The enzyme amylase catalyses the hydrolysis (breakdown) of starch to a reducing sugar. You are required to: identify which solution, E1 or E2 contains the highest enzyme concentration by estimating the concentration of reducing sugar produced by the action of E1 and E2 when breaking down starch prepare known concentrations of reducing sugar solutions to compare with the concentrations of reducing sugars produced by the action of E1 and E2 compare the concentrations of…

Biology 9700/36 — October/November 2013

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

questions

marks

120

minutes

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

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

The enzyme amylase catalyses the hydrolysis (breakdown) of starch to a reducing sugar.

You are required to:

identify which solution, E1 or E2 contains the highest enzyme concentration by estimating the concentration of reducing sugar produced by the action of E1 and E2 when breaking down starch
prepare known concentrations of reducing sugar solutions to compare with the concentrations of reducing sugars produced by the action of E1 and E2
compare the concentrations of reducing sugars by using the Benedict’s test.

For each of your Benedict’s tests you need to standardise the:

volume of Benedict’s solution
volume of the samples
temperature of the water bath.

(a)

(i)

State the:

volume of Benedict’s solution = ______ $\text{cm}^3$

volume of each of the samples to be tested = ______ $\text{cm}^3$

temperature of water bath = ______ $^{\circ}\text{C}$

(ii)

You are provided with:

labelled	contents	hazard	volume / $\text{cm}^3$
E1	amylase solution	harmful irritant	20
E2	amylase solution	harmful irritant	20
S	starch solution	none	50
G	0.4% reducing sugar solution	none	50
W	distilled water	none	100

Read steps 1 to 6 before proceeding.

Proceed as follows:

Put $1\ \text{cm}^3$ of E1 and E2 into separate beakers.
Put $10\ \text{cm}^3$ of S into each of the beakers with E1 and E2. Mix well.
Leave the beakers for 15 minutes.

During the 15 minutes you are required to:

set up a water bath ready for step 6 (on page 4)
prepare the known concentrations of the reducing sugar solution.

Set up the water bath to heat to the temperature as decided in (a)(i).

Decide which concentrations of the reducing sugar solution to make:

using serial dilution
using $20\ \text{cm}^3$ of the 0.4% reducing sugar solution, G, to start the serial dilution
reducing the concentration by half between each concentration.

Complete Fig. 1.1 to show how you will make three further concentrations.

Fig. 1.1

(iii)

Prepare all the concentrations of the reducing sugar solution, as in Fig. 1.1, in the containers provided.
You are now required to test for the concentrations of reducing sugars by using the Benedict’s test:
- test each sample separately using the volumes decided in (a)(i)
- record the time taken for the appearance of any colour change. If there is no colour change after 120 seconds record ‘more than 120’.

Prepare the space below and record only your results for the known concentrations of reducing sugars.

(iv)

Using your results for E1 and E2 complete the following.

The time taken for the first colour change in E1 was ______ .

The time taken for the first colour change in E2 was ______ .

Using these two times, state which of these two solutions has the highest concentration of reducing sugars.

..................................................................................................................................

Using the results from (a)(iii) estimate the concentration of reducing sugars in this solution.

..............................................................................................................................

(v)

Describe how you would modify this investigation to follow the time course of the hydrolysis of starch by enzyme E, without the use of Benedict’s solution.

(b)

A student investigated the effect of iron sulfate on the rate of amylase activity, using immobilised amylase in alginate beads.

The student prepared two types of alginate beads containing amylase:

with iron sulfate
without iron sulfate.

The student mixed the two types of beads together in varying proportions, for example 30 beads with iron sulfate and 70 beads without iron sulfate (30% with iron sulfate) as shown in Fig. 1.2. The student put the beads into beakers containing starch solution.

Fig. 1.2

Other variables were considered and kept to a standard.

The student measured the mass of reducing sugars produced by each mixture of beads in one minute.

The student’s results are shown in Table 1.1.

Table 1.1

percentage of beads with iron sulfate	mass of reducing sugar produced / $\mu\text{moles min}^{-1}$
0	60
10	25
20	12
30	5
40	2

(i)

Plot a graph of the data shown in Table 1.1.

Graph Grid

(ii)

Describe the trend shown by the data.

(iii)

Explain the reason for the difference in the results between 0 and 10 percentage of beads with iron sulfate.

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