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How different concentrations of the enzyme pectinase affect the degradation of the substrate pectin

Categories: ConcentrationEnzyme

Aim: My aim is to find out how different concentrations of the enzyme pectinase affect the degradation of the substrate pectin, and hence the volume of clarified apple juice produced in a specific duration of time.

Hypothesis: Will different enzyme concentrations have different effects on the yield of clarified apple juice obtained from apple pulp of equal masses, in a given time?

Biological Knowledge: Enzymes are a class of proteins, which are biological catalysts responsible for speeding up and controlling metabolic reactions, without their shape being altered by the reaction.

Pectinase (the enzyme I will be using for my investigation) is under the class of enzymes named hydrolases, which are responsible for catalyzing reactions between a substrate and water, and bind water to certain molecules. In this way, larger molecules are broken up into smaller units. This class of enzymes catalyses the cleavages of peptide bonds in proteins, glycosidic bonds in carbohydrates, and ester bonds in lipids. Enzymes are specifically designed for their target molecules, their substrates, by virtue of their shape, size, and the chemical charges that precisely fit and bond to the substrate molecule.

The section of the enzyme that binds the substrate is called the active site. An advantage of using enzymes is that they are specific and do not interact with other components in the food or beverage. Another advantage is that by catalyzing the same reactions repeatedly as long as the substrate is available (referred to as turnover), they can be used at very low concentration.

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Eventually, as the end products of the enzyme reaction increase in concentration, the reaction is temporarily inhibited by feedback inhibition. This shows how the catalysis of pectin by the enzyme pectinase occurs:

The cell wall of fruit comprises a complex mixture of various polysaccharides and some protein. Before fruit juice can be extracted from fruit, the cell wall has to be ruptured. This can be achieved by physically macerating the fruit or this can be accomplished by the use of enzymes. The cell wall contains cellulose, hemicelluloses, pectins and proteins. Pectinase specifically targets pectin, which bonds the long cellulose strands together, in the middle lamella, to form the outer core of the apple. Pectins are large polysaccharide molecules, made up (mainly) of chains of several hundred galacturonic acid residues.

Therefore, because fruit cell walls have such a complex structure of interwoven polymers, no single enzyme will break down cell walls. Complete liquefaction of a cell wall is likely to require a complex cocktail of carbohydrase enzymes. In fact, there is no single enzymes that will completely break down complex pectins. Commercial ‘pectinases’ are usually mixtures of enzymes such as polygalacturonases and pectin lyases, which result in the break down of pectins. Breaking down these bonds would mean the cellulose strand could move freely and therefore form a liquid.

Pectinases are obtained commercially from fungi, particularly species of Aspergillus and Penicillium. It is the bacteria and other fungal pectinases that help in the soft rot of fruits and vegetables, contributing to spoilage and decay. Hence pectinases break down the cell wall of plants (with no wood) this way, they are widely used in the fruit juice industry, where they are widely used to help extract, clarify and modify fruit juices. The optimal activity of these enzymes is at a pH of 5. 5 and generally below 50i??C, around 35i??C. Diagram of a Plant Cell Wall: Model Of Pectin Network In Plant Cell Wall:

Commercial Uses Of Pectinase:

* Clarification of juices/wines.

* Aiding the disintegration of fruit pulps.

* By reducing the large pectin molecules into smaller units, and eventually into galacturonic acid (compound become water soluble) loses their suspending powder.

* Viscosity is reduced.

* Insoluble pulp particles rapidly settle out. Factors That Affect The Enzyme Pectinase: Since pectinases are enzymes the same factors that affect enzymes also affect them.

These are:

* Fluctuations in temperature will affect the activity of the pectinase.

As temperature increases, the kinetic energy of the pectin and pectinase molecules increases and this results in an increase in movement. The faster these molecules move, the more frequently they will collide with each other, increasing the number of enzyme-substrate complexes formed and therefore increasing the rate of reaction. An increased rate of reaction would result in more pectin being catalysed in a given time, so leading to an increased rate of juice flow. Although highly unlikely in this experiment, significant increases in temperature would cause the atoms within the pectinase molecules to vibrate.

This breaks the hydrogen bonds holding the molecules in their shape and so altering the three-dimensional shape of the pectinase molecules to such an extent that their active sites no longer fit the pectin molecules. This would denature the pectinase and result in the lowering of the rate of juice flow.

* Variations in the pH will affect the activity of pectinase. Just like enzymes have an optimum temperature they also have an optimum pH. However, this value varies from enzyme to enzyme. For example in the stomach where the conditions are acidic the optimum pH level will be at a low pH value (pH2).

In the case of pectinase, which I will be experimenting on, the enzymes work best at a ph of 5. 5. At low pH levels the enzyme becomes inactive; however, this is a reversible situation since when the enzyme restores its optimum pH, the normal activity of the enzyme will resume. The result of an extreme pH, however, may cause an irreversible change to the enzyme. It may cause the enzyme to change: its protein structure, the binding of the substrate to the enzyme, the properties of amino acids or co-factors involved in the catalytic activity of the enzyme and the ionization of the substrate. The substrate concentration. Increasing the concentration of pectin, will increase the rate of reaction, providing not all active sites are in use, because the more pectin molecules there are in the solution, the more enzyme substrate complexes form. This will cause increased masses of apple juice and so increased flow rates to be obtained, as an increase in the rate of reaction will result in more pectin molecules to be catalyzed.

This remains true up to a point where all active sites are occupied and the volume of pectinase becomes the limiting factor causing no further increases in the rate of reaction.

Enzyme inhibition.

If there are more inhibitors, competitive or non-competitive, compared to the substrate concentration this will reduce the reaction rate in which juice flows. This is because competitive inhibitors compete for the active sites hence leaving less active sites for the pectins (the substrates) to get attached to and be catalyzed by the pectinases. However, as the substrate concentration increases, less inhibition will occur since there are more substrates meaning the substrates have a greater chance of finding a free active site, leaving fewer to be occupied by inhibitors.

Competitive inhibition is reversible, unlike non-competitive inhibition, where the structure of the enzyme is being denatured and hence having a very low reaction rate in which juice is produced. * The enzyme concentration, which will be the factor that I will be testing in my investigation. As the enzyme concentration increases, the rate of reaction also increases. This is because there will be more active sites that can catalyze substrates; thus causing an increase in the rate of activity. Therefore the enzyme concentration and the rate of reaction have a directly proportional relationship.

As the concentration of pectinase increases, the number of active sites will also increase. Provided that there is an excess of substrate molecules, the rate of reaction increases in proportion to the concentration of the enzyme, thus resulting in a higher yield of apple juice passing through the measuring cylinder. However if the enzyme concentration is very high in relation to the substrate concentration, there would not be any further increase in rate and it would remain constant. This is because even though there would be many active sites, there would not be enough substrates for catalysis. Read about test for presence of protein in food

Based upon all this biological knowledge I have attained, I want to find out how different pectinase concentration will affect the break down of the substrate pectin in the cell wall of equal masses of apple pulp, and how much clarified apple juice is produced in each pectinase concentration, in a given time. I believe that as the concentration of pectinase increases so will with the yield of apple juice produced until a point is reached where the pectin concentration and the pectinase concentration equal each other and no extra pectins will be degraded hence no extra apple juice will be produced.

METHOD OUTLINE:

– Apparatus:

* Knife. To cut the apple in small slices.

* Electrical balance. To measure the mass of the sliced apple pieces, so that the mass of apple used is the same for each concentration.

* Syringes, x2. To collect the exact volume of distilled water in one and the exact volume of pectinase in the other.

* Marking pen. To mark the beakers and measuring cylinders with their concentrations of pectinase.

* Water bath. In order to incubate the solutions at an optimum temperature for the enzymes.

Thermometer. To measure the temperature of the water so that the temperature does not increase exceptionally and denature the pectinase enzymes.

* Beakers, x2.

To keep the apple slices mixed with different concentration of pectinase solution.

* Glass rods, x2.

To stir the contents of the different concentrations.

* Filter papers.

To filter the apples slices and any other residue from mixing with the apple juice.

* Filter funnels, x2.

To keep the filter paper with the apple slices, the pectinase and distilled water in it. Measuring cylinders (10ml), x2. To collect the apple juice exiting the from the filter funnels.

* Stop clock.

To monitor for how long the pectinase acts on the apple pulp (1 minute).

* Tissue.

To wipe the wet apparatus with, so that there will not be any mixture of water with the chemicals, hence affecting my solutions and along with that my results.

* Goggles.

For eye protection from any unexpected mishaps.

* Lab coat.

In order to ensure that the enzyme solution will not accidentally fall on my clothes and ruin them.

* Gloves.

To prevent any possible contact of the pectinase solution with my hands, so as to prevent any skin irritations.

* Small towel.

In order to place and remove beakers in/out of the water bath, and prevent any possible burns.

Materials Used:

* Pectinase solution.

To test the effect of different enzyme concentrations, of the enzyme pectinase, on the rate of apple juice produced. I will be using the following concentrations: 0% of pectinase, 50%, 75% and 100% of pectinase because by using 0% of pectinase I will be able to see whether any apple juice may get produced without the presence of pectinase.

Then I will use the concentrations 50%, 75% and 100% of pectinase in order to see the change in the volume of apple juice produced as the percentage of pectinase increases, and hence we will be able to see how the pectinase concentrations affects the volume of apple juice produced in a specific period of time with equal amounts of apple pulp in every separate experiment.

* Distilled water.

Used in order to test the results when water is present alone and when water is present together with different concentrations of pectinase, to see how the results vary.

Apple. To cut into slices and add different concentrations of pectinase on it to see the rate at which juice is clarified. Apple is used because polygalacturonases, catalyzing the hydrolysis of glycosidic linkages next to the carboxyl groups, are present in some fruits such as bananas, cherries, pears, etc but not all fruits, like in apples and citrus fruits. This therefore means that since apples are absent of polygalacturonases, they cannot break down the pectins without an external support, such as pectinase, before ripening.

Hence we can use the enzyme pectinase to see how different concentrations of it may affect the yield of apple juice produced, being sure that any polygalacturonases present in apple will not affect the results obtained, as there are no polygalacturonases current.

Controlled Factors:

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

* 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 release of juice during the experiment and hence give different volumes of juice produced in a fixed time. This way I will be able to see how the different concentrations of pectinase affect the yield of apple juiced produced. * Volume of pectinase used and volume of distilled water used to dilute it should be constant. The total volume of pectinase and/or water I will be using is 4cm3, and accordingly I will use appropriate volumes of pectinase and/or water.

I will do so because this way my results will be accurate and reliable * Mass of apple pulp used should be the same for all different concentrations of pectinase (30g). This is because if the mass of apple pulp used for the different concentration of pectinase varies, this means that in concentrations with more mass of apple pulp the substrate concentration will be more, as there will be more pectins present, which are the substrates in this experiment, hence taking more time to break down the extra pectins and having a slow reaction rate.

If there is less apple pulp in other concentration of pectinase this will mean that there are less substrates (pectins), so there will be a faster reaction rate as there are less substrates to break down, hence giving me unfair results. Therefore by using an electrical balance I will make sure that all the different concentrations will have the same mass of apple pulp. * The duration of the experiment. Each experiment must be carried out for the same period of time (one minute) so that a clear trend can be shown on the graph and a comparison between the different concentrations of pectinase can easily be made.

I will make sure the duration of the experiments is the same by using a stop clock and taking the same period of time for each pectinase concentration. * The period of time in which the pectinase and fruit are left in the water bath before filtering (at 40i??C for 10 minutes). Leaving the pectinase and fruit in the water bath with a temperature higher than its optimum could result in the pectinase becoming denatured, so the water bath temperature will be monitored by a thermometer regularly. If they are left for less than 20 minutes than the pectinase will have had longer to work in some cases than in others.

This would make the test unfair; hence with the help of a stop clock, for once again, I will monitor the time for which the different concentrations remain in the water bath. * The electrical balance used needs to be the same. * The period of time left between readings. Inaccuracies in measuring the time period will affect the volume of juice produced after a particular time, this will result in unreliable results; so a stop clock should be used to monitor the time intervals precisely. * The temperature of the solution needs to be controlled.

This is because at low temperature the enzymes would not be as active as they would be at their optimum temperature. Also care should be taken that the enzymes are not kept under direct sunlight because then the enzymes may exceed their optimum temperature and therefore get denatured. Both cases would affect the rate of reaction of the enzymes, hence giving me anomalous results. In order to maintain a constant temperature, they will be kept in a water bath. * Use the same apple for all the tests, so that the volume of juice contained in the apple may not vary from fruit to fruit and thus affect the volume of fruit juice obtained. Distilled water must be used because tap water contains impurities, which could affect the permeability of the cell membrane in each different kind of fruit. This would affect the rate of release of fruit juice from the parenchyma. * The pH of the solution also needs to be maintained up to its optimum temperature (pH of 5. 5).

If the pH level of the solution is very high then it will have an irreversible effect on the enzymes. They would get denatured. If the pH level low the enzymes would become inactive. Both ways the yield of apple juice produced in a certain time would be affected, hence giving me anomalous results. Risk Assessment: I should not keep the distilled water or any solution near electrical points (such as that of the electrical balance), to prevent any electrical shocks or short circuits. * I have to handle the glass apparatus (such as the measuring cylinders, beakers, glass rods, thermometers and filter funnels) with care, so that they don’t break and cause any injuries.

* I ought to clean my hands if I have any contact with the pectinase solutions or take care the enzyme solution does not get spilled on my clothes or get in contact with my eyes, in order to ensure that I will be safe from any danger, so I will be using gloves, goggles and a lab coat. I need to pay attention not to touch the water bath when placing the beakers inside or take the beaker out of the water bath with bare hands, in order to avoid any possible burns, so I will be using a small towel to put them in and remove them from 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. * I need to use the knife with care, in order to prevent myself from getting cut.

Precautions: * I ought to be accurate when measuring the mass of the apple pulp, so that the substrate concentration in the apple (pectins) is not too much or too little, hence affecting the rate of reaction. * I have to wash the apparatus after every use. This is because there may occur a mixture of two chemicals, thus changing the solution and affecting the results. * I should dry the washed apparatus so that there will not be any mixture of water with the chemicals, hence affecting my solutions and along with that my results. I have to look at the lower meniscus of the solution in the measuring cylinder or syringe, so that I avoid any errors in the volume. * I require labelling the beakers and measuring cylinders with their enzyme concentration, so that I do not mix up the concentrations and cause any confusion. * I need to be precise with the time intervals whilst performing the experiment, in order to be able to meet an interval of one minute exactly in between each reading taken down for each enzyme concentration and the control. Method:

I will begin by labelling four beakers; one I will label ‘Control’, one ‘50% Pectinase’, one ‘75% Pectinase’ and the last ‘100% Pectinase’. Then I will set the water bath at a temperature of 40? C by using a thermometer. My next step will be to take the apple and cut it into very small pieces with the help of a knife, and by using an electrical balance then adjust the apple pieces so that the electrical balance shows a mass of 30g. Then I will take the 30g of apple pulp and place it in one beaker and with the same method I will weigh 30g of apple pieces and place them in the other beakers.

Following that I will use a syringe to collect 4cm3 of distilled water and then squirt it over the apple pieces in the beaker labelled ‘Control’. Using a different syringe I will suck up 2cm3 of the pectinase solution and 2cm3 of the distilled water and squirt it in the beaker labelled ‘50% Pectinase’. Then by the use of different syringes I will suck up 3cm3 of pectinase and 1cm3 of distilled water, and place the solution in the ‘75% Pectinase’ beaker, and I will suck up 4cm3 of pectinase solution with no water and place the solution in the ‘100% Pectinase’ beaker. Then I will stir the contents of each beaker with a separate glass rod.

The four beakers will then be incubated in a water bath of 40? C for about 15 to 20 minutes. Meanwhile I will label four measuring cylinders, one ‘Control’, one ‘50% Pectinase’, one ‘75% Pectinase’ and the other ‘100% Pectinase’. I will place a filter funnel in each measuring cylinder and then I will place a filter paper in each of the filter funnels. After 15-20 minutes I will remove the beakers with the help of a small towel, and then take the contents of the beakers and place them into the filter funnels of the appropriate measuring cylinders, and start the stop clock.

After ten minutes I will check the yield of apple juice produced for both experiments and record it. For my final experiment I will be finding the percentage change in comparison to my control (100% of water and 0% of pectinase). The formula which I will be using is the following: Average volume – average volume of the control X 100 Average volume of the control PILOT TEST: – I decided to have a pilot test in order to get an idea of the procedure of which I will perform and to see if the enzyme concentrations I have chosen are suitable and will work.

Method: I will label all my beakers with their according pectinase concentration (0%, 50%, 75% and 100%). I will add the appropriate water and pectinase amount for each concentration and then add the solutions in the suitably labelled beakers, which contain equal amounts of apple pulp (30g). I will stir them well with a glass rod and I will place them in a water bath for 15 to 20 minutes. I will then place the mixture on the filter paper within the filter funnel and note the volume of apple pulp entering the beaker every minute, for ten minutes.

Following that I will put my results in a table to compare the effect of the different concentrations of pectinase used in the experiment. I will do the experiments, two concentrations at a time in order to ensure that no inaccuracies with time occur. I will repeat the experiment for each concentration twice. Table to Show the Average Volume Produced At Different Pectinase Concentrations: Concentration Of Pectinase (%) Final Volume (ml) after 10 mins Average Volume (ml) 1 2 0 0 0 0 50 3. 75 3. 7 3. 73 75 5. 80 5. 80 5. 80 100 . 25 6. 30 6. 28 The results I obtained were exactly what I expected. This is because with only water and no pectinase the pectins would not be able to be degraded and hence no apple juice would be produced. However the experiment with the 50% of pectinase added to it had an average volume of 3. 73ml. This proves that with the addition of pectinase the pectins get degraded in the cell wall of the apple and hence juice is released.

With the addition of even more pectinase, 75%, there was more yield of apple juice produced, 5. 0ml. This shows that the higher the pectinase concentration added the more the enzymes there are to catalyse the pectins in the cell wall, hence producing more apple juice. For 100% of pectinase the apple juice produced was 6. 28ml, catalyzing even more pectins with an even higher enzyme concentration. Conclusion: Although my results for my pilot test were satisfactory, thus showing m method was correct and worked perfectly well, I have decided to make some changes for my implementation. The decisions I have taken are: I am going to use 5 different concentrations of pectinase in order to have a wider range of results to compare and to be more precise.

The concentrations will be 0% of pectinase (control), 20%, 40%, 60%, 80%, 90% and 100% of pectinase. This would help to study the effect of enzyme concentration on the same mass of substrate, and find at what exact point onwards, there is maximum yield of apple juice produced. * I will also use 10g of apple pulp rather than 30g in order for the pectinase to work best on a smaller surface area, and give even clearer results. I will also be repeating the experiment for each concentration thrice, in order to attain even more accurate results. OBTAINING EVIDENCE: – Apparatus: * Apple. * Knife. * Electrical balance. * Syringes, x2. * Marking pen. * Water bath. * Thermometer. * Beakers, x2. * Glass rods, x2. * Filter papers. * Filter funnels, x2. * Measuring cylinders (10ml), x2. * Stop clock. * Tissue. * Goggles. * Lab coat. * Gloves. * Small towel. Method: I began by labelling two beakers with care, like all the other glass apparatus I used during the experiment, so as not to break them and create any injures from the broken glass.

One I labelled ‘Control’ and one ‘20% Pectinase’. Then I set the water bath at a temperature of 40? C by the use of a thermometer. My next step was to take the apple and cut it into very small pieces, carefully so as to avoid cutting myself, with the help of a knife, and by using an electrical balance I then adjusted the apple pieces so that the electrical balance showed a mass of 10g. I measured the mass of the apple pulp as accurately as possible, so that the substrate concentration in the apple (pectins) is not too much or too little, hence affecting the rate of reaction.

Then I took the 10g of apple pulp and placed it in one beaker and with the same method I measured 10g of apple pieces and placed them in the other beaker. Following that I used a syringe to collect 4cm3 of distilled water and then squirted it over the apple pieces in the beaker labelled ‘Control’. I took care not to keep the distilled water or any other solution near any electrical points, in order to prevent any electrical shocks or short circuits. Using a different syringe I sucked up 0. 8cm3 of the pectinase solution and 3. cm3 of the distilled water and squirted it in the other beaker, labelled ‘20% Pectinase’. Then I stirred the contents of each beaker with a separate glass rod. The two beakers were then incubated in a water bath of 40? C for about 15 to 20 minutes, placing them inside the water bath using a small towel in order to avoid any possible burns. Meanwhile, I labelled two measuring cylinders, one ‘Control’ and one ‘20% Pectinase’. I placed a filter funnel in each measuring cylinder and then I placed a filter paper in each of the filter funnels.

After 15-20 minutes I removed the beakers with the help of the same small towel and took the contents of the beakers and placed them into the filter funnel of the appropriate measuring cylinder, and started the stop clock. At an interval of one minute, precisely, I recorded the volume of apple juice produced for each experiment, for ten minutes, taking care to look at the lower meniscus of the solution in the measuring cylinder, as I did when checking the volume within the syringe.

Once the experiment was over I disposed off the apple juice and pieces and repeated the same method for an extra two times on the same concentration of pectinase. This method described above was done for the rest of the pectinase concentration: 40%, 60%, 80%, 90% and 100% of pectinase, doing two concentrations at a time in order to be sure that I was being accurate with the time intervals. Table to Show the Average Volume Produced At Different Pectinase Concentrations: Concentration Of Pectinase (%) Final Volume (ml) after 10 mins Average Volume (ml) 1 2 3 0 0. 9 0. 9 0. 85 0. 88 20 . 0 4. 0 4. 0 4. 0 40 4. 4 4. 45 4. 4 4. 42 60 4. 7 4. 7 4. 75 4. 72 80 5. 1 5. 15 5. 15 5. 13 90 5. 2 5. 15 5. 2 5. 18 100 5. 2 5. 2 5. 2 5. 2 ANALYSING EVIDENCE:

– I have included all the results for my experiment in the obtaining evidence section, and I will now move on to analyzing them. Table To Show The Average Volume Produced And The Percentage Change At Different Pectinase Concentrations: Concentration Of Pectinase (%) Average Volume (ml) Percentage Change (%) average volume – average volume of the control X 100 average volume of the control 0 0. 88 0 20 4. 0 354. 55 40 4. 2 402. 27 60 4. 72 436. 36 80 5. 13 482. 95 90 5. 18 488. 64 100 5. 2 490. 91 It can be seen from the results that as the pectinase concentration increases so does the yield of apple juice produced, until a point is reached where all the substrates have occupied an active site, and so even if the pectinase concentration is increased, hence having more active sites, there are not enough substrates to occupy the extra active sites, therefore there will be no extra pectins in the cell wall of the apple slices to get catalysed and the yield of apple produced does not increase further.

We can see for the 0% of pectinase concentration, the volume produced is only 0. 88ml. It is the lowest value recorded because as there are no enzymes, hence no active sites, to catalyse the pectins on the apple slices’ cell wall, the pectins don’t get degraded and therefore there is an insufficient amount of apple juice produced. As for the 20% of pectinase concentration since there active sites that could accommodate the substrates there was apple juice produced. The pectins were accommodated in the active site of the pectinase enzyme and were therefore catalysed, and so juice was produced.

The amount of juice produced was 4. 0ml The 40% of pectinase concentration produced even more apple juice than the 20% of pectinase. This was because as there were more enzymes, there were also more active sites to carry out the process of catalysis and accommodate more substrates. Thus the time taken to catalyse the pectins was lower and therefore more pectins could be catalysed in those 10minutes, therefore producing a higher yield of apple juice, which was 4. 42ml. The 60% of pectinase concentration took even lesser time than the 20% and 40% of pectinase.

This was because as there were more enzymes, there were also even more active sites to carry out the process of catalysis and accommodate even more substrates. Thus the time taken was the lower, so as to be able to produce even more apple juice in the period of 10minutes. The apple juice produced in this case was 4. 72ml. The 80% of amylase concentration must have taken an even lower time to catalyse the pectins and as it had even more enzymes, and therefore more active sites, to catalyse the pectins.

This therefore meant that as there are more active sites there could be a larger amount of pectins catalysed in a period of time therefore having produced the most apple juice in comparison to the 20%, 40% and 60% of pectinase concentration. The apple juice produced in at this pectinase concentration was 5. 13ml. The 90% and the 100% of pectinase concentration although having more and more enzymes to catalyse the pectins, produced only a slightly more amount of apple juice.

The reason behind this is most probably because although the pectinase concentration was increased, the substrate concentration was kept constant; therefore reaching a point where although more enzymes were introduced, each substrate had already accommodated an active site. This meaning that there were enzymes that did not help in catalysis as they didn’t not have a substrate to catalyse, this connoting that no extra juice would get produced. So the 90% of pectinase concentration produced 5. 18ml of apple juice, and the 100% of pectinase concentration produced 5. ml of apple juice, which is practically equal to the apple juice produced by the 80% of pectinase. This must have meant that somewhere in between 60% and/or 80% of pectinase, a point was reached where the substrate concentration was equal to the enzyme concentration and therefore even if more enzymes were introduced there would be no more pectins to be degraded, and therefore no extra apple juice produced. From my study about enzymes and the degradation of pectins on the cell walls of plant materials, I knew that pectinase was the enzyme responsible for the catalysis of pectins.

Furthermore, I knew that as there will be more enzymes there would also be more active sites that are responsible for catalysis. From this I was able to make my hypothesis for the effect that enzyme concentration would have on the yield of apple juice produced. My hypothesis was that as the enzyme concentration would increase the time would decrease and the rate of reaction would increase, as there would be more active sites that would catalyze the pectins at a faster rate, therefore producing more apple juice in a period of ten minutes.

So as the enzyme concentration would increase, so would the apple juice produced until a point was reached where even if the enzyme concentration was increased more, all the substrates would be accommodated by an enzyme and no more apple juice would be produced as there would be no extra pectins to be degraded in order to release more juice. This meant that the 0% pectinase concentration would take the most time to degrade the pectins in a given time since it would have the least active sites, and so have the least apple juice produced.

My results also prove this, as there was no percentage change and only 0. 8ml of juice was produced. Then followed the 20% of pectinase with a smaller time duration than the 0% of pectinase concentration, since there would be more enzymes and hence more active sites for catalysis. This is why the 20% of pectinase had a 354. 55% increase in comparison to the control and noted 4. 0ml of apple juice production. The 40% of enzyme concentration came after the 20% of pectinase with a higher reaction rate and even lower time duration, and then followed the 60% and then the 80% of enzyme concentration with even lower time durations.

The 40% of enzyme concentration had 402. 27% increase with 4. 42ml of apple juice produced, the 60% had a 436. 36% with 4. 72ml of apple juice produced and the 80% had a 482. 95% increase having produced 5. 13ml of apple juice. This showing more and more juice was getting produced as the enzyme concentration increased because as the time duration decreased with more active sites being present, there would also be more juice produced in a specific period of time.

Then the 90% and 100% of enzyme concentration would degrade the pectins in the shortest duration of time, since it would have the most enzymes, but the yield of apple juice produced would only increase if the substrate concentration was also increased. However with the substrate concentration remaining the same there were no extra pectins to be degraded and therefore no additional apple juice was produced. So the percentage increase for 90% of pectinase is 488. 64%, with 5. 18ml of apple juice being produced, and 490. 1% increase for 100% of pectinase, with 5. 2ml of apple juice being produced. Analysis Of The Charts: From the line graph and bar chart you can tell that as the pectinase concentration increases, in the x-axis, so does the volume of clarified juice produced, in the y-axis. This is because as the concentration of enzymes increases, there are more active sites available and thus more substrates (pectins) can be degraded in a given period of time, as the extra active sites increase the rate of reaction, hence break down more substrates in less time.

This causes a sloped, upward line to be formed for the line graph, and bars to keep rising in height. However, as the concentration of pectinase reaches to 80%, from that point on the curve stabilises and give an almost straight line, or the bars begin to have a uniform height, and seem to be equal bars in a row. That is because from that point onwards, even though there are more enzymes, and therefore more active sites, the substrate concentration is constant and therefore all the pectins have already been accommodated by an active site to be degraded.

Hence, even if more enzymes are introduced there will be no change in the volume of apple juice produced, as the extra enzymes added will have no pectins to catalyse. This results in a straight line being formed after the 80% of pectinase concentration in the line graph, and bars of approximately the same height to be formed. Conclusion: Looking back at my results, I can say with confidence that my hypothesis was exactly right, as the rate of reaction did increase with the enzyme concentration, and hence the yield of apple juice produced as well increased.

This can also be portrayed through my graphs, as it is visible that I obtained the graphs that I had predicted. The fact that I managed to prove my predictions right, shows that my experiment was carried out correctly and with great care and precision. EVALUATING EVIDENCE:- The method that I used was easy to follow and therefore there were no errors 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 made use of were also very simple to handle and other than that, they were also simple to use and were the appropriate apparatus for this specific experiment. Hence the results that I obtained were satisfactory as the method which I followed and apparatus which I used were good and under safe, school laboratory circumstances. Limitations: As the mass of the apple may not have been measured accurately, this meant that there is a larger or smaller surface area for the enzymes to catalyse.

This may have altered the rate of reaction and hence the amount of apple juice produced and therefore this inaccuracy may have affected my results, hence making them less reliable. I may have been inaccurate when measuring the volume of pectinase solution and distilled water within the syringe. This may have resulted in the addition of more or less distilled water or pectinase hence either having a greater or lower enzyme concentration. Having a great concentration would give more apple juice, whilst having a lower enzyme concentration would give out less juice therefore giving me inaccurate results.

As I may have been inaccurate when noting down the reading from the measuring cylinder this may have meant that the results noted down were incorrect, making my entire experiment unreliable, with inaccurate results. Having three trials, although increasing the reliability of my results may have also slightly changed them, when taking their average. This is because the individual results are altered and therefore may result in a wrong reading. The most significant error in my method could be the fact that I did not rinse each lot of 10 grams of apple with distilled water before I added the pectinase enzyme.

This meant that the juice that had already come out of the apple pulp, before the pectinase was added, was not removed. This could have caused the volume of apple juice measured at each interval to be higher than would normally be expected and would reduce the reliability of my results. It was also difficult to have three experiments in progress at the same time, despite the fact that they were staggered by one minute, as two readings needed to be taken concurrently it still meant errors in reading could be made.

This could mean that some experiments were left slightly longer than they should have been, resulting in an unfair test. Therefore the volume measured at each interval may not be very reliable. The fluctuations in temperature of the water bath may have affected the activity of the pectinase enzyme; more of the pectin molecules in the cell wall may have been broken down in one repeat than in another causing a higher average result than expected.

As a result of all the above reasons my results may have varied a little between themselves, but this is why I repeated each concentration thrice and found its average volume, in order to avoid any variability in my results. I believe that my results are reliable because of this reason and also because my hypothesis was proved right and I received the expected results and expected graph trends. Improvements: I should be sure that I will measure the mass very carefully and with concentration.

I have to be more precise when measuring the volume of pectinase solution and distilled with the syringe, taking care look at the lower meniscus; and doing the same when noting down the result from the measuring cylinder. I ought to rinse the chopped fruit with distilled water before the pectinase enzyme is added would definitely make the results more reliable. This would ensure that the juice produced was due to the action of the pectinase enzyme. Next time I could do each concentration at a time, instead of doing two simultaneously.

Cite this page

How different concentrations of the enzyme pectinase affect the degradation of the substrate pectin. (2020, Jun 01). Retrieved from http://studymoose.com/different-concentrations-enzyme-pectinase-affect-degradation-substrate-pectin-new-essay

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