The enzyme, E , catalyses the hydrolysis (breakdown) of starch into glucose. You are required to investigate the progress of this enzyme-catalysed reaction by both testing for the disappearance of starch testing for the appearance of glucose by finding the time taken for the decolourisation of potassium manganate(VII) solution. To follow the progress of this reaction you will need to take samples from the reaction mixture at different times. (i) Decide how often you will…

Biology 9700/34 — May/June 2016

Cambridge AS Level · Advanced Practical Skills · worked solutions for every part, with the mark scheme

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marks

120

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Topics Presentation of Data and Observations · Manipulation, Measurement and Observation · Analysis, Conclusions and Evaluation · Use of the Light Microscope

Q1Manipulation, Measurement and ObservationPresentation of Data and ObservationsAnalysis, Conclusions and EvaluationFree sample

The enzyme, E, catalyses the hydrolysis (breakdown) of starch into glucose.

You are required to investigate the progress of this enzyme-catalysed reaction by both

testing for the disappearance of starch
testing for the appearance of glucose by finding the time taken for the decolourisation of potassium manganate(VII) solution.

To follow the progress of this reaction you will need to take samples from the reaction mixture at different times.

(a)

(i)

Decide how often you will take these samples including removing the last sample at 20 minutes.

State the times when you will remove the samples.

(ii)

Decide how you will test a sample to show that the starch has disappeared.

State the reagent you will use ______

Describe how you will test each sample to show that the starch has disappeared and state the colour of any results you might collect.

test ______

colour ______

(b)

To test for the production of glucose, the change in the colour of potassium manganate(VII) solution is:

purple $\longrightarrow$ colourless

Test-tube Z shows the colourless end-point.

You are provided with:

labelled	contents	hazard	volume / $\text{cm}^3$
E	amylase solution	harmful irritant	10
S	starch solution	none	40
A	sulfuric acid	irritant	20
P	potassium manganate(VII) solution	harmful	20

You are advised to wear suitable eye protection, especially when using the amylase solution, E and the sulfuric acid, A. If either E or A come into contact with your skin, wash off with cold water. P may stain your skin.

When carrying out a practical procedure the hazards of using the solutions need to be considered. Then the level of risk needs to be assessed as low or medium or high.

State the hazard with the greatest level of risk when using the solutions then state the level of risk of the procedure: low or medium or high.

hazard ______

level of risk ______

(c)

Proceed as follows:

Label the test-tubes with the sampling times that you stated in (a)(i).
Put $2.5\ \text{cm}^3$ of A into each test-tube.
Put $1\ \text{cm}^3$ of P into each test-tube and gently shake to mix with A.

Temperature may affect the rate of an enzyme-catalysed reaction.

(i)

Use the thermometer to measure the temperature of the room.

State the temperature of the room. ______

(ii)

Read step 4 to step 9 and note that the timer should not be stopped until you have the last end-point recorded.

The reaction will start as soon as E is added to S.

Put $30\ \text{cm}^3$ of S into a beaker.
Put $4\ \text{cm}^3$ of E into the beaker containing S.
Immediately stir the mixture in the beaker and start the timer.
At each of your sampling times:
- test for the disappearance of starch as described in (a)(ii)
- test for the appearance of glucose by removing $3\ \text{cm}^3$ of the mixture and putting it into the appropriately labelled test-tube, mixing well.
Record, in (c)(ii), the time shown on the timer when the colourless end-point is reached (raw result). Do not stop the timer.
Repeat step 7 and step 8 for each of the times decided in (a)(i) until you have recorded the end-point for the last sample, removed at 20 minutes.
Calculate the time taken to reach each end-point (processed results).

Prepare the space below to record your results for:

the test for starch
the raw results for the appearance of glucose
your processed results for the appearance of glucose.

(iii)

Now measure the temperature of the room once again.

State the temperature of the room. ______

State the difference between this temperature and the temperature of the room you recorded in (c)(i).

______

State whether temperature was a significant error in this investigation.

______

(iv)

Explain how a temperature rise of $30\ ^{\circ}\text{C}$ might have affected the mixture of starch and amylase.

(v)

This procedure investigated the progress of the hydrolysis of starch by amylase.

To modify this procedure for investigating a different variable, the time for the hydrolysis would be standardised.

Consider how you could modify this procedure to investigate the effect of pH on the activity of amylase.

Describe how the independent variable, pH, could be investigated.

______

Describe how the dependent variable could be measured.

(d)

A student investigated the effect of starch concentration on the initial rate of reaction of amylase.

Table 1.1 shows the results for this investigation.

Table 1.1

percentage concentration of starch	initial rate of reaction of amylase / arbitrary units
0.00	0
0.50	100
1.25	215
1.75	285
2.50	340
3.25	340

You are required to use a sharp pencil for graphs.

(i)

Plot a graph of the data shown in Table 1.1.

Fig. 1.1

(ii)

Use the graph to estimate the Michaelis-Menten constant ( $K_m$ ).

Show your working on the graph and in the space below.

$K_m$ = ______

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