In this experiment, I will study a single enzyme, Lactase, examining the effect of temperature conditions upon its stability, that is, its ability to catalyse a reaction in varying temperature conditions. Temperature has a complex effect on enzyme activity, a rise in temperature will increase the kinetic energy of enzyme and substrate molecules, and therefore increase the rate of a chemical reaction (The collision theory). However, increases in temperature will also affect the stability of the enzyme molecules also affecting the "lock and key principle", so enzymes will no longer be able to combine with its substrate therefore slowing enzyme activity. The overall rate of activity will depend on a balance between these two factors, Lactase will have its own optimum temperature, which will become apparent after my experiment. The average optimum temperature for most enzymes is 40oC.
Outline method
The presence of glucose can be detected by 'Diastix' glucose test strips, which colour code for the concentration of glucose. The milk which will be passed over the immobilised lactase beads, will be tested with these strips to determine the rate of lactase catalysing the hydrolysis of Lactose to glucose and galactose. The main problem that arose in previous experiments is the colour coding cannot give an exact figure of glucose present only a set bracket.
Plan: Key Variables
- The same milk must be used for all experiments.
So the level of lactose in the milk is kept constant.
- The same amount of milk must be used for all experiments.
This is so the amount of lactose is kept constant.
- The immobilised enzyme beads must be the same size for all experiments.
The size of the immobilised beads can affect the enzyme activity, and as this isn't a variable I will be testing it will remain constant.
- The same amounts and concentration of Lactase enzyme must be kept constant for each immobilised enzyme beads batch.
This is so the immobilised enzyme beads are all identical minimising differences between repeated experiments.
- The same amounts and concentration of sodium alginate solution must be kept constant for each immobilised enzyme beads batch.
This is also so the immobilised enzyme beads are all identical minimising differences between repeated experiments.
- The same amounts and concentration of calcium chloride solution must be kept constant for each immobilised enzyme beads batch.
This is also so the immobilised enzyme beads are all identical minimising differences between repeated experiments.
- The temperature that the beads are heated to must remain constant.
This is so the temperature won't drop as the beads are been left to equilibrate at the temperature.
- The milk must be passed over the lactase beads for the same amount of time at the same drop rate for every experiment.
This is so each experiment is identical minimising experimental errors between repeats.
Plan: Preliminary method
I will firstly immobilise 2cm3 of lactase (Beta galactosidase) by mixing it in a beaker with 8 cm2 of 2% sodium alginate solution, then adding this mixture drop wise to a new beaker of 100 cm3 of 1.5% Calcium chloride solution. The immobilised enzyme pellets should be allowed to harden for a few minutes, then rinsed thoroughly with distilled water before use. I will make 5 batches of these immobilised enzyme beads.
After the beads have set, I will place them in five 10cm3 open syringes, I will then put the syringes in beakers filled with water, and place these in water baths, at 20oC, 30oC, 40oC, 50oC and 60oC respectively, and give the immobilised enzyme beads 10 minutes to equilibrate at the temperature, they are stored at. I will then pass 20 ml of milk into the syringe (a small piece of gauze will be placed over the syringe opening so that it does not become blocked by enzyme beads), and is allowed to slowly perculate over the immobilised lactase beads, at a drop rate of 1 minute using a pipette and an adjustable clamp attached to the end of the syringe to obtain this, when the milk had been passed over the beads totally I will test it with Diastix glucose test strips for 30 seconds.
I will then immediately register the result, I will repeat this procedure with the 5 batches of beads heated to their set temperature. I will repeat the experiment 3 times, for optimal results.
As a control experiment, I intend to find out the best temperatures to use for the most efficient results.
Plan: Risk Assessment
Lactase enzyme: Observe standard handling procedure to avoid direct contact with the product or inhalation of dust from the dried product. In case of accidental spillage and contact with the skin or eyes, rinse promptly with water.
Sodium alginate: Low risk, avoid contact with eyes.
Calcium chloride: Low risk avoid contact with eyes.
Milk: Very low risk.
USE EYE PROTECTION
Plan: Pilot experiment, Range of readings
In order to determine suitable temperatures, for the immobilised lactase beads to be heated to, I carried out rough preliminary experiments heating the beads to different temperatures, I used the range I intended to use, 20oC, 30oC, 40oC, 50oC and 60oC respectively, and other sets of temperature ranges, 25oC, 30oC, 35oC, 40oC and 45oC, and 35oC, 40oC, 45oC, 50oC and 55oC. I then carried out experiments to determine which set of temperatures would give the best results.
Results of pilot tests, Range of readings
Using range of temps 20oC, 30oC, 40oC, 50oC and 60oC
Using range of temps 25oC, 30oC, 35oC, 40oC and 45oC, and 35oC
Using range of temps 35oC, 40oC, 45oC, 50oC and 55oC
From the results I obtained it was clear that the first range of temps, 20oC, 30oC, 40oC, 50oC and 60oC respectively gave the clearest and most varied results, and I could draw more conclusions from them, I therefore have chosen to use these range of temperature in my practical.
Method
Firstly, I immobilised 2cm3 of lactase (Beta galactosidase) by mixing it in a beaker with 8 cm2 of 2% sodium alginate solution, then adding this mixture drop wise to a new beaker of 100 cm3 of 1.5% Calcium chloride solution. I allowed the immobilised enzyme pellets to harden for a few minutes, then rinsed them thoroughly with distilled water before use. I made 5 batches of these immobilised enzyme beads.
After the beads had set, I put them in five 10cm3 open syringes, I then put the syringes in beakers filled with water, and placed these in water baths, at 20oC, 30oC, 40oC, 50oC and 60oC respectively, and gave the immobilised enzyme beads 10 minutes to equilibrate at the temperature, they were stored at. I then passed 20 ml of milk into the syringe (a small piece of gauze was placed over the syringe opening so that it didn't become blocked by enzyme beads, restricting the drop rate), and allowed it to slowly perculate over the immobilised lactase beads, at a drop rate of 1 minute using a pipette and a adjustable clamp attached to the end of the syringe to obtain this, I tested the pressure of the clamp beforehand to ensure it gave an accurate drop rate of 1 minute for all the milk to pass over the beads, and into the beaker.
When the milk had been passed over the beads totally, I tested it with Diastix glucose test strips for 30 seconds, I then immediately registered the result, I repeated this procedure with the 5 batches of beads heated to their set temperature. I repeated the experiment 3 times, for optimal results.
Set up Diagram
Results
Experiment 1
Experiment 2
Experiment 3
Results Graph
Conclusions
Main trends and patterns
My results show that the immobilised enzyme Lactase, catalyses the hydrolysis of Lactose to glucose and galactose most efficiently at 40oC, 15 mmoldm-3 of glucose is converted and at more extreme temperatures such as 20oC and 60oC it loses it stability can cannot catalyse as well, so only 5.5 mmoldm-3 of glucose is converted and at 60oC No catalysis is offered at all, and no glucose is converted.
The raw data shows that lactase is significantly less active at temperatures 20oc either way from its optimum temperature 40oc, at higher temperatures 50oC and 60oC the amount of glucose produced quickly decreases, indicating that lactase cannot function at higher temperatures. The most important trend is the steady rise and fall of the graph, demonstrating the enzymes optimum temperature clearly. The three experiments show slight differences but the general trend is the same. There seems to be no obvious anomalous results, as key variables were maintained throughout the experiment. However I am surprised that the enzyme didn't function more efficiently at higher temperature as it immobilisation should have offer greater stability, at 60oC the enzyme beads even began to melt and offer no catalysis at all, this may be an experimental flaw however I wish to investigate it further. At lower temperatures such as 20oC the stability wasn't affected however the catalysis was very slow and therefore less glucose was converted.
Explanation of results
Temperature has a complex effect on enzyme activity, a rise in temperature will increase the kinetic energy of enzyme and substrate molecules, and therefore increase the rate of a chemical reaction (The collision theory). However, increases in temperature will also affect the stability of the enzyme molecules also affecting the "lock and key principle", so enzymes will no longer be able to combine with its substrate therefore slowing enzyme activity. The overall rate of activity will depend on a balance between these two factors, Lactase will have its own optimum temperature, so the enzyme activity will increase until lactase's optimum temperature is reached which proved to be 40oC after this temperature is exceeded the enzyme will begin to denature and offer less catalysis until it loses its stability and no longer functions, in the case of Lactase at 60oC.
The kinetic behaviour of a bound enzyme can differ significantly from that of the same enzyme in free solution. The properties of an enzyme can be modified by suitable choice of the immobilisation protocol, whereas the same method may have appreciably different effects on different enzymes. These changes may be due to conformational alterations within the enzyme due to the immobilisation procedure, or the presence and nature of the immobilisation support.
Immobilisation can greatly effect the stability of an enzyme. If the immobilisation process introduces any strain into the enzyme, this is likely to encourage the inactivation of the enzymes under denaturing conditions (e.g. higher temperatures or extremes of pH). However, where there is an unstrained multipoint binding between the enzyme and the support, substantial stabilisation may occur. This is primarily due to the physical prevention of the large conformational changes within the protein structure which generally precede its inactivation.
Many successful covalent immobilisation processes involve an initial freely-reversible stage, where the covalent links are allowed to form, break and re-form until an unstrained covalently-linked structure is created, in order to stabilise the resultant immobilised enzyme. Additional stabilisation is derived by preventing the enzyme molecules from interacting with each other, and the protection that immobilisation affords towards proteolytic and microbiological attack. This latter effect is due to a combination of diffusional difficulties and the camouflage to enzymic attack produced by the structural alterations.
In order to achieve maximum stabilisation of the enzymes, the surfaces of the enzyme and support should be complementary with the formation of many unstrained covalent, or non-covalent interactions. Often, however, this factor must be balanced against others, such as the cost of the process, the need for a specific support material, and ensuring that the substrates are not sterically hindered from diffusing to the active site of the immobilised enzyme in order to react at a sufficient rate. Although my results show that immobilized lactase showed a significant decrease in activity at higher temperatures, I did not have the time to carry out a similar experiment with free lactase, to compare if immobilisation had an effect on its temperature stability, however, I would make an educated guess that it would have - suggesting that lactase must have fairly low temperature tolerance.
Evaluation: Experimental limitations
The most important limitation to this experiment is the inaccuracy of the Diastix glucose test strips as they only colour code for set amounts of glucose the exact amount cannot be determined, therefore, the results work on boundaries and although they may colour code for the same amount there may be a significant difference in the actual amount of glucose present. However, although this was an inaccurate method it was the only means of testing the milk for presence of glucose, and therefore there were no alternatives to improve this.
The other major limitation was the temperature regulation, the water baths were unreliable and took a lot of time to heat the immobilised enzyme beads to the required temperature, and even then the temperature couldn't be kept exact, there were especially problems heating the water to 60oC and cooling it to 20oC. If more advanced water baths that can heat and cool water to a desired temperature quickly were available this could have improved the accuracy of my results however there were not available, therefore I solved this problem by adding boiled water to the water in the beaker to reach the required temperature, however it was still very time consuming and still slightly inaccurate.
Time constraints limited the experiment greatly as there was no time for further work or to investigate anomalies, this resulted in drawing conclusions from guess work. However, if I had carried out the further experiments as I had wished I would have far exceeded the maximum time allocated to the experiment.
Overall the investigation did provide significant evidence to support my hypothesis that, the immobilised enzyme will be most stable at the optimum temperature for enzyme activity, about 40 oC. The enzyme should show a decrease in stability at more extreme temperatures,
20 oC and 60 oC. Immobilising the enzyme means it should stay fairly stable at slight changes in temperature, 30 oC and 50 oC, because of the protection offered by the inert matrix.
Evaluation: Further work
If the time were available, I would have liked to have carried out a similar experiment with free lactase to make comparisons on the immobilisation procedure on temperature stability, I would have also wished to have investigated why the enzyme denatured so quickly at high temperature - even though it had been immobilised - to see if this was the nature of the enzyme or an experimental flaw.
Also a greater range of temperatures would have given improved results to draw conclusions from, investigating the effects of colder temperatures on the enzyme activity.
