1. Leaving Certificate Biology Higher Level
Chapter 9: Enzymes
Leaving Certificate Biology
Higher Level

2. Cell Metabolism
Metabolism is the sum of all the chemical reactions occurring in a living organism
Organisms ultimately get their energy from the sun via photosynthesis
Plants → Animals → Humans
ATP and enzymes drive metabolism

3. Enzymes
Enzymes are folded proteins, that are globular in shape, and catalyse biochemical reactions without being used up in the reaction
Due to the protein nature and globular, folded shape of enzymes they are affected by two important environmental factors pH
Temperature

4. Enzyme Function Active Site Theory:
All enzymes have an active site where the enzyme combines with its specific substrate
When the substrate enters the active site of the enzyme the enzyme changes shape very slightly to accommodate the substrate better – this is called the induced fit model of enzyme action

5. Induced Fit Model Substrate enters active site
The enzyme changes its shape slightly to accept substrate
An enzyme-substrate complex is formed
Substrate is changed into product(s)
Product(s) exit the active site upon which point the enzyme returns to its original shape

6. Induced Fit Model 1. 4. 2. 3. Products Substrate Enzyme Enzyme
Enzyme shape changes slightly
Enzyme-substrate complex

7. Optimal Activity of an Enzyme
Each enzyme has conditions under which it works best
Generally, most human enzymes work best at 37 ºC and pH 7
Exceptions would be enzymes in the stomach that work at a pH of 1 – 2
Plant enzymes work best between the temperatures of 10 – 30 ºC depending on their natural habitat

8. Heat Denaturation of Enzymes
Enzymes become denatured at extreme temperatures
Denaturation involves a permanent change in the shape of an enzyme so that it does not act on its substrate
For example human enzymes will begin to denature at around 40 ºC
During infections the temperature of the human body can reach 42 ºC [the body’s cells produce heat shock proteins which protect the folded shape of important enzymes]

9. Bioprocessing
Bioprocessing is the use of living cells or their components, such as enzymes to make useful products
Examples:
Production of beer using yeast
Production of insulin using genetically-modified E coli bacteria
Production of cheese using the enzyme rennin
Production of fructose from glucose using glucose isomerase

10. Bioprocessing with Immobilised Enzymes
Immobilised enzymes are enzymes that are attached to or trapped in an inert insoluble material
Three ways in which enzymes are immobilised:
Carrier-binding method
Physical adsorption
Ionic binding
Covalent binding
Cross-linking method
Entrapment method

11. Uses of Immobilised Enzymes
Immobilised lactase breaks down lactose in milk for lactose-intolerant people
Immobilised rennin is used in the cheese-making process
Immobilised glucose isomerase is used in sweet manufacture as fructose is sweeter than glucose

12. Advantages of Immobilised Enzymes
Immobilised enzymes have advantages over free enzyme (enzyme in solution):
Immobilised enzyme is more stable
Efficiency of the enzyme is unaffected
Easy recovery of product and enzyme at end of reaction
Immobilised enzymes can be reused many times reducing costs to manufacturers

13. Bioprocessing with Immobilised Enzymes
Bioprocessing is carried out in bioreactors
A bioreactor is a vessel in which a product is formed by a cell or cell component, such as an enzyme
Immobilised enzyme and substrate are placed in the bioreactor and the bioreactor is kept very carefully at the correct temperature and pH in order to achieve the maximum amount of product

14. Bioprocessing
Bioprocessing is carried out using one of two general procedures:
Batch culture
Continuous-flow culture
A fixed amount of substrate is placed in bioreactor
Reaction is allowed to proceed
Product is collected at end of reaction
Bioreactor is then cleaned out for the next batch
Continuous-flow culture:

15. Batch Culture A fixed amount of substrate is placed in bioreactor
Reaction is allowed to proceed
Product is collected at end of reaction
Bioreactor is then cleaned out for the next batch

16. Continuous-flow Culture
Substrate is continually infused into the bioreactor
Reaction proceeds and conditions within are strictly controlled using sensors
Product is continually collected

17. Experiment: to investigate effect of pH on enzyme action
Three graduated cylinders with celery (catalase enzyme), pH buffer (4, 7, 13) and 1 drop washing-up liquid set up in 25˚C water bath.
Hydrogen peroxide added to all three.
Volumes in graduated cylinders noted at 0 min, 1 min and 2 min.
Rate of enzyme action calculated by 2 min volume minus 1 min volume.
Result: pH 7 graduated cylinder showed the most enzyme action.

18. Experiment: to investigate effect of temperature on enzyme action
Three graduated cylinders with celery (catalase enzyme), pH buffer 7 and 1 drop washing-up liquid set up in three separate water baths of 0˚C, 25˚C, 80˚C.
Hydrogen peroxide added to all three.
Volumes in graduated cylinders noted at 0 min, 1 min and 2 min.
Rate of enzyme action calculated by 2 min volume minus 1 min volume.
Result: 25˚C graduated cylinder showed the most enzyme action.

19. Experiment: to investigate effect of heat denaturation on enzyme action
Two graduated cylinders with celery (catalase enzyme), pH buffer 7 and 1 drop washing-up liquid set up in three separate water baths of 25˚C and 100˚C and allowed to reach temperature.
Hydrogen peroxide added to both.
Volumes in graduated cylinders noted at 0 min, 1 min and 2 min.
Rate of enzyme action calculated by 2 min volume minus 1 min volume.
Result: 25˚C graduated cylinder showed enzyme action and 100˚C graduated cylinder showed no enzyme action (denaturation).

20. Experiment: to immobilise an enzyme and examine its application
Yeast cells are immobilised using sodium alginate.
Yeast suspension is mixed with sodium alginate solution and dropped into calcium chloride to solidify.
Beads of alginate beads are washed three times and placed in a dropping funnel.
A sucrose solution is placed into funnel and immobilised yeast allowed to act on sucrose.
Product is released by opening tap.
Product is tested for reducing sugar (glucose and fructose) using clinistix or glucose test strips.
Result: Immobilised yeast converted the sucrose to glucose + fructose without contaminating the product.