The Effect of pH on Pectinase
The Effect of pH on Pectinase
Hypothesis
I plan to investigate the effects of varying pH on the enzyme 'Pectinase'. I will test the yield of apple juice from a set amount of pureed apple at different pH. I think the optimum pH for pectinase is likely to be around pH 5, an acidic pH, as many commercial pectinases are produced from fungi.
Before I look at the structure of pectinase, first it would seem sensible to look at the substrate that pectinase will be working on, Pectin. Starch and Pectin are very similar molecules. Starch and Pectin are both polysaccharides. However in Pectin the repeating unit is not glucose as in starch, but galacturonic acid. Galacturonic acid is very similar to glucose, except that the carbon with CH2-OH attached in glucose is replaced by a -COOH in galacturonic acid (shown below). Bonds between carbon 1 of the first galacturonic acid and carbon 4 of the next hold pectin chains together. This sequence continues down the chain.
Enzymes are biological catalysts; they alter the speed of reactions without being reactants themselves. There are two theories on how enzymes work. The first is the 'lock and key theory'. This theory suggests that substrate molecule fits perfectly into its specific enzymes active site, and only that enzyme. This can be seen below:
The second theory is the 'induced fit theory'. This suggests that the active site on the specific enzyme changes its shape to accept the substrate molecule. The diagram below shows this:
Pectinase is an example of an enzyme. Pectinase is often formed from more than one enzyme - polygalaturonase, pectinmethylesterase and pectin lysase are normally mixed to form pectinase. These three enzymes can assist the breakdown and modification of pectins from many plant materials. In industry they are used in the processing of grapes, apples and oranges (a citrus fruit). Commercial pectinase may contain cellulase and other cell wall degraders.
Pectin, which is found inside the apple puree I will be testing the pectinase on, can form gels that will bind up liquids. They do this by binding water. However they can only do this when the pectin molecules are very large, for example in fruits such as apples. In fruits pectin naturally functions as a type of 'glue' to help hold the cell wall together. The middle lamella in plant cells, which is between two cells, is densely packed with pectin, indicating that pectin helps hold cells together. This also suggests that digesting pectin by use of enzymes would also make access to the cellulose easier.
this diagram shows the stucture of plant cell, indicating the typical positioning of pectin within the cell - source www.bmc.co.uk
To begin the breakdown to pectin, therefore pectinase must split the bonds between the carbon 1 and the carbon 4 atoms of galacturonic acid, thus shortening the chain into smaller molecules. This type of reaction is known as a catabolic reaction, where large molecules are broken down into smaller ones.
PH is a measure of the number of H+ ions in a substance. There are 3 ways pH can alter enzyme activity. Every enzyme has an optimum pH. This is a pH level that the enzyme works fastest at. For example Peptidase, found in the acidic environment of the stomach has an optimum pH of 2.4, this is a highly acidic pH.
However, if the pH were to change from the optimum level then it would affect the charge on the active site of the enzyme, or the charge on the substrate molecules, in this experiment on the apple puree. Changes in charge on the active site or substrate molecules will slow down the rate at which the enzyme can form the enzyme-substrate complex. The diagram below shows how different pH can alter enzyme activity:
The second way that pH can alter enzyme activity is by changing the properties of the amino acids in the polypeptide chain that builds up each enzyme. Enzymes are just special globular proteins. If we were to look back at the secondary structure of globular proteins we could see that hydrogen bonding is important in the formation of proteins.
Amino acids, that build up proteins, contain both a -CO (carboxyl group) and an -NH (amino group). These groups are charged, -CO is slightly negative and -NH slightly positive, so they attract each other and allow for bonds between amino acids to occur. If pH were to change the charge on one of these groups then where the groups would previously attract each other they would now repel, and this would change the properties of the entire globular protein. Also because the -NH group is a base, it is able to pick up H+ ions, this could change ...
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Amino acids, that build up proteins, contain both a -CO (carboxyl group) and an -NH (amino group). These groups are charged, -CO is slightly negative and -NH slightly positive, so they attract each other and allow for bonds between amino acids to occur. If pH were to change the charge on one of these groups then where the groups would previously attract each other they would now repel, and this would change the properties of the entire globular protein. Also because the -NH group is a base, it is able to pick up H+ ions, this could change the type of amino acid that was in the polypeptide chain, and this would lead to the malfunction of the enzyme.
The final way that pH can alter enzyme activity is by changing the ionization of the substrate. In this is experiment; if the apple puree becomes ionized then it is likely that the reaction will slow down.
Apparatus
Water bath set to 40 C
Water bath set to 100 C
8 x 250cm? beakers
Thermometer
8 x Funnels
8 x 250ml measuring cylinders
5 ml each of Buffer solutions set at PH 3,4,5,6,7
5 x 100cm? apple puree
8 x 1g pectinase
Stopwatch
All this equipment will be needed for each repetition of the experiment
Method
Firstly I will place 100cm? of apple puree into each of my 250cm? beakers, one at a time.
Then I will place 1g of pectinase into each beaker that now contains the apple puree and then add buffer solutions set at different pH to each 6 beakers. Starting with pH3 running to pH7. In the 6th beaker I will place sand as a control, to prove the effects I am seeing are due to pH.
I will then transfer the 6 beakers into my water bath set at 40 C and allow the pectinase to incubate in the apple puree for 20 minutes.
After 20 minutes I will remove the beakers from the 40 C water bath.
I will then transfer the puree into the funnels set up above measuring cylinders; having first placed filter paper inside the funnels. This will stop the large lumps of puree from falling into the final beaker during the filtration, leaving only the fruit juice to filter through.
This process will be repeated for all 5 of the different pH beakers. I wish to repeat the experiment twice, three times if I have time, to try and remove any anomalous results. Due to time constraints I will only measure how much fruit juice is produced from each of the different samples in 5 minutes. This gives adequate time for fruit juice to pass through the filter.
I will take measurements at the beginning of the experiment to make sure I have 100cm? of apple puree. If there were differences in the amount of apple puree then it could lead to variations in the results due to the affects of different amounts of substrate.
I will also make sure I apply the same amount of pectinase to my apple puree. If this is not controlled then the variable of enzyme concentration will not have been controlled. If there were an increased number of enzymes then the reaction would take place faster than the other reactions and I would have made the test unfair as I could not tell if it was the pH concentration, or enzyme concentration that was affecting the rate of reaction.
I must make sure that I add the same amount of each concentration of pH to the 5 different apple puree samples. I plan to add 5ml of each pH to their respective apple puree and 5ml of sand to the 6th beaker. I'm using sand, as it is a neutral substance. If the amounts of pH added are not measured accurately the test will not have been fair.
During the experiment the main measurements to be taken will be during the incubation making sure that the temperature remains constant within the water bath at 40 C, and at the end of the experiment measuring the yields of apple juice produced.
I will arrange my results in three tables (1 for each experiment) as shown below:
Experiment number:
(1, 2 or 3)
PH Concentration
pH3
pH4
pH5
pH6
pH7
Sand
Yield of Fruit Juice/cm3
From the results I record in my pilot experiment in this table I will then decide which pH to measure again in my full experiment. It is vital therefore that my measurements are precise so my decision is not blurred by anomalous results.
Variable
There are 3 variables that must be controlled in this experiment to keep the test fair. The first variable is temperature. If temperature in an enzyme-controlled reaction increases then so does the kinetic energy of the enzyme. This causes the enzyme to move more quickly and increases its chance of colliding with other substrate molecules. Every enzyme has an optimum temperature. However if this optimum temperature is exceed then the extra heat will cause the enzyme to vibrate so much that the bonds that hold its structure together will break! By keeping all the beakers in the same water bath at 30 C then all the enzymes will be working at the same temperature and so this variable is being controlled.
The second variable that must be controlled is the concentration of the enzyme pectinase that will be added to the substrate, apple puree. If you increase the number of enzymes in a solution you increase the number of active sites available to substrate molecules. This means that the reaction could take place faster. It is obvious that this variable needs to be controlled; therefore on in each beaker I will place 1g of pectinase. This means the same amount of pectinase is available to each 100cm? of substrate apple puree.
Finally the substrate concentration must be controlled. If there is a small amount of substrate, the reaction will take place slowly because not all the active sites are in use. However as substrate concentration increases, so does the rate of reaction, until a point is met where all the active sites are in use and cannot work any faster. By using 100cm? of apple puree in all 6 beakers I can be sure that the substrate concentration is being controlled.
Risk assessment
In any experiment there are always hazards. When using pH one must be careful not to get any of the pH solution on their hands. This is due to low pH (i.e. pH3) being acidic and higher pH (i.e. pH10) being alkaline. Acids and Alkalis are harmful to human skin because
When using a water bath there is the risk of scalding. In my experiment I need to boil the beakers containing apple puree and pectinase, along with pH3, 4, 5, 6, 7 or distilled water, to stop the reaction. I must be careful not to scald myself, and take care whilst also being precise with my measurements. The well being of others must be taken into account so that I do not inadvertently scald other students whilst conducting this experiment.
Pectinase is an enzyme and some students may be allergic to some enzymes, they can produce skin rashes or affect eczema. I must take care not to spill any pectinase onto fellow students whilst conducting this experiment.
Social effects of experiment
There are no real social effects from this experiment. Although enzymes are extracted from living organisms this is not of significant effect to those organisms for there to be any problem.
Pilot Experiment
Our Pilot experiment took place on Wednesday 27th February. In my pilot experiment I experimented with a range of Buffer solutions at pH3 to pH7 in a water bath set at 40 C and used sand as my control. I used sand so that I could be sure that all the liquid that filtered through from the apple puree would be a product of the breaking down of the apple puree into fruit juices. If I had used distilled water for my control then the distilled water could have filtered through and given misleading results.
The results from my pilot experiment were as follows:
Experiment number:
Pilot
PH Concentration
pH3
pH4
pH5
pH6
pH7
Sand
Yield of Fruit Juice/cm3
24
28
30
28
25
24
From these results I could see that pH5 or pH6 appeared to be the optimum pH for pectinase to work at. I decided that in my full experiment I would use Buffer solutions at pH3 to give an acidic pH, ph5 to give a near neutral pH, and pH10 to give an alkaline pH. These 3 different buffers gave me what I felt was an adequate range of pH. I decided to continue using sand as my control as it appeared to work well in this situation. I decided that I would repeat my full experiment 3 times as this should remove any anomalous results I might be seeing.
Method (for full practical)
Firstly I placed 100cm? of apple puree into each of my 250cm? beakers, one at a time.
Then placed 1g of pectinase into each beaker that now contained the apple puree and then added buffer solutions set at different pH to each 6 beakers. Starting with pH3, then pH5 and finally pH10. In the 4th beaker I placed sand as my control, to prove the effects I saw were due to pH.
I then transferred all 4 of the beakers into my water bath set at 40 C and allowed the pectinase to incubate in the apple puree for 20 minutes. Once 20 minutes had passed I moved the beakers from the water bath to my desk where I had already set up 4 funnels with measuring cylinders beneath them, ready for the apple puree to be added.
I then transferred the puree into the funnels set up above measuring cylinders. As soon as I had placed the apple puree into each funnel, I quickly and efficiently started the stopwatch and let it run for 5 minutes.
After 5 minutes I removed the apple puree from above the measuring cylinders then measured accurately how much fruit juice had filtered through into each cylinder. I repeated my experiment 3 times in the practical, although I did think about doing 4 repetitions but decided that 3 would provide me with adequate results to draw reasonable scientific conclusions. Each time I took a set of beakers out of the water bath I replaced them with the already prepared next repetition, this allowed me to conduct my experiment in a time efficient manner.
Results
The results of my first experiment are below:
Experiment number: 1
PH Concentration
pH3
pH5
PH10
Sand
Yield of Fruit Juice/cm3
27
35
2
26
The results of my second experiment are below:
Experiment number: 2
PH Concentration
pH3
pH5
PH10
Sand
Yield of Fruit Juice/cm3
29
34
3
23
The results of my third, and final experiment are shown below:
Experiment number: 3
PH Concentration
pH3
pH5
PH10
Sand
Yield of Fruit Juice/cm3
26
34
4
26
After recording all three sets of results I decided to make a table of the average fruit juice production for each pH over the entire three experiment repetitions. The averages are shown in the table below:
Experiment number: Averages
PH Concentration
pH3
pH5
PH10
Sand
Yield of Fruit Juice/cm3
27.3
34.3
3
25
I drew graphs to show the effects of pH on pectinase for my pilot experiment, and my 3 practical repetitions. In the graph that represents the results taken in my pilot practical we can see that the yield of fruit juice produced by the breakdown of pectin by pectinase increases with the increases in pH as I predicted in my hypothesis until it reaches pH5. From pH6 onwards the amount of fruit juice produced began to fall. It is likely this reduction in the amount of fruit juice produced was due to the higher pH that was being experienced by the pectinase. I had predicted this to happen and so was happy to see this confirmed in my pilot practical. From the results of my pilot practical I decided to use only pH3, pH5 and pH10 in my full practical.
In my full practical the results that I took consistently supported my hypothesis that pectinase would operate most effectively in a buffer solution set at pH5. At the more acidic pH3, fruit juice production was only slightly more than that of the neutral sand that I used as my control. However at pH10, a highly alkaline buffer solution, fruit juice yield was very poor, with the average yield a mere 13ml?. The buffer solution set at pH5 produced a good yield consistently over the 3 repetitions of 35ml?, 34ml? and 34ml?. There did not appear to be any inconsistencies throughout my experiment, and all the results that were recorded supported my original hypothesis. It is likely that the highly alkaline buffer, set at pH10, denatured pectinase as it tried to breakdown pectin inside the apple puree, and so the pectin was not broken down catabolically into its subunits, thus giving a poor yield of fruit juice.
Evaluating Evidence and Procedures
The results I took in my experiment were correct to the nearest ml?. This was because of the limitations of the measuring cylinders available. I could not have made my results any more precise because the measuring cylinder was calibrated to measure only to the nearest ml?. It is important to know the potential percentage error from this measuring cylinder. Because the measuring cylinder did not measure decimal places, a reading of 26ml? is correct to ? 0.9ml?. This is because a reading of 26.4ml? would still show as 26ml? on the measuring cylinder due to the way its markers were spaced. To demonstrate the percentage error of pH3 on my pilot practical the working would be as follows:
Percentage Error = error x 100/reading
Percentage Error = 0.9ml? x 100/24ml?
Percentage Error = 3.75%
It is clear that in a classroom experiment the results taken are not as precise as those that can be recorded in the computerized laboratories of top biological research companies. However, this does not mean the results that are taken using the equipment provided are not accurate, and do not show trends and patterns. From using the simple equipment in this experiment I was able to prove my hypothesis to be correct and eliminate any anomalous results by carefully and efficient use of the materials. However if this experiment were to be repeating professionally it is likely that, rather than the mere 3 repetitions I was able to do in the practical time, the repetitions would be around 100, or possibly more even up to 1000 repetitions to fully prove the effects of pH on pectinase.
The results I took proved that extreme pH does affect enzyme activity and that the enzyme Pectinase does have a specific pH at which it prefers to work. From my results I was able to discover this pH to be the buffer solution set at pH5.
To further extend research into the effects of pH on enzymes there are many possible experiments. The breakdown of Pectin by Pectinase is an example of a catabolic reaction because a large molecule is broken down into its smaller subunits. This means that during the experiment there was still time for some of the pectin to be broken down before the pectinase was irreversibly denatured. However research into enzymes on the effect anabolic reactions, where small molecules come together to form larger ones would provide interesting results.
Research into the optimum temperate for an enzyme to operate, would give those in the biotechnologies industry the ideal information for the use of enzymes in for example washing up liquid. It would be very simple to modify the experiment I conducted that looked at the effects of pH, to look at the effects of temperature. If a range of water baths from 20 C to 80 C were used and pH, substrate concentration and enzyme concentration were controlled properly then I could have modified my experiment to look at the optimum operating temperature for pectinase. If chemists can discover the optimum temperature and pH for every enzyme in living organisms then it would give a great insight into reactions within the environment around us.
Currently scientists debate whether the induced fit theory, or the lock and key theory provides the answers to how enzymes work. No one knows exactly which of the two is correct but currently research is taking place into which of the two theories most accurately describes how enzymes operate and interact with their substrate molecules.