The action of lipase.

The action of lipase

AIM: Lipases hydrolize fats into glycerol and fatty acids1, therefor to investigate how different amount (1 cm3, 2 cm3, 4 cm3, 8 cm3, 12 cm3, 20 cm3) of 3% lipase solutions break down the fat in 5 cm3 of milk.

RESEARCH QUESTION: how fast can different amount (1 cm3, 2 cm3, 4 cm3, 8 cm3, 12 cm3, 20 cm3) of 3% lipase solutions break down the fat molecules in 5 cm3 of milk?

INDEPENDENT VARIABLE: amount of 3% lipase solution (1 cm3, 2 cm3, 4 cm3, 8 cm3, 12 cm3, 20 cm3)

DEPENDENT VARIABLE: the speed (measured in minutes) of hydrolizing fat molecules

CONTROLLED VARIANLE: concentration of lipase solution, amount of milk, amount and concentration of sodium carbonate solution

HYPOTHESES: • lipase hydrolize fats into glycerol and fatty acid1 therefore as the amount of lipase is increased, the fat molecules will be hydrolized faster and faster

• boiled lipase will not break down fat molecules, because the enzymes denaturate on high temperature

Materials: Equipments:

-milk -test tubes

-0.05M sodium carbonate solution -test tube rack

-phenolphthalein indicator -test tube holder

-3% lipase solution -dropping pipette

-graduated pipette

-Bunsen burner

-beaker

-watch

PROCEDURE:

Using a graduated pipette, 5 cm3 of milk is placed in seven test tubes.
7 cm3 sodium carbonate solution is placed in each test tube.
Using a dropping pipette phenolphthalein is add to each tube until the contests are bright pink. The test tubes are shaken well after adding each drop.
In a spare tube about 15 mm 3 % lipase solution is placed and the liquid is heated over a Bunsen burner until it boils for a few seconds. The tube is cooled and 1 cm3 of boiled liquid is transported ...

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-watch

PROCEDURE:

Using a graduated pipette, 5 cm3 of milk is placed in seven test tubes.
7 cm3 sodium carbonate solution is placed in each test tube.
Using a dropping pipette phenolphthalein is add to each tube until the contests are bright pink. The test tubes are shaken well after adding each drop.
In a spare tube about 15 mm 3 % lipase solution is placed and the liquid is heated over a Bunsen burner until it boils for a few seconds. The tube is cooled and 1 cm3 of boiled liquid is transported to test tube 1.
With a graduate pipette 1 cm3 unboiled lipase solution is placed in test tube 2.
With a graduate pipette 2 cm3 unboiled lipase solution is placed in test tube 3.
With a graduate pipette 4 cm3 unboiled lipase solution is placed in test tube 4.
With a graduate pipette 8 cm3 unboiled lipase solution is placed in test tube 5.
With a graduate pipette 12 cm3 unboiled lipase solution is placed in test tube 6.
With a graduate pipette 20 cm3 unboiled lipase solution is placed in test tube 7.
The test tubes is shaken to mix the contents.

DATA COLLECTION:

• Colour of the solution (milk, sodium carbonate solution): white

• Colour of the solution (milk, sodium carbonate solution, phenolphthalein): bright pink

• To the seven test tubes 5 0.5 cm3 milk and 7 0.5 cm3 sodium carbonate solution was placed.

Table 1. Observations of the action of lipase

Table 2. Observations of the action of lipase – Student A

Table 3. Observations of the action of lipase – Student B

• In each case the measurement of time started after placing the lipase solution, and shaking the test tube.

• Regarding the scale of numbers (from 3 to 40 in average - see Table 4.) it was uneccasary to measure the time with the accuracy of seconds.

• In each case the test tube containig the unboiled lipase solution (tube 1) did not reach the stage of becoming white during the time of lab (about 90 min), this is denoted with a ’-’ sign.

• The scaling of the graduated pipette was 1 cm3 therefor the accuracy might differ by 0.5 cm3 .

DATA PROCESSING AND PRESENTATION:

Table 4. The action of lipase - average time taken for the solution to become white

•The average is reported with half of the range (instead of standard deviation), because of the small number of replicates. The samll number of replicates did not allow to leave out any of the obtained data. – see conclusion and evaluation to see the possible way of avoiding this problem.

•Graph 1. represents the data of Table 4. plotted on a graph. On the x axis the independent variable (amount of lipase) and on the y axis the dependent variable (the time taken for the solution to become white) can be seen. The best-fit line is drawn linear, going through the range boxes. If the x and the y values were of the same distance from the origo, then the best-fit line would be 45o.

•Tube 1 – boiled lipase is not plotted on the graph because it was the contol test, to make sure that the colour change does not happen anyway and that the role of the lipase is responsible for the colour change, since the enzymes denaturate in high temperature, change shape, therefore do not work any more.

• The horizontal error bars represent amount of lipase (cm3) 0.5

• The vertical error bars represent the average time (min) half of the range (in case of

tube 2 – 2 cm3 the range was 0)

• No systematic error occured during this experiment, the random errors are discussed above – graduating pipette, watch

CONCLUSION AND EVALUATION:

• The hypotheses were supportive, because as it can be seen on Graph 1, as the amount of lipase was increased, the solution changed colour faster and faster, in sciantifically speaking more and more lipid was broken down to glycerol and fatty acid. The boiled lipid did not change to colour of the solution – no lipid was broken down.

• In this experiment the time taken for the solution (milk, sodium carbonate solution, phenolphthalein, lipase solution) to become white was tested. Phenolphthalein is and acid/base indicator. It starts to be bright pink because the carbonate ions in the sodium carbonate give the overall pH to be alkaline. It is transparent in acid solution. When the lipase breaks down the fat molecules (lipids) into fatty acid and glycerol, the pH decreases into the acid range and the colour of the phenolphthalein fades. This allows the white colour of milk to come through.

• The lipase in the controll test (tube 1) denaturated on high temperature, and could not function any more, so no fat molecule was broken down, therefore the pH of the solution did not change, and the colour of phenolphthalein did not indidcate the change of pH, and the white colour of milk could not come through. This is why it is dangerous for humans to have a raised temperature (fever), because the heat unables the enzymes to function, on which the human body is reliant. Lipase is used as biological washing powder (since it breaks down the fat molecules) but if it is put in hot wash, the fat does not come out of the clothes, because lipase denaturates.

• On Graph 1. it can be seen that the action of lopase is in inverse proportion to time.

SUGGESTIONS FOR FURTHER IMPROVEMENT:

• To calculate the average time taken for the solution to become white, more students’ data should be used.

• The action of lipase could have been tested in another way as well: the test tubes should be observed at a certain time, and the dependent variable would be the ratio of pink and white in the test tube. It would show that as the amount of lipase is increased, the ratio of white will be greater and greater. For the contol test – boiled lipase the ratio of pink and white would be 1:0. Although there are many source of error for this experiment, mainly measuring the ratio. I would suggest using ruler, but cause difficulties how to measure the u-shaped bottom of the test tubes.

• Testing tube 1 and 2 the conclusion was that the enzyme lipase denaturates on high temperature. To go further, I would suggest an experiment to test at what temperature does enzyme lipase denaturate. For this, lipase on room temperature (about 21 Co) and heated lipase (25, 30, 35, 40 and 45 Co) should be used. The independent variable: temperature of lipase, the dependent variable: time taken for the solution to become white, and the controlled variable: amount of solutions added, including lipase.

• I would suggest to use 1 drop of 1 M sodium hydroxide insted of sodium carbonate, because glycerol has three alcohol groups and each of these weakly dissociates to give acid character. The carbonate ions react in acid giving off CO2 and a froming a hydroxide after the hydrolysis of water molecules. These then neutralise the acid, so carbonate ions act like a buffer therefore the acid production cannot be registered as fast as it should be. Therefore 1 drop of

1 M sodium hydroxide should be use, to avoid the buffer effect.

• The random error of using graduated pipette cannot be avoided. Measuring the time could have been done with the accuracy of seconds, the reason for not doing so is discussed above in the data collection section.