Investigation into Pectinase

Plan:

An Enzyme is a biological catalyst. This particular enzyme, pectinase, provides an alternative ‘route’ for the reaction that has a lower activation energy ‘barrier’. Each type of enzyme has a specific 3D shape, which exactly fits the substrate with which it is reacting. The location on the enzyme where the substrate binds and the reaction takes place is known as the active site. This releases the product without damaging the enzyme so it is therefore ready to be used again. This is what happens with the enzyme when it meets and reacts:

Purified pectinase is a multi-component preparation highly effective in depolymerising plant pectin’s with varying degrees of esterification. Important enzymatic activities include pectin lyase (EC 4.2.2.10) and activities on polygalacturonic acid (pectin) and nonmethylated polygalacturonic acid (pectate). The result of adding Pectinase to fruit pulp means that the volume of juice taken from the fruit is increased. This is the result of the Pectin’s holding fruit cells together like a chemical ‘glue’. This means adding pectinase to your fruit pulp means you can extract more fruit juice than without it.

A number of factors affect the rate at which an enzyme works: concentration, temperature etc. PH levels also affect the enzyme, if the level is too acidic or alkaline the 3D shape of the enzyme is altered because the conditions are too corrosive for it to keep its shape. This deems the enzyme unable to react with its specific substrate because the two shapes will no longer be able to fit together because of the one altered shape.

I wish to investigate how changing the PH affects the working of the enzyme pectinase in the breaking down of fruit pulp, in this case apple and what the optimum PH level is for this particular job. I have carried out two preliminary experiments to help me decide which concentration of pectinase to use and which PH’s to use.

Experiment one:

I put 40g of apple pulp into each of three beakers. I then added 2cm³ of pectinase (5%, 20%, 100%) into three different beakers, to the pulp and stirred the mixture well. I then incubated the beakers in a water bath at 40ºC for 15 minutes and where then filtered the apple pulp in order to get the measurement of the juice. I then recorded the amount of juice produced at one-minute intervals for a total of 5 minutes.

The results are shown below:

The results of this preliminary experiment show that 100% concentration of Pectinase, ‘working enzyme’, worked most effectively. It produced the most apple juice from the fruit pulp. However, the lower concentrations of 20% also worked well and so did the 5 %. Considering 100% concentration is extremely expensive and can be potentially dangerous and 20% works just as well seeing as it is a fifth of the 100% concentration but produces higher results than a fifth I shall use it in my experiment.

Experiment two:

I repeated the exact experiment, but adding 2cm³ of a buffer as well as the enzyme to each beaker of apple pulp.

The results of this experiment are shown below:

These results show that pectinase works most effectively at PH 2. From my research I have found that the optimum PH for main pectinase activity is usually in the acidic range of 4.8 to 5.0. Although the results are quite similar extensive research has shown that the optimum is 4.8 to 5.0.

Hypothesis: The PH level will affect the amount of juice ...

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Experiment two:

I repeated the exact experiment, but adding 2cm³ of a buffer as well as the enzyme to each beaker of apple pulp.

The results of this experiment are shown below:

Hypothesis: The PH level will affect the amount of juice extracted from the pulp.

Prediction: Using the information from my experiments, secondary sources and my preliminary experiments I predict that PH5 will be the optimum PH for pectinase in apple pulp.

Method:

Equipment List:

20% pectinase enzyme
10 pieces of filter paper
2 funnels
Buffers of the following PH’s: PH2, PH4, PH7, PH9.
2 measuring cylinders
2 stop clocks
5 syringes
Distilled water
Apple pulp
Water bath
2 clamp stands
2 glass rods
10 beakers

I have come to the decision about the PH’s because of my preliminary experiments. My results showed me that optimum was around 5.00. Because I know that the optimum is 4.8-5.0 I wont got too far out of this range. This is an acidic optimum so I started at 4 and went up to 9 which is more acidic. This meant that I would have a wide range of results.

Diagram of how my apparatus will be set out:

I am going to repeat each PH twice so I can increase the accuracy of my results. If time allowed it I would test on a wider range of PH’s and treble check them etc. So I will complete my experiment as follows considering I have restrictions on the amount of time I have and the amount of apparatus and the accuracy of it:

I will stir the apple pulp to equally spread the juice throughout and then place 40g of apple pulp into two beakers. I will then add PH(*) to both beakers as I am backing up my results with two results. Then I will add 2cm³ of 20% pectinase enzyme so that the enzyme has not had time to work before there is buffer in the apple mixture. I shall then stir it 5 times with a glass rod. Both beakers will then be placed in the water bath at 40ºC and the clock started. The beakers will be kept in these conditions for 15 minutes and nothing will be changed. I shall then set up the filtering apparatus. I set up two stands, both holding funnels. I then fold the filter paper into cones and place them in the funnel. I then dampen them so they stick to the sides with distilled water and let them drain so no excess water gets into the measuring cylinders. The measuring cylinder will then be placed underneath the funnel, which will collect any produce from the apple pulp. I shall also prepare the next two beakers changing only the PH’s. Once the beakers in the water bath have been incubated for 15 minutes they will then be removed and the contents placed into both funnels so that the paper can get rid of any fruit pulp and just allow the juice to get through to the measuring cylinder. I will then record the amount of juice produced in the measuring cylinder at one-minute intervals for a total of 5 minutes because after this point the amount of juice being produced decreases and significantly slows down. I asked two other people to help me with this. I asked one to write down the results and watch the stop clock and the other to observe the other measuring cylinder. This helps the results to be more accurate.

In order ensure the accuracy of my results I must make sure that only the independent variable changes (the PH) and that everything else stays exactly the same. Therefore I must make sure that the same amount of apple pulp is put into each beaker, that all the beakers are incubated at the same temperature for the same period of time and that the same amount of pectinase and buffer is added to the apple pulp (2cm³).

To make sure I have accurate results I must use the equipment as accurately as possible. For example, when weighing out the 40g of apple pulp I should place the beaker onto the scales first, and then press the ‘tear’ button to return the measurement to nought and then add the apple. That way, my results are more accurate since I’m only measuring the apple pulp this way.

These might also affect the accuracy in my results. For example, there is a time gap between the enzyme and buffer being added to the pulp and the beakers being placed in the water bath. This means it will affect my results. As it will be difficult for me to stop and start the clocks at the accurate times, I will acquire some help from some fellow students which should help me be more accurate. Also the apple pulp mixture tends to stick to the side of the beaker so I would not be able to get everything into the filter paper, which would affect the results. As I do my experiment I will also look out for things that will affect my results.

Because Pectinase is an enzyme I will need to be very careful when working with it. I shall clear my table of books etc. and make sure stools and bags are tucked in so they are not in the way. I shall wear a lab coat, goggles and gloves to avoid getting the enzyme on me. If any does get on me I will wash it off and alert my teacher and also alert her if there is a spillage because this is potentially dangerous to the rest of my class.

I shall now conduct my experiment following the method above and considering all of the points I have made to make my experiment more accurate and successful.

Analysis

The graph I have produced demonstrates the trend in the results. It begins at PH2 with the average amount of juice being produced per min to be 1.8cm³, it then rises to 1.9cm³ at PH4 and reaches a peak at PH5 of 2.5cm³. Then, as expected the amount of juice produced per min falls to 1.96cm³ at PH7 and then sharply rises to 2.68cm³ at PH11. However, I state the result at PH11 to be anomalous, since it does not agree with the trend present in the graph, the prediction I made in my plan or other information I have collected. This graph clearly demonstrates an optimum at PH5 of 2.5cm³.

I already know that PH levels affect the rate at which enzymes work. Too acidic or alkali a PH can permanently distort the 3D shape of the enzyme therefore destroying it’s ability to react with it’s specific substrate. At PH5 however, conditions are correct for the largest amount of juice to be produced, the enzymes shape is not altered meaning the substrate can continue to react with the Pectinase and produce a large amount of juice at a faster rate. The PH levels are also known to change the binding of the substrate to the enzyme, changing the properties of amino acids involved in the catalytic activity of the enzyme and altering the ionisation of the substrate. At the optimum PH of Pectinase i.e. PH5, the enzyme is under the correct conditions, considering all other factors that effect enzyme activity are correct, and these functions can be correctly carried out.

EVALUATION

During the experiment I experienced a number of difficulties and unfortunately my results were not entirely accurate. I predicted the optimum PH of the enzyme Pectinase to be at approximately PH5, however my results demonstrated that the optimum was PH11. However, I assume there was a fault in my method due to the fact that there is a significant difference between the results of trials ‘a’ and ‘b’ for PH5. Trial ‘a’ resulted in 14cm³ of juice produced after 5 minutes, and trial ‘b’ resulted in only 11cm³ of juice produced after 5 minutes. This is a major fault, which would have greatly affected its results in comparison to the other PHs and the investigation as a whole. This error could have been due to a number of reasons, which are described below.

I found it difficult to manage the time I had been provided with, and often found I didn’t have enough time to complete the experiment accurately. I often put two groups of different PHs in the water bath with only a small time period between them. This meant it became increasingly difficult when it came to measuring the amount of juice; due to lack of apparatus it had to be thoroughly washed before the next PH could be recorded. Therefore, I discovered that at times I was left with only a minute or so to clean the recording apparatus before the next PH was due to be removed from the water bath and put into the funnels to be recorded.

Due to the pressure of time I often found myself under, the apparatus was often not correctly set up, for example I found for one PH that as I placed the apple pulp in the filter, the measuring cylinder was not placed under to collect the juice. This would have greatly affected the results I collected, as a large amount of juice would have been lost.

The water bath was also an issue. When I first started the experiment, the water bath was only at 33°C when it was supposed to be at 40°C, it then progressively increased in temperature so when I placed the apple pulp at PH11 in the bath, it was significantly warmer than the temperature at which the others had been in. As I know that temperature is one of the factors, which affects the rate at which an enzyme works, this would have greatly affected the final results.

There were also other difficulties when recording the amount of juice being produced. It was difficult to synchronise the two beakers of apple pulp being placed into the filters at the same time, this was due to lack of equipment meaning I had to use one stop watch for the beakers of each PH. This meant that one beaker full of pulp might have been put into the filter before the other. It was also difficult to start the stopwatch at the accurate time, however I tried to consistently start it when as much of the pulp had been removed from the beakers as possible. However, obviously there is a natural delay between starting the watch and the moment all the apple is removed, but because I was already holding the beaker and glass rod it was delayed further, because I had to place these on the bench in order to start the clock.

However, apart from the major points above, all other aspects of the experiment I completed sufficiently. I took all safety precautions i.e. I wore a lab coat and goggles at all times and wore gloves when handling syringes containing enzymes and PH solutions since both are irritants. I consistently weighed the apple in the same way; added ph solutions and enzymes to the pulp of both beakers at the same time and stirred the same amount of times for each PH. I was always to place and remove the beakers from the water bath at the same time as to make my results more accurate.

However, if I had the opportunity to repeat the experiment again, I would do a number of things differently. I would have made sure I had a sufficient amount of time to complete the experiment and enough apparatus to carry it out accurately. Ideally, I would have completed each PH at a time, but due to restrictions this was not possible. However, despite this fact I could have arranged my time more effectively to produce more accurate results.

The results I collected were not as I expected, they showed that the optimum PH for Pectinase is PH11, however I know this is incorrect. And as I explained in the first paragraph this is probably due to the fact that trials ‘a’ and ‘b’ for PH5 were significantly different therefore I have chosen to pronounce the results for PH11 anomalous.

Due to the number of faults in my method as described above, some of my results may not be sufficiently accurate or reliable. I could have completed the experiment at a much higher standard to produce more precise evidence, however due to lack of time and equipment this proved difficult.