Biology IA Task Wen Wen Teh 12E Investigating Factors that could Effect the Action of an Enzyme IntroductionEnzymes are proteins that have taken on a specific 3-dimensional shape. Enzymes function as biological catalysts to speed up chemical reactions in the body by lowering the activation energy. They do this by bring the substrate together and binding them to form the product. This substrate-enzyme interaction is best described with the induced fit model. The area in the enzyme known as the active site which changes its shape to fit the specific shape of the substrate. Lipase is the enzyme catalyzes the hydrolysis of lipids into glycerol and fatty acids. The lipids typically found in milk are triacyl glycerides. Fatty acids are acidic in nature, so when it is in water, it will donate hydrogen ions to the water thus increasing the hydrogen ion concentration and making it acidic, lowering the pH. So the faster the lipids are being catalysed, the faster the decrease in pH. The factors that affect the rate of reaction are the pH level, temperature, and the concentration of enzyme and substrate. Different enzymes work best at different pH levels, there is no set optimum pH level for all enzymes. However, as the rate of decrease in pH is the variable being measured, it cannot be the independent variable.. The enzyme and substrate concentration is also factor because higher enzyme or substrate concentration will increase the contact between enzyme and substrate, thus higher reaction rate. This experiment will investigate how the surrounding temperature affects the action of the enzyme. This is because the higher the temperature, the more energy the substrates and enzymes have to move around and come into contact with each other more often which increases the action of the enzyme. However after a certain temperature, the enzyme will start to denature and lose it shape, therefore unable to catalyse reactions anymore, as shown in the graph below. Figure 1: Relationship between rate of enzyme reaction and temperature Research question How does temperature affect the rate of reaction of lipase? Thermometers 8. Ice 5. If a higher concentration was used. The lipase solution with the milk is left in the water bath or ice beaker for 30 minutes for the lipase to act equally on the milk substrate. The same type and brand of milk was used because different types and brands of milk contain different amount of fats. which will affect the rate of reaction of the lipase. The amount of lipase solution used was also controlled by measuring 2cm3 into each test tube. the lipase will have less affect on the milk compared to the rest which will lead to unreliable results. The lipase catalyzes the reaction which turns the milk acidic and lowers the pH level. The milk used was full fat milk from the same package. it will affect the rate of reaction of the lipase on the milk and influence the results. Apparatus 1. If the boiling tubes were left for a lesser time. 30° C. 0. So the closer it is to the optimum temperature. 20° C. it will increase the rate of reaction and vice versa. 40° C. Full fat milk 4. The volume of milk which is the substrate was controlled at 5cm3 by measuring it out as accurately and precisely as possible with a measuring cylinder of a small deviation. Data logger 6. Water baths will be used to achieve temperatures 30° C. 5% lipase solution 2. 50° C. The investigation will test a range of temperatures: 10° C.Biology IA Task Wen Wen Teh 12E Variables Independent variable The independent variable will be the temperature at which the reaction takes place in. the greater the catalysis thus the faster the drop of pH. which will affect the results. 50° C. while beakers filled with ice for 10° C and 20° C.05 mol dm-3 Sodium bicarbonate solution (Na2CO3) 3. Sodium bicarbonate solution is added to the milk to make it more alkali to easier monitor the change in pH. Boiling tubes . Dependent variable The dependent variable measured will be the rate of reaction of the lipase. 40° C. If a different amount of lipase was used. pH probe 7. so the lipase will have equal amount of substrate to act on making it a fair test. Controlled variable The concentration of lipase used is set at 5% lipase solution to make sure the concentration is the same throughout. Test tubes 9. The change of pH will be measured with a pH probe and the data recorded on a data logger. 50° C. Hypothesis The higher the temperature. Measure out 2cm3 of lipase solution into a test tube. it should be alkaline. 13.Biology IA Task Wen Wen Teh 12E 10. 40° C. 5 Syringes. 6. 7. Test tube rack 11. . Set up data logger and pH probe. 2. the faster the rate of change of milk pH level. Add lipase to the boiling tube and start the stop clock. 10cm3 ± 0. Measure out 7 cm3 of sodium bicarbonate solution and add it to the milk. 10.001 cm3 measuring cylinders (milk) 16. Check the pH level with pH probe. Place the pH probe into the boiling tube and get the initial reading of pH level. 2cm3 14. 4. Repeat steps 7-11 for different temperatures. Repeat the whole experiment 5 times to ensure reliability of results. Incubate the boiling tubes with the milk and sodium bicarbonate solution in the water baths and ice until the desired temperature is reached by monitoring with a thermometer. 100cm3 beakers (act as water baths for temperatures below room temperature) Methods 1. 8. 11. Set up water baths at 10° C and 20° C with ice and water. Pipette 13. Record the pH of the milk using the data logger at every 30 seconds interval for 30 minutes. Glass rods 12. Measure out 5 cm3 of milk with the 5ml measuring cylinder into each boiling tube. 5. Then the rate drops as the enzyme denatures at higher temperatures. 12. 9. Stop clock/stopwatch 15. 3. Label the boiling tubes with the temperature to be investigated. Set up water baths at 30° C.1cm3 measuring cylinders (sodium bicarbonate solution) 17. until it reaches the optimum temperature where the rate of change of pH level is the highest. 5cm3 ± 0. 86 7.79 7.73 7.72 7.81 7.81 7.72 7.87 7.42 7.79 7.44 7.76 7.42 7.79 7.88 7.40 7.86 7.89 7.43 7.39 7.42 7.79 7.83 7.87 7.82 7.39 7.42 7.37 7.89 7.86 7.71 7.40 7.78 7.88 7.86 7.78 7.78 7.41 7.81 7.39 7.42 7.82 7.40 7.77 7.77 7.78 7.44 7.37 7.76 7.76 7.89 7.38 7.88 7.79 7.72 7.42 7.75 7.88 7.76 7.87 7.37 7.89 7.80 7.84 7.1) Test 4 Test 1 Test 2 Test 3 Test 4 7.84 7.87 7.85 7.85 7.73 7.87 7.82 7.83 7.76 7.44 7.44 7.79 7.82 7.86 7.88 7.86 7.Biology IA Task Wen Wen Teh 12E Results Collected Data .42 7.76 7.77 7.40 7.88 7.75 7.78 7.76 7.73 7.79 7.85 7.87 7.37 7.76 7.84 7.86 7.37 7.85 7.90 7.79 7.73 7.88 7.82 7.76 7.45 20 pH (± 0.39 7.43 7.82 7.72 7.87 7.72 7.41 7.79 7.82 7.74 7.72 7.86 7.78 7.73 7.42 7.80 7.77 7.38 7.86 7.38 7.75 7.76 7.78 7.83 7.71 7.42 7.76 7.41 7.41 7. Temperature (°C) Time (s) 10 Test 1 Test 2 Test 3 7.42 7.77 7.81 7.74 7.85 7.84 7.83 7.42 7.78 7.42 7.39 0 61 123 180 242 300 361 422 480 541 603 660 722 780 841 902 960 1021 1079 1140 1202 1260 1321 1382 1440 1501 1559 1620 1682 1740 1800 .45 7.39 7.70 7.89 7.83 7.76 7.44 7.77 7.75 7.74 7.83 7.83 7.87 7.72 7.41 7.38 7.79 7.86 7.86 7.78 7.45 7.86 7.42 7.41 7.40 7.83 7.85 7.87 7.82 7.88 7.86 7.87 7.85 7.83 7.86 7.87 7.77 7.83 7.77 7.88 7.83 7.72 7.40 7.76 7.83 7.78 7.89 7.73 7.87 7.81 7.80 7.42 7.78 7.88 7.73 7.88 7.81 7.90 7.88 7.89 7.79 7.38 7.74 7.82 7.78 7.77 7.40 7.84 7.75 7.37 7.73 7.83 7.87 7.87 7.83 7.77 7.41 7.41 7.74 7.82 7.87 7.81 7.75 7.89 7.79 7.75 7.89 7.38 7.85 7.73 7.78 7.88 7.83 7.38 7.44 7.76 7.38 7.83 7.71 7.77 7.45 7.78 7.39 7. 85 7.49 7.59 7.65 7.47 7.42 7.72 7.78 7.45 7.52 7.82 7.65 7.63 7.50 7.48 7.50 7.43 7.40 7.48 7.62 7.62 7.49 7.56 7.95 7.61 7.52 7.57 7.71 7.61 7.52 7.83 7.47 7.53 7.47 7.50 7.60 7.43 7.60 7.69 7.63 7.75 7.89 7.41 7.40 7.73 7.62 7.82 7.58 7.62 7.42 7.56 7.57 7.87 7.64 7.41 7.49 7.48 7.57 7.76 7.49 7.82 7.90 7.92 7.84 7.84 7.46 7.86 7.48 7.52 7.63 7.52 7.59 7.64 7.87 7.66 7.1) Test 4 Test 1 Test 2 Test 3 Test 4 7.68 7.90 7.52 7.62 7.47 7.64 7.64 7.60 7.68 7.83 7.74 7.64 7.48 7.58 7.45 7.61 7.52 7.48 7.50 7.39 7.46 7.58 7.89 7.50 7.45 7.63 7.60 7.84 7.47 7.46 7.49 7.81 7.86 7.51 7.56 7.40 7.92 7.69 7.49 7.50 7.50 7.85 7.60 7.47 7.64 7.69 7.50 7.64 7.58 7.59 7.63 7.55 7.67 7.45 7.47 7.44 7.40 7.62 7.87 7.43 7.67 7.57 7.48 7.58 7.72 7.66 7.59 7.73 7.83 7.45 7.49 7.60 7.48 7.59 7.60 7.73 7.52 7.75 7.84 7.50 7.49 7.67 7.61 7.75 7.70 7.87 7.67 7.47 7.65 7.49 7.84 7.74 7.74 7.58 7.50 7.82 7.50 7.49 7.46 7.47 7.56 7.76 7.90 7.59 7.61 7.47 7.62 7.47 7.60 7.42 7.42 7.76 7.54 7.81 7.89 7.59 7.75 0 61 123 180 242 300 361 422 480 541 603 660 722 780 841 902 960 1021 1079 1140 1202 1260 1321 1382 1440 1501 1559 1620 1682 1740 1800 .59 7.84 7.60 7.82 7.74 7.41 7.48 7.59 7.51 7.62 7.69 7.50 7.91 7.44 7.56 7.47 7.Biology IA Task Wen Wen Teh 12E Temperature (°C) Time (s) 30 Test 1 Test 2 Test 3 7.50 7.87 7.50 7.78 7.73 7.48 7.62 7.83 40 pH (± 0.89 7.47 7.76 7.75 7.57 7.54 7.65 7.42 7.82 7.59 7.38 7.87 7.48 7.58 7.77 7.86 7.63 7.63 7.52 7.74 7.60 7.87 7.60 7.41 7.87 7.61 7.62 7.49 7.92 7.68 7.62 7.57 7. 46 7.48 7.52 7.47 7.52 7.46 7.48 7.50 7.1) Test 2 Test 3 Test 4 7.49 7.47 7.52 7.49 7.81 7.50 7.50 7.80 7.44 7.47 7.48 7.51 7.47 7.78 7.45 7.48 7.47 7.51 7.78 0 61 123 180 242 300 361 422 480 541 603 660 722 780 841 902 960 1021 1079 1140 1202 1260 1321 1382 1440 1501 1559 1620 1682 1740 1800 Table 1: The original results of the change of pH under different temperatures over 1800 seconds (30 minutes) .51 7.78 7.47 7.51 7.81 7.48 7.49 7.50 7.52 7.50 7.52 7.51 7.46 7.79 7.80 7.46 7.79 7.51 7.48 7.45 7.50 7.47 7.48 7.80 7.51 7.80 7.47 7.47 7.47 7.48 7.53 7.47 7.76 7.80 7.47 7.59 7.48 7.50 7.78 7.46 7.48 7.47 7.80 7.52 7.76 7.48 7.48 7.78 7.51 Time (s) Test 1 7.82 7.82 7.50 7.50 7.81 7.51 7.47 7.48 7.45 7.47 7.52 7.Biology IA Task Wen Wen Teh 12E Temperature (°C) 50 pH (±0.49 7.46 7.46 7.51 7.48 7.46 7.45 7.81 7.50 7.49 7.47 7.45 7.48 7.78 7.46 7.52 7.75 7.80 7.51 7.78 7.81 7.48 7.46 7.82 7.79 7.48 7.51 7.48 7.47 7.47 7.47 7.46 7.50 7.81 7.79 7.51 7.85 7. the change of pH level over 1801 seconds .10 0 123 242 361 480 603 722 841 960 1079 1202 1321 1440 1559 1682 1801 y = -0.0003x + 7.745 Test 1 Test 2 Test 3 Test 4 Linear (Test 1) Linear (Test 2) Linear (Test 3) Linear (Test 4) y = -0.70 pH level 7.50 7.30 7.4121 y = 0.50 7.0001x + 7. the change of pH level over 1801 seconds The change of pH level is supposed to be negative because the lipase lowers the pH level of the milk.60 7.8862 Time (s) Graph 2: At 20 °C. However. Therefore.40 7.20 7.0004x + 7.0012x + 7.80 7.10 0 123 242 361 480 603 722 841 960 1079 1202 1321 1440 1559 1682 1801 y = 0.90 7.409 y = -0. the graph above shows increases in the pH level at 10°C which is against the supposed change.30 7.8712 y = -0.80 7. the results above are invalid.0008x + 7.20 7.60 7.7734 Test 1 y = 0.70 pH level 7.765 Test 2 Test 3 Test 4 Linear (Test 1) Linear (Test 2) Linear (Test 3) Linear (Test 4) Time (s) Graph 1: At 10 °C.8203 y = 0. Change of pH at 20°C 8.Biology IA Task Wen Wen Teh 12E Data Processing Calculating the trend Change of pH at 10°C 7.90 7.40 7.00 7.0007x + 7.0009x + 7.0011x + 7. 5245 y = -0.20 7.70 7.60 7.50 7.00 7.005x + 7.10 0 123 242 361 480 603 722 841 960 1079 1202 1321 1440 1559 1682 1801 y = -0.925 Time (s) Graph 3: At 30 °C.004x + 7. the change of pH level over 1801 seconds Change of pH at 40°C 8.90 7.Biology IA Task Wen Wen Teh 12E Change of pH at 30°C 8.80 7.30 7.50 7.80 pH level 7.4967 y = -0.60 7.0044x + 7.0019x + 7.40 7.0037x + 7.8662 Test 1 Test 2 Test 3 Test 4 Linear (Test 1) Linear (Test 2) Linear (Test 3) Linear (Test 4) Time (s) Graph 4: At 40 °C.7081 y = -0.0023x + 7.40 7.6317 Test 1 y = -0.5245 y = -0.30 0 123 242 361 480 603 722 841 960 1079 1202 1321 1440 1559 1682 1801 y = -0.0024x + 7.00 7.90 7.70 pH level 7.7003 Test 2 Test 3 Test 4 Linear (Test 1) Linear (Test 2) Linear (Test 3) Linear (Test 4) y = -0.0036x + 7. the change of pH level over 1801 seconds . 0012 0. due to fluctuations in the data collected.0024 30 0.00007 0.0003 0. However.20 0 123 242 361 480 603 722 841 960 y = -0. 1801 1079 1202 1321 1440 1559 1682 .0019 0.5032 y = -0. the rate of reaction cannot be calculated in this manner.0050 0.70 pH level 7.00002 0.90 7.0044 50 0.0044 0.0023 0.0024 0.Biology IA Task Wen Wen Teh 12E Change of pH at 50°C 7. Temperature Rate of reaction (r/s-1) (°C) Test 1 Test 2 Test 3 Test 4 Mean 20 0. the change of pH level over 1801 seconds The rate of reaction is calculated as the absolute change in pH divided by the time (s).0005 0.40 7.0040 0.0003 0. The negative gradient of the graph represents the decrease in pH.80 7.0036 40 0.474 Test 1 Test 2 Test 3 Test 4 Linear (Test 1) Linear (Test 2) Linear (Test 3) Linear (Test 4) Time (s) Graph 5: At 50 °C.0005x + 7.0003x + 7.4867 y = 7E-05x + 7.0036 0.0007 0.0009 0.0037 0.30 7. Therefore a trendline is added to best gauge the trend.60 7.50 7.0002 Table 2: This shows the rate of reaction at each temperature and the calculated average rate of reaction.7999 y = -2E-05x + 7. while the absolute value of the gradient represents rate of change which is also the rate of reaction. The highlighted values are anomalous. 005 0. .002 0.0005 0 0 20 40 Temperature (°C ) 60 Rate of reaction (r/s-1) Average Poly.004 0. (Average) Graph 6: This shows the shows the process data of the rates of reaction against various temperatures.0045 0.003 0.0015 0.001 0.Biology IA Task Wen Wen Teh 12E Presenting Processed data Average rate of reaction of lipase 0.0035 0.0025 0. Even so. Therefore it influences the final trend in graph 6 which plots the relationship of the rate of reaction against the temperature. The range of optimum temperatures are from 31°C to 49°C. which is the predicted result. The pH probe and data logger had a lot of fluctuations which greatly affected the results of the experiment. This is due to the rates of change of pH being very small and hard to detect. It can also be improved by using more precise instruments such as the pH probe and data logger. The temperature of the water bath changed slightly during the experiment and the temperature of the ice bath also increased when the ice started to melt. By adding more sodium bicarbonate to make the milk more alkali and adding more lipase. which shows that it is the optimum temperature for lipase activity. The results obtained for 10°C were shown to be invalid. A way to improve this would be by incubating the beakers so the temperature would stay constant. However. So there would be more time to conduct more repeats. The actual exact optimum temperature for lipase activity is 37°C. the trend was not even as there were a lot of uncertainties in the data itself. It also supports the hypothesis because the shape of Graph 6 is similar to the shape of the graph in figure 1. The fluctuations in the data could be due to the fact that the temperature could not be kept constant. Although there was a trend of general decrease in the pH for most of the temperatures. Evaluation The results of the experiment are valid because the appropriate variables were controlled and the results were consistent and reliable. Thus. The range chosen was also unsuitable as it produced too many results which then had to be filtered. the standard deviation cannot be calculated.Biology IA Task Wen Wen Teh 12E Conclusion The data showed that 40°C had the greatest rate of change of pH level. the reliability can be improved by doing more repeats as only 4 repeats were done due to insufficient time. the rate decreased. The experiment could be improved by conducting it over a shorter time period. This can be avoided in the future by conducting more initial tests to choose the appropriate temperature and sort out any systematic error. as there was insufficient data. This shows that the rate increased as the temperature increased until it reached the optimum temperature. as the change was quite constant throughout the 30 minutes. The original results were recorded with a few seconds . it cannot be proven that 40°C is the exact optimum temperature because the other temperatures close to 40°C were not investigated. This would effect the data obtained as temperature itself is the independent variable. the experiment could be improved to show a greater change and the results can be detected more easily. then as the temperature continues to increase. This makes the results more prone to human errors and bias. To improve this. . the data logger should be set to a more even and wider range.Biology IA Task Wen Wen Teh 12E intervals.