Temperature
This can be explained by the kinetic theory. In that as the temperature increases, the molecules vibrate faster due to the increase in energy. Therefore the number of collisions increases. However if the enzyme has reached its optimum temperature and the temperature continues to increase, the enzyme molecule starts to loose its shape, particularly the tertiary structure. Due to the increased vibrational energy the tertiary structure starts to break down (however the bonds can reform if desirable temperatures are re-stated) and the enzyme is said to be denatured as it has lost its 3D structure
Concentration
At ( a ) there is a les s amount of substrate molecule, therefore all the active sites can engage in catalysis. The rate of reaction is able to increase.
At ( b ) there are more substrate molecules. All the active sites are engaged in catalysis. This is the maximum rate of reaction.
At ( c ) there is a large number of substrate molecules, however all the active sites are engaged in a reaction. There the rate of reaction ceases to increase.
pH
The pH affects the charge of the amino acids at the active site. Thus the properties of te active site change and the substrate can no longer bind. A change in pH alters the ionic charge on either the acidic or basic group on the peptide chain. The change on the group reduces the affinity the active site has for the substrate.
N.B not all enzymes require the same pH. e.g. Pepsin prefers more acidic conditions, whereas trypsin favours alkaline conditions.
Enzymes and Denaturisation
Denaturisation of an enzyme is due to the loss of its 3D structure. It happens when the relatively weak bonds that maintain the tertiary structure are changed. It may be due to exposure to high temperatures, heavy metal ions, extremes of pH and some organic solvents.
Immobilisation of Enzymes
Immobilised enzymes are enzymes which are held in granules, or attached to fibres, then packed into a column through which a supply of the reactants is continually passed, to form the product.
There are different methods of enzyme immobilisation;
Entrapment; the enzyme is mixed with gel-forming ingredients and when the gel forms the enzyme remains "trapped" in the gel . The pores are large enough to let the substrate in, but not the enzyme out.
Covalent bonding; the enzyme is covalently bonded to a matrix, which prevents enzyme molecules from being leached away. Some enzyme molecules may be denatured by the bonding process.
Adsorption; the enzyme is Adsorbed to various surfaces, but because the attachment is not permanent this method is usually only used for scientific studies or for "disposable" enzymes.
Encapsulated in a compartment behind a semi-permeable membrane
Direct cross-linking; the enzyme molecule is covalently bonded with bridging molecules, reactants and products have easy access, but some enzymes might be denatured.
Advantages of immobilization
- Product is enzyme- free
- The enzyme can be re-used.
- The enzyme can be more stable and long lasting, as it is protected by the inert matrix.
However there are also disadvantages:
- diffusion of substrates and products may be hampered by partitioning of the enzyme in the immobilised layer
- the enzyme may have a more constrained conformation in the immobilised state, giving it a lower catalytic activity
Lactose and Lactase
Lactose (the carbohydrate found in milk or milk products) is a disaccharide consisting of a glucose molecule joined to a galactose molecule. As disaccharides can not be transported across the plasma membrane they first must be hydrolysed by specific disaccharidases, in this case ‘lactase’.
(Hydrolysis of lactose by lactase)
Method
(Aim: To investigate whether immobilised enzymes are less susceptible to denaturisation than non-immobilised enzymes by measuring the time taken at different temperatures for the enzyme to denature.)
As two types of the same enzyme are being compared to one another, there shall be two differing experiments.
Immobilised Enzymes
- Transfer 20 cm3 of 2% sodium alginate solution to a small plastic beaker
- Using another syringe transfer 2 cm3 of 2% lactase enzyme into the same beaker and mix thoroughly using a glass rod
- Suck up this mixture into a syringe
- Slowly add this mixture DROP BY DROP to 200cm3 of 1.5% calcium chloride solution in a large beaker.
- (The calcium chloride acts as a hardening agent). Allow the beads to settle and harden for 5-6 minutes.
- Using a strainer, rinse the beads thoroughly with water and transfer them to the different test tubes which are at different temperatures.
- Quickly, add 5cm3 of milk and a clinistix fixed against the side of test tube
- Start the timer. Record time taken for colour change ( = hydrolysis)
- If there is no colour change, it means the enzyme has been denatured.
Non - immobilised Enzymes
(1)
Predication
I think that the immobilised lactase will denature at higher temperatures than the non-immobilised enzyme therefore suggesting that the immobilised lactase is less susceptible to denaturisation.
As the enzyme is trapped in the gel-like substance, it allows the substrate to enter and exit however restricts the enzyme from leaving the entrapment substance. Therefore making the enzyme more stable, and providing it with a delicate barrier against its surroundings.
I think this delicate barrier will help protect the tertiary structure from losing its shape (to an extent) because some the increase in vibrational energy, caused by the increase in temperature, will be absorbed by the inert matrix rather than the enzyme itself. Therefore, helping to secure the active site of the enzyme from the detrimental effects of excessive heat energy.
The non immobilised lactase does not have this inert matrix to protect its active site/ tertiary structure therefore when excessive temperatures are present the vibrational energy will cause the weak bonds which hold the tertiary structure in place to change thus denaturing the enzyme.
This signifies, that immobilised enzymes are less susceptible to denaturisation