Use of immobilised enzymes

Introduction

Immobilised enzymes have a wide range of commercial applications, such as their use in the
production of lactose-reduced milk, using immobilised lactase (or β-galactosidase).

Immobilised enzymes are attached to inert, insoluble materials and have a number of
advantages over enzymes in free solution, including the ability to reuse the enzyme, which
reduces the overall cost of the process. Immobilised enzymes can also be used in continuous
processes, which can be automated, and some enzymes are more stable when immobilised
and are therefore less likely to be denatured. Enzymes can be immobilised in a range of
materials, including agar gels, cellulose and polyacrylamide.

The purpose of this practical is to produce immobilised lactase and to test whether or
not it is effective at digesting lactose present in milk.

Materials
 2 cm3 lactase,
 8 cm3 of 2 % sodium alginate solution, made up in distilled water.
 100 cm3 of 2 per cent calcium chloride solution in a plastic beaker
 Semi-quantitative glucose test strips such as clinistix
 Small piece of nylon gauze or muslin
 10 cm3 plastic syringe
 10 cm3 plastic syringe barrel
 Retort stand
 Short length of tubing, to fit plastic syringe, and screw clip
 100 cm3 beaker
 Glass rod
 Plastic tea strainer
 Distilled water
 Pasteurised milk

Method
1 Mix the sodium alginate solution with the enzyme solution in a beaker, then transfer
to a plastic syringe.

2 Add this mixture drop-wise to the calcium chloride solution. Alginate beads,
containing the immobilised enzyme, will form immediately. Leave to harden for 10 to
20 minutes.

3 Strain off the beads using the tea strainer and rinse with distilled water.

4 Put a piece of nylon gauze in a 10 cm3 syringe barrel, to prevent the beads becoming
stuck in the outlet, then add the beads to the syringe. Hold the syringe using a retort
stand.

5 Close the screw clip then fill the syringe with milk. Open the clip slightly, and test the
products for the presence of glucose. If you use quantitative test strips, such as Diabur
5000, you can investigate the relationship between time and glucose concentration.

[NB sometimes the screw clips can be very stiff and it might be easier to hold the
rubber tubing between your thumb and fore finger and just release a sample when
needed. Wear gloves].

Conclusion

Does the Diabur strip change colour when the milk is dripped on to it? If so, what colour does
it go?

Yes. Pink to purple.

Does the colour change as the length of time the milk is mixed with the enzyme increases?

Yes. In the length of the time it will be more purple(dark purple) and more base.

In Addition:

The experimental method could be modified to examine the effect of different sized beads on
rate of enzyme activity. What would the IV be in this case and how would you measure it?

1- Mix the sodium alginate solution with the enzyme solution in a beaker, then
transfer to 5 plastic syringe with different size like 0.5 cm3 – 1 cm3 – 2 cm3 –
3cm3 – 5cm3.
2- Add this mixture drop-wise to the calcium chloride solution. Alginate beads,
containing the immobilised enzyme, will form immediately. Leave to harden for 10
to 20 minutes.
3- Strain off the beads using the tea strainer and rinse with distilled water.
4- Put a piece of nylon gauze in a 10 cm 3 syringe barrel, to prevent the beads
becoming stuck in the outlet, then add the beads to the syringe. Hold the syringe
using a retort stand.
5- Close the screw clip then fill the syringe with milk. Open the clip slightly, and test
 the products for the presence of glucose. If you use quantitative test strips, such as
Diabur 5000, you can investigate the relationship between time and glucose
concentration.
6- the time of dropping milk on the PH paper should be constant for all of the
syringes.
7- Try the experiment for each syringe in 0 s – 20 s – 40 s – 60 s.

Materials are constant to first method as 2 ml lactose- 8 cm 3 of 2 % sodium alginate

solution- 100 cm3 of 2 per cent calcium chloride solution. except the size of plastic syringe
and syringe barrel.To have different size of beads we should use different size of syringe
for example the size of beads of 0.5 cm3 syringe is smaller than the size of the beads of 5
cm3 syringe. but the number of beads of 0.5 cm3 syringe is more than number of 5 cm3
syringe.

What is the advantage of using immobilised enzymes in industrial processes?

development of enzyme immobilization has led to the production of tailored enzymes that
are stable. They also enabled recovery and of enzymes, reduced contaminants in products,
and improved control of industrial processes. and its economically beneficially.