4.6 To prepare one enzyme immobilisation and examine its
application
Teacher Notes
Apparatus required per class group of 24 students
100 ml Beakers
48
Glass rods
36
500 ml Beakers
12
20 ml Syringes
12
Weigh boats
12
Electronic balances
3
Separating funnels
24
Thermometers
36
Retort stands
24
Straws
24
Labels
24
Spatulas
12
Wash bottles
12
Timers
12
100 ml Graduated cylinders
24
Hot water (30OC – 40OC)
Yeast (without CaSO4)
Sodium alginate
Calcium chloride powder
Sucrose
The apparatus for this experiment lends itself to being pre-prepared in a ‘box of
equipment’ – see Section 1.
Biology SLSS 2009
1
Advance preparation
o Prepare the hot water for the sucrose solution.
o Prepare the 1.4% calcium chloride solution.
Advance chemical preparation
(a) 1.4% w/v calcium chloride solution
o Weigh out 1.4 g of calcium chloride powder.
o Dissolve in distilled water, make up to 100 ml and stir.
(b) Sucrose solution
o Weigh 1 g of sucrose powder.
o Dissolve in distilled water and make up to 100 ml..
Safety precautions
o Sodium alginate is mildly hazardous. For further information see MSDS.
o Calcium chloride is harmful. For further information see MSDS.
Expected outcome of experiment
o After preparing the immobilised enzyme, beads will have formed.
o The free yeast will convert the sucrose into glucose faster than the immobilised
yeast. This is due to a greater surface area.
o The glucose drained from the immobilised yeast will be clear in colour while the
glucose drained from the free yeast will be cloudy.
Disposal and post-experiment work
o The immobilised enzyme beads can be stored in distilled water for a period of up to
two weeks.
o Excess calcium chloride can be dissolved in large quantities of water and run to the
foul water drain in excess water.
Biology SLSS 2009
2
4.6 To prepare one enzyme immobilisation and examine its
application
Student Notes
Apparatus required per group
o
4 x 100ml Beakers
o
2 Labels
o
3 Glass rods
o
Spatula
o
500 ml Beaker
o
Wash bottle
o
20 ml Syringe
o
Timer
o
2 Weight boats
o
2 100 ml Graduated cylinders
o
Electronic balance
o
Yeast
o
2 Separating funnels
o
Sodium alginate
o
3 Thermometers
o
Calcium chloride solution
o
2 Retort stands
o
Sucrose solution
o
2 Straws
o
Glucose test strips
o
Sieve
o
Distilled water
Assembled apparatus
Biology SLSS 2009
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Method
Preparation of the immobilised enzyme
1. Add 0.4 g of sodium alginate to 10 ml of distilled water in a 10 ml beaker.
Mix thoroughly.
2. Mix 2 g of yeast in 10 ml of distilled water in a second 100 ml beaker.
3. Pour 100 ml of the 1.4 % calcium chloride solution into a 500 ml beaker.
4. Add the yeast suspension to the sodium alginate solution and mix thoroughly
with a glass rod.
5. Draw all of the mixture into a 20 ml syringe.
6. From a height of 10 cm release the mixture from the syringe, one drop at a
time, into the calcium chloride solution. Beads containing yeast cells will form.
7. Leave the beads to harden for at least 10 minutes.
8. Filter the beads through a sieve with distilled water.
Assembled apparatus
Biology SLSS 2009
4
Application of the immobilised enzyme – production of glucose from sucrose
1. Mix another 2 g of yeast in 10 ml of distilled water.
2. Pour this yeast suspension into a separating funnel labelled ‘free yeast’.
3. Pour the beads into another separating funnel labelled ‘immobilised yeast’.
4. Place a straw in the centre of each funnel, directly above the tap.
5. Put 50 ml of sucrose solution into each of 2 beakers.
6. Pour the sucrose solution from each beaker into the separating funnels.
7. Using glucose test strips, immediately test samples from each funnel for
glucose.
8. Repeat the test at two minute intervals until glucose appears in both and
record results.
9. Run off the remaining product from each funnel into the beakers as shown in
the diagram.
10. Compare the turbidity of the solutions from both funnels.
11. Replicate the experiment or cross reference your results with other groups.
12. At the end of the experiment, clean all of the equipment and replace it in its
correct place.
Results:
Time
Free yeast – presence of glucose
(minutes)
Immobilised yeast – presence of
glucose
0
2
4
6
8
10
/////////////////////////////////////// Free yeast
Immobilised yeast
Turbidity of solution
Conclusion/Comment
Biology SLSS 2009
5