Practical 4 - Temperature and Enzyme activity

What is the effect of temperature on enzyme activity?

In normal reactions as temperature increases, the rate of enzyme activity increases.

In enzyme catalysed reactions the enzyme has an optimum temp at which it works best.

What happens to enzyme activity at low temperature?

• There is little kinetic energy

• The enzyme & substrate are not moving much

• Less collisions

• Fewer enzyme-substrate complexes form

• Low rate of reaction

What is the optimum temperature of the human body?

37 degrees celcius

What happens to enzyme activity as the temperture increases up to the optimum?

• The enzyme and substrate are gaining more kinetic energy.

• So, there are more collisions

• More enzyme-substrate complexes form.

What happens once the temperature goes above the optimum?

The reaction will start to decrease.

This is because…

• If there is too much thermal energy, the enzyme and substrate have too much kinetic energy, leading to violent collisions.

• This breaks the hydrogen bonds in the secondary and tertiary structure, which changes active sites’ shape, making the substrate no longer complementary.

What happens once the temperatures are too high?

The substrate can no longer fit into the actice site.

At very high temp this damage can be permanent and we say the enzyme has beome denatured.

What is the aim of this practical?

To investigate the effect of temperature on the rate of reaction of enzymes, we will be using lipase.

• By changing the temperature, we should notice a difference in the time taken for the fatty acids to be produced.

Why is lipase used?

Lipase is an enzyme the body uses to break down fats in food so they can be absorbed in the intestines.

• The full fat milk contains lots of fats

• When consumed, fats are broken down into fatty acids & glycerol.

What happenes once Lipase is added to full fat milk?

Adding Lipase to full fat milk a reaction will occur, and the fats will break down into fatty acids and glycerol.

• The pH will become more acidic after the reaction takes place.

By adding the phenolphthalein indicator, the pink will turn colourless as the fatty acids that are produced lower the pH.

By changing the temp we should notice a difference in time taken for the fatty acids to be produced.

What is the enzyme activity like from 0 - 40 degrees celcius?

As the temp increases the kinetic energy increases.

• Means there are more collisions bwteen the enzyme active site and the substrate (lipids).

• There are more enzyme-substrate complexes forming so this increases the rate of reaction.

What is the enzyme activity like 40 degrees celcius and above?

As the temperature continues to increase, above optimum, this causes the enzyme to become denatured.

• This changes the shape of the active site so the substrate (lipids) no longer fits

• So there are less enzyme substrate complexes forming therefore the rate of reaction decreases.

What is the hypothesis?

As the temperature increases, the time taken for the lipase to break down the fat in milk will decrease, so the rate of reaction will increase.

This will happen because…

• As the temp increases the kinetic energy increases. (0-40)

• Meaning there will be more collisions between the enzyme active site & the substrate (lipids)

• More enzyme-substrate complexes will form, so this will increase the rate of reaction.

• As the temperature continues to increase above optimum, this causes the enzyme to become denatured. (Above 40)

• This changes the shape of the active site so the substrate (lipids) no longer fits

• So there are less enzyme substrate complexes forming; therefore, the rate of reaction decreases.

What are the independent variables?

Temperature: 20, 40, 60 (degrees celcius)

What are the dependent variables?

Quantitative (numerical data) - Time in seconds

Qualitative (non-numerical data) - Pink to colourless

What are the control variables?

Volume of milk - measuring cylinder/ 5ml syringe

Volume of lipase - 1 ml syringe

Volume of sodium carbonate - 10 ml syringe

What were the hazards, risk ,precautions and emergency procedure of this practical? (Health & Safety)

Hazard - Risk - Precaution - Emergency procedure

• Phenolphtalein - Laxative - Do not ingest - Seek first aider

• Sodium carbonate - Low hazard - Do not ingest/avoid spillage - Clean up

• Glassware - Smashing may cause cuts - Place away from bench edge/handle with care - Seek first aid/teacher for clean up.

• Milk - Low hazard/allergies - Do not ingest - If allergic, seek first aid.

• Lipase 1% - Low hazard - Do not ingest - Seek first aid if consumed.

• Hot water bath - Burns/electrocution - Handle with care/disconnect when not in use - If burnt, seek first aider.

What was the equipment used?

• x6 Test tubes - to hold the temperatures of 20, 40, 60 & 3 boiling ones (degrees Celsius)

• Boiling tube rack - to hold the test tubes

• Sharpie - to label the temperature of each test tube.

• 5 ml syringe / measuring cylinder - To measure 5cm3 of milk

• 10 ml syringe - to measure out 7 cm3 of sodium carbonate

• 1 ml syringe - to measure 1 cm3 of lipase solution

• Phenolphthalein drops - add 4 drops to the test tube

• Thermometer - placed in the test tube to measure the temperature

• Water bath - to maintain the specific temperature needed

• Stirring rod - to stir the contents

Stopclock - begins once the test tube of contents is added to the lipase & stops once the phenolphalein loses its colour.

What was the method?

1) Label a test tube with the temp you will be investigating (20,40,60 degrees cel)

2) Measure out 5 cm3 of milk using a 5 ml syringe and add this to the test tube.

3) Measure out 7 cm3 of sodium carbonate using a 10 ml syringe and add this to the same test tube.

4) Add 4 drops of phenolphthalein to the same test tube. The soloution should now be pink (if not stir gently with glass rod)

5) Place a thermometer in the test tube. Take care as the equipment could topple over.

6) To a separate boiling tube add 1 cm3 of lipase solution using a 1 ml syringe.

7) Place both test tubes in the correct water bath and leave until the contents reach the same temperature as the water bath.

8) Remove the thermometer from test tube and add the contents of the test tube containing the milk, sodium carbonate and phenolphthalein to the test tube containing lipase and start the stopclock.

9) Stir the contenst of the test tube gently using a stirring rod until the solution loses its pink colour (and turns back to white milk colour)

10) Stop the clock/watch and note the time in a suitable table of results.

11) Repeat 3 times for each temperature.

12) Plot a graph of the rate of reaction to occur against temperature.

13) To convert time to rate of reaction you do 1/time for each of the mean temperatures.

What is the conclusion?

Same as hypothesis but with obervations and data included

How could you improve this experiment?

Do more repeats for the same temperature to give more reliable results.

To extend the range of results, investigate more temperatures, e.g. 5, 10, 15, 20, 25, 30, 40-80 (degrees Celsius)

How do you extend this experiment?

Repeat the experiment with different milk, semi-skimmed and skimmed.

Repeat the experiment with a different enzyme, for example, protease.

How did we enure accuracy of the volume of milk?

Used a 10 cm3 measuring cylinder.

Read bottom of meniscus.

At eye level.