|Sachet of yeast (without CaSO4)|
Calcium chloride powder
Glucose test strips
Hot water (30 °C 40 °C)
2 Graduated cylinders (100 cm3)
4 Beakers (100 cm3)
Beaker (500 cm3)
2 Separating funnels
3 Glass rods
2 Retort stands
Syringe (20 cm3)
- Familiarise yourself with all procedures before starting.
Preparation of the immobilised enzyme
- Add 0.4 g of sodium alginate to 10 cm3 of distilled water in a 100 cm3beaker. Mix thoroughly.
- Mix 2 g of yeast in 10 cm3of distilled water in a 100 cm3beaker.
- Prepare 100 cm3of a 1.4% w/v calcium chloride solution in the large beaker.
- Add the yeast suspension to the sodium alginate solution and mix thoroughly with a glass rod.
- Draw all of the mixture into a 20 cm3syringe.
- 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.
- Leave the beads to harden for at least 10 minutes.
- Filter the beads through a sieve and rinse with distilled water.
Application of the immobilised enzyme production of glucose from sucrose
- Mix another 2 g of yeast in 10 cm3of distilled water.
- Pour this yeast suspension into a separating funnel labelled `Free yeast' as shown in the diagram.
- Pour the beads into another separating funnel labelled `Immobilised yeast'.
- Prepare 100 cm3of 1% w/v sucrose solution with water warmed to about 40 °C.
- Pour 50 cm3of the sucrose solution into the yeast in each of the separating funnels.
- Using glucose test strips, immediately test samples from each funnel for glucose.
- Repeat the test at two-minute intervals until glucose appears in both.
- Record result.
- Run off the remaining product from each funnel into the beakers as shown in the diagram.
- Compare the turbidity of the solutions from both funnels.
- Replicate the investigation or cross reference your results with other groups.
|Time (minutes)||Free yeast presence of glucose||Immobilised yeast presenceof glucose|
|0|| || |
|2|| || |
|4|| || |
|6|| || |
|8|| || |
|10|| || |
|///////////////////////////////||Free yeast||Immobilised yeast|
|Turbidity of solution|| || |
Familiarise yourself with all procedures before starting
Follow instructions step by step
Listen to the teacher's instructions
Correct manipulation of apparatus
Use the balance
Use a graduated cylinder to measure volumes
Prepare solutions and mixtures
Draw the mixture of alginate solution and yeast suspension into the syringe
Release the mixture drop by drop into the calcium chloride solution
Transfer the beads to the separating funnel
Filter the beads
Rinse the beads with distilled water
Set up the separating funnels
Warm water to about 40 °C
Use a thermometer
Pour 50 cm3 of sucrose solution into each funnel
Use glucose test strips to test for glucose
Repeat at two-minute intervals using the timer
Note the clarity of both sucrose solutions
See beads forming
Observe any colour change using glucose test strips
Compare the turbidity of both end products
Write up the procedure
Tabulate the results
Record the time
Draw reasonable conclusions from your observations and results
Become aware of any other application(s) of what you learned in this activity
Exercise caution for your personal safety and for the safety of others
Work in an organised and efficient manner
Label as appropriate
Work as part of a group or team
Clean up after the practical activity
Alginates Most alginates are produced by brown algae (Phaeophyceae, mainly Laminaria spp.) whereas agars are produced from red algae (Rhodophyceae).
Immobilised enzymes An immobilised enzyme is one that has been made insoluble or held in place so that it can be reused many times. Once immobilised, an enzyme's stability is increased, possibly because its ability to change shape is reduced. One method of immobilisation is an entrapment method, in which the enzyme is enclosed within a gel (e.g. alginate beads). The openings in the encapsulating material have to be of sufficient size to permit the substrate or reactant to pass through to the enzyme without allowing the enzyme to leak out. The most common entrapment method is the formation of calcium alginate beads. The enzyme is mixed with sodium alginate, an acidic polysaccharide, and the mixture is dropped into a solution of calcium chloride. The calcium ions replace the sodium ions and crosslink the polysaccharide. The result is the production of insoluble calcium alginate beads containing trapped enzymes. Calcium alginate gel entrapment is a suitable method for this activity because it is simple to prepare and allows high enzyme activity and stability.
2 Na(Alginate) + Ca ++ Ca(Alginate)2+ 2 Na +
- Prepare the hot water required to make the sucrose solution.
- Sodium alginate is not readily soluble in water. Sodium alginate solution is best prepared by adding the powder to agitated water, rather than vice versa to avoid the formation of clumps.
- If left to stand, sodium alginate will absorb water and will dissolve more readily.
- Beads may be stored under distilled water for two to three days until ready to use.
- To avoid beads blocking the separating funnel, a plastic disposable pipette or straw may be inserted into the funnel before adding the beads.
- When using glucose test strips make sure to read instructions carefully as the procedure varies depending on the brand used.
- Only use yeast which does not contain calcium sulfate.
- For a better visual display, calcium chloride solution may be transferred to a clear graduated cylinder before adding yeast/alginate mixture.
- Cut glucose test strips in two, lengthwise, to make the supply last longer.
- Use separate weigh boats for the different chemicals to avoid calcium coming in contact with the yeast and alginate.
- Separating funnels with Teflon taps, rather than glass taps, are preferable as they are less likely to stick.
- Use wide-topped separating funnels as it is easier to introduce beads.