METHODS OF ENZYME IMMOBILISATION

Question 1

Why are enzymes useful in industry?

Answer 1

• Speed up rates of industrial processes = more product and hence money
• Do not get used up in the reaction = saves money
• Enzymes lower the Ea of a reaction allowing to occur easily at lower temp. = saves money

Question 2

Why are batch reactions inefficient?

Answer 2

Product mixed with enzyme
-Requires purification (downstreaming) = expensive and time consuming

Enzyme is lost
- = wastes money

Production not continuous

Question 3

What is enzyme immobilisation?

Answer 3

Enzyme is confined (cannot move) but can still act on substrate.

Question 4

Where is enzyme immobilisation most often used?

Answer 4

Column flow reactors to ensure max production
-Rates of flow, methods of immobilisation and environmental conditions are able to be controlled to ensure max. yield + efficiency

Question 5

Advantages of immobilisation?

Answer 5

1. More efficient that Batch
2. Enzyme is supported hence more stable
   - More thermostable
   - More active over a range of pH
3. Product is enzyme free
   - Simplifies purification
   - Saves money on purification
   - Avoids allergic reactions via purification
4. Enzyme can be reused
   - Saves money
5. Continuous production

Question 6

Disadvantages of immobilisation?

Answer 6

Reduces enzyme activity

Start up cost is expensive

Question 7

What is adsorption?

Answer 7

Enzymes attached to an inert substance (e.g glass or collagen matrix) via weak forces

Question 8

Advantages of adsorption?

Answer 8

• Easy to immobilise enzyme
• Cheap to immobilise enzyme
• Better than entrapment/encapsulation as substrate molecule do not diffuse through a membrane to reach active site

Question 9

Disadvantages of adsorption?

Answer 9

• Enzymes can be easily washed away
• Active sites can be blocked by adsorptive material therefore reducing activity
• some enzymes can denature via immobilisation process

Question 10

What is entrapment?

Answer 10

Enzymes are trapped within polymers (alginate beads or microspheres)

Question 11

Advantages of entrapment?

Answer 11

• Enzymes cannot leak out or washed away

- Active sites are not blocked

Question 12

Disadvantages of entrapment?

Answer 12

-Resistance to the diffusion of substrate in/product diffusing out

Question 13

What is encapsulation?

Answer 13

Enzymes are trapped inside a selectively permeable membrane e.g nylon

Question 14

Advantages of encapsulation?

Answer 14

-Enzymes not bound, hence active sites not blocked therefore activity not adversely affected

Question 15

Disadvantages of encapsulation?

Answer 15

• Some resistance to substrates diffusing in/ products diffusing out
• Enzymes can leak out through mesh

Question 16

Why does increasing the flow rate of substrate through a flow column reactor increase the amount of substrate unreacted?

Answer 16

As flow rate increases, the amount of enzyme remaining in the juice increases/the amount of enzyme (catechol oxidase) removed decreases;

at higher flow rates, there is insufficient time for substrate molecules to be catalysed by the enzyme/for substrates to bind to the active sites of the protease molecules

Question 17

What is cross-linkages?

Answer 17

Enzymes are covalently bonded to matrix (e.g cellulose) as a consequence of chemical reactions or each other via linking chemicals (e.g. glutaraldehyde)

Question 18

Advantages of Cross-linkages?

Answer 18

Enzymes are securely bound

• Resistant to pH change
• more thermostable
• not washed away

Question 19

Disadvantages of cross-linkages?

Answer 19

• Active sites may be blocked by binding chemical/matrix

- Active site may be damaged during chemical reactions in binding process

Question 20

What are clinistix

Answer 20

Regent strips that contain enzymes impregnated on them and are used to test for the presence of glucose in blood or urine (used in the diagnosis of Type 2 diabetes)

Question 21

What the advantages of using clinistix

Answer 21

Give results in minutes
Cheap
Quantitative

Question 22

Describe a Biosensor

Answer 22

During pregnancy the glycoprotein hormone hCG can be detected in urine.

The pregnancy test (a biosensor) has been developed to check for the present of this hormone and confirm a pregnancy

Specific antibodies are present on the paper at the bottom of the stick and bind to the hCG hormone once the urine sample is applied.

The hCG hormone bound to the antibody flows along the paper to the test region where there is a second antibody which traps the antibody bound hCG. An immobilised enzyme also present causes the dye to change colour to give a positive test for the pregnancy.

Unbound Antibodies progress up the stick to the control region where they are bound by a third type of antibody and this also causes a colour change
-this is proof that the antibodies are behaving properly, not proof of pregnancy.

Question 23

What can the pregnancy test be described as?

Answer 23

ELISA (enzyme linked immunosorbent assay)

Question 24

Describe an example of enzymes being used as biomarkers

Answer 24

In respiratory diseases, the damaging enzyme elastase is used as a biomarker.

Elastase is released by white blood cells as a result of a lung infection.
Elastase hydrolyses the elastin protein in the alveoli and airways

In healthy people, there are low levels of elastase.

Following any infection, the inhibitor A1AT produced from the lungs to inhibit elastase.

Smoking inhibits the production of A1AT which allows the enzyme to cause emphysema.

Question 25

How is inhibitors being used in drugs?

Answer 25

A1AT used in clinical trials for treating respiratory infections