METHODS OF ENZYME IMMOBILISATION Flashcards
Why are enzymes useful in industry?
- 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
Why are batch reactions inefficient?
Product mixed with enzyme
-Requires purification (downstreaming) = expensive and time consuming
Enzyme is lost
- = wastes money
Production not continuous
What is enzyme immobilisation?
Enzyme is confined (cannot move) but can still act on substrate.
Where is enzyme immobilisation most often used?
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
Advantages of immobilisation?
- More efficient that Batch
- Enzyme is supported hence more stable
- More thermostable
- More active over a range of pH - Product is enzyme free
- Simplifies purification
- Saves money on purification
- Avoids allergic reactions via purification - Enzyme can be reused
- Saves money - Continuous production
Disadvantages of immobilisation?
Reduces enzyme activity
Start up cost is expensive
What is adsorption?
Enzymes attached to an inert substance (e.g glass or collagen matrix) via weak forces
Advantages of adsorption?
- 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
Disadvantages of adsorption?
- Enzymes can be easily washed away
- Active sites can be blocked by adsorptive material therefore reducing activity
- some enzymes can denature via immobilisation process
What is entrapment?
Enzymes are trapped within polymers (alginate beads or microspheres)
Advantages of entrapment?
- Enzymes cannot leak out or washed away
- Active sites are not blocked
Disadvantages of entrapment?
-Resistance to the diffusion of substrate in/product diffusing out
What is encapsulation?
Enzymes are trapped inside a selectively permeable membrane e.g nylon
Advantages of encapsulation?
-Enzymes not bound, hence active sites not blocked therefore activity not adversely affected
Disadvantages of encapsulation?
- Some resistance to substrates diffusing in/ products diffusing out
- Enzymes can leak out through mesh
Why does increasing the flow rate of substrate through a flow column reactor increase the amount of substrate unreacted?
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
What is cross-linkages?
Enzymes are covalently bonded to matrix (e.g cellulose) as a consequence of chemical reactions or each other via linking chemicals (e.g. glutaraldehyde)
Advantages of Cross-linkages?
Enzymes are securely bound
- Resistant to pH change
- more thermostable
- not washed away
Disadvantages of cross-linkages?
- Active sites may be blocked by binding chemical/matrix
- Active site may be damaged during chemical reactions in binding process
What are clinistix
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)
What the advantages of using clinistix
Give results in minutes
Cheap
Quantitative
Describe a Biosensor
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.
What can the pregnancy test be described as?
ELISA (enzyme linked immunosorbent assay)
Describe an example of enzymes being used as biomarkers
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.
How is inhibitors being used in drugs?
A1AT used in clinical trials for treating respiratory infections
