Purification of soybean peroxidase
The materials required for the science fair project:
- 1 bottle of ethanesulfonic acid
- 1 packet of sodium chloride
- 1 bottle of pyrogallol
- 1 bottle of hydrogen peroxide
- 1 bottle of potassium phosphate
- 1 bottle of soybean peroxidase
- 1 unit of the BioCad Perfusion Sprint Chromatography system
- 1 anion exchange polymer (10mm x 50mm)
- 1 YM30 Amicon ultrafiltration disc
- 1 bottle of distilled water
- 1 Virtis Vacfreeze refrigerated condenser module
1. For this science fair project, the independent variable is the level of catalytic activity of the commercial and purified soybean peroxidase. The dependent variable is the catalytic activity of the soybean peroxidase. This is determined by the colorimetric measurement of oxidizing pyrogallol to purpurogallin. The constants (control variables) are the pH, amount of sodium chloride and temperature of the environment, which will remain at room temperature.
2. Obtain the commercial soybean peroxidase and purify it using the BioCad Perfusion Sprint Chromatography equipment and the anion exchange polymer (10mm x 50mm).
3. Perform the ultrafiltration to remove salt by using the YM30 Amicon ultra filtration disc. Wash the purified soybean peroxidase protein 6 times using the distilled water. Next, lyophilize the soybean peroxidase protein overnight using a Vitris Vacfreezer.
4. Test and compare the catalytic activity of the purified soybean peroxidase with the original commercial soybean peroxidase. Use a 0.01M potassium phosphate as a buffer at pH 6.0 together with 7.84M hydrogen peroxide at 20 deg C to oxidize 42.3 mM soybean peroxidase and measure the catalytic activity using a colorimeter. Record the results and measurements in a table, as shown below.