1. Immobilising Enzymes

2. Enzymes as Catalysts
Enzymes are used to speed up chemical (metabolic) reactions e.g. respiration or photosynthesis- so why use enzymes in industry?
They are specific
can catalyse reactions between specific chemicals, even in a large mixture
form fewer bi-products
less purification needed
Function well at relatively low temperatures saving money on fuel costs
Enzymes from thermophilic bacteria can be extracted and used at high temperatures

3. Isolating Enzymes
In some biotechnological processes, whole organisms are cultured to generate products, however when you need a single product, it is more efficient to isolate the enzyme
They can be isolated in large quantities
The extraction of enzymes from a fermentation mixture is known as downstream processing which is the separation and purification of any product of large scale fermentations

4. Immobilising Enzymes
Enzyme-substrate complexes must be formed in order to gain the products
The easiest way is to mix the isolated enzyme with the substrate, however the product must then be separated which can be a costly process
It is therefore possible to immobilise enzymes so they can catalyse the reaction without mixing freely

5. Methods of immobilising enzymes
Adsorption / carrier bound
Enzyme molecules are mixed with immobilising support e.g. glass beads or clay
Covalent Bonding / cross-linked
Enzyme molecules covalently bonded to a support

6. Methods of immobilising enzymes
Entrapment / inclusion
Enzymes trapped in their natural state in a gel bead
Reaction rate can be reduced as substrate needs to get through the trapping barrier
Membrane separation
Substrate separated from the mixture by a partially permeable membrane.

7. Problems that can occur with immobilised enzymes
Match the problems to the type of immobilisation and explain why the problem is caused
Enzyme can become detached (leakage)
Small amount of enzyme immobilised (depends on availability of cross-links)
Reaction rates can be reduced due to time take for E and S to interact

8. Advantages of immobilised enzymes
The advantages of using immobilised enzymes over enzymes in solution are
Immobilised enzymes can be reused
Product is enzyme free
Immobilised enzymes are more tolerant to pH and temperature changes

9. Advantages of Immobilised Enzymes
Enzyme not mixed with products so purification/ downstream processing costs are low
Enzymes available immediately for re-use which is good for continuous processes
The enzymes are more stable as the immobilising matrix protects the enzyme molecules – less likely to denature in extreme conditions (eg..?)
Eg Temp and pH

10. Disadvantages of Immobilised Enzymes
Additional time, equipment and materials needed, so expensive to set up
Can be less active as they do not mix freely with the substrate
Contamination can be costly if it occurs as the whole system needs to be stopped

11. Immobilising lactase in alginate

12. Immobilising lactase in alginate
The beads can be tightly packed into a column
The liquid substrate can be trickled over the beads
The product trickles out of the bottom of the column
The product is collected and purified.

13. Immobilisation in a Nutshell
Method
Description
Adsorption
Enzyme mixed with immobilising supports e.g. porous carbon, glass beads, clay & resins with hydrophobic interactions and ionic links*. Detachment is possible due to weak bonds but reaction rates are high if active site is displayed.
Covalent Bonding
Enzymes covalently linked to insoluble material e.g. clay using cross linking agent (gluteraldehyde/sepharose) Binding is strong, so very little enzyme leakage, but small quantities only
Entrapment
Enzymes trapped in a gel bead or cellulose fibre network. Active sites are not affected, but reaction rates reduced if substrate can’t get through trapping barrier
Membrane Separation
Enzymes separated by a partially permeable membrane. Enzyme on one side, substrate on another. Substrate molecules and products can pass across the membrane.
This is not exactly the same as the book.....so don’t feel you have to use this slide
*An ionic bond is a type of chemical bond formed through electrostatic attraction between two oppositely charged ions.

14. Immobilising Methods
4 possible methods: adsorption, covalent bonding, entrapment and membrane separation
Using OCR Biology p165 write about each type of immobilisation method
OR: Use ‘Putting enzymes to use’ worksheet.

15. Exam Practice – Unit 5 questions
Q12, 100, 157, 166, 167, 170 (e)