Macromolecules and Digestion Practical Investigation

MACROMOLECULES AND DIGESTION PRACTICAL INVESTIGATION

Ruby MacMillan, Stage 1 Biology

Abstract

The purpose of this practical investigation was to observe the temperature at which an enzyme denatures and the rate at which the reaction occurs slows. Prior to the completion of this investigation, it was predicted that as the temperature increases, the speed at which the lipase solution breaks down the fat in the milk to fatty acids would also increase. This was determined by completing 4 different tests at different temperatures. The first test involved heating a water bath to 20°C, and creating a solution using 7 drops of phenolphthalein, 4 drops of sodium carbonate solution, and 0.5mL of full cream milk. This solution was heated to 20°C before 0.5mL of lipase solution was added to the test tube. Immediately after, the timer was started and was not turned off until the solution had turned completely white. This time was recorded in the figure 1.1 below. The second test involved the same process however the water bath was heated to a temperature of 40°C, while the third test involved heating a water bath to 60°C. The fourth test involved the completion of the same process with a water bath heated to 80°C. Each of the four tests was completed 4 times, making an overall sample size of 16. The results demonstrated that the hypothesis was supported, showing that as the temperature increases, the rate at which a reaction takes place will also increase.

Introduction

Enzymes are biological catalysts that increase the rate at which a reaction takes place by providing an alternative reaction pathway of lower activation energy (Rsc.org 2016). The shape of an enzyme and the shape of the active site is specific to a substrate, meaning that only that specific substrate will fit in the active site. Enzyme denaturing is the process in which the shape of the active site changes, causing it to no longer function. This can be caused by high temperatures or extreme changes in pH. In this investigation, the high temperatures cause the enzyme to denature thus the reaction will not take place.

As the temperature increases, the rate of reaction also increases until the temperature is too high and the enzyme denatures. The enzyme activity slowly increases until it reaches the optimum temperature of approximately 37°C, or body temperature. After this point, as the temperature continues to rise, the rate in which the reaction takes place decreases rapidly, as the heat denatures the enzyme, as shown in figure 1 below.

[pic 1][pic 2]

Phenolphthalein is a weak acid which dissociates in water and under alkaline conditions, forming pink anions. This occurs at a pH10. When the pH of a solution drops below pH8.3, the phenolphthalein turns colourless. In this investigation, the phenolphthalein in the solution turns colourless, indicating that a reaction has taken place and the fat in the milk has been broken down into fatty acids.

Aim

To observe the temperature at which an enzyme denatures and the rate of reaction slows.

Hypothesis

As the temperature increases, the speed at which the lipase solution breaks down the fat in the milk to fatty acids will also increase.

Variables

Independent Variable

The independent variable in this practical investigation is the temperature of the water bath.

Dependent Variable

The time it takes for the lipase solution to break down the fat in the milk to fatty acids, shown via a colour change from pink to white.

Controlled Variables

• The pH of the solution

• Volume of lipase solution (5 drops)

• Volume of Phenolphthalein (10 drops)

• Volume of full fat milk (3mL)

• Volume of Sodium carbonate solution (10mL)

• Volume of water in the beaker (500mL)

• Stopwatch

• Size of test tubes

• Thermometer

Materials

• 90mL Sodium Carbonate solution (0.05 mol per L)

• 45 drops 5% Lipase solution

• 27mL full fat milk

• 90 drops Phenolphthalein

• 3x 10mL measuring cylinder

• 3x Stirring rods

• 9x Test tubes

• 3x Test tube racks

• 3x water bath

• 3L water

• 3x Thermometer

• 3x Timers

• Kettle

Method

1. All materials were gathered.
2. 600mL of water was boiled and added to the water bath.
3. 400mL of tap water was added to the water bath and left to sit until the water reached 60°C.
4. Using the 10mL-measuring cylinder, 3mL of full fat milk was measured by leveling the bottom of the meniscus with the line in order to avoid parallax.
5. The milk was added to the test tube using a funnel to avoid spillage.
6. Using a 10mL measuring cylinder, 10mL of sodium carbonate solution was measured by leveling the bottom of the meniscus with the line in order to avoid parallax.
7. The sodium carbonate solution was added to the test tube using a funnel to avoid spillage.
8. Using the dropper on the phenolphthalein, 10 drops of phenolphthalein were added to the test tube.
9. The test tube was placed in the beaker with 60°C water and the thermometer was used to measure the temperature of the solution.
10. Once the solution reached 60°C, 1mL of the 5% Lipase solution was measured using a volumetric pipette.
11. 5 drops of the 5% lipase solution was added to the test tube in order for a reaction to take place.
12. The stopwatch was immediately started and a glass stirring rod was used to combine all solutions.
13. Immediately after the solution turned a clear colour, the stopwatch was stopped and the time was recorded in table 1.1 below.
14. Steps 1-13 were repeated 2 further times.
15. Steps 1-14 were repeated with 20°C water, by using 1L of tap water and no boiling water.
16. Steps 1-14 were repeated with 40°C water, by using 700mL of tap water and 300mL of boiling water.

Risk Assessment

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