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Assessment Task 3

Open Ended Investigation

Year 12 HSC Biology

2018

30031024

Aim:

To investigate how the concentration of lipase affects the rate at which it catalyses the hydrolysis of the lipids found in full fat milk.

Background Information:

Enzymes have a tertiary structure that’s held together by ionic bonds, disulphide bridges and hydrogen bonds. The molecules have a small region that is, in fact, function, despite the large size. This is known as the active site, and it is where the substrate molecule is help by bonds that form temporarily between the groups of amino acids on the active site and groups on the substrate. The enzyme is then able to break the bonds holding the substrate together, thus making the substrate molecule break apart into smaller ‘products’. This is known as the ‘lock and key’ theory.

Figure 1: The ‘Lock and Key’ Theory

Lipase is a type of hydrolase enzyme that catalyses the hydrolysis of triglycerides into fatty acids and glycerol. It is referred to as a hydrolase as the reaction that it catalyses is a hydrolysis reaction, which is where molecules are broken down into smaller ones with the addition of water. Lipids are compounds that are made up of hydrogen, oxygen and carbon which can be extracted, due to its property of being non-polar, using non-polar solvents such as alcohol and ether. Lipids greatly assist with heat insulation, energy storage and the production of steroids and cholesterol.

Triglycerides (substrate) are the most common type of lipid, and it consists of one glycerol molecule and three fatty acid molecules bound together by ester bonds. They are the main constituents of body fat in humans and in many other animals, as well as vegetable fat. They are present in the blood stream in order to allow a bidirectional transfer of fat and blood glucose from the liver, and they are a major element of oils in the human skin. This means that excess calories from foods that are not needed for quick energy will turn into triglycerides and then proceed to be stored in fat cells to be used later. Therefore, eating more calories than you burn could result in high triglycerides. Atherosclerosis is the thickening of artery walls, which leads to stroke and diseases of the heart, and sometimes cases of acute pancreatitis. When in triglyceride form, lipids cannot be absorbed by the duodenum, which is the first section of the small intestine in most higher vertebrates.

Stephen Lucas’s ‘A2 Biology Coursework’ experiment has influenced my experiment greatly as it has provided current, reliable, accurate information that has assisted with my decisions of how to structure the method and how to set up my experiment. His ‘investigation into how the volume of lipase affects the rate of hydrolysis of lipids’ is thorough and very impressive, however he used bile salts in his tests. His experiment states that “Bile contains several salts derived from cholesterol including sodium glycocholate and sodium taurocholate. These salts act like detergents and help to emulsify fats, breaking fat droplets in the lumen of the small intestine into tiny globules.” I would like to test the rate at which lipase catalyse the hydrolysis of lipids without the emulsification from the bile salt.

Variables:

Independent: The concentration (volume) of lipase

Dependent: The resulting pH level

Equipment List:

Method

The pH metre was calibrated by placing the electrode in the buffer solution with a pH of 9, and it was allowed to stabilise to read ‘9.00’. The electrode was then removed from the buffer solution, rinsed with deionised water and dabbed with a paper towel.

Then the metre was then placed in the the buffer solution with a pH of 5, and it was allowed to stabilise to read ‘5.00’.The electrode was then removed from the buffer solution, rinsed with deionised water and dabbed with a paper towel.

The water bath was turned on and set to a temperature of 37˚C.

5mL of milk was measured from a beaker into a measuring cylinder using a pipette. This was then transferred into a test tube. This was repeated 4 more times in separate test tubes each time, labelling each of them from numbers 0 to 4.

Step 4 was repeated, however with 3mL of sodium carbonate and 1mL of phenolphthalein, and then the solution was stirred with a glass rod for 5 seconds

With one test tube acting as the control, 1mL, 2mL, 3mL and 4mL of lipase was added to the test tubes labelled ‘1’, ‘2’, ‘3’ and ‘4’, respectively.

Test tube ‘0’ was placed into the metal test tube rack in the water bath, measuring at a temperature of 37˚C, and the pH metre was placed with the electrode submerged in the solution. The stopwatch was started as soon as the test tube was in.

The pH level was measured every minute for 10 minutes and both qualitative and quantitative results were recorded at each minute interval. After 10 minutes the test tube was removed from the water bath

Steps 7 & 8 were repeated with test tubes ‘1’, ‘2’, ‘3’ and ‘4’, however the pH metres was rinsed with distilled water and re-calibrated between tests.

Diagrams:

Figure 2: Calibration of pH metre setup

Figure 3: Set up of water bath

Risk Assessment:

*NOTE: Sources directly from RiskAssess.com

Results:

Qualitative:

no change to the physical state of the solution (no gas, bubbles)

Pigmented, opaque pink colour of the initial solution fades gradually

No other physical change to the liquid solution

Figures 4-7: Gradual fade of the pink pigment in the solution

Quantitative:

Trial 1

Trial 2

Trial 3

Average

*NOTE: All values are shown (rounded) to 2 decimal places due to the pH meters only showed values of pH up to 2 decimal places.

Graphs:

Discussion

It is clear from the graphs, tables and statistical processes carried out that increasing the volume (concentration) of lipase increases the change in pH of the solution and also leads to an increase the rate of change of the pH of the solution. This has been proved by the larger maximum change in pH for the higher volumes of lipase and also by the shape of the graphs above. The only differences between each volume of lipase are the number of enzymes and therefore active sites present in the solution. The reason for the higher volumes of lipase, causing a decrease in the pH more quickly and by a larger amount, can be explained by the number of enzymes present and the hydrolysis reaction taking place between the lipids present in the full fat milk. As said in the Background Information, lipase oxidises triglycerides, using three molecules of water to break the 3 ester bonds and to produce a glycerol molecule and three fatty acids.

In all of the graphs it is evident that there was a rapid increase in reaction in the 1.00mL concentration between the 1 to 2 minute mark. I would have counted it as an anomaly, but it occurred in all 3 trials, thus I decided to keep it within the results. There is a decrease in pH over the 10 minute period for the 4.00mL concentration and the 1.00mL concentration, however the pH levels of the other concentrations increase. Regarding the gradients of the graphs, we can see that the 4.00mL concentration has a steeper slope, suggesting their either an excess or not enough of one of the volumes was measured out for the 4.00mL test. A similar conclusion can be drawn in regards to the gradient of the 1.00mL. Between the Trials 1, 2 and 3, and the Average graph, all of the results do seem to be very similar and when compared, move in a parallel motion.

Reliability refers to consistency, meaning it can be judged upon how many repeats were performed. My test was very reliable, as the experiment had 3 trials of each of the variations of lipase concentration, although there were some slight anomalies within the trials, so repeating the experiment a couple more times would provide a clearer average result, however due to the limit of time, this was unable to be done. As all of my results are so similar, it is proof that the results from my experiment are extremely reliable. The repetition, however, only deems the experiment reliable, as opposed to improving it. The repetition minimises the effect of random errors/outliers and allows them to be removed or disregarded, however the anomalies that were recorded, particularly for the 1.00mL concentration, as there was a common anomaly. In terms of improving the reliability, despite using the same type of all of the equipment I used and being as accurate as possible with all of my measurements, errors still could have occurred, such as the solution leaving residue on the inside of the measuring cylinder, meaning not all of the required volume would be transferred into the test tube. This is a minority in the experiment but possibly may have had an impact.

Accuracy is the quality or state of being correct or precise. My experiment proved to be accurate due to the readings/results being digital, rather than analogue. The results were also very close to the true value as the pH metres were calibrated between each test, in order to give the most precise reading. The use of the pH metres was a better decision as Universal Indicator would not have provided the most accurate result. The measuring cylinder used was accurate in that the gradations of the 10mL cylinder are small than those of a 50mL cylinder. The constant deionisation of the equipment used between experiments, done by washing all apparatus with distilled water, prevented cross contamination between lipid concentrations, and separate test tubes were used for each trial and concentration, with the pH metre remaining the same. As stated previously, the volumes were measured out at eye level, however a parallax error may still have occurred because there is no direct way of knowing when my eyes are exactly in line with the graduated millilitre mark I was aiming for. Regarding equipment, the use of a burette or an electronic pipette as an more accurate and consistent measurement of the volumes required. An electronic pipettes would have eradicated these kinds of errors due to their ability to detect the volume of a substance, down to micro-litres, thus giving a more precise measurement than the human eye can.

Validity is the quality of being logically or factually sound. My experiment can be deemed valid as the experiment does address the aim and clearly shows a display of data that is related to the nature of enzymes and is supported by the background information.  The test also uses very suitable equipment, however improvements could have been made, as stated above, regarding how precise the measurements were. The variables in my experiment were controlled to the best ability possible. This was due to using the exact same types of materials across all of the tests and on each of the separate ratios. Only one independent variable was also tested, which meant that the experiment was kept fair. The volumes of the components of the solution were measured at the bottom of the meniscus, at eye level, which is the best form of measurement when dealing with liquids. Controlled variables include the volumes of milk, phenolphthalein and sodium carbonate, which was managed as all substances will be measured using a pipette and a 10mL cylinder. When measuring, the bottom of the meniscus of the liquid is the measurement at which will be recorded, and the temperature at which the solution of lipase, sodium carbonate, milk and phenolphthalein is kept which was maintained as when measuring the pH level of the substances, the test tubes they are contained in will remain within a water bath regulated at 37˚C for the entire period of the testing.

Conclusion

The results of my experiment did agree with my background research, despite the minor anomalies. As the volume of lipase utilised in the experiment was increased, so too was the maximum change in pH. This means that the catabolic reaction between the substrate and the lipase enzymes was responsible for the solution’s decrease in the pH of the solution.As the volume of lipase was increased so too were the number of enzymes and thus active sites capable of hydrolysing the triglycerides to fatty acids and glycerol. The increase in hydrogen ion content/activity was then detected by the electronic pH meter, showing that overall, thus increasing the volume of lipase increases the change in pH and thus increases the rate at which the substrates are broken down. This common trend is exemplified by the time taken for the phenolphthalein to turn clear, the graphs and statistical processes carried out on all of the data harvested. The trend however is not as perfect as we would anticipate, suggesting that several anomalies have occurred during the procedure. In summary, the aim of my investigation on how the concentration of lipase affects the rates at which it catalyses the hydrolysis of the lipids found in full fat milk was effectively and well demonstrated within my experiment.

Bibliography

Baker Heart and Diabetes Institute. (2013). Baker Institute fact sheets. Retrieved May 24, 2018, from https://www.baker.edu.au/health-hub/fact-sheets/cholesterol-triglycerides-your-health

Department of Health & Human Services. (2014, February 28). Triglycerides. Retrieved May 25, 2018, from https://www.betterhealth.vic.gov.au/health/conditionsandtreatments/triglycerides

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El-Sawah, M. M., Sherief, A. A., & Bayoumy, S. M. (n.d.). Enzymatic properties of lipase and characteristics production by Lactobacillus delbrueckii subsp. bulgaricus. Retrieved May 24, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/7574552

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Yon-hui Lin, Moreau, R., & Anthony H. C. Huang. (1982). Involvement of Glyoxysomal Lipase in the Hydrolysis of Storage Triacylglycerols in the Cotyledons of Soybean Seedlings. Plant Physiology, 70(1), 108-112. Retrieved from http://www.jstor.org/stable/4267452

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