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Specification: 2.1.3

Learners should be able to demonstrate and apply their knowledge and understanding of:

(d) (i) the effects of pH, temperature, enzyme concentration and substrate concentration on enzyme activity

(ii) practical investigations into the effects of pH, temperature, enzyme concentration and substrate concentration on enzyme activity

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Learning Outcomes:

  • MUST (D): Consider independent, dependent and control variables in enzyme investigations. 
  • SHOULD (C): Describe results and draw accurate graphs to show results. Calculate initial rates of reaction from graphs.
  • COULD (B): Explain results using key terminology. Describe how a serial dilution is produced and why it is relevant. Explain why rates of reaction slow as a reaction proceeds.

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Investigating Enzyme Action

Possible independent variables?

  • Temperature
  • pH
  • Substrate concentration
  • Enzymes concentration

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How could each of these be varied?

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Investigating Enzyme Action

Possible dependent variables?

  • Concentration of product produced/substrate used up in a fixed time
  • Readings of product formation/substrate used up at time intervals

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How can rate then be calculated?

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What happens to rate of reaction as the reaction proceeds? Why?

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Initial Rate of Reaction

CGP p 106-107

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Graphs

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Investigating Enzyme Action

What variables must be controlled?

  • Temperature
  • pH
  • Substrate concentration
  • Enzyme concentration

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How would each be kept constant?

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Investigating Enzyme Action

What is a control experiment?

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Why is a control experiment needed?

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PAG 4.2 Trypsin and casein

Key points

  • Casein is milk protein
  • Trypsin is a protease enzyme
    • hydrolyses peptide bond
  • Casein is milky white colour
    • when digested it is translucent
    • time how long it takes for milk to clear
  • When clear a reference point behind the tube can be seen.

Colour standard for hydrolysed casein

Milk-trypsin mixture is compared to colour standard

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Investigating Enzyme Action

Independent variable:

  • Enzyme concentration

How could enzyme concentration be varied?

Dependent variable:

  • Time taken for cross to become visible

How can rate be calculated?

Control variables:

  • Temperature
  • pH
  • Substrate concentration

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Results table

Enzyme Concentration

Time taken for x to disappear (s)

Mean time taken (s)

Rate of reaction n (1/t)

Mean rate of reaction (1/t)

Try 1

Try 2

Try 3

Try 1

Try 2

Try 3

0.13

​

​

​

​

​

​

​

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0.25

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​

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0.50

​

​

​

​

​

​

​

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1.00

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2.00

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​

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Extension Questions:

1. What variable were controlled in this practical activity?

  • Substrate concentration
  • Volume of enzyme
  • Volume of substrate
  • pH
  • Temperature

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2. What could you have done to improve the validity of your results?

  • Ideally each concentration would have been replicated at least three times and a mean time calculated.
  • A way to standardise mixing the solutions on addition of the enzyme.

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Extension Questions:

3. Discuss this activity with the rest of the class. Did everybody get a similar result?

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4. What conclusions would you draw from this practical activity?

  • As enzyme concentration is increased the (initial) rate of reaction increases. There are more active sites available for catalysis of the reaction.
  • Beyond a certain concentration the rate does not increase further. Substrate in fixed concentration has all been used up.

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Demo Method

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Results table

Substrate concentration (vol)

Volume of gas collected (cm3) after ….. (s)

30

60

90

120

150

180

210

240

270

300

2

​

​

​

​

​

​

​

​

​

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4

​

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​

​

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8

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16

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20

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Mean and Standard Deviation

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Results table

Concentration of hydrogen peroxide (vol.)

Time (s)

Volume of oxygen produced (cm3) after ‘x’ (s)

30

60

90

120

150

180

2

1

 

 

 

 

 

 

2

 

 

 

 

 

 

3

 

 

 

 

 

 

Mean

 

 

 

 

 

 

SD

 

 

 

 

 

 

4

1

 

 

 

 

 

 

2

 

 

 

 

 

 

3

 

 

 

 

 

 

Mean

 

 

 

 

 

 

SD

 

 

 

 

 

 

etc.

 

 

 

 

 

 

 

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Check list:

  • Used as much of the page as possible?
  • Plotted data points spread out across the available space? (Appropriate scales)
  • Independent variable on X axis?
  • Dependent variable on Y axis?
  • Labelled axis with units?
  • Used a sharp pencil for the points (X)
  • Range/error bars?
  • Line of best fit (use a plastic ruler) – NOT dot to dot?
  • Circled any “outlier” results which are way off the line of best fit?

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Graph

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Graphs

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Extension Questions:

  1. What is the word equation for the reaction controlled by catalase?

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Hydrogen peroxide -> Water + Oxygen

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2. Give one reason why it was important to keep all cylinders the same size?

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In order to ensure the same amount of catalase is present.

Valid comparisons can then be made.

Substrate concentration is only independent variable.

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Extension Questions:

3. Explain the shape of the graph you have drawn using biological ideas and relevant enzyme theory.

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Increase in rate of gas production with an increase in substrate concentration.

More substrate for the enzyme to work on so more product is formed more quickly.

Levelling off, as enzyme cannot work more quickly and the substrate molecules effectively have to “queue up” for an enzyme to become free. So the rate levels off. All active sites are occupied at any one time.

QUOTE DATA

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Extension Questions:

4. (a) State two limitations of the experiment.

(b) State ways to overcome the limitations you have mentioned in (a).

  • Gas may escape while trying to put the bung in and start the stopclock at the same time…. So should use a gas syringe or a result may be anomalous
  • Difficult to read meniscus in measuring cylinder
  • Cylinders of potato different thicknesses/lengths… should measure mass
  • Random errors….. do replicates at each concentration.

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Starter

  1. Describe the structure of starch (6)

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  • What solution is used to test for starch? (1)

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  • What colour will this solution be in the presence of starch? (1)

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  • What will happen to the starch when adding amylase? (2)

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Design an experiment…..

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Investigating Enzyme Action

Independent variable:

  • Temperature

How can we vary temperature?

Dependent variable:

  • Time taken for starch to be hydrolysed

How can rate be calculated?

Control variables:

  • Enzyme concentration and volume
  • pH
  • Substrate concentration and volume

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Starch and amylase

Key points:

  • Starch is a storage polysaccharide
    • composed of amylose and amylopectin
  • Amylase digests α 1🡪4 glycosidic bond
    • produces maltose
  • Follow reaction by measuring time for
    • starch to disappear
    • maltose to appear
    • former is easiest
      • starch/iodine test

0:00

0:30

1:00

1:30

2:00

2:30

3:00

3:30

4:00

4:30

5:00

5:30

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Basic Method: Starch and Amylase

  1. Allow both starch and amylase to equilibrate at 5 different temperatures.

At each temperature:

  1. Mix starch and amylase together at 0 seconds.
  2. Every 30 seconds take a small sample and add to iodine solution in one well of a dropping tile.
  3. When the iodine solution stays brown the starch has been hydrolysed.

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Method

  1. Add 2 drops of iodine to every well in a spotting tile.
  2. Label a test tube with the temperature 20oC.
  3. Add 5cm3 starch to the test tube and allow to equilibrate in the water bath.
  4. Add 3cm3 amylase to SEPARATE test tube and allow this to equilibrate in the same water bath.
  5. Simultaneously start the stopclock and add the amylase to the starch. Shake gently to mix.
  6. Every 30 seconds use a CLEAN pipette to remove a few drops of reaction mixture and add this to a well in your spotting tile.
  7. Record the time taken for the starch to be hydrolysed (the time taken for the iodine solution to stay yellow/brown).
  8. Repeat experiment at 40, 50, 60 and 80 oC

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Temperature (oC)

Time taken for starch to be hydrolysed (s)

Try 1

Try 2

Try 3

Mean

20

240

270

240

​

30

90

60

90

​

40

30

30

30

​

50

180

210

150

​

60

300

270

240

​

80

X

X

X

X

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Temperature (oC)

Time taken for starch to be hydrolysed (s)

Rate (1/time)

Try 1

Try 2

Try 3

Mean

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20

180

180

180

180

0.00555

26

180

180

180

180

0.00555

40

90

90

90

90

0.011111

50

60

60

60

60

​

60

90

75

90

85

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80

X

X

X

X

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Temperature (oC)

Time taken for starch to be hydrolysed (s)

Rate 1/t

20

540

​

40

120

​

60

240

​

80

600 – no change

0

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Check list:

  • Used as much of the page as possible?
  • Plotted data points spread out across the available space? (Appropriate scales)
  • Independent variable on X axis?
  • Dependent variable on Y axis?
  • Labelled axis with units?
  • Used a sharp pencil for the points (X)
  • Range/error bars?
  • Line of best fit (use a plastic ruler) – NOT dot to dot?
  • Circled any “outlier” results which are way off the line of best fit?

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Graph

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Graphs

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Extension Questions:

  1. Evaluate the method you designed to carry out this activity. Could it be improved? What were the limitations of your investigation?

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Any reasonable response based on the students practical, for example related to timings, volumes, repeats etc.

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2. Why was it important to consider all the variables?

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Valid comparisons can then be made.

Temperature is only independent variable.

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Extension Questions:

3. Explain the shape of the graph you have drawn using biological ideas and relevant enzyme theory

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Graph should show an increase in the rate of reaction with an increase in temperature until the temperature is high enough to denature the protein ( ̴70°C and above depending on the source of the amylase).

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This is because the collision rate increases with increasing temperature so enzyme-substrate complexes form more frequently.

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Students could quote data from their table / graph.

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Extension Questions:

4. Discuss the success of this activity with the rest of your class. Consider what worked well and what did not for different individuals/groups in the class.

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This will be dependent on the experiments designed and the results obtained by the class.

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Discussions might include comment on how measurements were taken, the frequency of measurements, the detail included in students’ methods etc.