by bill barber
Aim: To investigate how the enzyme activity of Pectinase is affected by different temperatures when digesting the Pectin present in Apple Pulp.
The enzyme Pectinase has been used in the production of fruit juice for three decades; this is because it effectively digests the Pectin, found in the fibres of Apple Pulp. This pectin is located mainly between the plant cells, however it can also be found within both layers of the thin cell walls. The primary layer contains the fibres of cellulose, which holds the Pectin, requiring the use of the enzyme Pectinase to burst the cell and release the Pectin.
In order to do this the apples must be crushed in a mill to extract as much juice as possible before the pulp can then be stirred for around 20 minutes to oxidise the enzyme inhibitors. These inhibitors are naturally occurring and will generally slow the reaction. The pulp can then be heated to its optimum temperature of 30oC when Pectinase is used, and the enzyme activity would be expected to be at its most efficient. In the absence of Pectinase, a higher temperature would be required, usually of around 55oC. The Pectinase and Apple Pulp can then be incubated, allowing the juice to flow freely, however the age and brand of the apple will affect this.
There are many variables which must be controlled if the experiment is to be fair and provide accurate results, these include:
·Ensuring the enzyme and concentration of it is consistent in each of the experiments, as this will have an effect on the overall result. In my experiment I will use only the enzyme Pectinase, however I will also use distilled water, which will act as a control. When large amounts of the enzyme is added in one experiment, compared to that used in another, the rate of reactions will be very different and will therefore become unreliable.
·The amount of Apple Pulp I use in each experiment must be identical as larger amounts would mean a larger quantity of Pectin present and only the same amount of Pectinase to digest it as there is in another experiment with much less Apple Pulp, assuming that variable was controlled. This could mean Pectin still remains in the pulp, due to the lack of enzymes available to digest it, affecting the final results.
·The age and brand of apples used. Some varieties of apples contain Pectin, which is more difficult for the Pectinase to break down and digest, so using a number of brands would negatively affect the results, as they are more likely to differ. Also, the older the apple pulp is, the easier it is to obtain juice, due to the Pectin becoming progressively soluble and softening the tissue. Another factor, which could affect the results, is the place of storage of the apples and if they are tinned or not.
·The amount of time the Pectinase and Apple Pulp are incubated for must be controlled. Each experiment must allow the temperature to remain constant for the same amount of time to ensure the enzyme activity can be recorded accurately. If this condition was not controlled, the Pectinase may not be provided with enough time to digest all the Pectin and so there may be traces of the carbohydrate remaining. The beakers should be incubated for around 15-20 minutes each to ensure the enzyme has broken down the insoluble Pectin and also degraded the soluble Pectin in the Pulp. This encourages the flow of juice as breaking down the cellulose means the cell walls can no longer function and disintegrates.
·The temperature of the incubators will have a huge impact on the rate of reaction and enzyme activity. An optimum temperature provides energy for the enzymes to function at their best due to them being specified for a particular function in certain conditions, and by using a range of temperatures, as I will do in my experiment, I will be able to prove which temperature is most suited for the Pectinase to digest the Pectin and which temperatures cause the enzyme to denature.
·The PH used in the experiment will also affect results as the enzyme Pectinase has an optimum PH of around 6. If a PH above this was used, the Pectinase may become denatured as it is unable to function in extreme conditions, however if the PH was lower than this, the rate of reaction is most affected; as the PH increases, remaining below the optimum, the rate of reaction does too.
·The method used to measure the fruit juice at the end of the experiment. A burette or measuring cylinder can be used, but only one should be chosen as they have different levels of accuracy. The burette is much more accurate and clearer to read.
In addition to this, in order to ensure a fair test, I must avoid contamination of the equipment and materials I will be using. To do this I must keep the apparatus clean, using distilled water if I need to reuse the equipment, as this will limit the chance of transferring enzymes from one experiment to another for example.
Due to the high concentration of the enzyme I am using in the experiment, and because of the preparation of the juice itself, it should not be consumed. Much smaller concentrations are used on the size apple pulp that I will be using in commercial Apple juice production, and so in my experiment the Apple juice produced will be poisonous. I will also have to ensure care is taken when using the knife to chop the apple. The enzymes should also be handled carefully to avoid spillages, however if these occur, they should be promptly wiped away and distilled water used to wash the area thoroughly in order to avoid contamination.
From the experiment I have planned, I predict that the largest producer of Apple juice will be the Apple Pulp with the oxidised inhibitor and Pectinase at a temperature of 30oC. I have predicted this as the optimum temperature of Pectinase is 30oC and so the enzyme will be working at its most efficient, producing a larger yield of juice. The natural inhibitor in the Apple Pulp will be present in two of my three the experiments- containing only Pectinase and the other with only distilled water, which will slow down the rate of reaction as an inhibitor will block access to the enzymes active sites. However, this won’t be the case when the inhibitor is oxidised in the last of my experiments. I think the Apple Pulp and distilled water will produce only a small amount of fruit juice as there will be no enzyme present to act as a catalyst in the reaction and a natural inhibitor will also be present. I think the experiment with the inhibitor and Pectinase will produce more Apple juice than the beaker containing the distilled water, but not as much as I predict will be produced by the Pectinase and oxidised inhibitor. This is because the inhibitor in this beaker will be successful in making the reaction slow down, however I think the rate of reaction will increase as the temperature does, slowing down when it exceeds that of the optimum temperature. I think this will be similar in the beakers containing Pectinase and the natural inhibitor and Pectinase and the oxidised inhibitor. On the other hand, as there is no enzyme added to the distilled water, I predict the rate of reaction to increase very slowly, continuing even when the others have slowed or stopped due to denaturing enzymes, and produce more juice as the temperature reaches 55oC.
150g Apple Pulp
Water baths at temperatures 10oC, 20oC, 30oC, 40oC and 55oC
Clamp and Stand
1.Use the knife to cut the Apple up into smaller pieces and weigh it out to be slightly more than I need before putting it in the blender.
2.Use the balance to measure the weight of the beaker I will use to hold the chosen amount of Apple Pulp. This will be recorded and the balance ripped. I will then weigh out the correct amount of Apple Pulp by placing it in the beaker. The first weight recorded will then be deducted form the second weight to find the exact weight of the Apple Pulp.
3.Using a pipette, I will measure out the correct amount of Pectinase and add this to one of the beakers. Using a different pipette I will add the same amount of distilled water to another beaker of Apple Pulp. I will stir the last beaker with a glass rod for 15-20 minutes to ensure the inhibitor is oxidised, before adding Pectinase.
4.In turn, I will incubate each of the beakers, wrapped in cling film, at one of the temperatures provided, using ice to lower the temperatures from that of the room for around another 15-20 minutes.
5.After that time, I will use a burette, filter and filter paper to accurately measure the amount of fruit juice produced.
6.This will be repeated for each beaker three times in each of the temperatures available in order to find an average yield.
7.Each time the burette is used, I will use the stop clock to record how much juice is produced at certain intervals, every three minutes.
I have chosen to use this method in my experiment as I think it will be the most effective. Although the filter paper is expected to absorb a small amount of the juice that will be produced, this will be used in each experiment to ensure a fair test. The use of the filter will also mean it is able to sit in the burette, whereas other methods may require the equipment to be