Biology IA Task Wen Wen Teh 12E Investigating Factors that could Effect the Action of an Enzyme Introduction Enzymes 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 o f 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?
Biology IA Task Wen Wen Teh 12E Variables Independent variable The independent variable will be the temperature at which the reaction t akes place in. The investigation will test a range of temperatures: 10° C, 20° C, 30° C, 40° C, 50° C. Water baths will be used to achieve temperatures 30° C, 40° C, 50° C, while beakers filled with ice for 10° C and 20° C. Dependent variable The dependent variable measured will be the rate of reaction of the lipase. The lipase catalyzes the reaction which turns the milk acidic and lowers the pH level. So the closer it is to the optimum temperature, the greater the catalysis thus the faster the drop of pH. The change of pH will be measured with a pH probe and the data recorded on a data logger. Sodium bicarbonate solution is added to the milk to make it more alkali to easier monitor the change in pH. Controlled variable The concentration of lipase used is set at 5% lipase solution to make sure the concentration is the same throughout. If a higher concentration was used, it will increase the rate of reaction and vice versa, which will affect the results. The amount of lipase solution used was also controlled by measuring 2cm 3 into each test tube. If a different amount of lipase was used, it will affect the rate of reaction of the lipase on the milk and influence the results. 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, so the lipase will have equal amount of substrate to act on making it a fair test. 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 milk used was full fat milk from the same package. 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. If the boiling tubes were left for a lesser time, the lipase will have less affect on the milk compared to the rest which will lead to unreliable results. Apparatus 1. 5% lipase solution 2. 0.05 mol dm-3 Sodium bicarbonate solution (Na2CO3) 3. Full fat milk 4. Ice 5. Data logger 6. pH probe 7. Thermometers 8. Test tubes 9. Boiling tubes
Biology IA Task Wen Wen Teh 12E 10. Test tube rack 11. Glass rods 12. Pipette 13. 5 Syringes, 2cm3 14. Stop clock/stopwatch 15. 5cm3 ± 0.001 cm3 measuring cylinders (milk) 16. 10cm 3 ± 0.1cm3 measuring cylinders (sodium bicarbonate solution) 17. 100cm 3 beakers (act as water baths for temperatures below room temperature) Methods 1. Set up data logger and pH probe. 2. Set up water baths at 30° C, 40° C, 50° C. 3. Set up water baths at 10° C and 20° C with ice and water. 4. Measure out 5 cm3 of milk with the 5ml measuring cylinder into each boiling tube. 5. Measure out 7 cm3 of sodium bicarbonate solution and add it to the milk. Check the pH level with pH probe, it should be alkaline. 6. Label the boiling tubes with the temperature to be investigated. 7. 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. 8. Place the pH probe into the boiling tube and get the initial reading of pH level. 9. Measure out 2cm3 of lipase solution into a test tube. 10. Add lipase to the boiling tube and start the stop clock. 11. Record the pH of the milk using the data logger at every 30 seconds interval for 30 minutes. 12. Repeat steps 7-11 for different temperatures. 13. Repeat the whole experiment 5 times to ensure reliability of results. Hypothesis The higher the temperature, the faster the rate of change of milk pH level, until it reaches the optimum temperature where the rate of change of pH level is the highest. Then the rate drops as the enzyme denatures at higher temperatures.
Biology IA Task Wen Wen Teh 12E Results Collected Data .
Temperature (°C) Time (s)
10
20 pH (± 0.1)
Test 1
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
Test 2
Test 3
Test 4
Test 1
Test 2
Test 3
Test 4
7.77
7.76
7.45
7.76
7.87
7.90
7.71
7.43
7.76
7.78
7.42
7.82
7.86
7.89
7.74
7.40
7.76
7.78
7.42
7.83
7.87
7.89
7.75
7.42
7.77
7.76
7.42
7.83
7.88
7.88
7.77
7.40
7.79
7.76
7.42
7.82
7.87
7.89
7.74
7.40
7.78
7.78
7.42
7.82
7.87
7.87
7.75
7.41
7.78
7.76
7.42
7.83
7.86
7.88
7.75
7.41
7.78
7.76
7.41
7.82
7.86
7.89
7.73
7.40
7.79
7.77
7.41
7.83
7.87
7.88
7.73
7.39
7.79
7.77
7.42
7.83
7.86
7.88
7.73
7.39
7.79
7.77
7.42
7.83
7.86
7.88
7.72
7.41
7.78
7.76
7.42
7.84
7.86
7.89
7.75
7.40
7.79
7.75
7.42
7.84
7.87
7.88
7.72
7.39
7.79
7.78
7.42
7.83
7.86
7.89
7.73
7.39
7.81
7.76
7.41
7.85
7.85
7.87
7.73
7.38
7.81
7.76
7.40
7.83
7.87
7.87
7.74
7.38
7.81
7.77
7.40
7.83
7.86
7.87
7.74
7.37
7.79
7.76
7.41
7.83
7.85
7.88
7.73
7.38
7.80
7.75
7.41
7.85
7.86
7.87
7.73
7.37
7.80
7.76
7.42
7.82
7.85
7.88
7.72
7.37
7.81
7.77
7.42
7.82
7.86
7.89
7.71
7.38
7.78
7.75
7.44
7.82
7.86
7.88
7.72
7.37
7.80
7.78
7.44
7.83
7.85
7.88
7.74
7.37
7.79
7.78
7.44
7.82
7.86
7.89
7.73
7.38
7.79
7.78
7.43
7.83
7.87
7.89
7.72
7.38
7.79
7.77
7.44
7.83
7.84
7.87
7.72
7.38
7.79
7.78
7.45
7.83
7.85
7.87
7.73
7.37
7.79
7.77
7.45
7.83
7.85
7.88
7.71
7.38
7.81
7.77
7.44
7.82
7.84
7.88
7.72
7.39
7.81
7.78
7.44
7.82
7.84
7.90
7.70
7.39
7.81
7.76
7.45
7.83
7.86
7.86
7.72
7.39
Biology IA Task Wen Wen Teh 12E Temperature (°C) Time (s)
30
40 pH (± 0.1)
Test 1
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
Test 2
Test 3
Test 4
Test 1
Test 2
Test 3
Test 4
7.67
7.62
7.90
7.62
7.51
7.50
7.57
7.89
7.76
7.64
7.95
7.72
7.52
7.52
7.54
7.87
7.71
7.62
7.92
7.73
7.55
7.50
7.52
7.87
7.69
7.63
7.92
7.70
7.52
7.48
7.53
7.86
7.69
7.63
7.92
7.72
7.52
7.50
7.50
7.87
7.66
7.62
7.90
7.69
7.52
7.45
7.52
7.84
7.66
7.63
7.91
7.67
7.50
7.47
7.49
7.82
7.67
7.62
7.89
7.68
7.50
7.49
7.50
7.84
7.64
7.61
7.89
7.69
7.52
7.45
7.52
7.82
7.65
7.62
7.90
7.67
7.49
7.47
7.50
7.81
7.64
7.58
7.87
7.68
7.48
7.45
7.49
7.82
7.64
7.58
7.87
7.63
7.47
7.43
7.49
7.81
7.61
7.59
7.87
7.68
7.50
7.42
7.48
7.78
7.64
7.58
7.85
7.65
7.50
7.43
7.47
7.78
7.64
7.57
7.86
7.63
7.50
7.42
7.47
7.75
7.63
7.60
7.85
7.65
7.47
7.44
7.47
7.76
7.60
7.60
7.87
7.63
7.48
7.43
7.49
7.75
7.61
7.59
7.89
7.65
7.49
7.41
7.50
7.76
7.62
7.57
7.86
7.64
7.50
7.45
7.51
7.75
7.60
7.59
7.87
7.61
7.46
7.41
7.49
7.74
7.62
7.56
7.84
7.61
7.49
7.42
7.49
7.74
7.62
7.60
7.84
7.60
7.47
7.42
7.49
7.73
7.60
7.60
7.83
7.61
7.48
7.40
7.48
7.73
7.60
7.58
7.83
7.59
7.47
7.39
7.48
7.75
7.59
7.57
7.82
7.62
7.46
7.42
7.47
7.76
7.59
7.57
7.83
7.59
7.45
7.41
7.50
7.74
7.58
7.58
7.84
7.59
7.47
7.40
7.47
7.73
7.56
7.56
7.84
7.60
7.44
7.40
7.48
7.77
7.57
7.54
7.82
7.59
7.47
7.40
7.48
7.74
7.58
7.56
7.82
7.62
7.48
7.38
7.48
7.74
7.60
7.56
7.83
7.59
7.46
7.41
7.46
7.75
Biology IA Task Wen Wen Teh 12E Temperature (°C) Time (s)
50 pH (±0.1) Test 1
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
Test 2
Test 3
Test 4
7.85
7.53
7.59
7.52
7.81
7.51
7.48
7.48
7.82
7.51
7.52
7.48
7.80
7.48
7.50
7.47
7.81
7.48
7.50
7.48
7.78
7.50
7.51
7.49
7.78
7.49
7.52
7.46
7.79
7.48
7.49
7.48
7.82
7.46
7.51
7.47
7.79
7.48
7.47
7.48
7.76
7.48
7.47
7.47
7.80
7.46
7.46
7.45
7.79
7.45
7.52
7.46
7.78
7.47
7.46
7.47
7.75
7.48
7.52
7.45
7.78
7.44
7.47
7.47
7.80
7.46
7.51
7.46
7.76
7.45
7.49
7.47
7.78
7.47
7.50
7.48
7.80
7.46
7.52
7.48
7.81
7.45
7.50
7.47
7.81
7.47
7.48
7.46
7.79
7.48
7.51
7.47
7.80
7.50
7.51
7.46
7.80
7.51
7.51
7.47
7.78
7.48
7.51
7.48
7.81
7.49
7.52
7.47
7.82
7.50
7.50
7.47
7.81
7.47
7.52
7.50
7.80
7.50
7.51
7.50
7.78
7.47
7.51
7.51
Table 1: The original results of the change of pH under different temperatures over 1800 seconds (30 minutes)
Biology IA Task Wen Wen Teh 12E Data Processing Calculating the trend
Change of pH at 10°C y = 0.0004x + 7.8203
7.90
y = 0.0011x + 7.7734
7.80
Test 1 y = 0.0001x + 7.765
7.70
Test 2
l 7.60 e v e7.50 l H p
Test 3 Test 4
7.40 y = 0.0008x + 7.4121
7.30 7.20
Linear (Test 1) Linear (Test 2)
7.10 0 3 2 1 0 3 2 1 0 9 2 2 4 6 8 0 2 4 6 7 0 1 2 3 4 6 7 8 9 0 2 1 1
1 2 3 1
0 4 4 1
9 5 5 1
2 8 6 1
1 0 8 1
Time (s)
Linear (Test 3) Linear (Test 4)
Graph 1: At 10 °C, 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. However, the graph above shows increases in the pH level at 10°C which is against the supposed change. Therefore, the results above are invalid.
Change of pH at 20°C 8.00
y = -0.0003x + 7.8862
7.90 7.80
y = -0.0007x + 7.8712
7.70
Test 1 Test 2
y = -0.0009x + 7.745
l e v7.60 e l H7.50 p
Test 3 Test 4
7.40 7.30
Linear (Test 1) y = -0.0012x + 7.409
Linear (Test 2)
7.20
Linear (Test 3)
7.10 0 3 2 1 0 3 2 1 0 9 2 2 4 6 8 0 2 4 6 7 0 1 2 3 4 6 7 8 9 0 2 1 1
1 2 3 1
0 4 4 1
9 5 5 1
2 8 6 1
1 0 8 1
Linear (Test 4)
Time (s)
Graph 2: At 20 °C, the change of pH level over 1801 seconds
Biology IA Task Wen Wen Teh 12E
Change of pH at 30°C 8.00
y = -0.0036x + 7.925
7.90 7.80 l e7.70 v e l H7.60 p
7.50
Test 1 y = -0.004x + 7.7081
Test 2 y = -0.0044x + 7.7003
Test 3 Test 4
y = -0.0024x + 7.6317
Linear (Test 1) Linear (Test 2)
7.40
Linear (Test 3)
7.30 0 3 2 1 0 3 2 1 0 9 2 2 4 6 8 0 2 4 6 7 0 1 2 3 4 6 7 8 9 0 2 1 1
1 2 3 1
0 4 4 1
9 5 5 1
2 8 6 1
1 0 8 1
Linear (Test 4)
Time (s)
Graph 3: At 30 °C, the change of pH level over 1801 seconds
Change of pH at 40°C 8.00 7.90
y = -0.005x + 7.8662
7.80 Test 1
7.70 l e7.60 v e l H7.50 p
7.40
Test 2 Test 3
y = -0.0019x + 7.5245
Test 4 y = -0.0023x + 7.5245 y = -0.0037x + 7.4967
Linear (Test 1)
7.30
Linear (Test 2)
7.20
Linear (Test 3) Linear (Test 4)
7.10 0 3 2 1 0 3 2 1 0 9 2 2 4 6 8 0 2 4 6 7 0 1 2 3 4 6 7 8 9 0 2 1 1
1 2 3 1
0 4 4 1
9 5 5 1
2 8 6 1
1 0 8 1
Time (s)
Graph 4: At 40 °C, the change of pH level over 1801 seconds
Biology IA Task Wen Wen Teh 12E
Change of pH at 50°C 7.90 7.80 7.70 l e7.60 v e l H7.50 p
y = -0.0003x + 7.7999
Test 1 Test 2
y = -2E-05x + 7.5032
Test 3
y = -0.0005x + 7.4867
Test 4 Linear (Test 1)
y = 7E-05x + 7.474
7.40
Linear (Test 2)
7.30
Linear (Test 3)
7.20 0 3 2 1 0 3 2 1 0 9 2 2 4 6 8 0 2 4 6 7 0 1 2 3 4 6 7 8 9 0 2 1 1
1 2 3 1
0 4 4 1
9 5 5 1
2 8 6 1
1 0 8 1
Linear (Test 4)
Time (s)
Graph 5: At 50 °C, 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). However, due to fluctuations in the data collected, the rate of reaction cannot be calculated in this manner. Therefore a trendline is added to best gauge the trend. The negative gradient of the graph represents the decrease in pH, while the absolute value of the gradient represents rate of change which is also the rate of reaction. -1
Temperature Rate of reaction (r/s ) (°C) Test 1 Test 2 Test 3 Test 4 Mean 20 0.0007 0.0003 0.0009 0.0012 0.0024 30 0.0044 0.0024 0.0036 0.0040 0.0036 40 0.0023 0.0037 0.0019 0.0050 0.0044 50 0.0003 0.0005 0.00002 0.00007 0.0002 Table 2: This shows the rate of reaction at each temperature and the calculated average rate of reaction. The highlighted values are anomalous.
Biology IA Task Wen Wen Teh 12E
Presenting Processed data
Average rate of reaction of lipase 0.005 ) 1 -
0.0045 0.004
s / r ( 0.0035 n 0.003 o i t c 0.0025 a e r 0.002 f o 0.0015 e t a 0.001 R
Average Poly. (Average)
0.0005 0 0
20
40
60
Temperature (°C )
Graph 6: This shows the shows the process data of the rates of reaction against various temperatures.
Biology IA Task Wen Wen Teh 12E Conclusion The data showed that 40°C had the greatest rate of change of pH level, which shows that it is the optimum temperature for lipase activity. It also supports the hypothesis because the shape of Graph 6 is similar to the shape of the graph in figure 1, which is the predicted result. This shows that the rate increased as the temperature increased until it reached the optimum temperature, then as the temperature continues to increase, the rate decreased. Even so, it cannot be proven that 40°C is the exact optimum temperature because the other temperatures close to 40°C were not investigated. The range of optimum temperatures are from 31°C to 49°C. The actual exact optimum temperature for lipase activity is 37°C. Evaluation The results of the experiment are valid because the appropriate variables were controlled and the results were consistent and reliable. However, the reliability can be improved by doing more repeats as only 4 repeats were done due to insufficient time. Thus, the standard deviation cannot be calculated, as there was insufficient data. The experiment could be improved by conducting it over a shorter time period, as the change was quite constant throughout the 30 minutes. So there would be more time to conduct more repeats. Although there was a trend of general decrease in the pH for most of the temperatures, the trend was not even as there were a lot of uncertainties in the data itself. This is due to the rates of change of pH being very small and hard to detect. The pH probe and data logger had a lot of fluctuations which greatly affected the results of the experiment. By adding more sodium bicarbonate to make the milk more alkali and adding more lipase, the experiment could be improved to show a greater change and the results can be detected more easily. It can also be improved by using more precise instruments such as the pH probe and data logger. The fluctuations in the data could be due to the fact that the temperature could not be kept constant. 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. This would effect the data obtained as temperature itself is the independent variable. A way to improve this would be by incubating the beakers so the temperature would stay constant. The results obtained for 10°C were shown to be invalid. Therefore it influences the final trend in graph 6 which plots the relationship of the rate of reaction against the temperature. This can be avoided in the future by conducting more initial tests to choose the appropriate temperature and sort out any systematic error. The range chosen was also unsuitable as it produced too many results which then had to be filtered. The original results were recorded with a few seconds
Biology IA Task Wen Wen Teh 12E intervals. 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.
