The Effect of Temperature on the Activity of Pectinase

The Effect of Temperature on the Activity of Pectinase
Aim: ? To investigate how temperature affects the rate of reaction of pectinase on its substrate apple fruit pulp. Introduction: - === Enzymes are proteins that catalyze reactions in biological systems by reducing the activation energy needed for any given reaction. They do this by temporary binding to one or more reactants of the reaction they catalyze. By doing so, they lower the amount of activation energy needed and thus speed up the reaction. The activation energy is the amount of energy needed for molecules to react when they collide. The fact that molecules need to collide in order to react is known as the collision theory. [image] Most enzymes are simple globular proteins, and the tertiary structure of the enzyme protein is well developed. Enzymes are specific which means a particular enzyme only works on one substance known as its substrate.
The precise folding of the polypeptide chains ensures that each globular protein has its own 3D shape or confirmation. Substrates bind with an enzyme at a specific spot on the enzyme called the active site. The combination of an enzyme with its substrate is known as the enzyme-substrate complex. This formation is explained by two theories. The Lock and Key hypothesis suggests that the substrate fits into the active site like a ?lock and key? where the substrate is the lock and the enzyme is the key. The Induced Fit theory suggests that the shape of the active site changes shape when the substrate molecule attaches to it. Pectins are large polysaccharide molecules, made up (mainly) of chains of several hundred-galacturonic acid residues, and they are found in plant cell walls. Pectin is able to form jellies (e.g. jam) but in fruit juices and other liquids it is undesirable. Pectinases are enzymes that break down complex pectin molecules to shorter molecules of galacturonic acid, as pectinase causes the disintegration of the cell wall, and so allowing the cell sap (juice) to flow out. This is why the biggest industrial use for pectinases is in the extraction and clarification of fruit juices; as some fruit juices e.g. apple juice may have bits of apple in it. This is where pectinase is used to catalyze the breakdown of the solid apple bits into apple juice, thus clarifying the apple juice. Pectinases are obtainable from fungi, such as species of Aspergillus and Penicillium, and also from bacterial organisms. [image] Temperature has a complex effect on enzyme activity. The "optimum temperature" of an enzyme is the temperature in which the enzyme functions most efficient. An increase in temperature below the optimum results in an increase in the kinetic energy of enzymes. This leads to higher efficiency of enzyme-substrate complex formation; therefore a higher rate of reaction. It differs in each enzyme, depending on its nature and structure. At extreme temperatures above the optimum, the increased kinetic energy disrupts the bonds holding the active site; the enzyme is unstable and the shape of its active site changes. This means that the enzyme is less efficient and successful at enzyme-substrate complex formation. The enzyme is said to be denatured; it has lost its ability to catalyze reactions. [image] Effect of temperature on rate of reaction Hypothesis: - ===== As temperature of pectinase rises, the rate of reaction will rise till 40?C. As it rises above 40?C the rate of reaction falls. The rate of reaction will increase as the temperature of pectinase increases because kinetic energy of pectinase increases, thus it moves faster and it is more likely to collide with apple fruit pulp. This results in a greater frequency of collisions. This will result in the rate of reaction increasing. Pectinase works best within the range (18?C ? 40?C). Rises in temperature above 40?C will cause the rate of reaction to fall because pectinase will denature. The shape of the active site of pectinase will have changed and will lack the ability of degrading the pectin in apple fruit pulp to apple juice. The higher the temperature above 40?C, the lower the rate of reaction. Below is the predicted graph for the temperature of pectinase against the rate of reaction: [image] Materials: - Equipment used Quantity Volume in Cm3 (if applicable) Diluted fungal pectinase 1% 1 50 Filter funnel 2 Filter paper 10 Test tubes 10 Test tube rack 2 Cut-off Syringe Apple fruit pulp 1 5 Water bath 1 60 Pipette 1 Scalpel spoon 1 5 Glass rod for mixing 1 Stopwatch 1 Measuring cylinder 1 Permanent marker 2 5 Aluminum foil 1 Method: - 1. I will label each of the following temperatures on 2 test tubes: 25?C, 30?C, 35?C, 40?C, and 45?C. 2. In each test tube, I will pipette 3cm³ of pectinase and syringe 6cm³ of fruit pulp. 3. I will stir the mixtures in each test tube thoroughly with the glass rod, then I shall place the test tubes in a test tube rack. 4. I will put the 2 test tubes labeled 25?C in the water bath at 25?C for 15-20 minutes. Then I will cover the remaining test tubes with aluminum foil until needed. 5. While waiting for the test tubes to reach the desired temperature, I shall set up the 2 5cm³-measuring cylinders and place the filter funnels on top with the filter paper folded in them. I will place the stopwatch in front of it for time recording. 6. I will then filter the solutions of the 2 test tubes into the 5cm³-measuring cylinders, and start stopwatch immediately. 7. I will record the volume of the juice collected in each measuring cylinder at 1-minute intervals for 10 minutes. 8. Finally, I will repeat steps 4-7 with the following temperatures: 30?C, 35?C, 40?C, and 45?C. The constant factors in this experiment are the concentrations of the enzyme & substrate, their volumes and the pH at which the reaction takes place in. For maintaining pH I will not use pH buffer solutions but I will use only one apple as it will have a specific pH and it will be constant throughout the experiment. I will be using fungal pectinase at 1% concentration, which will be prepared by mixing 49.5cm³ of distilled water to 0.5cm³ of the 100% concentrated fungal pectinase. I have decided to use the temperatures 25?C, 30?C, 35?C, 40?C, and 45?C because in my preliminary work I found out that these temperatures are adequate for showing how the rate of reaction is affected by changes in temperature. I will use a permanent marker to label the beakers and test tubes to avoid confusion. I will use a water bath to maintain the temperature of each test tube throughout the experiment, and a thermometer to check the temperature of the water bath before placing any test tube in it. I will use 3cm³ of pectinase and 6cm³ of apple fruit pulp in one test tube. This is because my preliminary experiments showed that using 1cm³ of pectinase with 5cm³ of fruit pulp would take too long to show an increase in volume over 1-minute time intervals. Using 2cm³ of pectinase with 8cm³ of apple fruit pulp made up a 10cm³ solution or mixture, which took too long to change temperature when placed in a water bath. Measuring the volume of juice at 1-minute intervals is appropriate as in my preliminary work I found out that measuring the volume at 5-minute intervals would take too long, and at 30 second intervals the measurements barely showed any variations in volumes. When mixing pectinase with apple juice in the beakers, I will use a glass rod to assure that they have been mixed together properly. For precise volume measurement of pectinase (enzyme) I will use a 5cm³ pipette and for the apple fruit pulp (substrate) I will use a cut-off syringe. I intend to use scalpel spoons for aid in collection of fruit pulp within a syringe. I will use a 5cm³-measuring cylinder because, as I found out in my preliminary experiments, it gives the most precise volume measurements at 1-minute intervals. I will use a stopwatch to attain accurate time recordings for my experiment. Finally, for accurate results I will carry out this experiment twice for each temperature, and average the results afterwards. Safety: - The enzyme pectinase may cause sensitization when inhaling its fumes / vapor, so to ensure that a very minimum amount is inhaled I will wear a mask. Most of the materials I will use are of low risk but I will wear safety glasses and wash my skin if anything spills on me. I will take extreme precautions when using glassware and I will be very careful not to break any of it. I will take caution not to put my hand in the water bath, as it may be extremely hot. I will wipe any enzyme spills and wash the area with water if a spillage occurs. The product (fruit juice) is not suitable for consumption, as it will contain the enzyme within it. Analysis ==== From the results obtained it is clear that for each temperature, the volume of juice rises after each minute. The amount of juice collected after 10 minutes for each temperature must be greater than 3cm³ because the enzyme alone takes up 3cm³ of the mixture. This is because enzymes, like chemical catalysts, are not used up in the reaction they catalyze and can therefore be collected after product formation. Below is a table showing the volume of juice extracted after 10 minutes for each temperature i.e. the total volume minus the amount of enzyme 3cm³. Temperature (?C) Volume (cm³) 25 1.5 30 1.6 35 1.8 40 2.7 45 1.5 The volume of juice produced after 10 minutes rises as the temperature rises, because an increase in temperature would lead to an increase in kinetic energy of pectinase. This means that it is more efficient in enzyme-substrate complex formation. Thus, the amount of juice produced is directly proportional to temperature; the higher the temperature, the higher the amount of juice produced. From the graph of total volume against temperature we can see that the highest volume of juice extracted was at 40?C. The volume produced at 25?C was 4.5cm³, and then it rises with temperature until it reaches its maximum at 40?C. This happens because at low temperatures the pectinase has less energy, so it will collide with the substrate (fruit pulp) less often, meaning the frequency of collisions is low, and the rate of reaction is low. At higher temperatures, the rate of reaction is higher because the enzyme has higher kinetic energy so it will collide more often with the substrate. After 40?C the enzyme denatures because it becomes unstable and the shape of its active site changes. Therefore, the rate of reaction falls; hence less juice is produced. After conducting the experiment I found the following anomalous results:
The total amount of juice produced after 10 minutes for the third
test at 30?C and the fourth and fifth tests at 35?C are lower than
3cm³, which in theory is not possible because the amount of
enzyme used is 3cm³, so the total amount of juice after 10 minutes
must be above 3cm³. This does not affect the analysis as the
readings were ignored when taking averages.
On the graph of total volume vs. temperature, the total volume for
30?C was 0.6cm³ below the curve and the total volume for 35?C was
0.7cm³ below the curve. This was taken into account when I drew
the line of best fit by using error bars for these temperatures.
Evaluation == The results obtained for each temperature show a very slight variation e.g. at 30?C the total volume of juice was 1.6 whereas at 35?C it was 1.8; a 0.2cm³ difference. This may be due to the following: the mixing time for each solution at preparation was different as in the method it was not specified for how long the mixtures were to be stirred. The method also states that the test tubes containing the mixtures are to be placed in the water bath for 15-20 minutes. Some mixtures may have been placed for 15 minutes and some for 20 minutes. An apparent anomaly occurred for the results of the third test at 30?C and the fourth and fifth tests at 35?C. The total amount of juice produced after 10 minutes was lower than 3cm³, which in theory is not possible because the amount of enzyme used is 3cm³, so the total amount of juice after 10 minutes must be above 3cm³. This may have occurred because the solutions weren?t mixed long enough while being prepared. This does not affect the analysis as the readings were ignored when taking averages. The other anomalous result was on the graph of total volume vs. temperature, where the total volume for 30?C was 0.6cm³ below the curve and the total volume for 35?C was 0.7cm³ below the curve. This was taken into account when I drew the line of best fit by using error bars for these temperatures. For each temperature, I have a minimum of 3 results that aided in achieving a better average. At 35?C I felt that there was a need for repetition as the total volume of juice for the first three sets of results was less than the total volume at 30?C. This contradicts the theory about the effect of temperature on enzyme activity: An increase in temperature below the optimum would result in an increase in the kinetic energy of enzymes. This would lead to higher efficiency of enzyme-substrate complex formation; therefore a higher rate of reaction. Because the third test at 30?C was anomalous, I repeated the experiment at that temperature twice as the more the experiment is repeated, the more precise the average. The results obtained in my experiment can support my hypothesis that as the as temperature of pectinase rises, the rate of reaction will rise till 40?C. As it rises above 40?C the rate of reaction falls. The amount of juice extracted may have varied by an insignificant amount, but as the temperature increased the amount of juice produced increased till 40?C, after which it fell. The only limitations I encountered while conducting my experiment were that different apples were used to prepare the substrate. Although they were of the same type, different apples tend to have different pH values. Also, after removing the mixtures from the water bath they were filtered and then the volume was taken at 60-second time intervals, but the temperature may have slightly dropped 5 minutes after removal. If I were to do the experiment again I would try even more temperatures because this would enable me to find the optimum temperature or range, and I would also obtain more results for each temperature to achieve high precision in my averages. A different substrate e.g. banana fruit pulp, and/or different concentrations of pectinase could be used to see how this would affect the rate of reaction and the amount of juice extracted.

The Effect of Temperature on the Activity of Pectinase 8.5 of 10 on the basis of 1952 Review.