INVESTIGATING THE EFFET OF pH ON THE ACTIVITY OF IMMOBILISEDAMYLASE

If the temperature is reduced to near or below freezing point, the enzymes will become inactive, but not denatured. They will regain their catalytic influence when a higher temperature is restored. In this experiment, I will control the temperature through out the experiment to make sure that it does not exceed the optimum temperature of the enzyme. I will keep the temperature of this experiment at 40oC, because the enzyme amylase can be used between 20 and 60oC and its optimum is 50oC, therefore the enzyme will work perfectly well as the temperature  I want to use is not far above or far below its optimum.

pH

The pH is one of the most important factors that affect the rate of an enzyme controlled reaction and this is what I will be investigating. Enzymes function most efficiently over a particular pH range. The optimum pH of the enzyme is that at which the maximum rate of reaction occurs. Changing the pH below or above its optimum, the rate of the enzyme activity diminishes.

As pH decreases, acidity increases and the concentration of H+ ions increases. This increases the number of positive charges in the medium. Changes in pH alter the ionic charge of the acidic and basic groups and therefore disrupt the ionic bonding that helps to maintain the specific shape of the enzyme.

Changing the pH above the optimum alters the number of free hydrogen (H+) or hydroxyl (OH-) ions. This affects the charge on the amino acid residues that make up the enzyme, and this might sometimes lead to the denaturation of the enzyme and change in shape of the active site of the enzyme. In summary, changes in pH leads to alteration of the enzyme shape including its active site.

Enzyme Concentration

If the substrate concentration is maintained at a high level, and other factors like pH, temperature are kept constant, the rate of the reaction is proportional to the enzyme concentration. Therefore as the enzyme concentration increases so will the rate of the reaction. Therefore this factor will also be kept constant.

Substrate Concentration

The concentration of the substrate is another important factor to consider in this experiment. The rate of the enzyme reaction increases with increasing substrate concentration. But the theoretical maximum (Vmax) rate is never obtained. As the substrate concentration increases, it gets to a point where there will be no significant change in the reaction rate. This is because if the concentration of the substrate is very high, the active site of the enzyme molecules becomes saturated with substrate, therefore the remaining substrate has to wait until the enzyme-substrate complex releases the products before entering into the active site of the enzyme. I will also control the concentration of the substrate through out the reaction because any changes in the substrate concentration will alter my results in the end.

In this experiment, starch is the substrate I am going to use. Starch is a main carbohydrate food reserve in plants. It is polymer made up of long chains of ά-glucose molecules with branches in places. The chains are folded and packed together in spherical plastids to form starch grains. Starch is formed by condensation and polymerization reactions. It is a mixture of two different compounds which are Amylose and Amylopectin.

• Amylose is a linear, unbranched polymer which makes up about 80% of starch 
• Amylopectin is a polymer with some 1,6 linkages that give a branched structure. It makes up about 20% of starch

Starch can be tested by adding drops of Iodine solution to it which it turns blue-black. It can be hydrolyzed by Amylase to maltose. The best way to know when it is completely hydrolyzed is when it starts going brick red instead of blue-black when tested with Iodine solution.

This is a rough sketch of what my graphs would look like after the experiment.

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MATERIALS / APPARATUS

These are the materials I will use for my preliminary experiment. And I will also use them for the main experiment though I might change some of the volume/quantities of the chemicals because I am carrying out the preliminary experiment mainly to determine the quantities and masses of the chemicals and substances to use.

1. 7.5g Starch for the main experiment + 4.5g for the preliminary experiment
2. 2cm3 Bacterial Amylase solution (Manufacturers; Phillip Harris, Instructions: use at pH 4.0 to 8.0, optimum pH: 6.0, Use at 20 to 60oC optimum: 50oC, Batch No: H030)
3. 2cm3 Amylase (concentration unknown)
4. 2.8g Calcium Chloride in 200 cm3 distilled water
5. 6g Sodium alginate in 200cm3 distilled water
6. Anhydrous Disodium Hydrogen Phosphate buffer
7. Citric Acid buffer
8. Buffers 4.0, 9.2, and 7.0
9. Distilled water
10. Water Bath
11. Measuring Cylinder (100cm3)
12. Boiling Tubes
13. Iodine
14. Spotting Tray/White Tiles
15. Teat Pipette
16. Syringes 2.5ml, 10ml, and 20ml
17. Mortar and pestle
18. beaker
19. Stirring rod
20. Stopwatch
21. Mesh/Sieve
22. pH meter
23. Weighing boat
24. Top pan balance

PRELIMINARY EXPERIMENT

A did a lot of preliminary work on this experiment, I was not very sure if the beads made with the syringe barrel would work quickly, secondly, I wasn’t sure of the temperature to use . This pilot experiment also helped me in determining how to make all the pH’s I want. Since I want to investigate the effect of pH on immobilized amylase, I need to have a range of pHs. But we don’t have the pHs I need, like pH 5.5 and 6.0; therefore, I need to make them up my self. This preliminary experiment helped to know the volume of citric acid and disodium buffers to make up the pH I wanted.

Below is a brief summary of how I carried out my preliminary experiment.

• I made up the sodium alginate solution by dissolving 6g sodium alginate in 200cm3 hot distilled water. Then I added 2.1ml Bacterial Amylase to the sodium alginate solution.
• Then I made up the calcium chloride solution by dissolving 2.8g calcium chloride in 200cm3  distilled water.
•  I used a 10ml syringe to collect the sodium alginate solution = amylase and dropped it into the calcium chloride solution to make the beads.
• I dissolved one tablet of pH 9.2 in distilled water and added 3g starch to it, and then I boiled it to dissolve the starch.
• I placed the 10cm3 of the starch solution and the 10cm3 beads I made in a 40oC water bath and allowed it to equilibrate.
• Then I dropped a few drops of Iodine on a spotting tray, I mixed the immobilised enzyme beads with the starch solution and started the stop watch, then I used a pipette to add a drop of the starch solution to the spotting tray after every 30 seconds. Then I recorded the time it took for the starch solution in the test tube to stop going blue-black.

I used 3g starch and found out that it took up to 27 minutes for the starch to be completely hydrolyzed on pH 4. Therefore I had to reduce the mass of the starch to 1.5 and the rate of starch hydrolysis became faster.

From my preliminary experiment, I noticed that the results of the experiment would not be very reliable and accurate if I only drop the starch solution into the spotting tray after every 30 seconds, therefore I have decided that in my main experiment, I will reduce the time to 10 seconds as soon as I notice any slight change in the color on the spotting tray.

I also tried using the syringe barrel to make the beads I want and they worked as I expected, so I believe that my experiment will be successful. This preliminary experiment also helped to figure out how to measure the size o the bead I used. I wanted to use a microscope, but the bead was bigger than the field of view, so I used a millimeter rule to measure four different beads from the beads I made and worked out the average. This is the result of my preliminary experiment.

CONSTANT FACTORS

The following factors will be kept constant through out this experiment.

Concentration of Starch

Concentration of Amylase (2.1ml Concentration unkown)

Concentration of Sodium Alginate

Concntration of Calcium Chloride

Concentration of Iodine

Temperature (40oC)

The size of the Immobilized amylase beads (4.9mm)

The only factor that will be changed is the pH of the buffer in the starch solution

FAIR TEST

 I will make this experiment a fair test by only changing one factor in the experiment which will be the pH. This will be a fair test if only the pH is changed because each test is identical except for the factor that was being investigated. One factor that must be kept the same is the bead size because it can greatly affect the rate of the reaction. This is because the size of the beads also affects the rate of the reaction.  The smaller the beads, the greater their surface area.

Surface area is one of the factors that affect an enzyme controlled reaction. If the bead size is changed it will alter the rate of the reaction, either by increasing or decreasing it.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          

PRECAUTIONS

Iodine is a dangerous chemical, I will wear safety glasses to protect my eyes from the iodine and I will take great care not to prevent spillage of iodine on my skin because it is irritant. And I will wash it off immediately with cold water if it does come in contact with my skin.

Calcium Chloride is irritating to the eyes; again I will wear safety glasses. Again I will avoid contact with my skin because it is irritant and I will wash it off immediately with cold water if it does come in contact with my skin. I will also avoid breathing the dust.

Bacterial Amylase solution is also dangerous so I will avoid breathing the gas/fume/vapour/spray of Amylase. I would avoid contact with my skin and eyes. I would wear suitable gloves and safety glasses. I would avoid inhaling the amylase because it may cause sensitization by inhalation.

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IMPLEMENTATION

I only changed the concentrations of the Starch I used in my preliminary Experiment. Apart from that every other thing is the same. I used the same bead size as the one I used in my preliminary experiment.

METHOD

I made up the sodium alginate by adding 200cm3 hot Distilled water to 6g sodium alginate in a 250 cm3  beaker, then I stirred it until the mixture became soluble. I used hot distilled water because Sodium alginate will take a long time to dissolve in cold distilled water. With a 2.5ml syringe, I added 2.1ml Bacterial Amylase to the sodium alginate solution.

I made up the calcium chloride solution by adding 200cm3 Distilled water to 2.8g Calcium Chloride and stirred until it dissolved completely.

To make the beads, I used a 20ml syringe to suck up the Sodium alginate solution which had the Amylase in it, and dropped it one drop at a time to another beaker containing 200cm3 calcium chloride. When I finished dropping all the Sodium alginate + Amylase solution into the calcium chloride, I left it for 15 minutes to solidify. Then I used a sieve to filter it and then I rinsed it with distilled water.

I made up the first pH solution by grinding 1 tablet of buffer 4.0 with a mortar and pestle. Then I added 100cm3 distilled water. I added 1.5g starch to the pH solution in a beaker and heated the mixture with a Bunsen burner until the starch dissolved completely. I repeated the same procedure for pH 7.0 and 9.2. To make up the 5.5 pH solution, I added 50cm3 Citric Acid buffer to 64cm3 Anhydrous Disodium Hydrogen Phosphate buffer, and measured it down to a 100cm3 . Then I added the 1.5g starch and heated it to make the starch completely soluble. I repeated the same procedure for pH 6.0, adding 42cm3 Citric Acid to 70cm3 Disodium Hydrogen phosphate. I made sure that the pH solutions were accurate by measuring them with a pH meter.

Then I set up a water bath at 40oC. I measured 15cm3  of the beads I made with a 15ml syringe into a boiling tube. And then I measured 15cm3 starch + 4.0 pH solution into another boiling tube, and placed both boiling tubes in the water bath for 7 minutes to equilibrate with the temperature of the water bath. I used a 40oC water bath because the instruction on the Bacterial Amylase pack said it should be used at 20 to 60oC. I made sure the temperature was correct by using a thermometer to read the temperature of the water bath.

While the two boiling tubes were still in the water bath, I put 1 drop of Iodine into each well in the Spotting Tray. After the two boiling tubes had equilibrated with the temperature of the water bath, I mixed them together and started the stopwatch instantly. I used a pipette to add one drop of the starch solution (enzyme-substrate solution) to the Iodine in the spotting tray every thirty seconds until I noticed a change, then I decreased the interval to every ten seconds until there was no further change. I did two other experiments on this pH, and repeated exactly the same procedure on all the other 4 pH solutions I made, carrying out three experiments in each case. 

RESULTS

CONCLUSIONS

Main trends and patterns

The results of this experiment show that there was a major difference in the response of the Bacterial Amylase enzyme to the different pH levels. The tabulated result also shows that the enzyme was less active in some of the pH levels.

Explanation of Results

The overall result of my experiment was what I expected. The results are very reliable and had no obviously anomalous results however. In order for me to know the rate of the whole reaction, I made another table form the results I had, this time I divided all the results I got from my experiment by 1. Then I plotted a graph on the Rate of the Reaction against the pH.

I said that the results are reliable because the error bars on the graph are no wide, but they are narrow, this shows proximity of the three results in each experiment. At pH 7.0, there were no error bars, because the time recorded for the three experiments on that pH was the same. The narrower the error bars, the more reliable the result, so I think my result is reliable.

The graph of the rate of the reaction against the pH show the lump in the line which looks out of place, from this graph, it is very obvious that the optimum pH of this enzyme is 6.0. This is because at pH 6.0, the rate of the reaction was fastest. It is very clear that the rate of the reaction was slow below its optimum pH, for example at pH 4 the rate of the reaction was slow. As the pH increased so did the rate of the reaction, but when the pH increased above the optimum pH (6.0), there was a rapid decrease in the activity of the enzyme.

 

At pH 7.0, the pH was above the optimum and that was when the reaction rate started decreasing.  This is because at pH 7.0, the ionic bonds that hold the shape of the enzyme were affected, and so was the ionisation of the R groups of Amino acids in the active site which form temporary bonds with the substrate (starch), therefore the enzyme-substrate complex could not be formed as fast as it was formed at pH 6.0. Since the bonds that hold the active site were affected, they could no longer hold much substrate in the active site, therefore reducing the rate of the reaction.

The error bars on this shows that the results of the experiment is reliable, as the bars are not wide therefore the differences between the Average result isn’t much.

The time against pH graph was what I expected. It is almost the same as the one I sketched in my hypothesis, you could see that the time taken for the hydrolysis of starch was lowest at pH 6.0.  Again this makes it obvious that its optimum pH is 6.0.  In this experiment the enzyme was not denatured but it even at pH 9.2.

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Denaturing unfolds the enzyme and the specific shape of the active site is lost. The active site changes due to the interruption of the hydrophobic interactions and hydrogen bonds, the enzyme will become unable to combine with the substrate because substrate will no longer fit into the active site. At pH 9.2, the enzyme was inactivated but not denatured, because the pH was above the optimum for Amylase and this altered the number of free hydrogen (H+) or hydroxyl (OH-) ions, therefore the  charge on the amino acid residues that make up the enzyme, therefore the enzyme became deactivated. The enzyme would become reactivated if the pH is reduced, but it will get denatured if the pH was increased further.

Again at pH 4.0, the enzyme was also deactivated due to increases in acidity and then increase in the concentration of H+ ions. This is why it took a long time for the reaction to take place at this p. The rate of the reaction would increase if the pH was increased further.

EVALUATION

The experiment was reliable; it corresponded with my earlier hypothesis. From my results, it was very obvious that the optimum pH was 6.0.

Even though my results were reliable, I had a few limitations when carrying out this experiment. The time at which the drops were taken and the total time recorded was not very accurate because there was a delay between taking the solution into the teat pipette and dropping it into the spotting tray. This could be improved by putting the solution + the beads into a burette and dropping it into the spotting tray from there. This can also improve the overall accuracy because it would be easy to drop the solution every five seconds. This limitation affected the conclusions made on this experiment. This is because the whole result of the experiment is based on the time taken for the iodine colour to change and if there is a limitation in recording the time taken, it will definitely affect the reliability of the results and it will also affect the final conclusions.

Another limitation was the difficulty in noticing the change of the iodine solution form blue-black to reddish brown, brick red. Colours may be misjudged since different people have different kind of sights. This could be prevented by using a device (such as calorimeter) to monitor when the starch is completely hydrolysed.

Another limitation could have risen form the stopwatch I used. Due to the large amount of work involved in this experiment, sometimes it is difficult to know when it is thirty seconds on the stop watch. This could be prevented by sing an alarmed stop watch that will make a sound at every 30 seconds.

This experiment was accurate but could be improved further by using different sizes of beads with a larger surface area. The reason for this is that the time taken for a reaction with smaller beads would be faster, and this method of using syringe sized beads may not be accepted in large sized industries where the chemicals need to be processed quickly.

The experiment could also be improved by using different temperatures to know the temperature that the enzyme works best on. Starch concentration could also be varied this is because the rate of the reaction increases as the substrate concentration increases.

Different concentrations of Amylase could also be used to improve the experiment. It could also be improved by increasing the range of pH’s used, in order to know when the enzyme will actually be denatured.