A control experiment will also be set up where 5g of apple pulp will be added to 2.5ml of distilled water. A control experiment is necessary to see if it is the pectinase that effects the volume of juice obtained. The results of this control will determine what degree of effect the pectinase has on the apple pulp and the juice produced if any. This control will also be repeated 3 times and an average will be taken for accuracy.
Key Variables:
Independent Variable
This is the variable that is to be changed in order to see the effect it has on the experiment. In this experiment it is the concentration of pectinase that will be changed. This will be achieved by altering the volume of enzyme and adding certain volumes of distilled water. This will be done as shown in the table earlier. I have chosen to use 5.00%, 2.50%, 1.00%, 0.50%, 0.25% and 0.10% pectinase solutions. This is a wide range of concentrations that should give a definite indication of the effect of concentration.
Dependent Variable
This is the variable that the concentration of pectinase will have an effect on. The dependent variable is the volume of juice produced through time. This variable should be solely due to the concentration of pectinase added to the apple pulp.
Control Variables
In order to make the experiment fair and obtain accurate results, certain factors must be kept the same. These are the factors that would effect the experiment if they were not controlled.
Enzymes work at optimum temperatures and therefore work better at some temperatures than they do at others. A high temperature like 60ºC is enough to stop the enzyme working at all. This is because heating changes the shape of the enzyme molecules and prevents them from working (denaturation). Temperature effects the rate at which an enzyme operates and therefore needs to be controlled when investigating the effect of concentration. Using a water bath and a stopwatch will do this. All the test tubes will be placed in a 40ºC water bath for exactly 20 minutes. This will prevent significant changes in temperature between the test tubes. The filtering process will also be carried out as soon as the test tubes are removed from the water bath.
In this experiment the substrate is pectin, which is found inside the apple pulp. Therefore in order to keep the substrate concentration as constant as possible, the same mass of apple pulp must be used inside each of the test tubes. Using an electronic balance to weigh out exactly 5g of apple pulp will do this. A higher substrate concentration in one test tube will increase the rate of reaction and therefore more juice will be produced from the apple pulp.
Every enzyme also has its own pH range. Excessive acidity or alkalinity could denature them and therefore effect the rate of reaction. The pH of the enzyme will not be altered and therefore the pH will be standard for all the experiments.
- Enzyme-Substrate Contact Time
Another factor that will effect the volume of juice produced from the apple pulp is the length of time the pectinase is in contact with it. If one test tube was left to stand for 10 minutes longer with the pectinase in it than another test tube, then it will have more time to react and therefore more pectin will be broken down. In order to eliminate this factor from effecting the experiment, the test tube will be placed immediately into the water bath after the pectinase is added. Another factor linked to this is the agitation of the pectinase solution and apple pulp. If one test tube was shaken and stirred vigorously and one was not, the energy given to the molecules would be higher and therefore they would have more kinetic energy to react and the rate of reaction would be quicker. In order to combat this factor, the test tube will be carefully handled and each experiment will be stirred gently with a glass rod for exactly 10 seconds.
Apples contain certain chemicals that act as active site-directed inhibitors of pectinase. This means that it competes with the pectin substrates for the active site of the pectinase molecules. This therefore prevents the pectinase from functioning as required and therefore the rate of reaction will be affected. These inhibitors will reduce the volume of juice produced and more inhibitors in one test tube than other will also effect the reliability of the results. In order to minimise the effect of this factor as much as possible, the apple pulp will be left to stand for 10 minutes before the pectinase solution is added. This is because the chemicals that contain the inhibitors lose their effectiveness when they are oxidised by the air. The rest of the apple pulp will then be covered with clingfilm in order to prevent any more oxidation while the experiment is carried out.
The surface area of the pieces of apple pulp will also effect the rate of reaction if it is not kept constant. A larger surface area will mean that there are more exposed substrates of pectin and therefore more successful collisions with the pectinase molecules can take place, which in turn increases the rate of reaction. This is a difficult factor to control as the pieces of apple pulp are very small. In order to reduce this factor as much as possible, the apple will be blended for exactly 10 seconds on the lowest setting in a food processor. All the apple pulp will be made at the same time for all the experiments so variation is eliminated.
In order to reduce other factors such as pressure, light and atmospheric conditions from interfering with the results of the experiment, the final main experiment will be carried out on one day. This should reduce the size of these factors as much as possible.
Safety/Risk Assessment:
It is extremely important to wear a labcoat and safety goggles at all times during the experiment. This is because enzymes are being used and therefore any spills onto skin or into eyes can cause severe damage. Pectinase should especially be handled with care, as it is a proteolytic enzyme that irritates skin, eyes and mucous membranes. This also means that unnecessary equipment such as bags and coats must be put well out of the way to avoid trips and accidental spills. Careful attention must be paid when using the knife, as it is very sharp and could cause severe pain. The juice obtained from the apple pulp should not be consumed. This is because it has not been treated to remove harmful bacteria and the equipment used is not hygienic enough. The apples used in the experiment will be bought from a supermarket that sells them for consumption. This will be done instead of picking apples from any trees that could lead to effects on organisms’ habitats and other wildlife. The food processor must also be operated carefully following any instructions directly and the blade should also be handled carefully. Overall this experiment is fairly safe if all the above risks are adhered to and correct conduct is exercised during the experiment. It is also important to clean up any spillages as soon as they occur and make sure apparatus is left in safe areas after the experiment is finished.
Pilot Experiment:
In order to make sure my outline method is sufficient enough to produce a range of results and that the experiment can be carried out safely and accurately, a pilot experiment was done. During this experiment it was hoped that any errors or improvements to the method were found out. The experiment was only done once for each concentration in order to obtain a rough set of results, however the main reason was to improve the method. Before carrying out this experiment it was evident that the apple needed to be peeled before starting. It was also noticed that while making the apple pulp, juice was being produced and that this needed to be drained away before adding the pectinase solution. This is because the experiment is designed to find out how much juice is produced from the apple pulp itself. This is probably the reason for the fairly high volumes after 5 minutes in the table below. Using filter paper and a beaker will drain the excess juice away. Whilst watching the test tube in the water bath, it occurred that some of the apple juice being produced could be evaporating away and therefore some sort of seal needed to be put over the test tube. A small sheet of clingfilm will be placed over each test tube.
Results of Pilot Experiment:
This is a table of the results obtained in the pilot experiment:
Having done the experiment and looked at the results obtained, it was noticed that after 5 minutes there was still some unfiltered juice left in the funnel. In order to find out the volume of all the juice produced by the apple pulp, a will be used to suck out all of the remaining juice and the final volume of the juice will be noted. This piece of apparatus was chosen to finish off the experiment, as there is only a limited amount of time in which to complete the experiment and this will avoid waiting until all the juice has filtered through. This final volume will not be needed in order to calculate the initial rate of reaction, but is worth recording in order to see how much juice the apple produces and if the method is successful in producing apple juice.
Detailed Method:
First of all, one ‘Bramley’ apple is carefully peeled using a potato peeler and the core is removed. It is then carefully chopped up into small pieces using a knife and a tile. The small pieces of apple are placed into a food processor and mixed for exactly ten seconds on the lowest speed. The contents are then put into a mortar and ground up using a pestle. This is done until an apple pulp is produced. The apple pulp is then poured onto a filter paper with a funnel and beaker. The excess juice is filtered out and the pulp remains in the paper. The apple pulp is then left to stand for exactly 10 minutes. A test tube is weighed using the electronic balance and 5 grams of the apple pulp is weighed inside it, using a spatula. 2.5ml of 5.00% pectinase is then syringed into the test tube and mixed gently using a glass rod for exactly 10 seconds. A small sheet of clingfilm is placed on the test tube to cover it and prevent the loss of any juice. The test tube is then placed in a 40ºC water bath for exactly 20 minutes. Meanwhile a 5ml measuring cylinder with 0.1ml intervals, filter paper, and funnel is set up to collect the apple juice produced from the pulp. After 20 minutes, the test tube is removed from the water bath and the contents are emptied onto the filter paper and funnel. A stopwatch is started once the test tube is empty and the volume of juice produced is measured every thirty seconds. This is done for 5 minutes. After 5 minutes, the filter paper is placed on the and the pump is squeezed until no more juice enters the conical flask. The volume of the juice is added to the volume in the measuring cylinder. 5 grams of apple pulp is then measured out in the same way but this time added to 1.25ml of pectinase and 1.25ml of distilled water to make a pectinase concentration of 2.5%. The rest of the experiment is carried out in exactly the same way. This is done for each of the different concentrations according to the table shown in the outline method. The whole experiment is then repeated 3 times and an average for every 30 seconds is taken for each concentration. The times of putting the test tubes in the water bath are carefully staggered in order to be more efficient with time.
This experiment is carried out with all the apparatus thoroughly washed with distilled water and the pectinase syringe prewashed with 5% pectinase. A labcoat and safety goggles are also worn at all times and careful attention is paid to the safety and risks mentioned earlier.
Results:
These tables show the results obtained from the experiment:
5.00% Pectinase
2.50% Pectinase
1.00% Pectinase
0.50% Pectinase
0.25% Pectinase
0.10% Pectinase
Control Experiment (2.5ml distilled water)
This is a graph to show the above results more easily.
The average volumes for every 30 seconds have been plotted for each concentration, up to and including 300 seconds.
Here is a table of the rate of the rate of reactions for each concentration. The rate was calculated by finding the gradient of each of the lines in the above graph. A computer did this in order to obtain the most accurate results as possible. The lines of best fit are not shown above for clarity.
This is a graph of the results in order to easily find any trends and patterns.
The tables for each concentration and the first graph clearly show that a greater volume of juice is produced in the first five minutes at a higher concentration of pectinase. The average volume produced after 5 minutes using 5.00% pectinase was 2.93ml, whereas the equivalent result for 0.25% pectinase was 1.20ml.
The total volumes achieved for each concentration show that this trend continues. All these volumes do not of course contain totally pure apple juice. The volumes all contain some of the pectinase solution that was added to the apple pulp. However the volume added was kept constant at 2.5ml and therefore the differences in volumes are due to the concentration of pectinase alone. The graphs for 5.00%, 2.50% and 1.00% show most clearly that the volume obtained rises in a linear fashion with time. However it is also clear that at approximately 240 seconds, the graph begins to taper off. Slight resemblance to this trend can also be seen in the other concentrations.
These graphs and tables alone do not show the rate of reaction for the five minutes. This is of course necessary in order to answer the hypothesis. Therefore a line of best fit and the gradient of that line was calculated was on the computer. The gradient gives the rate of reaction. These tabulated results were also graphed in order to make it easier to spot trends and patterns. The second graph shown above shows an almost straight line that indicates enzyme concentration is directly proportional to the rate of reaction. The graphs only just started to taper off for each concentration and therefore it was reliable to take the gradient of the line of best fit to find the initial rate of reaction.
There are some anomalies and inconsistencies in the tables and graphs produced above. It is clear that for the 2.5% pectinase solution there are slight inconsistencies in the volume produced at 30 and 60 seconds. This is also true for the graph for 1.0% pectinase. Slight anomalies can also be found elsewhere. The anomalies in the first graph will have an effect on the second graph and therefore explains why there is not a definite straight line. The anomalies obtained in this experiment will be explained later on in the evaluation.
As mentioned earlier, pectinase degrades pectin to form shorter molecules of galacturonic acid. It works by breaking the bonds between the galacturonic acids to shorten the pectin chain into many smaller units. Pectin forms a gel in the cell walls of plants that holds together cellulose microfibrils. As this pectin disintegrates, the juice that is eventually secreted becomes thinner and therefore more juice can be extracted. This is how the addition of pectinase causes the apple to produce more juice.
The results confirm that the hypothesis stated earlier is correct. It is clear that as the concentration of pectinase increases, the rate of reaction also increases. In the reaction that takes place between pectinase molecules and the pectin inside the cell walls of the apple, the pectin molecules bind with the pectinase to form an enzyme-substrate complex. This reaction takes place and then the product leaves the enzyme, which in this case is the smaller units of galacturonic acid. Each enzyme molecule has an active site that allows the substrate to combine to. Due to the tertiary structure of the pectinase molecule, its active site has a certain shape. This shape is complementary to that of pectin and therefore these two molecules are able to fit together. It is all this that means an increase in enzyme concentration has an effect on the rate of the reaction.
Increasing the enzyme concentration effectively means that there is an increase in the number of enzyme molecules in the same volume. This therefore means that there is now an increase in the number of active sites present. If there are more molecules in the same volume, this means that there is a greater chance of the pectin molecules and pectinase molecules colliding. It is now established that a higher concentration of enzymes gives them more of a chance to collide with the substrate molecules. Not all collisions between the substrate molecules and enzyme will be successful, as they must be in the right orientation when they collide and also at the right speed. However more collisions in turn means that there will be more successful collisions. The increase in the number of active sites gives a greater chance for the pectin molecules to bind with them and therefore increase the rate of reaction.
It is now clear that an increase in enzyme concentration brings about an increase in the rate of reaction. However this only happens if there are enough substrate molecules to begin with. If there were plenty of enzyme molecules and not many substrate molecules, then after all the substrate had been used, the rate of reaction would slow down and eventually cease altogether. This was not the case in this experiment as the rate of reaction was still rising significantly with 5.00% pectinase.
The first set of graphs showing the volume produced for each concentration shows that the graph slightly tapers off towards the end of the 5 minutes. This is because the enzyme was starting to have had its full effect on the substrate molecules. At this point the number of pectin molecules binding with the enzyme molecules was starting to decrease. This means that less pectin can be converted into shorter units of galacturonic acid and therefore the rate at which the juice could be extracted began to decrease.
This is a general graph showing the effect of enzyme concentration on the rate of the reaction:
This graph shows that with ample substrate molecules, the enzyme concentration is directly proportional to the rate of reaction. This means that doubling the concentration of the enzyme, doubles the rate of reaction.
Evaluation/Limitations:
The method chosen to investigate the effect of concentration on pectinase had some limitations and could be improved. The results obtained are not entirely accurate and therefore the experiment can be questioned on how reliable it is. The degree of accuracy can clearly be seen from the two graphs produced. The first graph shows a general decrease in the volume produced compared with the concentration of pectinase. However the differences between the lines produced shows that there are some anomalies in the results obtained. The result obtained for 2.50% pectinase is slightly higher than are expected. This is then reflected in the second graph where the rate of reaction for this concentration is also slightly higher than the straight line that theoretically should be obtained. The other concentrations below 2.50% pectinase also show a value slightly higher than the theoretical values in the rate of reaction graph. As well as slight overall anomalies, there are also individual recordings that appear to be anomalies. These can be seen in the first minute of most of the concentrations. For example, the earlier readings taken when using 1.00% pectinase seem to be slightly too high and this is also evident for 0.25% and 0.10% pectinase. These anomalies show that some of the data is inconsistent, however the extent of these anomalies is not so great as to effect the validity of the experiment. Therefore the conclusions drawn from these results are still justifiable. There are improvements that could be made to this experiment in order to improve the consistency of the results, however this involves more repeats and maybe additional experiments. Overall these slight anomalies proves that one single experiment is not particularly useful in drawing accurate and fully confident conclusions.
Having seen that improvements to the method could bring about more reliable data and fewer anomalous results, it is important to see where and how these improvements can be made. One reason why some of the individual recordings may be slightly high is because of the excess juice that filtered out quickly as soon as the test tube was emptied into onto the filter paper. Another reason could be that the time at which the stopwatch was started meant that it was more than 30 seconds of juice obtained. The stopwatch was started after all the contents of the test tube were emptied, which took a few seconds and during this time juice was filtering through. Therefore an improvement to this part of the method would be to find a way of emptying the test tube in one go. Using a slightly bigger test tube or even a boiling tube could do this. The higher than expected values for the rate of reaction could also try and be rectified by increasing the length of time that measurements were taken. This would have provided more data and therefore the graphs and rate of reaction would be more accurate. However this could only be done with more time in which to carry out the experiment. A more general improvement to the experiment would be the precision of the apparatus used. Although the apparatus used was the most accurate available, a more accurate water bath could have been used and also a clearer and more accurate measuring cylinder.
Further work to do with this investigation and exploring other factors relating to this experiment can also be carried out in order to add weight to the conclusions made earlier. It is possible to go on and look at the effect of temperature on pectinase and also substrate concentration, however the latter would be fairly difficult to measure. An investigation to look at the effect of pectinase on other fruits such as plums or grapes or investigate changes in concentration of other enzymes such amylase or catalase. These investigations would extend the enquiry of the enzyme pectinase and also into the substrate it is having an effect on.
