The pH at which an enzyme works best is called it optimum pH. pH is a measure of hydrogen ions (H+), or the number of hydrogen ions in a certain volume. The concentration of hydrogen ions affects the bonds and ionic bonds. If the pH changes much from the optimum, the chemical nature of the amino acids can change. This may result in a change in the bonds and so the tertiary structure may break down. The active site will be disrupted and the enzyme will be denatured.
Some enzymes are exceptions to these temperatures and pHs. Some enzymes can live cold environments, since there are more cold environments than hot ones, for example, oceans, which have average temperatures from 1-3°C. These organisms are called psychrophiles. Also some organisms like the warmer climate, they are called thermophiles, they can grow and reproduce in temperatures higher than 45°C, for example in hot springs. Similarly some enzymes like acidic conditions, with a pH lower than 5, they are called Acidophiles. Some enzymes also prefer alkaline environments with a pH above 9 they are called alkophiles.
Enzyme and substrate concentration can also affect enzyme activity. At low enzyme concentrations there is great competition for the active sites and the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate. Eventually, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor. We call this the point of optimal concentration. At a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low. When more substrate molecules are added, more enzyme-substrate complexes can be formed. As there are more active sites, and the rate of reaction increases. Eventually, increasing the substrate concentration yet further will have no effect. The active sites will be saturated so no more enzyme-substrate complexes can be formed. We call this the point of optimal concentration.
Enzymes are extremely valuable biological catalysts to living organisms as they control biological processes and allow them to take place in the conditions, which occur inside living organisms.
AFFECTS OF TEMPERATURE ON THE DIGESTION OF STARCH
PAUL MITCHELL
SCIENCE COURSEWORK
Affects of Temperature On The
Digestion of Starch
· Introduction
For my experiment, I will be looking at enzymes; more specifically, the enzyme amylase. Amylase breaks down starch into glucose. I will be looking at how temperature affects this conversion.
Enzymes exist in all living things. They are composed of polypeptide chains of amino acids and are produced in living cells. Each cell contains several hundred enzymes, which catalyse a vast number of chemical reactions.
Enzymes are known as 'Biological Catalysts' as they dramatically affect the rate at which reactions occur within living organisms, without being 'used up' or effecting the reaction in any other way. Enzyme catalysis saves the need for an increase in temperature in order to speed up reactions within living things. Such an increase in temperature would be lethal to the organism. Enzymes only control the rate of one specific reaction inside the body, as only one specific substrate can fit into its active site; therefore we say the enzyme is specialized.
There are two main types of enzyme:
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Intracellular enzymes, which control reactions that occur inside cells.
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Extracellular enzymes, which control reactions that occur outside cells
All enzymes are affected by temperature and pH. They have an optimum range of the two, and anything outside this range will denature the enzymes "active site." The enzymes that are used in reactions inside our bodies have an optimum temperature of 37oC. The enzyme can withstand a few degrees either side of this temperature; but any higher will cause the protein structure to break apart and stop working, because the bonds that hold the amino acids together are relatively weak; therefore, a high temperature would cause the atoms to excite and the bonds to break.
The enzyme will become denatured permanently and no longer functional. The optimum pH for an enzyme depends upon where about in the body it is working. Generally, most enzymes have an optimum neutral pH of around 7, but enzymes that work in the stomach, for instance, have a much lower optimum pH of 2 due to the acidic environment.
· Prediction:
I predict that as the temperature increases, so will the rate of the reaction. I think this because when heat is applied, it excites the particles more and as a result, they collide faster and so the reaction rate increases. When a particular temperature is reached, I think the rate of reaction will dramatically decrease because the amylase is working outside its optimum temperature range. Usually a rise of about 10oC will double the rate of reaction. After the enzyme reaches its limit, which I predict to be 40oC, I think the reaction rate will decrease rapidly and then stop. I think this because the enzyme has become denatured and the bonds holding the enzyme together have started to break; therefore denaturing the active site.
Variables:
To keep my experiment as fair as possible and to achieve the best possible results, I will keep the following aspects of my test identical:
Amount and concentration of starch solution, Amount and concentration of amylase solution, amount of iodine solution, amount of benedict's solution added and pH.
The only variable in my experiment will be the temperature change, as this is the objective of my test.
· Safety Precautions:
Throughout my experiment I will be handling chemicals; some of which are dangerous. I will also be using a Bunsen burner throughout the experiment so I will be taking certain precautions. First of all I will wear goggles from the start of my test to the very end. I will use tongs to handle the boiling/test tubes when they are taken out of the hot water bath. If any other safety precautions arise during the experiment, I will take certain measures to make them as safe as possible.
· Method:
I will set up my experiment as instructed below:
(All boiling tubes above have the same amount of starch and amylase present)
- Using a 100ml measuring cylinder, I will measure out 20ml of both Starch and Amylase solution.
- Put the 20ml of amylase and starch solutions in separate test tubes.
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Next, I will cool/heat an ice/water bath to 5oC (or the chosen temperature) and add a thermometer.
- Wait for the temperature to reach a constant.
- Add both test tubes of amylase and starch into the ice/water bath.
- Wait until both solutions reach the temperature of the ice/water bath.
- When both test tubes are at the correct temperature, add them together into a boiling tube.
- Place the boiling tube into the ice/water bath to keep the temperature constant.
- Once the amylase and starch solutions have been added together start the stopwatch.
- Every 30 seconds, using a pipette, take a sample from the boiling tube and add it into a spotting tile.
- Add 2 drops of iodine solution to test for the presence of starch and wait for the solution to change to a black/purple colour.
- Repeat this every 30 seconds until the solution no longer changes colour; therefore indicating all the starch being converted into glucose.
- Once the iodine stops indicating a black colour, stop the stopwatch.
- Record the indicated time on the stopwatch into the results table below.
- Then, add a few drops of Benedict's to the solution and put the boiling tube into a hot water bath.
- Wait until the solution has turned a brick red colour; thus indicating all the starch being turned into glucose.
The same process as above should be carrie
: : : : : Determine the optimum temperature at which the enzyme amylase digests starch into glucose residues through making predictions and doing practical experiments.
Biology Experiment-Investigation into Enzyme Amylase
Aim
The aim of my experiment is to determine the optimum temperature at which the enzyme amylase digests starch into glucose residues through making predictions and doing practical experiments.
Introduction
After great study into catalysts, I found that enzymes are nothing more than protein molecules that reduce activation energy and form products extremely quickly without having their own molecular structure transformed or broken down. I have also found that enzymes are extremely sensitive in the sense that they have a particular shape in which a substrate fits in to, like a key that fits into a lock. This brings reason into why Amylase is only an enzyme that breaks starch into glucose residues and no other substance.
Scientific theories are known that the most common enzymes works best at around 40.c. This is there optimum temperature they react. If this temperature begins to rise, the reactions slow down; this will continue to happen until the enzyme is denatured. It becomes denatured at normally 50.c, this happens because the enzyme becomes misshapen. This means that the enzyme will no longer work because it will no longer fit, the active site will be ruined. The enzymes react like a lock and key, only one lock fits the key. When the two join then you get the chemical reaction. When too much heat gets applied then the lock gets changed and so the key doesn't fit the lock so there is no reaction.
I have also found that enzymes are extremely sensitive to the surrounding in which they are in. A slight adjustment into the temperature or Ph can seriously reduce the amount of enzyme-substrate complex being formed and therefore less products being formed. It is therefore acceptable to believe that enzymes all follow these rules:
1. Enzymes have a specific shape only allowing the substrates they are designed to digest, to fit in. (Excluding discussion into competitive inhibitors…etc)
2. Enzymes have a certain temperature in which they are able to work to the maximum. This temperature in known as the optimum temperature. If the temperature is increased above this optimum temperature, then it is possible that the enzyme has become denatured and reducing the temperature there after can never bring the enzymes to work at the optimum again. In fact the enzymes will not work at all.
3. Enzymes are Ph sensitive. Increasing or decreasing the Ph can easily cause denaturing to occur.
Prediction
I predict that by adjusting the temperature lower then that of the optimum will slow down the formation of enzyme-substrate complex because of very little collisions occurring between the enzyme and substrate. Every time a substrate hits an enzyme there is a high chance of an enzyme-substrate being formed and therefore a product will be made. If the collisions are reduced by lowering the temperature then very little products are going to be made.
This brings us to the next question of where the optimum temperature can be found. There is no doubt that the human body also consumes starch and needs to digest these large molecules in to smaller residues before absorption into the blood system. The enzyme amylase is therefore also found in the human body in the regions of the mouth and after food leaves the stomach. The human body is at a constant temperature of 37 degrees and this leads me to believe that the optimum temperature could be at 37 degrees.
I also think that the enzyme will work best between 37.c to 40.c I predict this because the optimum temperature for most natural enzymes is 40.c, because this is a chemical enzyme; it will work best a little higher. If this temp is exceeded then I think that it will take longer to work because it will not be at the optimum temp, or it will not work at all because it has become denatured. An enzyme cannot recover from this state because the hydrogen bonds holding the protein structure together burst and are not able to hold on to the structure anymore. Below this temperature I think it will be slowed down but will not be denatured because the hydrogen bonds remain in tact at low temperatures and only break at high ones. They are fewer collisions between the amylase and starch and therefore less products.
Once the enzyme has been added to the starch buffer solution at 40.c and has been given time to digest the enzyme, to find out if the enzyme amylase has digested starch into glucose iodine solution is going to be added to the glucose solution. I am sure it will become blue or a black colour if glucose is not produced as this is to show a positive reading if starch is still there and it will become a brown colour if glucose is produced. This is only qualitative data to show whether glucose is present or not and the quantity of starch cannot be determined.
Fair test
To make this experiment as a fair test I will only change the temperature in the experiment as this is the independent variable. I will keep the following the same. These are the constant variables:
· The volume of starch solution (10 cubic centimetres) and amylase (10 cubic centimetres).
· The volume of water to keep the starch and amylase at a constant temperature (250 cubic centimetres).
· The apparatus, test tube, beaker, thermometer (shown above).
· The time to allow the starch and amylase to mix and to record results is up to 20 mins.
· I will use the same batch of iodine solution for testing the starch to see if it has broken glucose.
I will keep all of these things in the same order and in the same amount including any volumes specified above.
Apparatus
Test tube
Beaker
Stop watch
Thermometer
Iodine solution
Amylase solution
Starch solution
Water
Bunsen burner
Tripod
Gauze
Measuring cylinder
Pipette
Method
Method
For the following experiment I had do the follow to participate in the experiment:
· To start I will set up the apparatus carefully as shown in the diagram.
· Then I will measured 10ml of starch with a measuring cylinder and poured it into the test tube and repeated the same procedure for the amylase solution. (Hence I have two test tubes: one with starch inside and one with amylase)
· The I will half fill the beaker up to 250 cubic centimetres and place this on top of the Bunsen burner
· Then I will put both of the test tubes in the beaker which will contain water and light the Bunsen burner.
· I will also put a thermometer inside the beaker and check the temperature occasionally until the required temperature has been reached after heating.
· As the heating process is due to take time I will leave the Bunsen burner to heat the beaker for five minutes and return to see if the required temp has been reached.
· During this time I will get a white tile and drop iodine solution equally spaced on the tile. This is a special sort of tile that has equally placed gaps in-between them, in which the starch-amylase mixed solution will be put into, after certain time intervals that will be mentioned later. (Hence after the mixed solution is placed into the iodine solution a brown colour or a blue/black colour will appear through which I will determine if glucose has been produced or not)
THIS IS THE CRUCIAL PIONT. ONCE THE TWO SOLUTIONS, AMYLASE SOLUTION AND STARCH SOLUTION ARE MIXED, THE STOP WATCH MUST BE STARTED IMEDIATELY. I WILL GET A FRIEND/TEACHER TO START TIMING AS I POUR THE TWO SOLUTIONS TOGHETHER AS SPLIT SECONDS CAN MAKE A BIG DIFFERENCE IN THE PRODUTION OF GLUCOSE.
· Once five minutes have passed and the required temp has been reached I will take both test tubes and pour any one solution into the other test tube and mix together and start the stop watch.
· Once I have done that, after every 30 second intervals I will by using a pipette pour a drop of the amylase and starch solution on the drops of the iodine solution until 5 minutes have passed. (Hence I would have by now taken 10 readings). After 5 minutes have passed I will then take readings after 1 minute intervals.
· I will repeat the process of dropping the solution of amylase and starch on the iodine solution after every one minute for up to 20 minutes.
· I will repeat this process to get more reliable results and record my results in a table.
Results
The results below were obtained from the experiment. These are the average results after I did the experiment three times. To find the mean I used this formula:
Add up the three times in minutes
Mean (Average) = ---------------------------------------------
3
Temperature (degrees) Time (minutes)
20 16
25 12
30 8
35 4
40 3
45 6
50 10
55 18
60 26
70 no result
The results have been plotted on graph paper to show any patterns or and to give an outline of the activity of the enzyme amylase. However even after this graph was plotted it only showed to a certain extent the optimum temp at which the amylase works, but gave no idea of what the rate of reaction was like. The rate of reaction is necessary at this point to give a suggestion at the rate at which the amylase works at different temperatures and what temp its rate is best at.
To calculate the rate of reaction I used the following formula:
1
Rate of reaction = -------------------------------------------------
The time it took to break down starch
Hence the (1) is a constant in the formula which is then divided by the time it took for the starch to break down in to glucose.
Temperature Rate of Reaction
20 0.06
25 0.08
30 0.13
35 0.25
40 0.33
45 0.17
50 0.1
55 0.06
60 0.04
Conclusion
My results proved my prediction to be correct. The breakdown of starch to glucose accelerates as the temperature increases until the optimum temperature after which it begins to slow down and comes to a halt. Temperature influences the rate of enzyme activity as my graph show a gradual increasing gradient and after the optimum temp was exceeded a steep negative gradient becomes visible.
Usually a 10°c rise doubles the rate of enzyme activity. Hence the Q10 is roughly equal to 2 which describe this to be a chemical reaction. This means that every time the temperature raises by 10 degrees the rate of reaction doubles. This is only true up to an optimum temperature, however beyond this point (usually 40°c) the shape of the active site becomes distorted and the enzyme becomes inactive.
Although the theory of collisions supports the fact that the rate of reaction should increase constantly as the temperature increases but this statement only remains true up until 40 degrees. The hydrogen bonds that break down in the secondary structure of the enzyme amylase due to heat increasing cause the enzyme to become denatured. This does not allowing the break down of starch. It can also be understood from this investigation why the temperature rising above 40 degrees in the body could be lethal and a killing factor and why at the same time the temp falling below can be a killing factor of the human body.
Cooling or even freezing does not destroy enzymes, though it slows down their activity almost to a halt. From studying the graph and our results table we can see the enzymes optimum temperature (40°c). It was after 50°c where we could see a decrease in the enzymes activity. A rise in temperature increases the rate of most chemical reactions and a fall in temperature will slow them down.
Evaluation
The results are reliable, but the experiment was not completely accurate. This is because of the cooling down of water and having to heat it up to the right temperature frequently slightly adjusted the required temperature.
Also it was not completely accurate because the colours of the iodine are different due to the nature of different people seeing things differently, like Black and purple are hard to differentiate and one person might write down black when another writes purple. There are some odd results in my graph, which was made from human error because of my opinion in colour being different.
If I were to do this experiment again I would use the most sophisticated equipment available because you can't record things well enough yourself. I would use a constantly heated water bath that was always at the correct temperature and I would use an electric thermometer too. I would do this and I would make sure everything was perfect before every temperature was recorded. I would also change my variations in the temperature so that I had a clearer idea of the exact optimum temperature. I would record no denatured results. These are all the changes that I would enjoy making.
I also believe there to be a technical way of deciding how much starch has been digested. I have read through surfing the internet that a device called a colorimeter can be used and a complete different method can be applied to gain more accurate results through which human error can be diminished. Hence I could ascertain the amount of starch digested through which only qualitative data is not only recorded but quantity can be measured too.