Investigating the Effect Of Changing Temperature on the Activity of the Enzyme Pectinase

Investigating the Effect Of Changing Temperature on the Activity of the Enzyme Pectinase
Planning Hypothesis When I increase the temperature of the pectinase, its activity will also increase, which will be shown by the yield of juice. Due to the molecules warming up and then moving around more, the frequency of collisions between particles will increase. However this will be restricted, , after its peak of 40ºC, the Pectinase would start to denature and the rate of juice production will fall drastically. By 80ºC I predict the enzyme will be completely denatured, which will be shown by a low yield of juice. Biological catalysts called enzymes control the metabolic reactions.
Pectinase is the enzyme in my investigation. It is used as an industrial enzyme for the production of fruit juice, sometimes along with cellulase. It works by hydrolyzing the pectin?s which hold the cells of the pulp together, making the cells fall apart and release the juice from inside the vacuole. During the fermentation of the fruit juice, Pectinase removes the cloudiness of the solution caused by the presence of pectin. Apparatus - 500g of apple pulp. The pulp will be prepared before the experiment with the use of a potato masher. - 2% pectinase solution - 2 conical flasks - Weighing scales - 2 ice cream tubs. I will use an ice cream tub for my water bath and keep refilling it with water of the required temperature. - 1 thermometer. A 100ºC thermometer will provide temperature results of a sufficient accuracy (to ½ ºC). - Filter paper. - 2 Filter funnels - 2 measuring cylinders. I have chosen to use a measuring cylinder to measure the yield of juice as the results should be easy to observe. - 1 pipette - 1 spatula Preliminary work I will perform a preliminary experiment to find out how long it takes for incubating the apple pulp in the water bath for it to take affect. The reason for me performing this is experiment is so that I will find out how long I should incubate the solution in the water bath during the actual experiment. For this experiment I will use the same amount of Pectinase and apple pulp as I will use in the main experiment to make my reading more accurate. Preliminary work results The water bath in the experiment was at 24ºC ? room temperature. It took 5 minutes for the temperature of the solution to heat up; however the results seem variable as it cooled to 24ºC at 10 minutes, then went back up to 25ºC at 15 minutes. Time/ Minutes Temperature/ oC of solution 0 20 5 26 10 24 15 25 20 24 25 24 30 24 The water bath in this experiment was at 15 ºC. Not once in the experiment did the water stay at 15ºC, the temperature which I had attempted to keep it at. Time/ Minutes Temperature/ ºC of solution 0 17 5 16 10 16 15 16 20 17 25 17 30 16 The water bath in this experiment was at 70 ºC. In this experiment it was found that maintaining the high temperature difficult as it rapidly decreased. Time/ Minutes Temperature/ ºC of solution 0 20 5 29 10 40 15 46 20 55 25 60 30 65 From my preliminary work, I have decided to keep my experiments for 20minutes in the water bath. Although the experiment at 70 ºC was not fully heated, I had to make a compromise, as it would take too much time if I were to leave the solution in the water bath any longer. It was also discovered that the method of maintaining the water bath at a certain temperature exceedingly hard, as the water temperature was hard to control; thus the temperature of the solution would also vary because the water affects the heat of the solution. Therefore I have decided to make an alteration to my method of maintaining the water temperature to try to make the temperature of the solution consistent as well. Altered Apparatus List Every apparatus and amount will remain the same except for the use of the ice-cream tubs. From my preliminary experiments I found that refilling the ice cream tubs was too inaccurate, as the temperature would vary when the water was put in. In their place I will use a thermostatically controlled water bath as I believe it would be more constant in temperature it maintains the temperature more accurately by electronic means. I will also require 2 clamp stands to hold the conical flasks in the water. Method - Place 50g of apple pulp with the spatula into the conical flask on the weighing scales; take into consideration the weight of the conical flask. - Add 2 cm3 of diluted pectinase enzyme to the conical flask. If any enzyme is spilt, it should be wiped away quickly and the area washed with water. - Stir the contents of the conical flask with the glass rods. - Incubate the flask in the water bath for 20 minutes. Hold the conical flasks in the water with use of clamp stands - Filter the juice from the apple pieces, through the funnels into the measuring cylinders. - Record the volume of juice obtained from both lots of apples at 5-minute intervals. - For saving time make up two experiments at once, the initial experiment and the repeat. - Repeat with the above steps but increase the temperature of the water bath by 20ºC. Diagram [image] Safety Note Pectinase is a biologically active protein and should be handled with care because it can irritate the eyes, skin or mucous membranes, goggles therefore should be used whilst handling the enzyme. After using the Pectinase it would be wise for the individual undertaking the experiment to wash their hands lest any of the substance gets on them. This experiment uses very high enzyme concentrations that are toxic. In the juicing factories, 130 cm3 of enzyme was added to one ton of apples. Therefore, the apple juice that I have made will not be consumed. Also if I spill some pectinase I will wipe it away immediately lest it dries and forms dust which could be inhaled. Variables To make the test reliable I will keep other factors which can increase the yield of apple juice under control, such as the concentration of the enzyme ?I will use a constant amount, the concentration of the substrate ? I will always use 50cm3 of apple pulp. I will also keep the amount of apple pulp and pectinase used the same during the experiment. Results In a water bath of 80ºC Time/ Minutes Yield of Juice/ cm3 Time/ Minutes Yield of Juice Mean Yield of Juice/ cm3 0 3 0 2 3.5cm3 5 5 5 4.1 5.5cm3 10 6 10 5 6.5cm3 15 7.5 15 7.2 7.8cm3 20 8 20 7.6 8.3cm3 In a water bath of 60ºC Time/ Minutes Yield of Juice/ cm3 Time/ Minutes Yield of Juice/ cm3 Mean Yield of Juice/ cm3 0 2 0 1.0 1.5cm3 5 5.5 5 3.7 4.6cm3 10 8 10 5.0 6.5cm3 15 8.6 15 7.0 7.8cm3 20 9.4 20 10.5 10.5cm3 In a water bath of 40ºC Time/ Minutes Yield of Juice/ cm3 Time/ Minutes Yield of Juice/ cm3 Mean Yield of Juice/ cm3 0 2 0 1.8 2.0cm3 5 4.2 5 5.2 4.5cm3 10 6.1 10 7.1 6.7cm3 15 8.0 15 9.4 8.7cm3 20 13.3 20 12.8 12.9cm3 In a water bath of 20ºC Time/ Minutes Yield of Juice/ cm3 Time/ Minutes Yield of Juice/ cm3 Mean Yield of Juice/ cm3 0 1.0 0 0.5 0.8cm3 5 4.1 5 4.5 5.5cm3 10 6.3 10 5.9 6.6cm3 15 6.8 15 6.0 6.9cm3 20 7.0 20 7.1 7.6cm3 In a water bath of 30ºC Time/ Minutes Yield of Juice/ cm3 Time/ Minutes Yield of Juice/ cm3 Mean Yield of Juice/ cm3 0 1.0 0 0.7 0.9cm3 5 2.5 5 1.9 2.2cm3 10 4.1 10 3.8 4.0cm3 15 4.4 15 4.2 4.4cm3 20 5.1 20 4.6 4.9cm3 Analysis As stated in the hypothesis, the enzyme Pectinase works most efficiently at 40ºC, in consequence the most juice would be produced. This is demonstrated in the graph titled ?A graph to show the mean amount of Juice produced at different temperatures,? the yield of juice peaks at 12.9cm3 at 40ºC. At the peak temperature the enzyme would be working at its most effective, the molecules would be fast moving and with the collisions the substrates could bind with every active site. The action of the enzyme can be explained in the analogy of a lock and key, the key (apple pulp) fits into the key hole (the active site) of the lock (pectinase enzyme). As expected the yield of juice produced in experiments over 40ºC start to decrease, at 60ºC 10.0cm3 of juice is produced and at 80ºC it falls to 8.3cm3. This is due to the denaturing of the pectinase; the enzyme loses it shape resulting in the substrate becoming incapable of reacting with the active site. However fruit juice would still be produced as enzymes only speed up a reaction, with the enzyme becoming denatured the reaction will only slow down. In the experiments at 20ºC and 60ºC there is a large margin of error; the experiment at 20ºC has a 14% error margin and the experiment at 60ºC has an error margin of 10%. There is no apparent cause for this anomaly; however the inactivation of the enzyme may occur at different rates before and after the optimum temperature. Pectinase reacts slower at cooler temperatures than 40ºC because the particles in the solution have less kinetic energy to react with; above 40% Pectinase becomes misshapen due to the higher temperatures, which distort by the increase in kinetic energy breaking the bonds of the amino acids which form the enzyme, as a result the substrate cannot bind with the active site. The error bar for each of the other experiments, demonstrated on the graph, have only a small margin of error. Such as at 40ºC the error margin is only 2% and the experiment at 80ºC has an error margin of 7%. From this it could be deduced that the experiments were accurate and consistent. The experiments at 30oC did not go as expected as the yield of juice produced drops to 4.9cm3. The cause of this anomaly is the fact that the experiment was undertaken five days after the other experiments, in that time the prepared 2% pectinase solution had weakened its effectiveness, thus resulting in a slower experiment. The anticipated result would have been higher than 7.6cm3 yield of juice in the experiment at 20ºC. The reason for this is as the temperature increases towards pectinase?s peak of 40ºC the rate of collisions of the apple substrate and pectinase also increase. Evaluation The analysis of the results indicates that temperature affects the activity of the enzyme. The experiments at 20ºC, 40ºC, 60ºC and 80ºC went according to plan, as there were no anomalies. From the data fruit juice manufacturers could see that 40ºC should be the temperature in which they use pectinase to make juice. For practical reasons the simple apparatus were not very reliable. The measuring cylinders used in the experiments went up in 1cm3. If this experiment is performed again it would be more accurate to use a measuring cylinder, which measures to 0.1cm3. The thermostatically controlled water bath was not fully reliable, as it would waver a few ºC above and below the required temperature, this would produce an inaccurate amount of juice. Next time the experiment was performed it would be easier to use a more stable thermostatically controlled water bath. In using a pipette to distribute the pectinase solution was made the experiment inconsistent as it was impossible to give the exactly the same amount it could vary by 0.5cm3 of Pectinase solution. Even though this appears to be only a insignificant amount, it must be taken into consideration that a juicing company would only add 130cm3 of Pectinase to a ton of apples; therefore even if there is only a slightly higher amount of enzyme in one experiment a higher yield of juice would be produced, thus causing an inaccuracy in the data obtained. In addition this explains the fact that there are error margins because experiments that contain the same amount of enzyme and apple should produce practically the same amount of juice. On the other hand the apple cells of one experiment may be more turgid than those of another, with more juice inside the vacuole. It is evident that if another experiment has to take place after the initial experiments, a fresh batch of enzyme should be made. The anomaly result for the experiment at 30ºC clearly indicates that pectinase gets weaker as the enzyme gets older. This would slow the rate of reaction of the experiment and in consequence less juice would be produced. To make the creation of the apple pulp easier next time it is carried out would be to use a liquidizer instead of mashing it with a masher. In this way more time would be saved as mashing the apple to a pulp takes almost an hour, while liquidizing the apple takes less than five minutes. The results gained may not be accurate as nothing was done to control the acidity of the solution. Pectinase works best at a pH of 7; as the pH is decreased or increased, the overall shape structure of the enzyme becomes distorted causing the active site to become misshapen and the substrate becomes incapable of binding with it. In a further investigation a buffer could be used to control the pH of the solution. This would check the reliability of the results gained from the temperature, as it would be only factor that would change. If the enzyme was kept at the optimum pH of 7.0 then the yield of juice would increases, which indicates that the reaction would take place faster. Another factor, which should be taken into consideration when looking at the reliability of the results, is of inhibitors to the enzyme. Cells possess regulatory chemicals which slow down enzyme reactions; there are three types of inhibitors. Competitive inhibitors are a similar shape to the substrate; the reaction is slowed because when the inhibitor is in the active site the substrate cannot react with it. Non-competitive inhibitors bind with another part of the enzyme other than the active site; they distort the enzyme active site, which no longer allows binding of the substrate. End-product inhibitors, which are the produce of the first reaction, shaped to fit into the allosteric site of the enzyme and change the active sites shape, inhibiting the first reaction and those that follow.

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