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Assays for the Detection of Rubber Oxygenase Activities
橡胶加氧酶活性测定实验   

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参见作者原研究论文

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BMC Microbiology
Jun 2016

Abstract

Microbial biodegradation of rubber relies on extracellular rubber oxygenases that catalyze the oxidative cleavage of the double bond of the polyisoprene backbone into oligo-isoprenoids. This protocol describes the determination of rubber oxygenase activities by an online measurement of molecular oxygen consumption via a non-invasive fluorescence-based assay. The produced oligo-isoprenoid cleavage products with terminal keto- and aldehyde-groups are identified qualitatively and quantitatively by HPLC. Our method allows for the characterization of homologue rubber oxygenases, and can likely be adapted to assay other oxygenases consuming dioxygen. Here we describe the determination of rubber oxygenase activities at the examples of the so far two known types of rubber oxygenases, namely rubber oxygenase A (RoxA) and latex clearing protein (Lcp).

Keywords: Latex clearing protein (Lcp) (乳胶清除蛋白(Lcp)), Rubber oxygenase (橡胶加氧酶), Dioxygenase (双加氧酶), Polyisoprene (聚异戊二烯), Rubber (橡胶), Oxygen monitoring (氧监测)

Background

Oxygen is a key element in aerobic metabolism and is essential for the catabolism of hydrocarbons. Therefore the quick and accurate measurement of oxygen concentrations in solutions is of interest to monitor oxygen-dependent processes in biotechnology or reactions in biochemistry. Usually a Clark electrode is employed for these purposes. In many cases, however, the use of a Clark electrode is limited due to the necessity of a direct contact of the electrode with the analyte. Some examples comprise turbid solutions in monitoring fermentation processes or complex matrices like colloid latex emulsions as investigated by our research group. Another important aspect distinguishing this protocol from other oxygen detection assays is the very small amount of only 500 µl sample volume required for the in vitro assay. This protocol allows for the rapid and reproducible determination of the oxygen concentration of latex emulsions but likely is transferable to many other applications. In this non-invasive assay, a small sensor spot with a diameter of only ~4 mm is placed in the reaction vessel (cuvette) and comes into contact with the analyte. The sensor spot is excited by light that is emitted by the transmitter unit and guided to the sensor spot via a light conducting cable. The emitted fluorescence light is quenched by dioxygen and the signal intensity is proportional to the concentration of dioxygen. Since oxygen is the co-substrate of polyisoprene cleavage by rubber oxygenases, the activity of the enzymes can be calculated by determination of the oxygen consumption. The second assay describes the extraction of enzyme-produced oligo-isoprenoids with ethyl acetate and their qualitative and quantitative determination by high pressure liquid chromatography (HPLC). For information on the biochemical and molecular biological properties of rubber oxygenases we refer to the following references for RoxA enzymes (Braaz et al., 2004 and 2005; Schmitt et al., 2010; Birke et al., 2012 and 2013; Seidel et al., 2013) and for Lcp (Hiessl et al., 2014; Birke and Jendrossek, 2014; Birke et al., 2015; Watcharakul et al., 2016; Röther et al., 2016).

Materials and Reagents

  1. Duct tape (e.g., Tesa, extra Power universal)
  2. 50 ml Falcon tubes (e.g., SARSTEDT, catalog number: 62.559.001 )
  3. 15 ml Falcon tubes (e.g., SARSTEDT, catalog number: 62.554.502 )
  4. 2 ml reaction tubes (e.g., SARSTEDT, catalog number: 72.695.500 )
  5. 0.3 ml limited volume inserts (e.g., Brown, catalog number: 155650 )
  6. Hot glue
  7. Silicone Compound (e.g., RS Components, catalog number: 692-542 )
  8. 1.5 ml tubes (e.g., SARSTEDT, catalog number: 72.690.001 )
  9. Protein determination assay (e.g., Pierce BCA Kit, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
  10. Latex milk, 60%, low ammonia (e.g., Weber&Schaer, Hamburg, catalog number: RSS LA )
  11. Purified rubber oxygenases, Lcp or RoxA of known concentration
  12. Methanol for HPLC (e.g., VWR, catalog number: 20864.320 )
  13. Ethyl acetate (e.g., Carl Roth, catalog number: 7338 )
  14. Nitrogen gas
  15. Monopotassium phosphate (KH2PO4) (e.g., Carl Roth, catalog number: 3904 )
  16. Dipotassium phosphate (K2HPO4) (e.g., Carl Roth, catalog number: 6875 )
  17. Nonidet P-40 (e.g., Sigma-Aldrich, catalog number: 74385 )
  18. Sodium phosphate buffer (KP), e.g., 100 mM, pH 7 (see Recipes)
  19. Nonidet P-40 solution (see Recipes)
  20. Natural rubber latex (see Recipes)
  21. Diluted colloidal latex buffer (see Recipes)

Equipment

  1. Oxy-4-mini transmitter (PreSens, catalog number: 200000767 )
  2. Sharp scissors
  3. Absorption cells (cuvettes), semi-micro, light path 10 mm, volume 1,400 µl, optical special glass, 4 pieces (e.g., Hellma analytics, catalog number: 114-10-20 )
  4. Cuvette rack 16 x 1 cm (e.g., Carl Roth, catalog number: CNP5.1 )
  5. Oxygen Sensor Spots, SP-PSt3-NAU-D5-YOP (PreSens, catalog number: 200000023 )
  6. 3 mm power drill
  7. Light conducting cable (2.5 m, 4 pieces, POF-L2.5-1SMA) (PreSens, catalog number: 200000241 ; designated in the manual as ‘polymer optical fiber’)
  8. Cryo storage box (e.g., Greiner Bio one International, catalog number: 802 202 )
  9. Magnetic micro stirrer 5 x 2 mm, 4 pieces (e.g., Carl Roth, catalog number: 0955.2 )
  10. Centrifuge (e.g., Eppendorf, model: 5417 C)
  11. Autoclave
  12. Heat-stable container
  13. Pipette  
  14. Beaker
  15. HPLC (e.g., Agilent Technologies, model: Agilent 1100 series; DAAD detector at 210 nm)
  16. LiChrospher 100 RP-8 5 µm endcapped HPLC column (e.g., Trentec, catalog number: 124LP-85ER used in this assay; or alternatively EMD Millipore, catalog number: 150827 )
  17. HPLC glass vial (e.g., Brown, catalog number: 155710 )
  18. Fume cabinet
  19. Laboratory thermometer (e.g., VOLTCRAFT, model: DET1R )

Software

  1. Oxy4 V2 (Presens, Regensburg, Germany)
  2. Excel (Microsoft, Redmont, USA)
  3. Chem Station for LC 3D systems (Agilent Technologies, Waldbronn, Germany)

Procedure

Note: Ensure that all safety instructions for the handling of hazardous compounds and for waste management are properly considered; since these may vary in different countries the following protocol does not provide any instruction on these issues.

Part I. Oxygen consumption assay

In solutions, the oxygen concentration can be monitored in real time and allows for the biochemical characterization of oxygenases. Here, we describe an assay to analyze the oxidative cleavage of rubber (polyisoprene) by rubber oxygenases such as rubber oxygenase RoxA or latex clearing protein (Lcp).

  1. Preparations
    1. Prepare the oxygen monitoring instrument according to the instructions supplied by the respective manufacturer. (e.g., PreSens Oxy-4-mini, https://www.presens.de/products/o2/meters.html)
    2. The sensor spots are excited by light and therefore should be kept in a dark environment for long time storage when not in use, otherwise their reactivity and sensitivity will decrease.
    3. Cut the sensor spot in shape using sharp scissors. Don’t cut away too much to ensure a surface as big as possible but still fitting into the cuvette, here ~4 x 5 mm. Use long, slender tongs to reach into the cuvette.
    4. Pay attention on the orientation of the sensor spot, in case of PreSens spots, the red side comes in contact with the silicon compound and the glass wall of the cuvette whereas the black side represents the inside and comes in contact with the reaction mix later. Be careful not to put silicone compound on the black, reactive side of the sensor spot.
    5. Glue one sensor spot to the inner surface of each cuvette 5 mm above the bottom. Use the specified silicone compound as instructed by the manufacturer. For a video see: http://www.presens.de/support/presens-tv/how-to/o2-spot-integration-part1-glassvials.html. Dry over night at room temperature in the dark. Prepare four cuvettes, compare Figure 1.


      Figure 1. Oxygen monitoring cuvettes. The total volume per cuvette is 1.4 ml. A PreSens oxygen sensor spot (red) was cut and glued in place as described above.

    6. To align the light cable with the sensor spots in the cuvettes, a self-made, modified cuvette rack is employed that is not commercially available. Instruction for assembly:
      1. Cut the 16 slot cuvette rack in the middle (to give two equal sized halves).
      2. Seal the bottoms of the four front slots with duct tape.
      3. Fill the cavity completely with hot glue, wait until the glue is solid.
      4. Cut away the bottoms of the four remaining slots (see Figure 2) so that the cuvettes fit in completely.
      5. Use a 3 mm power drill to drill a hole from the front of the cuvette rack to the back.
        Note: Be very careful not to bend the light conducting cable, otherwise it might break and become dysfunctional.
      6. Insert the light conducting cable so that the interface of the cable is correctly aligned with the sensor spot in the cuvette, compare Figure 2.
      7. Secure the light conducting cable against mechanical stress with a drop of hot glue to the cuvette rack.
      8. Repeat this procedure to prepare four slots.
      9. To finish the apparatus, glue the prepared block on the lid of a cryo storage box as a strain relief for the light conducting cable.
      10. Now your device should look comparable to the one pictured in Figure 2.
      11. Upon completion, position the device on a magnetic stirring unit and set it to ~750 rpm to mix the reagent solutions with a magnetic micro stirrer (5 x 2 mm) in each cuvette.


        Figure 2. Images of the finished cuvette holder used to align the sensor spots glued into the cuvettes with  the light conducting cable. The apparatus is placed on a magnetic stirring device to mix the reaction.

        This simple and cheap measuring station has been set up because, to our knowledge, no small scale measuring vessels are available; furthermore, in protein assays often only a very small amount of sample is available and our set up saves reagents and enzymes. Alternatively, one might employ a poly-carbonate block and mill the cavities into the block or to use a 3D-printer to assemble a cuvette holder. The assembly can easily be adjusted for another number of cuvettes. Other variants and sizes of cuvettes can be employed for the assay as well.

  2. Measurement of oxygen consumption
    1. Measurement of protein concentration
      1. Determine the protein concentration of the oxygenase to be characterized in this assay exactly by a suitable method, e.g., using the Bradford assay, the BCA assay, or, as in our case, spectroscopically via the known specific extinction coefficient of the heme containing rubber oxygenases.
        For example: spectroscopic determination of a LcpK30 protein concentration c
        E412 = ε412 x c x d or c = E412 x ε412-1 x d-1
        Where,
        ε412: extinction coefficient of LcpK30, 80,000 M-1 x cm-1 [L mol-1 cm-1] at 412 nm
        E412: absorbance measured spectroscopically at 412 nm [no unit]
        d: path length of the cuvette, usually 1 cm
        LcpK30 molecular weight ~43,300 Da = 43,300 g x mol-1
      2. Experimental data: measure the extinction of your protein of interest with a photometer in a cuvette with a path length of 1 cm in a suitable dilution; in the following example a value for the extinction at 412 nm of 0.5 was recorded for a 1:10 dilution of a purified LcpK30 solution.
        E412 = 0.5; dilution = 1:10, path length = 1 cm.
      3. Calculation:
        c = 0.5/80,000 x 1 [M x cm/cm] = [M]
        c = 0.5/80 [mM] = 500/80 [µM] = 6.25 µM
        1 M LcpK30 = 43,300 g/L
        1 µM LcpK30 = 0.0433 g/L = 43.3 mg/L
        6.25 µM LcpK30 = 271.6 mg/L, since the protein was 1:10 diluted the concentration of the undiluted protein solution is 2716 mg/L or ~2.7 mg/ml
    2. Washing of latex milk
      1. Stock latex milk containing approximately 60% polyisoprene latex is usually stabilized with ammonia that has to be removed by washing.
      2. Prepare 200 ml 0.1% wt/vol Nonidet P-40 by solving the detergent in deionized water.
      3. Pour 25 ml stock latex milk into a 50 ml Falcon tube and add 25 ml Nonidet solution.
      4. Invert the tube 10 times. Do not vortex or shake to prevent coagulation of the polymer.
      5. Centrifuge the tube at 11,000 x g at 4 °C for 1 h to separate the latex from the solution.
      6. Remove the white latex on top with a spatula into a new 50 ml tube.
      7. Discard the washing solution.
      8. Add Nonidet solution to the latex up to 50 ml.
      9. Resuspend the latex by inverting the tube several times.
      10. Repeat the washing procedure three times.
      11. After the last centrifugation step, weigh the remaining latex and dilute it to a concentration of 60% wt/vol with Nonidet solution.
      12. The washed natural rubber latex can be autoclaved (liquid cycle 121 °C for 20 min) in a heat-stable container and stored at ~5 °C for approximately 6 months.
    3. Preparation of diluted colloidal latex buffer
      1. For use in the oxygen consumption assay, dilute the washed natural rubber latex to 0.2% wt/vol with air-saturated potassium phosphate buffer (KP, see Recipes) at the desired pH, usually pH 7.
        For example, pipette 6 ml KP buffer into a 15 ml Falcon tube and add 20 µl of the washed natural rubber latex in Nonidet solution, invert the tube 5 times.
      2. Incubate the buffer-diluted latex emulsion until the desired temperature (e.g., room temperature, 23 °C) is constantly reached; alternatively the whole system can be placed in a room with an elevated temperature, e.g., 30 °C). This is important since oxygen saturation depends on the temperature.
    4. Prearrangements for measuring oxygen saturation.
      1. Pipette 500 µl freshly prepared diluted colloidal latex buffer into each cuvette.
      2. Add a magnetic stirring rod to mix the emulsion at 750 rpm.
      3. Equilibrate for 2 min.
    5. Operation of the software
      1. Start the program for monitoring, in our case we use ‘oxy4v2’ for the measurement of the dissolved oxygen concentration in the cuvettes via the transmitter oxy4-mini.
    6. Calibration
      1. Select the tab labeled ‘Calibration’.
      2. Press the button labeled ‘All Channels’.
        Calibrate the diluted colloidal latex buffer to 100% saturation by pressing the button ‘CAL 100 %’. This corresponds to an oxygen concentration of ~8.3 mg per L at 23 °C. The slight difference in oxygen concentration between distilled water and the buffered assay emulsion used in this study is negligible. A second calibration with 0% oxygen is not necessary.
    7. Data logging
      1. Select the tab labeled ‘logging’.
      2. Press the button labeled ‘All Active Channels’.
      3. Pick a distinct file storage location and file name, e.g., ‘date_1Lcp_2Lcp_3blank_4blank_pH_temperature’.
      4. Press the button ‘save’.
    8. Setup for measurements
      1. Select the tab labeled ‘Measurement’.
      2. Pick a suitable sampling rate, e.g., 15 sec, equal for all cuvettes.
      3. Select 23 °C as the channel temperature when measuring at 23 °C room temperature.
      4. Choose ‘% air saturation’ as the oxygen unit.
      5. Press the button ‘All channels’ to start logging of acquired measurement data.
      6. Pay attention that the amplitude is in the green range, indicating that the light conducting cable and the sensor spot in the cuvette are properly aligned.
    9. Execution of the measurement
      1. Select the tab labeled ‘All Channels’ to view the saturation graph for all channels.
      2. Measure the baseline for up to 10 min until linearity of the oxygen saturation has been reached. The value should be at ~100 ± 2% for all channels.
      3. Add the desired amount of enzyme to the assay mixture in each cuvette with a pipette or Hamilton syringe. Usually, a concentration of 4 µg/ml rubber oxygenase is sufficient to achieve a decrease of the oxygen concentration with a measurable slope in ~10 min.
        For example: enzyme in the assay/protein concentration x assay volume = amount:
        4 µg/ml/0.25 µg/µl x 0.5 ml = 8 µl.
        Note: The decrease in oxygen concentration should be linear for at least 5 min. For routine assays, the cuvettes do not need to be sealed because oxygen consumption by the enzyme is much faster than diffusion of oxygen into the assay mixture.
      4. Stop the measurement by selecting the tab ‘Measurement’ and press the button ‘All Channels’. A representative dataset with a blank without enzyme (blue), wild type Lcp as reference (black) and two mutein samples (red and green) is pictured in Figure 3. Data was also shown in the supplementary materials of Röther et al., 2016.
    10. Termination and cleaning of the device
      1. Carefully remove each cuvette from the rack and pour the assay solution into a beaker to avoid loss of the magnetic stirrer. Wash the cuvette and stirrer with deionized water 3 times and dry the cuvettes in a dark environment.
      2. After prolonged measurements with diluted colloidal latex buffer, some precipitation of latex in the cuvettes might occur. The residual latex can be removed with a 2 mm spatula from the glass. Pay attention to the sensor spot to avoid damage. Do not use solvents to clean the inside of the cuvettes since they might dissolve the silicone compound securing the sensor spot in place. The spots can be reused as long as the amplitude is still indicated as sufficient by the software. If necessary, the sensor spot can be removed with a spatula and remains of the silicone compound can be removed with acetone. After drying the cuvette, a new sensor spot can be glued into the cuvette as described above.
      3. From the logged saturation data files, the slope of the decrease can be calculated using e.g., Excel as shown in the data analysis section. For data analysis and determination of specific enzyme activities, see below. Figure 3 shows a representative dataset for a measurement with four cuvettes.
         

        Figure 3. A representative dataset showing the monitoring of the oxygen saturation of diluted colloidal latex buffer. A blank without enzyme (blue) represents a baseline. After ≈ 4 min, wild type Lcp as reference (black) and two mutein samples (red and green) were added separately. The data are also shown in the supplementary materials of Röther et al., 2016.

  3. Inhibitor studies
    This assay allows for the rapid and easy determination of enzyme activities depending on the measurement of oxygen saturation. It is possible to compare different enzymes, muteins, or to investigate the effect of potential inhibitors. As an example, 0.1% SDS inhibits oxygen consumption by Lcp.

Part II. HPLC based separation of rubber cleavage products

After the oxidative polyisoprene cleavage occurred, the produced oligo-isoprenoids can be extracted from the reaction mixture and subsequently separated by HPLC for analysis as described below.

  1. Preparations
    1. Set up the HPLC apparatus according to the instructions supplied by the manufacturer and install the RP-8 reversed phase column.
    2. Degas deionized water.
    3. Supply the water as first mobile phase and methanol as the second.
    4. Program a flow rate of 0.7 ml/min at 23 °C.
    5. Program the injection volume of 20 µl sample per run.
    6. Program the solvent gradient:
      Minute
      Water (%)
      Methanol (%)

      0-5
      50
      50
      isocratic
      5-25
      0
      100
      gradient
      25-35
      0
      100
      isocratic
      35-36
      50
      50
      gradient
      36-40
      50
      50
      isocratic

  2. Oxidative polyisoprene cleavage assay
    1. Label 2 ml reaction tubes and place them in a rack.
    2. Pipette 700 µl diluted colloidal latex buffer (0.2%, see Part I, step B3) into each tube and adjust the temperature.
    3. If applicable, add the inhibitors at desired concentrations to the solution.
    4. Add the designated amount of enzyme, usually the final concentration is 4 µg/ml.
    5. Incubate the mixture with the lid open at the desired temperature, usually for 2 h for polyisoprene cleavage, shake softly every 30 min.
    6. Always prepare a blank without enzyme and treat it equally.

  3. Extraction of oligo-isoprenoids
    1. Work in a fume cabinet without ignition sources nearby when using ethyl acetate or methanol.
    2. Add 1,000 µl ethyl acetate to each tube containing 700 µl reaction volume.
    3. Close the lid and vortex the tube for 20 sec, white latex will coagulate.
    4. Centrifuge all tubes for 2 min at 20,000 x g at room temperature.
    5. Label 1.5 ml tubes according to the assay and remove 900 µl of the upper solvent phase carefully to avoid mixing and spillage. Discard the 2 ml tubes.
    6. Evaporate the ethyl acetate completely overnight in the fume cabinet or enhance evaporation by flushing carefully with nitrogen gas at ~0.2 L/min.
    7. Add 100 µl methanol to the tube and rinse the walls of the 1.5 ml tube by pipetting the solution up and down 5 times to dissolve the cleavage products. Transfer the methanol-product solution into an HPLC glass vial supplemented with a 0.3 ml small volume insert and screw on the lid.
    8. Apply all samples to HPLC analysis.

Data analysis

Part I. Oxygen consumption assay

  1. Evaluation
    1. The measurement of the oxygen concentration is very sensitive but also error prone to several factors.
    2. Due to the temperature and pressure dependent solubility of oxygen, measurements have to be performed at a constant temperature and analyzed with respect to the pressure. Slight variations in atmospheric pressure are within the margin of error of the method, however.
    3. Prolonged practical use of the method showed that each experiment should be repeated three times with data acquired for six measurements.
    4. The derived routine assay with four cuvettes is as follows:
      1. Blank without enzyme.
      2. Wild type as reference.
      3. Mutein sample A.
      4. Mutein sample B.
      Three repetitions correspond to 6 files for the mutein and 3 baseline files as well as 3 datasets for the wild type protein as a reference, requiring statistical similarity of the samples among each other (mean ± 3 x standard deviation).

  2. Calculation of specific enzyme activities
    1. After finishing the described oxygen monitoring, a text file in the *.csv format is saved automatically for each channel separately in the specified location.
    2. It contains a summary of information about the oxymeter and the chosen parameter settings. The results of interest for this analysis are logged as a semicolon (;) separated dataset.
    3. Open the *.csv file in a suitable calculation software e.g., MS Excel.
    4. Select the first column and press the tab ‘data’. Select ‘Text in columns’. Choose ‘Separated’ and choose semicolon (;) as the separator. This should separate the first column into several columns, one for each parameter.
    5. To analyze the oxygen consumption, only two columns are of interest. The time as the x-axis and the ‘Oxy/% air sat.’ as y-axis. Extract both columns into a new spreadsheet.
    6. Repeat these steps to process the data for all channels and paste the values into the spreadsheet.
    7. In the new spreadsheet, plot a graph using the X-Y scatter chart with lines option. The plot should look like the one shown during the measurement with the oxy-4v2 software.
    8. Since we calibrate the oxygen saturation to 100% using air-saturated diluted colloidal latex buffer, the measurement of the baseline is in the range of 100% prior to enzyme addition. Thereafter, a decrease of oxygen due to enzymatic reaction is visible and we use the slope of the linear decrease to calculate the specific enzyme activity.
    9. To determine the slope via linear regression, the easiest way is to copy the data of the linear decrease into a new series in the same diagram. For this line, we calculate the slope using the ‘trend line’ feature. Specify ‘linear’ and check the boxes to show the formula and the coefficient of determination (1 indicates best fit) for the fit. The unit of the slope is % O2 min-1.
    10. At 23 °C and 1,013 mbar, 1 L air-saturated diluted colloidal latex buffer contains ~8.3 mg O2. This is equivalent to 259 µmol O2 = 100%. Therefore, a decrease in oxygen saturation by 1% corresponds to ~2.6 µmol dioxygen (2.6 µmol %-1)
    11. To calculate the molar oxygen consumption rate, multiply the calculated slope and the factor: 2.3% min-1 x 2.6 µmol %-1 ~6.0 µmol min-1. This value corresponds to a volume of 1 L and is equal to 3.0 nmol min-1 in the 0.5 ml volume of the cuvette.
    12. To determine the specific enzyme activity, divide the molar oxygen consumption rate by the enzyme quantity used for the determination of the slope, in this case 2 µg to get 1.5 nmol min-1 µg protein-1 or 1.5 µmol min-1 mg-1 (corresponding to 1.5 U/mg).
    13. The specific activity of wild type LcpK30 (Lcp from Streptomyces sp. strain K30) is 1.5 U/mg at 23 °C (Watcharakul et al., 2016) correlating with 1.3 U/mg determined for a homologue Lcp from Gordonia polyisoprenivorans strain VH2 (LcpVH2) investigated by another group (Hiessl et al., 2014).
    14. Repeat this evaluation for all measurements, the values for replicates should be within the margin of standard error.
      Note: The decrease in oxygen concentration often slows down after an initial constant decrease (Figure 4) or even slightly increases again. This can be explained by diffusion of oxygen into the (not sealed) assay buffer. In case you wish to monitor the oxygen consumption rate for a longer time period you have to seal the cuvette or you should use a larger volume of the assay mixture.


      Figure 4. Example calculation of the specific activity of LcpK30. In this experiment an assay volume of 1 ml was used. The values for time (X) and oxygen saturation (Y) are measured as described and extracted from the resulting *.csv file into a spreadsheet. The data was visualized in an X-Y scatterplot. The addition of enzyme after 12 min resulted in a decrease of the oxygen saturation due to incorporation of dioxygen during cleavage of polyisoprene. The slope can be fitted and is proportional to the enzyme activity. By multiplying the proportionality factor and dividing by the enzyme quantity used in the measurement, the specific enzyme activity can be calculated as described.

Part II. HPLC based separation of oligo-isoprenoids

After the analysis of each sample by HPLC, a report file is saved that can be evaluated using an appropriate software, in our case Agilent Chem Station.

  1. For data processing, only a simple baseline correction by subtracting a blank run without enzyme from each result is sufficient. An example of a successful analysis is pictured in Figure 5.
  2. The identification of the peaks is possible according to their respective retention time in this assay. Analysis by HPLC MS was used to identify the corresponding oligo-isoprenoid molecules in previous studies (Braaz et al., 2005; Birke and Jendrossek, 2014).
  3. Quantification is possible by calculating the peak areas and by comparison to equally treated enzymes of known activity and concentration to determine relative activities.
  4. For RoxA proteins, usually only one major degradation product (n = 2) is detected. In contrast, Lcp cleaves rubber to a variety of oligo-isoprenoids with different numbers (n) of isoprene units (Figure 5).


    Figure 5. HPLC chromatogram for Lcp (black) and RoxA (red) derived cleavage products after subtraction of a blank run without enzyme. The formula of generated oligo-isoprenoid products is shown at the top right. The number of isoprene units in the cleavage products is indicated by the value of n above the identified peaks.

Recipes

  1. Sodium phosphate (KP) buffer
    1. Prepare 100 mM solutions of KH2PO4 and K2HPO4 separately
    2. Mix both solutions to adjust the pH of the resulting KP buffer, usually pH 7.0
    3. To prepare a buffer of pH 7 mix 5 volumes of the alkaline K2HPO4 solution with 4 volumes of the acidic KH2PO4 solution
  2. Nonidet P-40 solution
    Add 0.1% wt/vol of the detergent to deionized water
  3. Natural rubber latex
    Prepare as described above (Procedure Part I, step B2)
  4. Diluted colloidal latex buffer
    Prepare as described above (Procedure Part I, step B3)

Acknowledgments

We thank the Deutsche Forschungsgemeinschaft (DFG Je152/17 and Je152/18) for funding, PreSens, Germany, Weber and Schaer, Germany and IBA Life Sciences, Germany for supplying sensor spots, polyisoprene latex and Strep-Tactin columns, respectively.

References

  1. Birke, J., Hambsch, N., Schmitt, G., Altenbuchner, J. and Jendrossek, D. (2012). Phe317 is essential for rubber oxygenase RoxA activity. Appl Environ Microbiol 78(22): 7876-7883.
  2. Birke, J. and Jendrossek, D. (2014). Rubber oxygenase and latex clearing protein cleave rubber to different products and use different cleavage mechanisms. Appl Environ Microbiol 80(16): 5012-5020.
  3. Birke, J., Röther, W. and Jendrossek, D. (2015). Latex clearing protein (Lcp) of Streptomyces sp. strain K30 is a b-type cytochrome and differs from rubber oxygenase A (RoxA) in its biophysical properties. Appl Environ Microbiol 81(11): 3793-3799.
  4. Birke, J., Röther, W., Schmitt, G. and Jendrossek, D. (2013). Functional identification of rubber oxygenase (RoxA) in soil and marine myxobacteria. Appl Environ Microbiol 79(20): 6391-6399.
  5. Braaz, R., Armbruster, W. and Jendrossek, D. (2005). Heme-dependent rubber oxygenase RoxA of Xanthomonas sp. cleaves the carbon backbone of poly(cis-1,4-Isoprene) by a dioxygenase mechanism. Appl Environ Microbiol 71(5): 2473-2478.
  6. Braaz, R., Fischer, P. and Jendrossek, D. (2004). Novel type of heme-dependent oxygenase catalyzes oxidative cleavage of rubber (poly-cis-1,4-isoprene). Appl Environ Microbiol 70(12): 7388-7395.
  7. Hiessl, S., Bose, D., Oetermann, S., Eggers, J., Pietruszka, J. and Steinbuchel, A. (2014). Latex clearing protein-an oxygenase cleaving poly(cis-1,4-isoprene) rubber at the cis double bonds. Appl Environ Microbiol 80(17): 5231-5240.
  8. Röther, W., Austen, S., Birke, J. and Jendrossek, D. (2016). Cleavage of rubber by the latex clearing protein (Lcp) of Streptomyces sp. strain K30: Molecular insights. Appl Environ Microbiol 82(22): 6593-6602.
  9. Schmitt, G., Seiffert, G., Kroneck, P. M., Braaz, R. and Jendrossek, D. (2010). Spectroscopic properties of rubber oxygenase RoxA from Xanthomonas sp., a new type of dihaem dioxygenase. Microbiology 156(Pt 8): 2537-2548.
  10. Seidel, J., Schmitt, G., Hoffmann, M., Jendrossek, D. and Einsle, O. (2013). Structure of the processive rubber oxygenase RoxA from Xanthomonas sp. Proc Natl Acad Sci U S A 110(34): 13833-13838.
  11. Watcharakul, S., Röther, W., Birke, J., Umsakul, K., Hodgson, B. and Jendrossek, D. (2016). Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp. BMC Microbiol 16: 92.

简介

橡胶的微生物生物降解依赖于细胞外橡胶加氧酶,其催化聚异戊二烯骨架的双键氧化裂解成低聚异戊二烯。该方案描述了通过基于非侵入性荧光的测定在线测量分子氧消耗来测定橡胶加氧酶活性。通过HPLC定性和定量鉴定产生具有末端酮基和醛基的寡聚异戊二烯切割产物。我们的方法允许表征同系物橡胶加氧酶,并且可能适用于测定消耗双氧的其他加氧酶。在这里,我们描述了迄今为止两种已知类型的橡胶加氧酶,即橡胶加氧酶A(RoxA)和胶乳清除蛋白(Lcp)的实例的橡胶加氧酶活性的测定。

背景 氧气是有氧代谢中的关键因素,对碳氢化合物的分解代谢至关重要。因此,在生物技术或生物化学反应中监测氧依赖性过程是非常重要的。通常,Clark电极用于这些目的。然而,在许多情况下,由于电极与分析物的直接接触的需要,Clark电极的使用受到限制。一些实例包括监测发酵过程中的混浊溶液或由我们的研究组调查的复合基质如胶体乳胶乳液。将该方案与其他氧气检测测定法区分开的另一个重要方面是体外实验所需的仅500μl样品体积的非常少的量。该方案允许快速且可重复地测定胶乳乳液的氧浓度,但可能转移到许多其它应用中。在这种非侵入性测定中,将直径仅为约4mm的小传感器斑点放置在反应容器(比色皿)中并与分析物接触。传感器点由发射器单元发射的光激发,并通过导光电缆被引导到传感器点。发射的荧光被双氧淬灭,信号强度与二氧化碳的浓度成正比。由于氧是由橡胶加氧酶裂解的聚异戊二烯的共同底物,因此可以通过测定氧消耗来计算酶的活性。第二个测定法描述了用乙酸乙酯提取酶产生的寡聚异戊二烯,并通过高压液相色谱(HPLC)定性和定量测定。关于橡胶加氧酶的生物化学和分子生物学性质的信息,我们参考以下RoxA酶的参考文献(Braaz等人,2004和2005; Schmitt等人, 2010年; Birke等人,2012和2013; Seidel等人,2013)和Lcp(Hiessl等人,2014年; Birke和Jendrossek,2014; Birke等人,2015; Watcharakul等人,2016;Röther等人,2016)。

关键字:乳胶清除蛋白(Lcp), 橡胶加氧酶, 双加氧酶, 聚异戊二烯, 橡胶, 氧监测

材料和试剂

  1. 导管胶带(例如,,Tesa,通用额外功率)
  2. 50ml Falcon管(例如,SARSTEDT,目录号:62.559.001)
  3. 15ml Falcon管(例如,,SARSTEDT,目录号:62.554.502)
  4. 2ml反应管(例如,SARSTEDT,目录号:72.695.500)
  5. 0.3ml有限体积的插入物(例如,Brown,目录号:155650)
  6. 热胶
  7. 硅酮化合物(例如,RS Components,目录号:692-542)
  8. (例如,SARSTEDT,目录号:72.690.001)
  9. 蛋白质测定法(例如,Pierce BCA试剂盒,Thermo Fisher Scientific,Thermo Scientific,商品名:23225)
  10. 乳胶乳,60%,低氨(例如,Weber& Schaer,Hamburg,目录号:RSS LA)
  11. 已知浓度的纯化橡胶加氧酶,Lcp或RoxA
  12. 用于HPLC的甲醇(例如,VWR,目录号:20864.320)
  13. 乙酸乙酯(例如,Carl Roth,目录号:7338)
  14. 氮气
  15. 磷酸二氢钾(KH 2 PO 4)(例如,Carl Roth,目录号:3904)
  16. 磷酸二钾(K 2 HPO 4)(例如,Carl Roth,目录号:6875)
  17. Nonidet P-40(例如,Sigma-Aldrich,目录号:74385)
  18. 磷酸钠缓冲液(KP),例如100mM,pH7(参见食谱)
  19. Nonidet P-40溶液(见配方)
  20. 天然橡胶乳胶(见食谱)
  21. 稀释胶体胶乳缓冲液(见配方)

设备

  1. Oxy-4迷你变送器(PreSens,目录号:200000767)
  2. 夏普剪刀
  3. 吸收细胞(比色皿),半微量,光路径10mm,体积1,400μl,光学特殊玻璃,4片(例如,Hellma分析,目录号:114-10-20)
  4. 比色杯架16×1厘米(例如,Carl Roth,目录号:CNP5.1)
  5. 氧传感器点SP-PSt3-NAU-D5-YOP(PreSens,目录号:200000023)
  6. 3 mm电钻
  7. 导光电缆(2.5m,4片,POF-L2.5-1SMA)(PreSens,目录号:200000241;手册中指定为"聚合物光纤")
  8. 冷冻储存盒(例如,,Greiner Bio one International,目录号:802 202)
  9. 磁性微型搅拌器5×2mm,4片(例如,Carl Roth,目录号:0955.2)
  10. 离心机(例如,Eppendorf,型号:5417C)
  11. 高压灭菌器
  12. 耐热容器
  13. 移液器
  14. 烧杯
  15. HPLC(例如,Agilent Technologies,型号:Agilent 1100系列; 210nm处的DAAD检测器)
  16. LiChrospher 100 RP-8 5μm封端的HPLC柱(例如,Trentec,目录号:124LP-85ER,用于该测定;或者EMD Millipore,目录号:150827)
  17. HPLC玻璃小瓶(例如,Brown,目录号:155710)
  18. 烟柜
  19. 实验室温度计(例如,,VOLTCRAFT,型号:DET1R)

软件

  1. Oxy4 V2(Presens,雷根斯堡,德国)
  2. Excel(Microsoft,Redmont,USA)
  3. 化学工作站用于LC 3D系统(Agilent Technologies,Waldbronn,德国)

程序

注意:确保妥善考虑处理危险化合物和废物管理的所有安全说明;因为这些在不同的国家可能会有所不同,以下协议不会就这些问题提供任何指导。

第一部分。氧气消耗测定

在溶液中,可以实时监测氧浓度,并允许氧化酶的生化表征。在这里,我们描述了一种分析橡胶加氧酶如橡胶加氧酶RoxA或胶乳清除蛋白(Lcp)对橡胶(聚异戊二烯)的氧化裂解的分析。

  1. 准备
    1. 根据各制造商提供的说明准备氧气监测仪器。 (例如,,PreSens Oxy-4-mini, https://www.presens.de/products/o2/meters.html
    2. 传感器点被光激发,因此在不使用时应保持在黑暗环境下长时间存放,否则反应性和灵敏度会降低。
    3. 使用锋利的剪刀切割传感器的形状。不要切割太多,以确保表面尽可能大,但仍适合比色杯,这里〜4 x 5毫米。使用长而细长的钳子进入比色皿。
    4. 注意传感器光点的方向,在PreSens光斑的情况下,红色面与硅化合物和反应杯的玻璃壁接触,而黑色侧面表示内部并与反应混合物接触。请勿将硅胶复合物置于传感器的黑色反应端
    5. 将一个传感器点胶粘到每个比色皿的内表面上方5毫米以上。按照制造商的指示使用指定的硅胶。有关视频,请参阅: http://www.presens.de/support/presens-tv/how-to/o2-spot-integration-part1-glassvials.html 。在黑暗中室温干燥过夜。准备四个比色皿,比较图1.


      图1.氧气监测比色皿。每个比色皿的总体积为1.4 ml。如上所述,将PreSens氧传感器点(红色)切割并胶合到位。

    6. 为了将光缆与传感器光点对准,使用自制的改进的比色皿支架,这是不可商购的。装配说明:
      1. 在中间切割16槽样气瓶架(给两个相等的一半)。
      2. 用胶带密封四个前槽的底部。
      3. 用热胶水填充空腔,等待胶水固定。
      4. 切掉四个剩余插槽的底部(见图2),使比色皿完全适合。
      5. 使用3毫米的电钻从比色杯架的前面向后钻一个孔 注意:注意不要弯曲导光电缆,否则可能会损坏并变得功能失调。
      6. 插入导光电缆,使电缆的接口与比色皿中的传感器光点正确对齐,比较图2.
      7. 使用一滴热胶将光导电缆抵抗机械应力固定在比色杯架上。
      8. 重复此过程准备四个插槽。
      9. 要完成设备,将准备的块粘在冷藏储藏盒的盖子上,作为导光电缆的应变消除。
      10. 现在您的设备应该与图2所示的设备相当。
      11. 完成后,将设备放置在磁力搅拌单元上,并将其设置为〜750 rpm,将试剂溶液与每个比色皿中的磁性微型搅拌器(5 x 2 mm)混合。


        图2.完成的比色皿支架的图像,用于将传感器光点与导光电缆粘合到比色皿中。将设备放置在磁力搅拌装置上以混合反应。 />
        这个简单而便宜的测量站已经建立,因为据我们所知,没有小规模的测量船可用;此外,在蛋白质测定中,通常只有非常少量的样品可用,我们的设置可节省试剂和酶。或者,可以使用聚碳酸酯块并将空腔研磨到块中或使用3D打印机来组装比色皿固定器。组装可以轻松调整另外数量的比色杯。也可以使用其他变体和大小的比色皿进行测定。

  2. 测量氧气消耗量
    1. 蛋白质浓度测定
      1. 通过合适的方法,例如使用Bradford测定法,BCA测定法,或者如我们的实例,通过已知的特异性的光谱法确定在该测定中要表征的加氧酶的蛋白质浓度含有橡胶加氧酶的血红素消光系数。
        例如:LCPK30蛋白质浓度c
        的光谱测定 412 = 412 xcxd或c = E 412 412 -1×d-1
        哪里,
        ε412范围:Lcp K30的消光系数,80,000M ×cm [L mol -1 cm -1 ]在412 nm
        在412nm处光谱测定的吸光度[无单位]
        d:比色杯的路径长度,通常为1厘米 L30 K30分子量〜43,400Da = 43,400g×mol -1
      2. 实验数据:用适当稀释度的路径长度为1厘米的比色皿中的光度计测量您感兴趣的蛋白质的灭绝;在以下实施例中,对于纯化的LCP K30 溶液的1:10稀释度记录了在412nm处的消光值为0.5。
        412 = 0.5;稀释= 1:10,路径长度= 1厘米
      3. 计算:
        c = 0.5/80,000×1 [M×cm/cm] = [M]
        c = 0.5/80 [mM] = 500/80 [μM] =6.25μM
        1 M Lcp K30 = 43,400g/L
        1μMLcp K30 = 0.0433g/L = 43.3mg/L
        6.25μMLcp K30 = 271.6mg/L,由于蛋白质为1:10稀释,未稀释的蛋白质溶液的浓度为2716mg/L或〜2.7mg/ml
    2. 洗乳胶乳
      1. 含有约60%聚异戊二烯胶乳的乳胶乳通常用必须通过洗涤除去的氨来稳定
      2. 通过在去离子水中溶解洗涤剂,制备200ml 0.1%wt/vol Nonidet P-40
      3. 将25毫升乳胶乳倒入50ml Falcon管中,加入25毫升Nonidet溶液
      4. 反转管10次。不要旋转或摇动以防止聚合物凝结。
      5. 将管以11,000 x g在4℃离心1小时以将胶乳与溶液分离。
      6. 用抹刀将顶部的白色乳胶移至新的50ml管中
      7. 丢弃洗涤液。
      8. 向乳胶添加Nonidet溶液至50 ml。
      9. 通过反转管重悬胶乳几次。
      10. 重复洗涤程序三次。
      11. 在最后一次离心步骤之后,称重剩余的胶乳,并用Nonidet溶液将其稀释至浓度为60%wt/vol。
      12. 洗涤的天然橡胶胶乳可以在热稳定的容器中进行高压灭菌(液体循环121℃,20分钟),并在约5℃下储存约6个月。
    3. 稀释胶体胶乳缓冲液的制备
      1. 为了用于氧气消耗测定,将空气饱和的磷酸钾缓冲液(KP,参见食谱)稀释至0.2%wt/vol,在所需的pH(通常为pH7)下。 例如,将6ml KP缓冲液移液到15ml Falcon管中,并在Nonidet溶液中加入20μl洗涤的天然橡胶胶乳,倒置5次。
      2. 将缓冲液稀释的胶乳乳液孵育直到达到期望的温度(例如室温,23℃);或者,整个系统可以放置在具有升高的温度(例如30℃)的房间中。这是重要的,因为氧饱和度取决于温度。
    4. 测量氧饱和度的预先设置。
      1. 将500μl新鲜制备的稀释胶体胶乳缓冲液吸入每个比色杯。
      2. 加入磁力搅拌棒,以750rpm混合乳液
      3. 平衡2分钟
    5. 操作软件
      1. 启动监控程序,在我们的例子中,我们使用"oxy4v2"通过变送器oxy4-mini测量比色皿中的溶解氧浓度。
    6. 校准
      1. 选择标有"校准"的标签。
      2. 按下标有"所有频道"的按钮。
        通过按下"CAL 100%"按钮将稀释的胶体胶乳缓冲液校准至100%饱和度。这对应于在23℃下约8.3mg/L的氧浓度。本研究中使用的蒸馏水和缓冲测定乳液之间氧浓度的微小差异可以忽略不计。不需要用0%氧气进行第二次校准。
    7. 数据记录
      1. 选择标记为"日志记录"的标签。
      2. 按下标有"所有活动频道"的按钮。
      3. 选择不同的文件存储位置和文件名称,例如。''date_1Lcp_2Lcp_3blank_4blank_pH_temperature'。
      4. 按"保存"按钮。
    8. 测量设置
      1. 选择标记为"测量"的标签。
      2. 选择合适的采样率,例如,,15秒,所有比色杯相等。
      3. 在23°C室温下测量时,选择23°C作为通道温度
      4. 选择"%空气饱和度"作为氧气单位。
      5. 按"所有通道"按钮开始记录采集的测量数据。
      6. 注意振幅在绿色范围内,表示导光电缆和比色皿中的传感器光点正确对齐。
    9. 执行测量
      1. 选择标记为"所有通道"的选项卡可查看所有通道的饱和度图。
      2. 测量基线长达10分钟,直到达到氧饱和度线性。所有通道的值应为〜100±2%。
      3. 使用移液管或汉密尔顿注射器将所需量的酶添加到每个比色皿中的测定混合物中。通常,浓度为4μg/ml的橡胶加氧酶足以在〜10分钟内以可测量的斜率实现氧浓度的降低。
        例如:酶在测定/蛋白浓度x测定体积=量:
        4μg/ml /0.25μg/μl×0.5ml =8μl 注意:氧浓度的降低应为线性至少5分钟。对于常规测定,比色皿不需要密封,因为酶的氧消耗比氧气扩散到测定混合物中要快得多。
      4. 通过选择"测量"选项来停止测量,然后按"所有通道"按钮。具有无酶(蓝色),野生型Lcp为参考(黑色)和两种突变蛋白样品(红色和绿色)的代表性数据集如图3所示。数据也显示在Röther等的补充材料中。,2016.
    10. 设备的终止和清理
      1. 小心地从架子上取出每个比色皿,并将测定溶液倒入烧杯中以避免磁力搅拌器的损失。用去离子水洗涤比色皿和搅拌器3次,并在黑暗的环境中干燥比色皿
      2. 在用稀释的胶体胶乳缓冲液进行长时间测量之后,可能会出现一些在比色皿中的胶乳沉淀。可以用玻璃上的2mm刮刀去除残留的胶乳。注意传感器点避免损坏。不要使用溶剂来清洗比色皿内部,因为它们可能会溶解固定传感器光点的硅胶。只要振幅仍被软件充分说明,这些点就可以重新使用。如果需要,传感器点可以用刮刀去除,硅酮化合物的残留物可以用丙酮除去。干燥后,如上所述,可以将新的传感器点粘贴到比色皿中
      3. 从记录的饱和度数据文件中,可以使用数据分析部分所示的Excel例如计算减少的斜率。对于特定酶活性的数据分析和测定,见下文。图3显示了使用四个比色皿进行测量的代表性数据集。
         

        图3.显示监测稀释的胶体胶乳缓冲液的氧饱和度的代表性数据集。没有酶(蓝色)的空白代表基线。 ≈4分钟后,分别加入野生型Lcp作为参考(黑色)和两种突变蛋白样品(红色和绿色)。数据也显示在Röther等人的补充材料,2016年。

  3. 抑制剂研究
    该测定允许根据氧饱和度的测量快速且容易地测定酶活性。可以比较不同的酶,突变蛋白,或调查潜在的抑制剂的作用。例如,0.1%SDS抑制Lcp的氧消耗。

第二部分。基于HPLC的橡胶裂解产物的分离

在发生氧化聚异戊二烯裂解之后,可以从反应混合物中提取所产生的低聚异戊二烯,随后通过HPLC分离以进行如下所述的分析。

  1. 准备
    1. 根据制造商提供的说明设置HPLC装置,并安装RP-8反相柱。
    2. 去离子水。
    3. 供应水为第一流动相,甲醇为第二
    4. 在23°C下计算流速为0.7 ml/min。
    5. 计划每次运行20μl样品的注射体积。
    6. 编制溶剂梯度:
      分>
      水(%)
      甲醇(%)

      0-5
      50
      50
      isocratic
      5-25
      0
      100
      渐变
      25-35
      0
      100
      isocratic
      35-36
      50
      50
      渐变
      36-40
      50
      50
      isocratic

  2. 氧化聚异戊二烯切割测定
    1. 标记2 ml反应管,并将它们放在架子上
    2. 移取700μl稀释的胶体胶乳缓冲液(0.2%,见第一部分,步骤B3),并调节温度。
    3. 如果适用,将所需浓度的抑制剂加入溶液中
    4. 添加指定量的酶,通常最终浓度为4μg/ml
    5. 孵化混合物,盖子在所需的温度下打开,通常为聚异戊二烯切割2小时,每30分钟轻轻一摇。
    6. 一定要准备一个没有酶的空白,同样对待。

  3. 低聚异戊二烯的提取
    1. 在使用乙酸乙酯或甲醇时,在附近没有点火源的烟雾柜内工作。
    2. 向每个含有700μl反应体积的管中加入1,000μl乙酸乙酯
    3. 关闭盖子并旋转管子20秒,白色胶乳会凝结
    4. 在室温下以20,000×g离心所有管2分钟。
    5. 根据测定标记1.5ml管,并小心地去除900μl上溶剂相,以避免混合和溢出。丢弃2 ml管。
    6. 将乙酸乙酯在烟气柜中完全蒸发过夜,或者以〜0.2L/min小心地用氮气冲洗加强蒸发。
    7. 向管中加入100μl甲醇,并通过将溶液上下吸取5次冲洗1.5ml管的壁以溶解切割产物。将甲醇 - 产物溶液转移到补充有0.3ml小容量插入物的HPLC玻璃小瓶中,并将其拧在盖子上
    8. 应用所有样品进行HPLC分析。

数据分析

第一部分氧气消耗测定

  1. 评价
    1. 氧浓度的测量非常敏感,但也容易出现几个因素
    2. 由于氧气的温度和压力依赖的溶解度,必须在恒定温度下进行测量并且相对于压力进行分析。然而,大气压力的微小变化在方法的误差范围内。
    3. 该方法的长期实用性表明,每个实验应重复三次,获得六项测量数据
    4. 用四个比色皿进行常规测定如下:
      1. 空白无酶。
      2. 野生型作为参考。
      3. 突变蛋白样品A.
      4. 突变蛋白样品B.
      三个重复对应于突变蛋白和3个基线文件的6个文件以及野生型蛋白的3个数据集作为参考,需要彼此之间的样本的统计相似性(平均值±3×标准偏差)。

  2. 特定酶活性的计算
    1. 完成描述的氧气监测后,* .csv格式的文本文件将自动保存在指定位置的每个通道。
    2. 它包含有关氧量计和所选参数设置的信息的摘要。该分析的兴趣结果以分号(;)分隔的数据集记录。
    3. 以合适的计算软件,例如,MS Excel。
      打开* .csv文件
    4. 选择第一列,然后按选项卡"数据"。选择"文本列"。选择"分离"并选择分号(;)作为分隔符。这应该将第一列分成几列,每个参数一列。
    5. 为了分析耗氧量,只有两列是令人感兴趣的。时间为x轴,"Oxy /%空气坐"为y轴。将两列提取到新的电子表格中。
    6. 重复这些步骤处理所有通道的数据,并将值粘贴到电子表格中。
    7. 在新的电子表格中,使用带有线选项的X-Y散点图绘制图形。该图应该与使用oxy-4v2软件的测量过程一样。
    8. 由于我们使用空气饱和的稀释的胶体胶乳缓冲液将氧饱和度校准为100%,所以在加入酶之前,基线的测量值在100%的范围内。此后,酶反应引起的氧气减少是可见的,我们使用线性降低的斜率来计算特定的酶活性。
    9. 为了通过线性回归确定斜率,最简单的方法是将线性减少的数据复制到同一图中的新系列中。对于这一行,我们使用'趋势线'特征来计算斜率。指定"线性",并选中框以显示公式,并确定合适的系数(1表示最适合)。斜率的单位为%O 2 min -1
    10. 在23℃和1,013毫巴下,1L空气饱和的稀释的胶体胶乳缓冲液含有〜8.3mg O 2。这相当于259μmolO 2%= 100%。因此,氧饱和度降低1%对应于〜2.6μmol二氧(2.6μmol% -1
    11. 为了计算摩尔氧消耗速率,乘以计算的斜率和因子:2.3%min -1 x2.6μmol%-1.0μmol/min 。该值对应于0.5ml体积的比色杯中的1L的体积并且等于3.0nmol min -1。
    12. 为了确定具体的酶活性,将摩尔耗氧量除以用于测定斜率的酶量,在这种情况下为2μg,得到1.5nmol/min的蛋白质 - 1 或1.5μmolmin -1 mg -1(对应于1.5U/mg)。
    13. 在23℃下野生型Lcp K30(Lcp来自链霉菌属菌株K30)的比活性为1.5U/mg(Watcharakul等人,与另一组(Hiessl等)研究的来自Gordonia polyoprenivorans菌株VH2(Lcp VH2)的同源物Lcp测定的1.3U/mg相关联,2014)。
    14. 对所有测量重复此评估,重复的值应在标准误差的范围内。
      注意:在初始恒定下降后,氧浓度的降低通常会减慢(图4)或者再次稍微增加。这可以通过将氧扩散到(未密封的)测定缓冲液中来解释。如果您希望在较长时间内监测氧气消耗率,则必须密封比色皿,或者应使用较大体积的测定混合物。


      图4. LcpK30的比活性的实施例计算。在该实验中,使用1ml的测定体积。测量时间(X)和氧饱和度(Y)的值,如从所得到的* .csv文件所描述和提取的那样进入电子表格。数据在X-Y散点图中可视化。在12分钟后添加酶导致在聚异戊二烯裂解过程中掺入二氧氧化碳降低了氧饱和度。斜率可以拟合并与酶活性成正比。通过将比例因子乘以除以测量中使用的酶量,可以如所述计算特异性酶活性。

第二部分基于HPLC的低聚异戊二烯分离

在通过HPLC分析每个样品后,可以使用适合的软件(在我们的安捷伦化学工作站)中评估报告文件。

  1. 对于数据处理,仅通过从每个结果中减去没有酶的空白运行的简单基线校正就足够了。成功分析的一个例子如图5所示。
  2. 根据其在该测定中的相应保留时间,峰的鉴定是可能的。以前的研究(Braaz等人,2005; Birke和Jendrossek,2014),使用HPLC MS分析鉴定了相应的寡聚异戊二烯分子。
  3. 通过计算峰面积和通过与已知活性和浓度的均等处理的酶进行比较来确定相对活性,可以进行定量
  4. 对于RoxA蛋白质,通常只检测到一种主要降解产物(n = 2)。相比之下,Lcp将橡胶切割成各种具有不同数量(n)异戊二烯单元的寡聚异戊二烯(图5)。


    图5.在没有酶的空白运行之后减去Lcp(黑色)和RoxA(红色)衍生的切割产物的HPLC色谱图。
    产生的寡聚异戊二烯产物的配方显示在右上角。裂解产物中异戊二烯单元的数量由所标识峰的n值表示。

食谱

  1. 磷酸钠(KP)缓冲液
    1. 分别制备100mM KH 2 PO 4和K 2 HPO 4的溶液
    2. 混合两种溶液以调节所得KP缓冲液的pH值,通常为pH 7.0
    3. 为了制备pH7混合物的缓冲液,将5体积的碱性K 2 HPO 4 O 4溶液与4体积的酸性KH 2 PO 3 > 4 解决方案
  2. Nonidet P-40解决方案
    向去离子水中加入0.1%wt/vol的洗涤剂
  3. 天然橡胶乳胶
    如上所述进行准备(步骤Ⅰ,步骤B2)
  4. 稀释胶体胶乳缓冲液
    准备如上所述(程序第一部分,步骤B3)

致谢

我们感谢德国Forschungsgemeinschaft(DFG Je152/17和Je152/18)为德国PreSens,德国,韦伯和德国Schaer以及德国IBA生命科学公司提供资金,分别提供传感器点,聚异戊二烯胶乳和Strep-Tactin色谱柱。

参考文献

  1. Birke,J.,Hambsch,N.,Schmitt,G.,Altenbuchner,J.and Jendrossek,D。(2012)。< a class ="ke-insertfile"href ="http://aem.asm。 org/content/78/22/7876.short"target ="_ blank"> Phe317对于橡胶加氧酶RoxA活性至关重要。应用环境微生物78(22):7876-7883。
  2. Birke,J。和Jendrossek,D。(2014)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/24907333"target ="_ blank" >橡胶加氧酶和胶乳清除蛋白将橡胶切割成不同的产品,并使用不同的切割机制。应用环境微生物 80(16):5012-5020。
  3. Birke,J.,Röther,W.and Jendrossek,D。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25819959"目标="_ blank">链霉菌的乳胶清除蛋白质(Lcp)菌株K30是一种b型细胞色素,其生物物理性质与橡胶加氧酶A(RoxA)不同。应用环境微生物81(11):3793-3799。
  4. Birke,J.,Röther,W.,Schmitt,G.and Jendrossek,D。(2013)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/23934498"target ="_ blank">土壤和海洋粘菌细菌中橡胶加氧酶(RoxA)的功能鉴定。应用环境微生物79(20):6391-6399。 />
  5. Braaz,R.,Armbruster,W.and Jendrossek,D。(2005)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15870336"目标="_ blank">黄单胞菌属的血红素依赖性橡胶加氧酶RoxA 通过双加氧酶机制切割聚(顺式-1,4-异戊二烯)的碳骨架。应用环境微生物71(5):2473-2478。
  6. Braaz,R.,Fischer,P.and Jendrossek,D。(2004)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15574940"目标="_ blank">新型血红素依赖性加氧酶催化橡胶(聚顺式-1,4-异戊二烯)的氧化裂解。应用环境微生物70(12):7388 -7395。
  7. Hiessl,S.,Bose,D.,Oetermann,S.,Eggers,J.,Pietruszka,J.and Steinbuchel,A。(2014)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/24928880"target ="_ blank">胶乳清除蛋白 - 在顺式双键上切割聚(顺式-1,4-异戊二烯)橡胶的加氧酶。 em> Appl Environ Microbiol 80(17):5231-5240。
  8. Röther,W.,Austen,S.,Birke,J。和Jendrossek,D。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27590810"target ="_ blank">由Streptomyces sp。的乳胶清除蛋白(Lcp)切割橡胶。菌株K30:分子洞察。 Appl Environ Microbiol 82(22):6593-6602。
  9. Schmitt,G.,Seiffert,G.,Kroneck,PM,Braaz,R.and Jendrossek,D。(2010)。来自黄单胞菌属(Xanthomonas)的橡胶加氧酶RoxA的光谱性质,一种新型的双加性双加氧酶。微生物学第156(Pt 8):2537-2548。
  10. Seidel,J.,Schmitt,G.,Hoffmann,M.,Jendrossek,D.and Einsle,O。(2013)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/23922395"target ="_ blank">来自黄单胞菌属的进行性橡胶加氧酶RoxA的结构 sp。 Proc Natl Acad Sci USA 110(34):13833-13838。
  11. Watcharakul,S.,Röther,W.,Birke,J.,Umsakul,K.,Hodgson,B.and Jendrossek,D。(2016)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/27215318"target ="_ blank">来自新分离的降解玫瑰色红球菌菌株RPK1的纯化乳胶清蛋白(Lcp)的生物化学和光谱表征揭示Lcp的新特性。 BMC微生物 16:92.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Röther, W., Birke, J. and Jendrossek, D. (2017). Assays for the Detection of Rubber Oxygenase Activities. Bio-protocol 7(6): e2188. DOI: 10.21769/BioProtoc.2188.
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