.5 M sodium phosphate buffer (pH 7.50)

    Prepare 0.5 M solutions of NaH2PO4 and Na2HPO4

    Mix 20% (v/v) NaH2PO4 and 80% (v/v) Na2HPO4

    Adjust pH to 7.5

  5. 0.1 M sodium phosphate buffer (pH 7.50)

    Dilute 0.5 M sodium phosphate buffer 1:5 with deionized water

    Store at room temperature

  6. 0.1 M PMSF

    Dissolve 0.174 g PMSF in 10 ml ethanol

    Store at -20°C

  7. 60 mM Na2S2O4/NaHCO3

    Prepare 120 mM solutions of Na2S2O4 and NaHCO3

    Mix 50% (v/v) Na2S2O4 and 50% (v/v) NaHCO3

    Note: Prepare fresh

  8. 1% (w/v) sulfanilamide solution

    Dissolve in 3 M HCl

    Note: Prepare fresh and store at 4°C in the dark

  9. 0.01% (w/v) NED solution

    Dissolve in deionized water

    Note: Prepare fresh and store at 4°C in the dark

Acknowledgments

This protocol was adapted from a previously published paper by Tan et al. (2020). This research was supported by the National Natural Science Foundation of China (31430004, 31830002, 81671988, 91751000, 91951000 to G.-P. Z.), the Research Unit Fund of Li Ka Shing Institute of Health Sciences (7103506 to G.-P. Z.), and the Hong Kong Health and Medical Research Fund (12110622 to G.-P. Z.).

Competing interests

The authors declare that they have no conflicts of interest.

References

  1. Kuypers, M. M. M., Marchant, H. K., and Kartal, B. (2018). The microbial nitrogen-cycling network. Nat Rev Microbiol 16(5): 263-276.
  2. Morozkina, E. V., and Zvyagilskaya, R. A. (2007). Nitrate reductases: structure, functions, and effect of stress factors. Biochemistry (Mosc) 72(10): 1151-1160.
  3. Tan, W., Liao, T. H., Wang, J., Ye, Y., Wei, Y. C., Zhou, H. K., Xiao, Y., Zhi, X. Y., Shao, Z. H., Lyu, L. D. and Zhao, G. P. (2020). A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation. J Biol Chem 295(15): 5051-5066

简介

[摘要]硝酸盐是微生物主要的无机氮源之一。许多细菌和古菌谱系可以表达细胞质同化硝酸盐还原酶 (NAS),其催化硝酸盐同化途径中硝酸盐到亚硝酸盐的限速还原。在这里,我们提出了一个详细的协议,用于测量体外硝酸还原酶(NAR)的NAS活性酶从Mycolicibacterium耻垢使用生理和非生理的电子供体的粗提取物。

[【背景】硝酸盐(NO 3 - )除了是植物的重要氮源外,由于竞争激烈,普遍用作微生物的无机氮源,尤其是放线菌等土壤细菌,以及蓝藻等海洋细菌。自然环境中有限的营养成分(Kuypers等人,2018 年)。在原核生物中,硝酸盐通过两个连续步骤被还原为氨 (NH 3 ),其中第一个关键步骤由 NAS 催化。原核NAS的是基于生理上归类使用电子给体(Morozkina和Zvyagilskaya,2007;谈。等人,2020) ,我。电子。, 黄素氧还蛋白或铁氧还蛋白依赖性 NAS 和 NAD(P)H 依赖性 NAS ; ħ H但是,迄今为止,已经有一些报道检测体外细菌NAD的NAR活性(P)H依赖性的NAS的使用它们的生理电子供体。相反,甲基紫精(人工化学还原剂)是最常见的LY使用的电子给体为体外NAR活性测定。

在该协议中,采用优化的检测系统,可以在耻垢分枝杆菌粗细胞提取物中测量 NAD(P)H 依赖的 NaR 活性。简而言之,在厌氧条件下制备的酶样品可以使用 NADPH 和 NADH 作为电子供体进行体外催化。硝酸盐还原产生的亚硝酸盐与磺胺和 N-(1-萘基)乙二胺二盐酸盐形成有色偶氮化合物,可通过其在 540 nm 处的吸光度进行测量。该方法适用于细菌NAS酶体外NaR活性的测定。

关键字:耻垢分枝杆菌, 同化硝酸还原酶, 体外硝酸还原酶活性测定, NADPH, NADH, 甲基紫精


材料和试剂

1.耻垢分枝杆菌mc 2 155     
2. Middlebrook 7H10 琼脂(BD Biosciences,目录号:262710)     
3. Middlebrook 7H9 肉汤(BD Biosciences,目录号:271310)     
4.甘油(Sigma-Aldrich,目录号:G5516)     
5.吐温80(Sigma-Aldrich,目录号:P1754)     
6.乙醇(Sigma-Aldrich,目录号:51976)     
7.苯甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)     
8. Bradford 蛋白质测定试剂盒(Beyotime,目录号:P0006)     
9.硝酸钠(NaNO 3 )(Sigma-Aldrich,目录号:S5506)     
10.亚硝酸钠(NaNO 2 )(Sigma-Aldrich,目录号:S2252) 
11.磷酸氢二钠(Na 2 HPO 4 )(Sigma-Aldrich,目录号:255793) 
12.磷酸二氢钠(NaH 2 PO 4 )(Sigma-Aldrich,目录号:S3139) 
13.烟酰胺腺嘌呤二核苷酸磷酸(NADPH)(Sigma-Aldrich,目录号:N7505) 
14.烟酰胺腺嘌呤二核苷酸(NADH)(Sigma-Aldrich,目录号:43420) 
15.甲基紫精(Sigma-Aldrich,目录号:856177) 
16.黄素单核苷酸(FMN)(Sigma-Aldrich,目录号:F2253) 
17.黄素腺嘌呤二核苷酸(FAD)(Sigma-Aldrich,目录号:F6625) 
18.连二亚硫酸钠(Na 2 S 2 O 4 )(Sigma-Aldrich,目录号:71699) 
19.碳酸氢钠(NaHCO 3 )(Sigma-Aldrich,目录号:S6014) 
20.磺胺(Sigma-Aldrich,目录号:S9251) 
21. N-(1-萘基)乙二胺二盐酸盐(NED)(Sigma-Aldrich,目录号:222488) 
22. 7H10 琼脂(见食谱) 
23. 7H9 培养基(见配方) 
24. MPLN 培养基(见食谱) 
25. 0.5M的小号裂果磷酸盐缓冲液,pH 7.5(参见食谱) 
26. 0.1M的小号裂果磷酸盐缓冲液,pH 7.5(参见食谱) 
27. 0.1 M PMSF(见配方) 
28. 60 mM Na 2 S 2 O 4 /NaHCO 3 (见配方) 
29. 1% (w/v) s ulfanilamide 溶液(见配方) 
30. 0.01% (w/v) NED 溶液(见配方) 

设备

移液器套件(Gilson,目录号:F167300)
高速离心机(Beckman,目录号:AVANTI JXN-26)
超声波细胞破碎仪(Beyotime,目录号:E0385)
配备脱气模式的超声波浴(Beyotime,目录号:E0439)
250 毫升和 2 升锥形烧瓶
培养皿(直径 90 毫米)
37℃培养箱
孵化器摇床
水浴
分光光度计
充满混合气体(氮气,9 5 %;氢气,5%)的厌氧室(手套箱)
注:将吸管工具包,超声波细胞破碎,和厌氧室内水浴。

软件

微软Excel

程序

M 的制备。耻垢文化
条纹中号。耻垢上冷冻培养一个7H10琼脂平板上,并培育需氧在37 ℃下进行3个d AYS直至菌落是可见的。
将单个菌落接种到 250 毫升锥形瓶中的 50 毫升 7H9 培养基中,并在 37 °C下有氧孵育16 小时,以 180 rpm振荡(过夜培养)。
通过以 3,000 × g离心10 分钟从过夜培养物中收集细胞。用无氮 MPLN 培养基和离心机如上所述洗涤沉淀物两次。将细胞重悬在50 ml MPLN培养基中作为接种培养物。
再接种10毫升接种培养到1L MPLN介质supplem在2用10mM硝酸盐ented -升锥形烧瓶中,并温育需氧条件下于37 ℃下振荡培养至晚对数期(OD 30小时600 0.8 - 1.0)。

M 的制备。smeg matis粗提物
在 4 °C 下以 5,000 × g离心10 分钟收集细胞。
用30ml洗涤细胞沉淀两次脱气的0.1M小号ö dium磷酸盐缓冲液(pH 7.5)。
转移的细胞沉淀进入厌氧室,重悬在20毫升脱气的0.1M小号补充有10%v / v甘油和1mM PMSF裂果磷酸盐缓冲液(pH 7.5)。
通过超声处理(脉冲:0.3 秒,停止:0.7 秒)在冰上破坏细胞 10 分钟,并将裂解物转移到厌氧室内的离心管中。
在厌氧室外在 4 °C 下以 16,000 × g离心1 小时。
将离心管带回厌氧室,收集上清液(细胞质部分)作为粗酶提取物。
注意:在测定过程中保持在冰上,并在 24 小时内使用。
确定的总蛋白浓度的细胞质˚F raction使用Bradford方法。

的制备将亚硝酸盐标准曲线
在去离子水中使用亚硝酸钠 (0、3、30、60、120、240 和 300 μM)制备亚硝酸盐稀释系列。 
吸管0.5毫升亚硝酸盐稀释系列(试验样品)到1.5毫升试管小号。使用 0.5 ml 10 mM硝酸钠作为对照(空白样品)。  
向所有管中加入 0.5 ml s ulfanilamide 溶液和 0.5 ml L NED 溶液。
通过旋转混合并在 25 °C下孵育10 分钟。
将 1 ml测定混合物转移到比色皿中。
使用分光光度计测量540 nm 处的可见吸收。
使用以下公式计算吸光度变化:Δ A 540 nm (样品)= A 540 nm (测试)- A 540 nm (空白) 
绘制与亚硝酸盐浓度的标准曲线上的水平轴和吸光度变化上的垂直轴(图1)。

C:\Users\Madandan\Desktop\2003763--1861 Wei Tan 884996--合作期刊The Journal of Biological Chemistry\Figs tif\图1.tif
图 1. 亚硝酸盐标准曲线。误差棒表示三个生物学重复的平均值 ± SD (n = 3)。根据标准曲线,回归方程为 x (µM) = (y-0.0204)/0.0092,其中 x 代表亚硝酸盐浓度,y 代表Δ A 540 nm (样品)。

体外NaR 活性测定
用于制备反应混合物之前将酶测定,脱气在测定中和对所用的溶液花边在使用前厌氧室中。
移液管以下的试剂的还原硝酸盐反应(表小号1和2)进入该厌氧室内部的1.5毫升试管中:

表 1.测定NAD(P)H 依赖的 NaR 活性的测定混合物

化验样品

测试
空白的
去离子水
0.090 毫升
0.100 毫升
磷酸钠缓冲液 (0.5 M)
0.100 毫升
0.100 毫升
NaNO 3 (50 mM)
0.100 毫升
0.100 毫升
NADPH (2 mM)或 NADH (2 mM)
0.100 毫升
0.100 毫升
FMN (0.25 毫米)
0.050 毫升
0.050 毫升
时尚 (0.25 毫米)
0.050 毫升
0.050 毫升
粗酶提取物
0.010 毫升
——
步骤1:轻轻混匀,在厌氧室内30 ° C孵育10分钟
1% (w/v) s磺胺溶液
0.500 毫升
0.500 毫升
0.01% (w/v) NED 溶液
0.500 毫升
0.500 毫升
步骤2:轻轻混匀,在25孵育℃下在黑暗中厌氧室内部10分钟

表 2.用于确定甲基紫精依赖性 NaR 活性的测定混合物

化验样品

测试
空白的
去离子水
0.090 毫升
0.100 毫升
磷酸钠缓冲液 (0.5 M)
0.100 毫升
0.100 毫升
NaNO 3 (50 mM)
0.100 毫升
0.100 毫升
甲基紫精 (0.75 mM)
0.100 毫升
0.100 毫升
Na 2 S 2 O 4 /NaHCO 3 (60 mM)
0.100 毫升
0.100 毫升
粗酶提取物
0.010 毫升
——
第 1 步:轻轻混匀并在厌氧室内30 °C孵育10 分钟
步骤 2:在厌氧室外的露天环境中剧烈搅拌,直至蓝色消失,停止反应
1% (w/v) s磺胺溶液
0.500 毫升
0.500 毫升
0.01% (w/v) NED 溶液
0.500 毫升
0.500 毫升
步骤3:轻轻混匀,在25孵育℃下在黑暗中厌氧室外部10分钟
注意:加入 Na 2 S 2 O 4 /NaHCO 3 后,测定混合物变为深蓝色,表明甲基紫精被减少。

将1 ml测定混合物转移到比色皿中。
测量测试样品和空白样品在 540 nm 处的可见吸收。
使用以下等式计算NaR 比活度:

NaR比活(nmol/min/mg)=C亚硝酸盐DF/T/C酶/V酶

w ^这里,
C亚硝酸盐(µM) = [ A 540 nm (Test) - A 540 nm (Blank)-0.0204]/0.0092,测定中形成的亚硝酸盐浓度
DF:牛逼,他稀释倍数
T(分):Ť他的反应时间用于测定硝酸盐还原
注意:10分钟的n孵育时间足以测量体外硝酸还原酶活性。
Ç酶(毫克/毫升):Ť他的蛋白质浓度的粗酶提取
V酶(毫升):Ť他的体积的在测定中使用粗酶提取

食谱

7H10琼脂
将19 g 7H10粉末溶解在 1 L 去离子水中
添加 4 ml 50% (v/v) 甘油和 5 ml 10% (v/v) Tween 80
在 121 °C 下高压灭菌15 分钟
7H9 中号
将 4.7 g 7H9 粉末溶解在 1 L 去离子水中
添加 4 ml 50% (v/v) 甘油和 5 ml 10% (v/v) Tween 80
在 121 °C 下高压灭菌15 分钟
无氮 MPLN 培养基( pH 6.6 )
36.7 mM KH 2 PO 4
7.7 mM 柠檬酸钠
5 mM 硫酸镁4
60 µM FeCl 3
20 µM NaMoO 4
3.4 µM 氯化钙2
3.5 µM 硫酸锌4
4 µM 硫酸铜4
2% (v/v) 甘油
0.05% (v/v) 吐温 80
在 121 °C 下高压灭菌15 分钟。
0.5M的小号裂果磷酸盐缓冲液(pH值7.5 0 )
准备 0.5 M 的 NaH 2 PO 4和 Na 2 HPO 4溶液
混合 20% (v/v) NaH 2 PO 4和 80% (v/v) Na 2 HPO 4
将pH 值调整为 7.5
0.1M的小号裂果磷酸盐缓冲液(pH值7.5 0 )
用去离子水以 1:5 的比例稀释 0.5 M 磷酸钠缓冲液
在室温下储存
0.1 M PMSF
将 0.174 g PMSF 溶解在10 ml 乙醇中
储存在-20 °C
60 mM Na 2 S 2 O 4 /NaHCO 3
准备 120 mM Na 2 S 2 O 4和 NaHCO 3溶液
混合 50% (v/v) Na 2 S 2 O 4和 50% (v/v) NaHCO 3
注意:准备新鲜
1% (w/v) s磺胺溶液
溶于 3 M HCl
注意:准备新鲜并储存在 4 °C的黑暗中
0.01% (w/v) NED 溶液
溶于去离子水中
注意:准备新鲜并储存在 4 °C的黑暗中

致谢

该协议改编自 Tan等人先前发表的论文。(2020)。本研究得到国家自然科学基金(31430004、31830002、81671988、91751000、91951000 to G.-PZ)、李嘉诚健康科学研究所研究单位基金(7103506 to G.-PZ)的支持和香港健康与医学研究基金 (12110622 to G.-PZ)。

利益争夺

作者宣称,他们有没有冲突小号的兴趣。

参考

Kuypers, MMM, Marchant, HK 和 Kartal, B. (2018)。微生物氮循环网络。Nat Rev Microbiol 16 (5) : 263-276。
Morozkina, EV 和 Zvyagilskaya, RA (2007)。硝酸还原酶:压力因素的结构、功能和影响。生物化学 (Mosc) 72(10) :1151-1160。
Tan, W., Liao, TH, Wang, J., Ye, Y., Wei, YC, Zhou, HK, Xiao, Y., Zhi, XY, Shao, ZH, Lyu, LD和Zhao, GP (2020) . 最近进化的含双黄素的单体硝酸还原酶负责高效的细菌硝酸盐同化。J Biol Chem 295(15) :5051-5066。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tan, W., Shao, Z. and Zhao, G. (2021). In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract. Bio-protocol 11(14): e4098. DOI: 10.21769/BioProtoc.4098.
  2. Tan, W., Liao, T. H., Wang, J., Ye, Y., Wei, Y. C., Zhou, H. K., Xiao, Y., Zhi, X. Y., Shao, Z. H., Lyu, L. D. and Zhao, G. P. (2020). A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation. J Biol Chem 295(15): 5051-5066
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。