Experiment to investigate the effect of temperature on enzyme activity

5/2/02 Experiment to investigate the effect of temperature on enzyme activity

Aim

The effect of temperature on enzyme activity can be tested in different ways using different enzymes. In this experiment we will be testing the effect of temperature on the activity of Rennilase in the clotting of milk. We will test the time it takes for the milk to clot with this enzyme at different temperatures.

Background Information

Enzymes are biological catalysts.

The way enzymes work can be described as the lock and key theory

Enzymes are affected by concentration
Enzymes are affected by PH
Enzymes are affected by temperature

Enzymes are Biological Catalysts

Enzymes speed up chemical reactions without changing them (making them a catalyst as this is what a catalyst does).
Reactions that build up larger molecules are called Anabolic reactions.
Reactions that break larger molecules to smaller molecules are called catabolic reactions.

The way enzymes work can be described as the lock and key theory

An enzyme provides a place for a reaction to take place. This is called the active site.

The molecule that is going to react is called a substrate molecule. It fits exactly into the active site (hence the lock and key theory the active site is the lock and the substrate molecule the key).

The enzyme then tweaks the substrate molecule so that they form a product.

The product leaves the active site and the enzyme can be used again.

Affected Enzymes are by PH

As the concentration of enzyme increases, the rate of reaction increases. But only up to a certain point where the rate of reaction levels off.
The rate levels off because as many enzyme-substrate collisions as possible are occurring, so adding more enzyme will not speed up the process. Increasing the substrate concentration will produce a similar shaped graph.

4) Enzymes are affected by PH

Enzymes are affected by the PH of the medium in which they are working.
Most enzymes will work most effectively at PH 7 (neutral), a few will work at other PH’s. Such as Pepsin which works in the stomach and prefers
PH 2.
If an enzyme is not working in an environment with the correct PH it will be temporarily denatured, but it will work again in the correct PH.

Temperature affects enzymes

Most chemical reactions happen faster when the temperature increases.
At higher temperatures molecules more around faster making it easier for them to react.
Normally when no enzyme is involved a 10°C rise will double the rate of reaction.

No Enzyme:

There are no enzymes involved in this
Example.
The molecules reacting move faster and have more energy at higher
Temperatures.

Enzyme catalysed reaction:

Between 0-40°C the rate of reaction rises in the same way as a reaction containing no enzyme. It does this for the same reason.

At 40°C the enzyme starts to become damaged and so the reaction slows down.
-By 60°C the enzyme is completely destroyed.

40° is the optimum temperature for this enzyme (the temperature at which the rate ...

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There are no enzymes involved in this
Example.
The molecules reacting move faster and have more energy at higher
Temperatures.

Enzyme catalysed reaction:

Between 0-40°C the rate of reaction rises in the same way as a reaction containing no enzyme. It does this for the same reason.

At 40°C the enzyme starts to become damaged and so the reaction slows down.
-By 60°C the enzyme is completely destroyed.

40° is the optimum temperature for this enzyme (the temperature at which the rate of reaction is the greatest).

Enzymes are very sensitive to high temperatures. Once the enzyme starts to become damaged at about 40°C it cannot catalyse reactions as well and when this happens the rate of reaction slows down and we say the enzyme has been denatured. At even higher temperatures the reaction will stop completely because the enzyme has been destroyed.

Strategy

In this experiment we are only investigating temperature and so the other factors that I have mentioned previously must be kept constant.

The concentration of enzyme is 1cm³ to every 9cm³ of milk. In the experiment the milk will be added to the rennilase because there is more milk than rennilase and so if a little milk is lost in the pouring it would not matter as much in comparison to loseing some rennilase which would make a big difference.

The PH of milk is 7.

The PH of Rennilase is 7.

The temperature is the variable.

Apparatus

30 test tubes

Thermostatically controlled water baths

Rennilase

Whole fat Milk

Measuring Pipette

Thermometers

Test tube rack

3 large beakers

Ice

Stop clocks

Hypothesis

I predict that from 20°C upwards the milk will clot when the rennilase has been added and as the temperature increases the time in which the milk clots will decrease. When the temperature reaches 50°C the time it takes for the milk to clot will decrease and at 60°C upwards no clotting will occur as the enzyme will be denatured and the rate of reaction will have decreased.

I predict this because in a previous experiment involving starch, iodine and amylase, where the time it took for the amylase to react with the starch causing the liquid to return to clear again was recorded, the optimum temperature was 40°C and below this the rate of reaction decreased with the temperature. The rate of reaction at 0° was 0,at 25° it was 0.1, at 35° it was 0.6 and at 45° it was 4.3. This is because as the temperature increases the enzyme and substrate molecules have more energy and move faster. This causes more enzyme-substrate reactions to occur, but once the optimum temperature is exceeded some of the enzymes become damaged and the others just move around faster. Therefore it may appear that the optimum temperature is higher but if heated to about 60° the enzymes are all permanently denatured.

From previous experiments as the temperature increases, the rate of reaction will increase but after 40°C the enzyme will start to become damaged and the rate of reaction will slow down and at 60°C the enzyme will be destroyed.

In this experiment I will be investigating the effect of temperature on enzymes.

I think the way I have planned to do this is a good way because it only investigates temperature and everything else is kept constant. I will only be testing temperatures from 0°-70° because previous experiments have shown that this is the largest range in which enzymes can work. I have used 0° to prove that very cold temperatures will make enzymes dormant as they cannot work in these mediums. I will be testing at 70° to prove that all the enzymes have become destroyed at 60° and none can survive such high temperature as 70°.

Diagram

Safety

Safety glasses were worn throughout the experiment to protect our eyes from the rennilase and other substances.

Lab coats were worn also to protect us from rennilase as it can digest protein and therefore could be irritable to our skin.

If our skin came into contact with rennilase it was washed off immediately.

Method

Water baths for the temperatures 30,40 and 50°C were prepared.
For 0°C ice was placed in one of the large beakers.
For 60 and 70°C water of those temperatures were placed in the two other beakers.
All of these baths had their temperatures checked and maintained through out the experiment.
6 test tubes were placed in a test tube rack.
1cm³ of rennilase was added to 3 of them.
9cm³ of milk was added to the other 3.
All six test tubes were placed in the beaker containing ice and the temperatures of the milk and rennilase monitored.
When the temperatures of the contents in the 6 test tubes reached the temperature of the water in the water bath, the contents of the 3 test tubes containing milk were added to the 3 test tubes containing rennilase and a timer was started.
The contents of the test tubes were observed and when the milk clotted the timer was stopped and the time it took for the milk to clot was recorded.
This was repeated for each of the water baths although points 2-4 are not necessary for thermostatically controlled baths.

Conclusion

The results matched my prediction because after about 20°C the milk clotted when the rennilase was added and as the temperature increased the time for the milk to clot decreased. When the temperature reached 50°C the time it took for the milk to clot reached a minimum and from 60°C upwards no clotting occurred because the enzymes were fully denatured and so the rate of reaction decreased as the temperature increased above 50°C.

From the experiment I conclude that the optimum temperature for rennilase is approximately 41°C. Below this temperature the rate of reaction is increasing slowly as the temperature increases. From 0°C-20°C there is almost no change in the rate of reaction and from 20°C- 30°C the change is 0.087 but from 30°C-40°C the rate of reaction triples, the change is 0.295.

I would expect that the optimum temperature in this experiment is about 41°C, because if you look at the graph, the rate of reaction increases up to about 41°C and then it starts to level out and drop after 50°C. This means that the enzymes are becoming damaged after about 41°C and at 50°C most of them are damaged and becoming denatured and by 60°C they are all destroyed. So for temperatures above the optimum temperature the general trend indicates that as the temperature increases the rate of reaction decreases.

This all happens because as the temperature increases, the molecules have more energy and move around faster. This causes more enzyme-substrate collisions(enzyme-substrate reactions). After the optimum temperature the enzymes are starting to denature. The ones that are left continue to move faster as the temperature increases, so the rate of reaction still increases because some are still reacting but as more and more are denatured, the rate of reaction goes down until they are all destroyed.

My prediction regarding the optimum temperature was correct although one degree Celsius higher. This difference is well with in the accuracy I would expect for this experiment.

Evaluation

The results can be misleading. The graph shows that the temperature at which the rate of reaction is at its highest is 50°C which you would consider to be the optimum temperature because this is the point where the rennilase is reacting fastest(the definition of optimum temperature). However, this is not the case because after about 40°C the enzymes are becoming damaged due to the high temperature and some are denatured. Not all of the enzymes have been denatured and so enzyme-substrate reactions are still occurring causing the rate of reaction to peak at a temperature which is above its optimum temperature.

This presents problems for anyone trying to obtain accurate results for this experiment because at these temperatures two things are happening, the rate of reaction is increasing but yet the rennilase is being damaged and denatured due to the higher temperature. This makes it difficult to determine the optimum temperature because these results all depend on how long you leave the rennilase in the water bath before mixing it with the milk.

To improve this experiment I would make sure that the rennilase was kept in the water baths for a standard time because above a certain temperature (between 40°C and 50°C), if left long enough, all the rennilase will be denatured and no clotting will occur.

This is can be shown by the difference in the 3 results regarding 50°C. They vary widely because of the denaturing of the rennilase.

Adequacy of Evidence

The accuracy of the graph is limited because more results are needed. With more results you could determine the optimum temperature much more accurately. I would propose results, as a minimum, at 35°C,45°C and 55°C.

There was a wide variation in the times recorded for each of the 3 samples at each temperature. In order to obtain more accurate averages a larger number of samples are required at each temperature.

Equipment

The water baths were useful as they were thermostatically controlled and so the temperature of the water within them was kept constant. However, for 0°C, 60°C and 70°C thermostatically controlled water baths were not available and so it was difficult to ensure that the water was kept at a constant temperature.

Subjective Judgement/Reliability

To determine the time taken until clotting occurred it was necessary to visually assess this process. Due to time limitations it was necessary for two people to take results and therefore there may have been variation in the opinions of what the milk and rennilase looked like when it had clotted. This means that the accuracy of the times recorded may have varied. If I was doing the experiment again I would make sure that only one person was assessing this.

Limitations

If I could repeat the experiment I would give myself a lot more time. I would do this because it was difficult to monitor the results from several sections of the experiment running at the same time. If there had been more time the problem of subjective judgement would have been reduced because one person could obtain all the results.

There were insufficient stop watches to monitor each sample.

There were insufficient water baths as there were not ones for 0°C, 60°C and 70°C.

Further research

If I had unlimited time I would investigate the following because I believe they may have an effect on the reaction time:

Types of milk.
PH -Buffer solutions to make different acidities of the medium. Rennilase works in the stomach, which has a PH of about 2. Where as in this experiment the medium in which the rennilase was working had a PH of 7. I would investigate the effect of PH on rennilase.

Anomaly

In this experiment we had to repeat one of the results for 50°C because the rennilase denatured before we mixed it with milk and so no clotting took place. This confirms that while the reaction rate was maximum at 50°C it is not the optimum temperature.