Using this protocol, we can easily produce quite a number of flavonols from flavanones in one pot. This technique is labor-and time-saving, highly cost-effective, and easy to control, so it has a huge industrialization potential. Yes, this method can provide insight into economic production of other secondary metabolites, but it needs some modifications when applied to it.
He will usually get nothing or very low yield. My advice is to handle recombinant enzymes on ice and add 10%glycerin to the stock solution of enzymes. That's because only through visual demonstration can a new hand understand the important details and truth affecting the successful execution of the experiment.
First prepare buffers. Make two times synthetic buffer by dissolving appropriate amount of TRIS-base, sodium ascorbate, alpha ketoglutaric acid into de-ionized water, and pipette appropriate amount of glycerol to the solution. Use a pipette to add hydrochloric acid to adjust the pH to 7.2, and adjust the volume by adding de-ionized water.
Store the two times synthetic buffer at four degrees Celsius for future use. Then make a 100 times stock solution by dissolving ferrous sulfate heptahydrate in deionized water. Make a stock solution of 25 millimolar flavonoid by dissolving flavonoid in methanol, and store the solution at minus 20 degrees Celsius.
The most critical step is to set up a synthetic system. To set up a synthetic system to produce a flavanol from a flavanone, first prepare the synthetic system in a two milliliter tube according to the reagents listed in this table. Place the open tube in a shaking heat block at 40 degrees Celsius, at 600 rpm for forty minutes.
After that, terminate the reaction by adding 10 microliters of acetic acid, and 100 microliters of ethyl acetate. Two hours later, use a pipette to tranfer the top organic phase to a 1.5 milliliter tube, and place the tube in a hood for air drying at room temperature. To perform TLC analysis, first retrieve the tubes from the hood, and redissolve the flavanoid powder in 160 microliters of methanol in a tube.
Prepare authentic flavonoid samples with serial concentrations, by diluting the 25 millimolar flavonoid stock solution in methanol. Load one microliter of the redissolved reaction sample, and one microliter of the authentic flavonoid sample, onto a polyamide plate. Do the same for the other concentrations of authentic flavonoid on the same plate.
Then in a fume hood, put the sample loaded plate in a chromatography cylinder filled with a solvent system. Attach the plate to the bottom of the cylinder, and run the plate in the solvent system for 20 minutes. Air dry the plates at room temperature.
With a sprayer bottle, spray the plates with 1%ethanolic solution of aluminum chloride, followed by air drying again at room temperature for 30 minutes. Visualize the spots on the plates under a UV light at 254 nanometers, and take images, then on the computer, open the software ImageJ. Click file, open, to open the image to be analyzed.
Click the left most rectangular selection tool in the ImageJ user interface. Outline the ROI in the image with the mouse, and press one to label the first ROI. Move the rectangular selection with the mouse right to the next ROI, and press two to label the second ROI.
Repeat to label all other ROIs by pressing two. Press three to generate profile plots for all ROIs, in a pop-up window. Then, use the straight line selection tool to drop baselines so as to define a closed area for each peak of interest.
Activate the wand tool, by clicking the corresponding icon in the ImageJ user interface. Click inside the peak to display results for all peaks in a pop-up window. Next, in Excel, plot the gray values from the pop-up window against the corresponding flavonoid concentrations, to make a TLC based standard curve of the authentic flavonoid.
Then calculate the yield of the flavonoid of interest produced in this protocol, according to the resulting formula. Use a syringe to process each sample sequentially through a 0.45 micrometer and 0.22 micrometer filters. Load the samples into an HPLC-MS system through suction nozzle, and separate the samples at 30 degrees Celsius using a C18 column.
Elute the column at one milliliter per minute by a gradient of 10 to 85 volume per cent acetyl nitrile in water, and monitor the absorbance of the eluate from 200 to 800 nanometers. Perform the LC-MS analysis in a negative ion mode, with a drying nitrogen flow of 10 liters per minute at 300 degrees Celsius, in a sheath gas flow of seven liters per minute at 250 degrees Celsius, and collect data using a built in software. To extract single wavelength chromatographs, open the qualitative analysis program, and click file, open datafile.
Select the file to be analyzed in the open data file window, and click open to open the file. Right click the mouse in the chromatogram results window and then extract chromatograms in a pop-up menu. In the type list, click on other chromatograms.
In the detector combo box, select DAD1, then click okay to display the HPLC results in the chromatogram results window. Click the manual integration icon docked at the top of the chromatogram results window. Draw baseline for the peak required for manual integration analysis with the mouse.
Click view, integration peak list, to display the results. Copy the results of the peak areas to Excel, and plot an HPLC based standard curve of the authentic flavonoid against the corresponding flavonoid concentrations, then calculate the yield of the flavonoid of interest produced in this protocol according the resulting formula. Then repeat the same procedures to extract single wavelength chromatograms to analyze the MS data for the exact mass of flavonoid compounds.
Click the range select icon on the chromatogram results toolbar. Select the peak of interest. Right click the mouse in the selected range, and click the extract MS spectrum in the pop-up menu to display the results in the MS spectrum results window.
To determine whether this in-vitro system can be used for the conversion of a flavanone into a flavonol, NRN was added into the system. There were two new spots emerged on a polyamide TLC plate. One spot showed a migration distance similar to that of DHK, and the others similar to that of KMF.
Further analysis by HPLC and LC-MS demonstrated that the new chemical showed a retention time of 12 minutes and 20 minutes respectively, in a quasi-molecular ion peak M over Z at 287 and 285 which were identical to those of DHK and KMF. To determine whether the system can be used for the conversion of other flavanones into their corresponding flavonols, ERD was added into the system. Two new spots on a polyamide TLC plate displayed a migration distance similar to that of DHQ and QRC respectively.
HPLC and LC-MS analyses demonstrated that these new chemicals revealed a retention time of 10 minutes and 16 minutes respectively, and a quasi-molecular ion peak M over Z at 303 and 301, which exactly corresponded to those of DHQ and QRC. When preparing the synthetic system, you should add the enzyme lastly, and add the ferrous sulfate second to last. Yes, it provides a guide for the economical production of other secondary metabolites.
