disulphide bonding
hydrogen bonding
The optimum temperature for most enzymes is 37°C. The enzymes in the body have this optimum temperature and the body has adapted to control its temperature so the enzymes are working at their best.
pH levels
Enzymes also have an optimum pH level, where they work best, any changes to this level will cause the enzymes to begin to denature. Denaturing of the enzyme is caused because changes in pH affect the ionic bonding which determines the tertiary structure of an enzyme. This change in the globular protein causes changes in the shape of the active site. Hence reduces the rate of reaction as less substrate-enzyme complex can be formed. The optimum pH for lactase is 6.5.
(Vmax is the point at which the maximum rate of reaction is reached)
This graph doesn’t apply for all enzymes as enzymes in the stomach have a low optimum pH and so are suited to the acidic conditions of the hydrochloric acid in the stomach. The bell shape either moves to the left or the right depending on the optimum pH for the enzyme.
Concentration of enzyme and substrate
As in most experiments the amount of substrate greatly outnumbers the amount of enzyme, an increase in enzyme concentration will cause the rate of reaction to increase proportionally to the increase in enzyme concentration. This is because more enzyme-substrate complex can be formed, as there are more enzyme active sites available.
An increase in the concentration of substrate has the same effect except that the increase in rate of reaction stops at a certain point. This is due to all the enzyme active sites being used. Any further increase in substrate concentration will have no effect of the rate of reaction as the enzyme can’t work any faster and more enzyme-substrate complex cannot form until there are active sites free.
Hypothesis
Immobilised lactase enzymes have a higher denaturing temperature and produce more glucose than free lactase enzymes. This is because the enzymes are trapped, so it will take more energy for the enzyme to move, and so ultimately more energy (heat) for them to vibrate so much that the bonds break, causing the enzyme to become denatured.
If this is true then I would expected to observe that the immobilised enzyme beads will produce a higher % of glucose (double the % of the free enzyme solution) and that it will be denatured at a much higher denaturing temperature (at least 5°C higher).
Prediction
I predict that a rise in temperature will cause a rise in the rate of reaction until 40°C, after which enzymes will denature so the rate will fall for the free enzymes. However the immobilized enzymes rate of reaction will continue to rise, as the enzymes will denature at a higher temperature.
There will be a rise in reaction rates because a rise in temperature will mean the lactose molecules are moving faster and are more likely to collide with the lactase enzymes resulting in a greater frequency of collisions. A higher temperature will also mean more lactose molecules will have an energy above the activation energy, so there will be more collisions with the right activation energy. This will result in the rate of reaction increasing for both free and immobilized enzymes.
Initially I would expect that the free enzyme solution would have a higher rate of reaction as the enzymes in the immobilised enzyme beads are trapped, so more energy is required to make them vibrate. Also the immobilised enzymes rely more on the substrate molecules colliding with the beads, as they are unable to move, so higher temperatures are needed to allow the substrate molecules to move enough.
After 40°C the rate will fall for the free lactase because the enzymes will denature. This is due to the attractions between amino acid molecules in the enzyme breaking and the enzyme will lose its shape. The active site of the enzyme changes so it cannot from the enzyme-substrate complex and break down lactose into glucose and galactose. As the temperature rises further the lactase will denature quicker and the rate of reaction will fall further.
The immobilised enzyme beads will follow the same pattern but as they’re more stable it will occur at a higher temperature.
The rise of rate of reaction is governed by the Q10 coefficient, which states that a 10°C rise will result in an approximate double of the rate of reaction.
Q10 = Rate of reaction at T + 10°C
Rate of reaction at T°C
Method
Preliminary work was undertaken to determine the amount of milk and enzyme, what temperature graduations and length of time to use.
Immobilised beads
-
4cm3 lactase (β-galactosidase)
-
16cm3 of 2% sodium alginate solution
-
200cm3 of 2% calcium chloride solution in a plastic beaker
- Semi-quantitative glucose test strips (Diabur 5000)
- Muslin gauze
-
10cm3 plastic syringe and barrel
- Clamp and clamp stand
- Short length of tubing, to fit plastic syringe and screw clip
- Glass rod
- Distilled water
- Pasteurised milk
-
100cm3 beaker
- Plastic tea strainer
- Test tubes
Free enzyme solution
-
5cm3 lactase
-
20cm3 distilled water
- Conical flask
-
10cm3 plastic syringe and barrel
- Clamp and clamp stand
- Short length of tubing, to fit plastic syringe and screw clip
-
Glass graduated pipette (10cm3) and pipette filler
- Electric heated water bath
- Pasteurised milk
- Stopwatch
- Glucose test strips
- Thermometer
Making the immobilised enzyme beads
- I will mix the sodium alginate solution with the enzyme solution in a beaker using a glass rod.
- I will then transfer the solution to a plastic syringe
- I will add the mixture drop wise to the calcium chloride solution. I will leave the beads to harden for 10 minutes
- I will strain the beads using the tea strainer and rinse with distilled water.
Making enzyme solution
-
I will use a clean graduated 10cm3 pipette to measure 5cm3 of lactase and place in a clean 50cm3 conical flask. I will measure out 20cm3 of distilled water using a clean graduated pipette and place in conical flask.
- I will place a bung on the conical flask and invert several times to ensure that the enzyme solution and the distilled water are well mixed.
Immobilised enzymes method
- The required temperature was set on the water bath and the test tube rack was placed in the water bath.
- The beaker of pasteurised milk was placed in water bath to heat.
- 25 enzyme beads were measured out and placed in a test tube.
- A small amount of distilled water was added to test tube – to ensure even heating and so enzymes heat quicker as air is a good insulator.
- The test tube was placed in test tube rack and then a thermometer was placed in this test tube. I will leave to heat to required temperature.
- A small piece of gauze was cut and placed in syringe with tubing attached. The gauze prevented the enzyme beads blocking the outlet. The screw clip was closed.
-
A glass-graduated pipette was used to measure out 10cm3 of milk. The measurement was taken from the bottom of the meniscus.
- The enzymes were removed from the water bath once they had reached the required temperature. The enzyme beads were transferred to the syringe barrel.
- The milk was added to syringe barrel and the stopwatch started.
- After 7 minutes the screw clip was slowly opened and the bottom half of the liquid in the syringe barrel was run off before being tested for glucose using the Diabur strips.
- The test strip was left for 2 minutes before the colour change was compared and the result recorded.
This was repeated 3 times at each temperature. The starting temperature was 30°C and the temperature was increased by 5°C increments up to 60°C. The results are as follows;
5 minutes
7 minutes
10 minutes
I found that 7 minutes was the best length of time as enough glucose was produced to get some readings but not so much that there is no differences between the temperatures. I also found that divisions of 5ºC were adequate as there was noticeable difference between the results at different temperatures. I also found the quantities were suitable (using 10cm3 of milk, 25 beads and 1cm3 of 20% lactase solution). A problem I found in my preliminary work is that due to using room temperature milk when it was added to the higher temperatures it cooled them down so the experiment did not happen at the correct temperature. To combat this I will also heat up the milk in the water bath so that temperature stays as close to the chosen temperature as possible. Another problem I noticed was that the immobilised beads would cause the distilled water in the test tubes to become cloudy (presumably due to enzyme leakage) after prolonged heating in the test tubes and both the free and immobilised enzymes became denatured at a much lower temperature than I expected. I research further the effects of heating on enzymes.
I found that the length of incubation time would decrease the denaturing temperature as the graph below shows. Long incubation times also reduce the maximum productivity of enzymes by around 10 %. These findings may explain why the denaturing temperature and the productivity was lower than expected.
—— short incubation period (2 minutes)
----- long incubation period. (10 minutes)
Due to this I will try to reduce the incubation time as much as possible. Instead of leaving the test tube of free enzyme or enzyme beads in the water bath while it heated to the required temperature, I will heat the water bath up and then place the test tube. I can still leave the milk in the water bath, as this takes much longer to heat as a larger quantity is needed.
Equipment
Immobilised beads
-
4cm3 lactase (β-galactosidase)
-
16cm3 of 2% sodium alginate solution
-
200cm3 of 2% calcium chloride solution in a plastic beaker
- Semi-quantitative glucose test strips (Diabur 5000)
- Muslin gauze
-
10cm3 plastic syringe and barrel
- Clamp and clamp stand
- Short length of tubing, to fit plastic syringe and screw clip
- Glass rod
- Distilled water
- Pasteurised milk
-
100cm3 beaker
- Plastic tea strainer
- Test tubes
Free enzyme solution
-
5cm3 lactase
-
20cm3 distilled water
- Conical flask
-
10cm3 plastic syringe and barrel
- Clamp and clamp stand
- Short length of tubing, to fit plastic syringe and screw clip
-
Glass graduated pipette (10cm3) and pipette filler
- Electric heated water bath
- Pasteurised milk
- Stopwatch
- Glucose test strips
- Thermometer
Making the immobilised enzyme beads
- the sodium alginate solution was mixed with the enzyme solution in a beaker using a glass rod.
- The solution was then transferred to a plastic syringe
- the mixture was added drop wise to the calcium chloride solution. the beads were left to harden for 10 minutes
- the beads were strained off using the tea strainer and rinsed with distilled water.
Making enzyme solution
-
A clean graduated 10cm3 pipette was used to measure 5cm3 of lactase and was placed in a clean 50cm3 conical flask. Do not blow through pipette to remove last bit of enzyme in pipette as it has been calibrated to compensate for this.
-
20cm3 of distilled water was measured out using a clean graduated pipette and placed in the conical flask.
- A bung was then placed on conical flask and inverted several times to ensure that the enzyme solution and the distilled water are well mixed.
Immobilised enzymes method
- The required temperature was set on the water bath and the test tube rack was placed in the water bath.
- The beaker of pasteurised milk was placed in water bath to heat.
- 25 enzyme beads were measured out and placed in a test tube.
- A small amount of distilled water was added to a test tube – to ensure even heating and so enzymes heat quicker as air is a good insulator.
- The test tube was placed in test tube rack and then a thermometer was placed in this test tube. This was then left to heat to the required temperature.
- A small piece of gauze was cut and placed in a syringe with tubing attached. The gauze prevented the enzyme beads blocking the outlet. The screw clip was closed.
-
A glass-graduated pipette was used to measure out 10cm3 of milk. The measurement was taken from the bottom of the meniscus.
- The enzymes were removed from the water bath once they had reached the required temperature. The enzyme beads were transferred to the syringe barrel.
- The milk was added to syringe barrel and the stopwatch started.
- After 7 minutes the screw clip was slowly opened and the bottom half of the liquid in the syringe barrel was run off before being tested for glucose using the Diabur strips. This is done as the enzyme beads float to the top and so the top section will be have the most product as it is unlikely that the bottom part will have chance to have formed enzyme-substrate complex as the enzyme and substrate have not mixed.
- The test strip was left for 2 minutes before the colour change was compared and the result recorded.
-
This was repeated 3 times at each temperature. The starting temperature was 30°C and the temperature was increased by 5°C increments up to 60°C.
Diagram
Free enzymes method
- The required temperature was set on the water bath and the test tube rack was placed in the water bath.
- The beaker of pasteurised milk was placed in water bath to heat.
-
1cm3 of enzyme solution was measured out using a clean graduated pipette and placed in a test tube.
- The test tube was placed in test tube rack and then a thermometer was placed in this test tube. This was then left to heat to required temperature.
-
A glass-graduated pipette was used to measure out 10cm3 of milk. The measurement was taken from the bottom of the meniscus.
- The enzyme test tube was removed from the water bath once it had reached the required temperature. The enzyme solution was transferred to the syringe barrel making sure the screw clip was closed.
- The milk was added to syringe barrel and the stopwatch started.
- After 7 minutes the screw clip was slowly opened and the bottom half of the liquid in the syringe barrel was run off before being tested for glucose using the Diabur strips.
- The test strip was left for 2 minutes before the colour change was compared and the result recorded.
-
This was repeated 3 times at each temperature. This was because this will provide an adequate average. The starting temperature was 30°C and the temperature was increased by 5°C increments up to 60°C. I chose 30°C for my start temperature as previous research showed that the optimum temperature for lactase is 48°C, so starting at this temperature will allow me to build up to the theortical optimum without having to repeat the experiment to many times. As this will increase the cost due to expense of Diabur strips etc.
Diagram
Fair testing
To make the test fair all other factors affecting the rate of reaction must be kept constant. This includes surface area of the immobilized beads; the concentration of the lactase solution and the pH of each experiment. The experiment will be kept a fair test by:
-
using the same volume of immobilized beads (1cm3 or 25 beads), to keep the volume of enzymes constant. So that the volume of enzyme would be the same in both the free and immobilised I measured the volume of 25 enzyme beads, which was 1cm3 and so this is the volume of free enzyme solution I used,
- using the same volume of milk so both experiments had equal amount of lactose,
- washing the equipment with distilled water to prevent changes in pH as tap water can be slightly acidic,
- using the same size beads with the same surface area as this is a factor which can affect the rate of reaction. To control this I will use the same syringe each time and apply a constant pressure so that the volume of each beads is the same,
- using the same type of milk, as different milk may have different amounts of lactase (concentration of substrate is a confounding variable),
- using the same concentration of lactase in immobilized beads and free solution which was 20%,
- recording the glucose reading on the Diabur strips as they take 2 minutes before the result can be determined. Taking the reading before or after 2 minutes may mean that too much or too little a % of glucose has been recorded.
- it must be ensured that when taking readings off the measuring cylinders and thermometers etc, the readings are taken from the bottom of the meniscus at eye level, so as to minimise the risk of inaccurate readings.
Safety
Only very few enzymes present hazards, because of their catalytic activity, to those handling them in normal circumstances but there are several areas of potential hazard arising from their chemical nature and source. These are allergies, activity-related toxicity, residual microbiological activity, and chemical toxicity. Due to this, dry enzyme preparations have been replaced to a large extent by liquid preparations.
All enzymes, being proteins, are potential allergens and have especially potent effects if inhaled as a dust. Once an individual has developed an immune response as a result of inhalation or skin contact with the enzyme, re-exposure produces increasingly severe responses becoming dangerous or even fatal.
- Any waste enzyme powder should be dissolved in water before disposal into the sewage system.
- Enzyme on the skin or inhaled should be washed with plenty of water.
- Liquid preparations are inherently safer but it is important that any spilt enzyme is not allowed to dry as dust formation can then occur.
- Wear safety goggles and tie back long hair.
- Taking care when handling hot water.
- Taking care with water bath as it is an electrical device so not to spill water on the plug.
- Taking care with the glassware, as it can be easily broken
- Harmful if swallowed
- May be harmful if inhaled or in contact with eyes
- Eye/skin/respiratory irritant
- Safety goggles to be worn
Results after 7 minutes
Free enzyme solution
Immobilized
The average shown is the mean. Below are the mode and median averages for the experiment.
Free enzyme solution
Immobilised beads
Conclusion
The results obtained during the course of the experiment seem to be quite conclusive. It possible to identify a pattern or trend in the average results obtained. From the rate of reaction graph, it is clear that there is an increase of glucose concentration with an increase in temperature. This is, however, only up to a certain point.
Free enzymes
The rate of reaction increases until 35ºC, after which it starts to fall. There is still some glucose being produced after 45ºC, but only very little. By 50ºC the average reading is 0% and the enzymes have been completely denatured.
Immobilised enzymes
The rate of reaction increases until 40ºC and has begun to fall by 45ºC. After 45ºC the average % glucose drops dramatically as the enzymes become denatured more quickly. The rate of reaction continues to decrease as the temperature increases until finally no more glucose is produced at 60ºC.
The results I obtained, do back my hypothesis as the rate of reaction did increase as the temperature increased up to a certain point. I had predicted that it would have taken a higher temperature to denature or begin to denature the immobilised enzyme but there was only 5ºC difference between the peak temperature for the free and immobilised enzymes. This difference is not significant enough to conclude that there is a difference due to errors in the experiment. Therefore, I can’t draw definite conclusions from this single experiment, as there was a large percentage procedural error.
I can conclude from my results that the enzyme activity or the rate of reaction increases with temperature up until around 40ºC (for immobilised) and 35ºC (for free) as the enzyme and substrate molecules gain more and more kinetic energy. As a result, the reactants move around with increased energy. This results in there being an increased number of effective collisions. Subsequently, the rate of reaction increases. After 40ºC (immobilised) or 35ºC (free), the rate of reaction deteriorates. As the graph shows it takes longer for the immobilised enzymes to have 0% glucose after 7 minutes.
As kinetic energy increases, in theory, the rate of reaction should keep on increasing, this is however not true. This due to the fact that after the optimum temperature for either the free or immobilised enzyme activity, the bonds that hold together the enzyme structure, start to break (this is especially true of hydrogen bonds), due to the increased kinetic energy, which causes vibrations which “shake” the bonds apart. Resultantly, the rate of reaction deteriorates as the enzyme becomes denatured due to the fact that its active site structure, is lost due to the breaking up of the bonds that hold it together. This means that the substrate molecule can no longer fit into the active site of the enzyme as the shape of the active site changes.
Immobilised enzymes have a higher optimum temperature possibly because the enzymes can’t move around as they are entrapped in the beads and so the substrate has to move around faster to have the same chance of colliding with the enzyme. Therefore it needs a higher temperature to move around faster.
Evaluation
Although the results obtained from the experiment support my prediction, I was not satisfied with the experiment, as the results obtained were limited as the experiment was only repeated three times. There were a lot of errors, both in the conducting of the experiment, and in the results obtained.
- I felt that the Diabur strips used to measure the % of glucose allowed a lot of room for error. The lowest percentage they would measure was 0.1% and after 1% they only went up in 1% differences. As they had to left for two minutes before a reading could be taken the wrong % may be recorded as if the reading was taken too early too low a percentage may be recorded but if taken too late too high a percentage may be recorded. The Diabur strips show different percentages by slightly different colour changes. Error can arise from this, as perception of colour is different from person to person and depending on the lighting.
- Another problem was that heat was lost to the surrounding so the temperature reduced during the duration of the experiment. To minimise this, if I were to repeat the experiment, I would insulate the syringe to try to maintain the desired temperature.
- I also assumed that the pH of the solutions would remain the same throughout, though this may not have been the case. To control this I would use a buffer solution at pH 6.5 which would maintain the pH of the solution and pH 6.5 is the optimum pH for lactase.
- I left the milk in the water bath so some may have evaporated which would increase the concentration of lactose in the milk and as the concentration of the substrate is a variable it may affect the results.
Considering the above, it is probable that the results obtained during the experiment are possibly unreliable. I would have to repeat my experiment, trying to eliminate the problems I highlighted above. Repeating results more times helps to obtain an average which is closer to the true average.
I have identified 2 anomalies for the immobilised beads experiment which are highlighted on my graphs. I concluded that these are anomalous results as they don’t fit the Q10 theory which states that the rate of reaction doubles for every 10°C increase in temperature. But my average results for 35°C and 40°C for the immobilised enzymes didn’t fit this. For 35°C the average % glucose was double the amount for 30°C (from 1% to 2%) and for 40°C the average % glucose after 7 minutes was triple that of 30°C (from 1% to 3%). Possible causes for these errors were that –
- The beads may have contained slightly more enzyme which would mean that there would be a higher possibility of forming enzyme-substrate as increased enzyme concentration is proportional to increased rate of reaction.
- The Diabur strips may have been left too long so the colour may correspond to a higher % glucose when read.
- The pH levels may have changed which may provide closer to optimum condition for the enzymes which may lead to an increase in the rate of the reaction.
- If the lactase and the sodium alginate weren’t mixed properly some beads may contain less or more lactase than others. This would either reduce or increase the rate of reactions.
- Some of the immobilised beads contained a lot of air as air entered the syringe when the suspension was being sucked up in the syringe. This would decrease the amount of enzyme and so would reduce the rate of reaction.
If I was to conduct the experiment again, I would make sure that it was more accurate overall. As I feel that the experiment doesn’t give accurate enough results, I would use a different experiment which would be easier to measure. An example of this would be using an enzyme that has a gas as a product, as this can be measured to the nearest 0.5mm3. Examples of enzymes which catalyse an experiment which has a gas as a product is catalase which speeds up the reaction of converting hydrogen peroxide into oxygen and water.
With the above taken into account, I would say that my conclusion is not very secure due to the fact that there were several errors. Subsequently, the results do not provide a very stable evidence for support of my hypothesis. Although the percentage error of individual equipment is small, the procedure error of the experiment is quite high as the results had to be determined by eye.