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Chloe Lupepe

Immobilizing enzymes
Aim: To successfully immobilize protease
Apparatus and Reagents:
1. Sodium alginate 50cm3
2. Protease enzyme 25cm3
3. Calcium chloride 50cm3
4. Albumin 15cm3
5. A glass rod
6. A spatula
7. Thermometer
8. 100cm3 beaker
9. 10cm3 syringe
10.6 test tubes and test tube rack
11.Beaker for water bath
12.Teat pipette
Method
1. Put all the sodium alginate and the entire enzyme
solution into a 100cm3 beaker and stir thoroughly with
a glass rod to make a mixture of sodium alginate and
enzyme.
2. Using a clean 10cm3 syringe, fill the syringe with the
sodium alginate and enzyme mixture.
3. Carefully add the mixture, drop by drop, to the calcium
chloride solution. Small jelly like beads should form.
4. When the surface of the calcium chloride solution is
covered in beads, carefully use a spatula to scoop out
the beads and place them in a test tube. Continue to
make beads until half of the test tube is filled with the
immobilized enzyme.
5. Ensure the calcium chloride solution is drained from the
test tube containing the immobilized enzymes
6. Prepare a water bath of between 25-30, and place the
test tube containing the immobilized enzyme into the water
bath, allow the enzymes to adjust to the temperature for 2
minutes

Chloe Lupepe
7. While the enzymes are adjusting to the temperature,
preform the biuret* test on the substrate, Albumin, to
test for the presence of protein.
8. After two minutes pour the albumin into the test tube
containing the immobilized enzymes, until the albumin
is covering all the beads. Start timing.
9. After 5 minutes use a teat pipette to draw 1cm3 of
solution from the test tube containing immobilized
enzymes, and preform the biuret test on the solution in
the pipette.
10. Repeat step 8 at 10minutes, 15minutes and
20minutes. Ensure the temperature of the water bath is
kept constant.
11. Compare the results of the biuret tests of albumin,
the solution at 5 minutes, 10 minutes, 15 minutes and
20 minutes.
Results with biuret test
Albumin gave a clear strong purple colour
After 5 minutes the colour was a lighter shade of purple
After 10 minutes a lilac colour was observed
After 15 minutes a faint lilac colour was observed
After 20 minutes the colour remained blue
Conclusions
The results of the biuret test showed that the amount of
protein decreased as time progressed, to there being no
protein present after 20minutes. Due to the fact that
protein decreased it can be concluded that the enzyme
was successfully immobilized. This is because if the
enzymes were not immobilized the test for protein would
have been positive after 20minutes, because enzymes are
biological protein catalysts, and so would have given a
positive result with the biuret test.