Investigating The Use of Pectinase

Investigating the use of pectinase in the

Production of fruit juice.

Introduction/Background Information

Enzymes are proteins, which act as the acceleration devices of biochemistry. This means that they are used to catalyze (speed up) reactions. Enzymes are specifically designed for a target substrate, according to their size, shape and chemical charges that precisely bond to its substrate molecule. The section of the enzyme that binds to the substrate is called the active site and an advantage of enzymes being so specific is that they will not interact with or disturb other components present during the reaction. The diagram shows the reaction between pectinase and pectin and also how they are specific to each other.

The enzyme that I will be using during my investigation is pectinase, which is the specific enzyme used in the breakdown of pectin, a polysaccharide substrate found in the cell walls of many plants.

In plants the pectin naturally functions as a glue to hold the cell wall together. Pectins are large polysaccharide molecules, similar to starch except that the repeating unit (residue) of pectin is galacturonic acid instead of the glucose in starch. Moreover, pectins are made up mostly of chains of several hundred galacturonic acid residues. This causes fruit cell walls to have a complex structure of interwoven polymers, which no single enzyme can fully break down. The complete breakdown of a cell wall would most likely need a complex cocktail of carbohydrase enzymes. Commercial pectinase is usually a mixture of enzymes such as polygalacturonases and pectin lyases. Furthermore, pectins are known to be very good at binding water, so, for example in jam or jelly, pectin is what makes it set. In the structure of the cell, the middle lamella, the part between neighbouring cells is rich in pectin, this means that pectin also helps to glue cells together.

Pectinases are obtained commercially from fungi, particularly Aspergillus. Pectinase is in a particular class of enzymes and is known as a hydrolase, these are enzymes that are present when catalyzing reactions between water and a substrate. During a process like this water is bonded to certain molecules and molecules are also broken up into smaller units (as shown in diagram).

Simply, pectin, along with cellulose and proteins, is found in the cell walls of such fruits as apples and oranges and the enzyme pectinase breaks down pectin. So it makes sense to say that pectinase breaks down plant cell walls, which, evidently produces more fruit juice, this way, they are widely used in the fruit juice industry, where they are commonly used to help extract, clarify and modify fruit juices.

The commercial uses of pectinases include the clarification of wines/juices and aiding the disintegration of fruit pulps. This is done by reducing the large pectin molecules into smaller units and the eventually into galacturonic acid, here, a compound has been made soluble in water.

Factors affecting the enzyme pectinase

Changes in temperature can affect pectinase; this is because all molecules are in motion, except when at absolute zero. For example, as the temperature increases, the kinetic energy of the molecules has been increased. So, in the case of enzyme catalyzed reactions, the speed of both the enzyme and substrate molecules have been increased. As this happens, the chances of collisions forming enzyme-substrate complexes also increases, this an increased rate of reaction, which would result in more pectin being catalysed in a given time, therefore leading to an increased rate of juice production. However, the temperature can only increase up to a certain point called the optimum temperature. For most enzymes, this is generally below 50°C and around 35°C, as the graph shows. Beyond this point, the rate of reaction begins to decrease. This is because an increasing temperature causes more and more hydrogen bonds to break, which changes the shape of enzyme molecules. Moreover, after an enzyme has passed its optimum temperature, it has been denatured and can never work as if new again as fewer enzyme molecules can fit with the substrate molecules at their active site. The reaction continues to decrease until the entire enzyme is denatured and the reactions come to an end.

Variations in pH will also affect the activity of pectinase. Just like enzymes have an optimum temperature, they also have an optimum pH. However, this value is different for each enzyme. For example, gastric acid in the stomach, where the conditions are clearly acidic, and the optimum pH level will be low, at pH2. In the case of pectinase, which I will be using during my experiment, the optimal activity of this enzyme is at pH5.5 (as shown in the graph). At low pH levels pectinase becomes inactive but this is a not a permanent problem such as high temperature, since the enzyme can be restored back to its optimum pH where normal activity will resume. However, an extreme pH may cause an irreversible alteration to the enzyme. This may be a change in its protein structure, causing binding problems between substrate and enzyme.

This graph shows that as the enzyme concentration increases, the rate of reaction also increases, so long as there is an excess of substrate, at constant concentration, available in order for the enzyme to continue working. This is a linear or directly proportional relationship. They both increase because of the greater presence of active sites that can catalyze substrates, thus causing an increase in activity.

Increasing the substrate concentration will increase the rate of reaction to a certain extent, as long as the concentration of the enzyme remains constant. This is because the more pectin molecules there are, the more enzyme-substrate complexes formed, which therefore, leads to more juice being produced. It is to a certain extent because at some point, increasing the substrate concentration will not affect the rate of reaction because the substrate molecules will have occupied all active sites within the enzyme; this point is labelled as Vmax on the graph.

Collision Theory

Whenever I have mentioned that an increase in kinetic energy leads to an increase in the rate of reaction, the collision theory has been the basis of my idea.

The collision theory states that chemical reactions are the result of reactants colliding with each other, with enough energy to form a chemical bond. When these bonds are formed, they are known as successful collisions. The energy in question is caused by different factors, such as, temperature or concentration. This energy causes molecules to move faster than usual (increased kinetic energy) and also brings a higher chance of collision.

Activation Energy

Activation energy is defined as the energy that must be overcome or the minimum energy needed in order for a chemical reaction to occur.

As I have ...

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Activation Energy

Activation energy is defined as the energy that must be overcome or the minimum energy needed in order for a chemical reaction to occur.

As I have previously explained, energy is needed for collisions to happen. Without a catalyst, there is only a small portion of particles with sufficient energy to react. Adding an enzyme does not actually lower the activation energy but, instead would provide an alternative route for the reaction to occur. This alternative route has a lower activation energy. So, many more particles can then react by taking this route. Therefore, in this experiment, pectinase would decrease the time taken to break down pectin in the cell walls of the apple pulp.

Aim

This practical offers a number of possibilities for individual investigations, for example, I can study the effects of the enzyme’s concentration, the type of apple to be used or even the type of fruit altogether. I have chosen to use apple as my fruit and also to focus on the temperature of the reaction. So, the aim of my investigation is:

To investigate the effects of the enzyme pectinase, more specifically the volume of juice produced at different temperatures, when acting on apple pulp.

Hypothesis

From the biological knowledge that I have collected, I can make a testable hypothesis. I believe that as the temperature increases, enzyme activity will also increase, causing an increased rate of reaction. I also believe that there will be an optimum temperature, most likely between 30°C and 40°C, that the enzyme will work best in and that beyond this point, enzyme activity will decrease and eventually come to a halt.

Apparatus

Electrical balance – needed to weigh quantity of fruit.
Syringes (5cm3) – to accurately measure out amounts of liquid materials used.
Water bath – to control the temperature of the reaction.
Thermometer – to measure temperature.
Measuring cylinders (10cm3)– to collect juice produced from the reaction.
Filter papers – needed to ensure only juice is allowed through to the measuring cylinders.
Filter funnels – to hold filter papers.
Beakers, small (50cm3) and large (250cm3).
Glass rods
Stop clock – to time the experiments.

Materials

Pectinase solution, I will use this because this is the correct enzyme for the particular substrate at hand. It will break down the apple pulp and then I will test how well this is done under different temperatures.

Apple pulp, this is needed as it is the focal fruit of my investigation. I have chosen to use apple because polygalacturonases are absent in them, similar to some citrus fruits and unlike other fruits such as bananas and cherries. This means that the pectins in their cell walls cannot be broken down without external support, such as pectinase, before ripening. Furthermore, I have chosen to use pulp so that surface area will not be a factor affecting the reaction, as it will be the same for each sample.

Distilled water, if I would need to use water in my experiments it would need to be distilled as normal tap water could contain impurities.

Buffer solution, these are solutions are which simply designed to adjust or prevent change in the pH of other solutions. They do this by resisting change in hydronium and hydroxide ion concentrations. I have chosen to include a buffer as a means of preventing the pH of any solution being an affecting factor in my experiment. This brings me onto deciding what I should control in my experiment…

Controls

To make this a fair test, the following factors must be kept constant each time the experiment is carried out:

Concentration of pectinase used, different concentrations of pectinase will affect the break down of pectins in the cell wall. This in turn will affect the production of juice during the experiment and hence give different volumes of juice produced in a fixed time. In order to decide what type of concentration to use, I will firstly conduct a pilot test.
Volume of pectinase used and volume of buffer solution used to keep pH constant. The total volume of pectinase and buffer that I will be using will be between 2cm3 and 5cm3. I will decide the actual amount later on, most likely in a pilot test. I believe that these are appropriate volumes of pectinase and buffer and also that it is important not to use too much liquid material so as not to distort my results.
The mass of apple pulp needs to be kept at a constant at all temperatures. If not, the reaction rate of reaction for each experiment could vary unfairly. For example, if there is an excess of apple pulp at 40°C, then the volume of juice produced is likely to be far greater than a lack of pulp at 20°C.
The duration of the experiment, each experiment must be carried out for the same period of time so that results can easily be compared. I will make sure the duration is always the same by using a digital stop clock and taking the same period of time for each pectinase concentration.
The same type of apple should be used, however, since I will use pre tinned apple pulp, I will ensure that the same brand is used.
The pH of the solution also needs to be maintained. If the pH level of the solution is too high, it will have an irreversible effect on the enzymes by denaturing them. If the pH level is set too low, the enzymes would become inactive. Either way, the yield of apple juice in a certain time would be affected, thus, giving unreliable results. As I have found out from my background information, the optimum pH of pectinase is pH5.5, so I have decided to use pH6 during my experiment.
Lastly, the concentration of the enzyme pectinase is one of the most important factors that need to be controlled. A change in the concentration of an enzyme can cause a great difference in its catalyzation, with the possibility of a reaction ending too quickly or taking too long. For this reason, I will conduct a pilot test that will help me to find out what concentration of pectinase to use.

Accuracy

In order to achieve reliable results, I must try to make my experiment as accurate as possible. I will do this by…

Accurately measuring the mass of the apple pulp, so that the substrate concentration in the apple is not too much or too little.

The electrical balance used needs to be the same each time so that any margin for error can be kept the same.

I will use clean, dry apparatus each time so that any water from washing will not affect results. I will ensure this is done by leaving wet apparatus to dry after every wash and interchanging between two sets, i.e. wash one set, use, then leave to dry, then use the other set, wash and leave to dry, then return to the initial set which should have dried completely in time for its next use.
I will also make measurements and take readings by looking at the lower meniscus of solutions in measuring cylinders or syringes; this will help to avoid any errors in the volume.
I will label beakers and measuring cylinders with the name of its solution or mixture, what concentration it is or what temperature it will be used at, so that I do not mix up the solutions and cause any confusion.
Lastly, I will be precise with the time intervals whilst performing the experiment.

Risk Assessment

I should not keep the buffer solution or any other solution near electrical devices or connection points, such as that of the electrical balance, to prevent any electrical shocks or short circuits.
I must handle the glass apparatus, such as beakers, glass rods and thermometers, with care, so that they do not break and cause any injuries.
I should wash my hands if I come into contact with the pectinase solutions or take care that it does not spill on any clothes or get in contact with my eyes.
I need to make sure that I do not touch the water bath when placing the beakers inside or taking them out with bare hands to avoid any possible burns, so I plan to use a small towel to handle them when interacting with the water bath.
By no means should I or any other unaware person consume the apple juice produced, therefore the apple juice produced should be disposed off as soon as the experiment is over.
Lastly, I must pay full attention to each experiment at all times for maximum safety.

Pilot Test

This preliminary test will help me to find out what the most suitable concentration of pectinase will be to use in my main experiment. This pilot test will also give me an insight to the type of procedure that I will be using later on. Furthermore, this test will be carried out in one day at room temperature.

During the test I will investigate four different concentrations:

100% pectinase – 2cm3 pectinase and 0cm3 water
75% pectinase/25% water – 1.5cm3 pectinase and 0.5cm3 water
50% pectinase/50% water – 1cm3 pectinase and 1cm3 water
25%pectinase/75% pectinase – 0.5cm3 pectinase and 1.5cm3 water

At each concentration, I will measure the length of time taken for the amount of juice produced to reach 1cm3, 2cm3, 3cm3 4cm3 and 5cm3. Therefore my pilot test will also help me to decide how long to run my experiment for.

Prediction

I believe that the lower the concentration, the longer it will take to reach each volume. This is because a lower concentration will lead to less substrate being available to be catalyzed by the enzyme pectinase and therefore a much slower rate of reaction.

Method

First of all, I will begin by labelling my beakers containing pectinase and distilled water and from these I will measure out the concentration of pectinase e.g. 75% pectinase/25% water (1.5cm3 and 0.5cm3 respectively) using a syringe.
I will then measure out 20g of apple pulp in another beaker and reset the stop clock. Then I will set up the apparatus consisting of the funnel containing a piece of filter paper in the measuring cylinder as shown in the diagram.
To start the experiment I will add the pectinase and distilled water to the apple pulp and begin the stop clock.
I will wait for 5 minutes for the enzyme to act and also stir the solution with a glass rod at one-minute intervals for 10 seconds.
After the 5-minute activity stage, I will stop the clock, pour the mixture into the filter funnel and restart the clock.
During this period of time I will continue to observe the volume of juice produced in the measuring cylinder and when it has reached the target volumes of 1cm3, 2cm3, 3cm3, 4cm3 and 5cm3, I will record the time on the stop clock. Once this was done for all concentrations, results were recorded into the table:
Results table for pilot test studying concentration

Using these results, I can also plot a graph of time taken against volume (on graph paper).

Conclusion

The results that I obtained were exactly as I had predicted. As the concentration of pectinase decreases, the rate of reaction also decrease as more time is taken to achieve the stated volume. For example, the concentration of ‘50% pectinase/50% water’ took over 4 minutes longer than ‘75% pectinase/25% water’ to produce 5cm3 of juice.

After doing an experiment on the last concentration of my pilot test, I noticed that its results were completely anomalous. These are the results that are marked in red and have been crossed out. Because of this I decided to repeat this particular experiment and ignore the previous one. The repeat follows the trend of the others and also helps to prove my prediction.

From this pilot test I can make decisions on what I will do during the main experiment, such as deciding on certain measurements and durations…

Firstly, I have decided to make the experiment last for a total of 10 minutes; 5 minutes for enzyme activity at the controlled temperature and another 5 minutes for data collection. Secondly and most importantly, the concentration that I have decided to use is 50% pectinase; however, the portion of water will be replaced by a buffer solution of pH6 in the main experiment. I will also keep the mass of apple as 20g as this seems to be appropriate. Lastly, the total volume of pectinase and buffer that I will use will be 4cm3, since the concentration is 50% pectinase/50% buffer, the volumes will be 2cm3 each.

The method of this pilot test was to record the length of time taken for the volume of juice produced to reach a total of 5cm3. However, for the main experiment, I will choose to change this into measuring the volume of juice produced within the time limit of 5 minutes. I have chosen to do this because the reaction time at some temperatures may be very long and could make some results difficult to compare.

In addition, since I will be investigating temperature change, I have chosen a range of 50°C, this being from 20°C to 70°C. I have chosen this range because I believe the experiment should span from an approximate room temperature to a temperature that the majority of enzymes would have denatured at.

Main experiment: Investigating temperature change on pectinase

Method

Before starting my experiment I will label a beaker containing pectinase and one containing buffer solution, pH5.5. I will also place an empty beaker on the electrical scale, ready for the apple to be weighed out. But before weighing the pulp, I will use the ‘tare’ function to reset the scale. This is to ensure that the mass of the beaker is not counted towards the mass of the apple pulp, which helps to keep my experiment accurate. Then the beakers, each monitored with a thermometer, will be placed in the water bath (as shown in the diagram) to incubate towards the desired temperature of 20°C, 30°C, 40°C 50°C, 60°C or 70°C, depending on the experiment.

Whilst this is happening I will also set up a measuring cylinder, filter funnel and filter paper. (The same as in the pilot test diagram).

Once the pectinase, buffer and apple have reached the target temperature, I will measure out the concentration of 50% pectinase (2cm3) and 50% buffer solution (2cm3). Then I will reset the stop clock and add the buffer firstly before adding the pectinase to the pulp and starting the clock at the same time. From this point, I will stir the mixture for a period 10 seconds every minute for 5 minutes, this process is all whilst the mixture is still in the water bath to keep the temperature constant.

When the 5 minutes are over, I will stop the clock, pour the mixture into the filter funnel and restart the clock. After doing this, I will observe the stop clock and record the volume of juice produced every 30 seconds for another 5 minutes. Once this is done for all temperatures, I will record the data for each temperature in separate tables.

Results

Analysis

We can see from the 20°C experiment that the total average volume of juice produced is 5.63cm3. This is minimal when compared to some of the other temperatures, as it is the least amount of juice produced before the enzyme denatures. Moreover, the first experiment and its two repeats are very similar with an average range of 0.46cm3.

This is the average distance between volumes at the time intervals, calculated by:

Looking at the experiment of 30°C, I can see that the results for this experiment have an average range of 3.51cm3 and from this I can see that the results of each repeat are not very similar. However, when an average is made from the three the total average volume of juice produced has risen by 0.77cm3 since 20°C. This is already an indication that increasing temperatures can increase enzyme activity provided the reaction is not affected by other factors such as concentration.

From the 40°C experiment, I can see that even though the total average volume of juice produced here is greater than that of 30°C, it has only increased by 0.33cm3 to 6.73cm3. This shows that 30°C and 40° are most likely either side of the optimum temperature as their results do not differ by very much. This supports my initial hypothesis. However, this experiment at 40°C has still produced the greatest volume of juice, so, considering my chosen range of temperatures, 40°C is the optimum temperature here. Therefore, out of the six experiments, this temperature has given the reaction the most kinetic energy which has created a better chance of enzyme-substrate complexes being formed from collisions and therefore caused the most catalyzation.

At 50°C, the total average volume of juice produced has decreased since the last experiment by almost 1cm3 to 5.77cm3. This shows that 50°C is greater than the optimum temperature. However, it is only slightly greater as this difference is similar to the difference between the 20°C and 30°C experiments when juice production was gradually increasing. This shows that even though the enzyme was beginning to denature, the majority of active sites had remained intact. This also supports my initial hypothesis, that after the optimum point, the activity rate of the enzyme will begin to decrease. Furthermore, the range of this experiment is 1.06cm3 so the results from repeats were not too different.

Looking at the experiment at 60°C, I can see that the total average volume of juice produced continues to decrease and almost by the same amount, 0.8cm3 to 4.97cm3. This is because a greater portion of the enzyme has been damaged due to high temperatures and the rate of catalyzation decreases even more. Also, the range of this experiment was very low at only 0.66cm3, so, the variation in repeats was not too much.

Lastly, the 70°C experiment did not produce any juice. From my observations, I noticed that the enzyme pectinase had turned very thick at this temperature. Therefore, the enzyme had been completely denatured and there were no active sites to occupy the substrate of pectin.

Looking at my results, I can clearly see that the total volume of juice produced increases as the temperature of the experiment increases. However, this is only up to 40°C, after this, the average volume of juice produced decreases. Therefore, I can state that the optimum temperature of pectinase is definitely between 31°C and 49°C inclusive. This is because an increase in temperature caused an increase in kinetic energy that led to a greater possibility of the enzyme and substrate binding. However, since high temperatures can spoil the active sites in an enzyme, when the optimum temperature was passed, the chance of substrates finding an undamaged active site reduces. This becomes a reduction in the rate of catalyzation and therefore production of juice decreases.

Conclusion

My hypothesis stated that enzyme activity would increase as temperature increases until a point between 30°C and 40°C is reached. This has been proven by my results as the greatest volume of juice produced was during the 40°C experiment. I also stated in my hypothesis that after this point (optimum temperature), the enzyme activity would continue to decrease and eventually stop. This is also backed up by my results as the volumes at 50°C and 60°C decrease until juice production eventually ceases at 70°C.

Rate of Reaction

To find out the overall rate of reaction when considering all temperatures during my experiment, I firstly found the rate of reaction for each temperature by using the formula: rate of reaction = 1/t. However, my experiment is investigating volume in a given time, so I will replace 1 with V, therefore, creating the equation:

Where V is the total average volume of juice produced and t is the total time, which is always 300 seconds. This gives the rate of reaction a unit of cm3s -1.

From these results, I can draw a graph for the rate of reaction of the enzyme pectinase (on graph paper). From the table and the graph, I can see that the rate of reaction steadily increases up to 40°C and then decreases from here fairly steadily. Also, the highest rate of reaction is at 40°C, which has been proven to be the optimum temperature in this experiment. Furthermore, I believe that there is a very steep decrease between 60°C and 70°C, so, the rate of reaction at 60°C should be lower as most enzymes would have denatured at this temperature.

Evaluation

Overall, I believe that my investigation was a success. I also believe that the procedure that I used was easy to follow and regulated, so I do not believe that I made many or any major mistakes whilst performing the experiment, nor did I have any difficulties performing it, as all the apparatus needed to successfully complete this test were available. The apparatus that I chose of were also very simple to use and apart from that, they were appropriate for this specific experiment. Hence, the majority of the results that I obtained were suitable as the method which I followed was also appropriate and safe.

However, during my pilot test, I encountered a problem. The last of its experiments with a concentration of 25% pectinase/75% water had results that came out completely anomalous as if there was 100% pectinase in the mixture. This mistake was most likely because of a measuring or calculation error on my part. For this reason I decided to ignore these results and repeat the experiment. The results from the new experiment were more in tune with the others.

I believe that the 20°C, 40°C and 50°C experiment were satisfactory but the others could have been better.

The first 30°C experiment before the two repeats was a bit strange as the total volume of juice produced was 8cm3 as if the experiment was at 40°C instead of 30°C. This may have been caused by a misreading of temperature. If I ignore this first one and take the average from the two repeats only, the total average volume of juice produced would be 5.6cm3 which may be considered to be more appropriate even though it is only 0.03cm3 from the total average volume of juice produced at 20°C.

I also believe that the 60°C experiment was also slightly anomalous. This is because from my background information, I learnt that the denaturing temperature of pectinase is around 60°, therefore, I think the total average volume of juice produced for this experiment should have been less, as most of the active sites in an enzyme at this temperature should have been damaged.

Even though the experiment at 70°C produced no juice, I believe that it was fine as the temperature was far too high in order for pectinase to act.

If I were to do this experiment again, I think I could have repeated the pilot test at least twice to obtain more accurate and consistent results. I also could be more precise when measuring out the volume of the pectinase and buffer solution with the syringe, taking more care to look at the lower meniscus and doing the same when noting down the result from the measuring cylinder.

In addition, during this experiment I could have added a control, which would have been another experiment where only distilled water would have been added to the apple instead of pectinase and buffer solution. This would have helped me to understand and make sure that the juice produced during the experiment was a result of enzyme activity rather than just the liquid material being filtered through.

Lastly, I could have tried to use a buffer solution of pH5.5 as this is the optimum pH for pectinase instead of pH6, because of this, the pH of the enzyme could have an affecting factor towards my results. However, this material was not available to me at the time.

In order to make my experiment more detailed when investigating pectinase, I could have had a wider range of temperatures, for example from 10°C to 70°C or even had a greater number of temperatures to investigate, such as doing an experiment at 5 degree intervals instead of 10 (25°C, 30°C, 35°C, 40°C, etc…). Furthermore, if I were to extend my experiment, I could even investigate other fruits, such as pears, to see if pectinase works in the same way. Other experiments that I could have carried out would include studying the concentration of pectinase or even a different enzyme altogether.