The overall goal of this procedure is to synthesize a my cellar, enzyme immobilization and stabilization polymer. To accomplish this first, combine the neon suspension with the cheal ammonium bromide salt, and vortex, the mixture in order to exchange the proteinated form of nafion with ammonium salts. The second step is to dry the resulting polymer by pouring the mixture into a whey boat and allowing the solvents to evaporate.
Next, soak the polymer and deionized water for 12 to 24 hours to remove excess salt After salt extraction, rinse the polymer with water and then allow the polymer to dry completely by evaporation. The final step is to transfer the polymer to a clean vial and resuspend it insolvent for use in enzyme Im mobilization. Ultimately, this my cellar enzyme immobilization polymer is used to improve the stability of enzymes in activity assays.
Generally, individuals new to this method will struggle because this procedure seems extremely straightforward, but the drying procedure is critical to the fabrication of a functional polymer. Demonstrating the procedure will be shu a graduate student in my laboratory To begin the procedure for modifying nafion with Nerium ammonium salts. Measure a threefold molar excess relative to the sul acid groups on the nafion polymer of the alk ammonium bromide salt.
Transfer the alk ammonium bromide salt to a glass vial vigorously. Shake a bottle of 5%weight per volume, nafion suspension for approximately 30 seconds to ensure that the neon is suspended uniformly in solution. Pipette out two milliliters of the now resuspended nafion and add it to the glass vial containing the AGU ammonium bromide salt.
Vortex the vial at 900 RPM for 10 to 15 minutes. Next, pour the viscous solution into a plastic weighing tray and use a pipette to transfer any residual solution from the vial to the weighing tray. In this example, the way boat is partially covered and left on the laboratory bench to allow the solvents to completely evaporate out of the weighing tray at a rate of evaporation that takes more than six hours.
Complete evaporation of all solvent at the appropriate rate of evaporation should leave behind a yellow or brown transparent film on the bottom of the weighing tray as shown here. If the solvent evaporates too fast, a crusty or crystalline material will form instead of a transparent film indicating that the mic cellar structure of the polymer has been destroyed. If solvent evaporation is too slow or the ammonium salt is allowed to react too long and take on too much water vapor, either a tacky orange gel or a white crystalline material will form.
In either case, the procedure must be restarted. After all solvent has evaporated at the appropriate rate of evaporation, fill the weighing tray with 10 to 20 milliliters of 18 mega ohm centimeter deionized water cover, and soak for 12 to 24 hours to remove excess alk ammonium bromide salts and HBR 12 to 24 hours later, pipette out the water and add enough deionized water to fill the tray. Be careful to always pipette away from the polymer film to avoid losing any of it.
Rinse a total of three times after the rinses. Allow the weighing tray to sit uncovered until the polymer is completely dry. When completely dry, the polymer should be a clear and somewhat brittle plastic film.
In contrast, polymers that were not dried properly will have a very different appearance after salt extraction as shown here. Using a spatula carefully remove the dried film from the weighing tray and transfer it into a clean glass vial. Add two milliliters of ethanol and three ceramic mixing beads and vortex for four hours or until the polymer film is completely resuspended.
The alk ammonium modified nathe on solution is now ready to be used for enzyme immobilization. If the enzyme to be immobilized is a dry enzyme, weigh out one milligram of the enzyme into a 1.5 milliliter micro centrifuge tube and add one milliliter of a hundred millimolar pH seven phosphate buffer to create a one milligram per milliliter enzyme solution. Transfer 120 microliters of the one milligram per milliliter enzyme solution to a new tube.
Add 60 microliters of the ALK ammonium modified nafion solution and vortex for 10 seconds. The enzyme to polymer solution ratio must be kept at two to one if this mixture needs to be scaled up for large numbers of replicates. Finally pipette 60 microliters of the enzyme polymer solution into the bottom of each of the three separate one square centimeter cuvettes and allow them to dry overnight.
The stability of the enzyme within the polymer film can then be assessed by enzymatic activity assays as demonstrated. Next to begin this assay, add the following reagents into the vet co casted with the modified nafion polymer and NAD dependent dehydrogenase enzyme. 1.3 milliliters of 50 millimolar sodium pyrophosphate, 1.5 milliliters of freshly prepared 15 millimolar nicotinamide adenine dinucleotide or NAD and 0.1 milliliters of water.
Place the vete in a UV visible spectrophotometer set to a wavelength of 340 nanometers zero. The spectrometer add 0.1 milliliters ethanol into the vete and mix reagents by gently pipetting the solution up and down five times for a blank use 0.1 milliliters of additional water. Instead of the 0.1 milliliters of ethanol, five minutes after the reagents were added to the vete record the absorbance at 340 nanometers.
Record the absorbance again 20 minutes after adding reagents to the qve, these two data points will be plotted to get a slope that can be used for activity calculations to assay for enzymatic specific activity of immobilized pq. Dependent dehydrogenases pipette these reagents into the qve co casted with the modified nafion polymer and PQQ dependent dehydrogenase. 1.5 milliliters of sodium phosphate and 200 microliters of 600 micromolar pH methyls sulfate or PMS.
Place the vete in a UV visible spectrophotometer set to a wavelength of 600 nanometers and zero. The spectrometer add 100 microliters of a mixture of 700 microliters to six chloro endemol or DCIP and 200 microliters of the substrate of interest. Mixed the reagents by gently pipetting the solution up and down five times for a blank.
Use 200 microliters of water instead of the substrate of interest. Five minutes after the reagents were added. Record the absorbance at 600 nanometers.
Record the absorbance again 20 minutes after adding the reagents to begin this assay. Add to the Q VET co casted with the modified nafion polymer and glucose oxidase. Two milliliters of a solution containing the following 0.2 molar para hydroxy benzoic acid 0.02%Sodium azide 128 units of peroxidase 0.3 millimolar, four amino anti pyre, one molar potassium phosphate, and 50 millimolars of glucose.
Makes the solution by pipetting up and down five times. Place the Q vet in a UV visible spectrophotometer set to a wavelength of 510 nanometers. Five minutes after the reagents were added to the qve.
Record the absorbance at 510 nanometers. Then record the absorbance again at 20 minutes after addition of reagents. This table shows assay results of NAD dependent glucose dehydrogenase activity immobilized and selected modified nafion polymers.
It was observed that immobilized NAD dependent glucose dehydrogenase had lower specific activity than the enzyme and buffer solution. This is because polymer membranes decrease transport of large molecules, and although glucose the substrate for the assay is small, the co-enzyme NAD needs to diffuse in and out of the membrane. For NAD dependent dehydrogenase, the transport limitation due to immobilization in the polymer decreases the observed enzymatic activity.
This next table shows assay results of PQQ dependent glucose dehydrogenase activity immobilized and selected modified nafion polymers. It's important to note here the higher activity of the enzymes immobilized on most nafion films versus the enzymes and buffer solution. Since PQQ dependent glucose dehydrogenase is a membrane associated protein, the hydrophobic modified my cellar, nafion provides a more membrane like environment than buffer for stabilizing the active PQQ dependent glucose dehydrogenase, thus enhancing the activity of the enzyme.
Lastly, the representative assay results of glucose oxidase specific activity. Immobilized and selected modified nafion polymers are shown in this table. The enzymatic assays for each of the polymers with each of the three enzymes show that the trends in relative specific activity compared to enzyme and solution is a function of the enzyme system.
Consequently, the exact polymer needed for each enzyme has to be optimized. While attempting this procedure, it is important to remember that the drying step is critical to this particular procedure.
