Investigating the Effect of pH on Enzymes

Plan:

My aim is to carry out an experiment that will let me deduce the affect that a varying pH will have on the rate at which the enzyme amylase will break down starch into its component parts, which are maltose and dextrins.

Background:

What is an Enzyme?

Enzymes are substances that act as catalysts and so they increase the rate of chemical reactions. In biological systems reactions may occur very slowly or even not at all unless a catalyst is present and this is why enzymes are required. With an enzyme the product of the reaction will occur far faster even to a factor of at least one million. Unlike inorganic chemical catalysts, enzymes are specific. This means that one enzyme normally is used for one reaction. This is as each enzyme has a particular shaped active site, which the substrate (the molecule that is being broken down) will combine with. The shape of the active site depends on the structure of the whole enzyme.

The Structure of Enzymes

All enzymes are globular proteins, and so have a primary secondary and tertiary structure. The primary structure of a protein is the number, type and sequence of amino acids that make up a polypeptide chain. In the case of the enzyme that we are using (alpha-amylase) the polypeptide chain is around 496 amino acids in length. Each of these amino acids has the general formula NH2.RCH.COOH, where the R group varies giving the 20 different individual amino acids. Two amino acids join together in a condensation reaction, where a molecule of water is removed, forming a dipeptide. The bond that joins them is called a peptide bond; more and more of these are formed forming polypeptide chains. The secondary structure is where these polypeptide chains arrange themselves into another shape, which could either, be into a spiral shape, the alpha helix or into a corrugated sheet structure called a beta pleated sheet. The structure of alpha amylase consists of a combination of alpha helices and beta pleated sheets. Both of these structures are held together by hydrogen bonding between the CO of one amino acid and the NH group of another amino acid that is further along the chain. The tertiary structure is the arrangement of the two secondary structure into a three dimensional shape. This is what determines the shape of the active site of an enzyme and so controls its function. The tertiary structure is held together by ionic bonding between oppositely charged R groups, disulphide bridges that form between R groups with sulphur in them as well as hydrogen bonds.

How do Enzymes work? – The Lock and Key Theory

This theory was put forward to explain why enzymes are specific, only work for a certain substrate. As the enzyme itself is usually bigger than its substrate only part of it comes into contact with it, this part of the enzyme is called the active site. Only around three to twelve amino acids make up the active site but it has a an exact shape so as the substrate can fit into it. The substrate is the key and the enzyme being the lock. When the two come together the form a temporary structure called an enzyme/substrate complex. The reaction occurs at the active site and this is where the new products are formed, these products don’t have the same shape as the active site and so repel and leave the active site empty, which is free then to react with more substrate molecules. The diagram below shows this process:

The induced fit theory is an updated version of the above lock and key theory. It puts forward the idea that the active site does not completely correspond to the shape of the substrate. Instead the active site is more flexible and is therefore able to mould itself around the substrate. When it has bound closely the reaction then can occur forming the new products, which will not fit into the active site and so will be repelled, the enzyme then goes back to its ‘relaxed’ state and so is able to react further with the substrate. This is what the diagram below shows: I have mentioned that enzymes are catalysts, and this means that they lower the activation energy. The activation energy is that needed to start a chemical reaction. This means that no other extra heat is needed to make the chemical reaction start, i.e. heat, so the cells in the body can work at lower temperatures. The energy diagram below will help explain activation energy.

By lowering the activation energy of a reaction, the enzyme provides a new route for the reaction to follow, so it reduces the input needed so the reactions can occur at temperatures normally found in the body.

The variables:

There are certain factors that can affect the way in which an enzyme works and these are:

Temperature: - Increasing temperature increases the rate of most reactions as it increases the speed at which the molecules move at and so increase the chance of collisions between reacting particles, meaning more reactions take place. Also, with greater energy the collisions are more likely to overcome the activation energy. This is also true of enzyme-controlled reactions, to a point, as up to around 40°C the rate of reaction is increase. This increase in rate of reaction can be shown as the temperature co-efficient Q10, the formula for which is

Q10 = rate of reaction at T + 10°C

Rate of reaction at T

Basically a 10-degree rise doubles the rate of reaction. The increase in rate of reaction does not occur all the time as an optimum temperature is reached, where the reaction is at its fastest. Past this temperature the rate of reaction drops quickly. This is as the heat given to the molecules also makes them vibrate and as the enzyme vibrates the bonds that hold its tertiary structure together begin to break, so losing its shape. This alteration in the three dimensional shape means he shape of the active site is changed so the substrate will no longer fit into the active site. When this happens the enzyme is said to have been denatured, which can be a permanent change lowering the rate of reaction. Cooling the enzyme to extremes will also slow reaction time down as the kinetic energy that is required to start the reaction, i.e. the collision energy, is not there in the form of heat but there is no permanent change. In my experiment the temperature will be kept constant by carrying all experiments out at room temperature. This will ensure a fairer experiment as only one variable will be changed, to do this I will keep the enzyme and starch/enzyme solutions away from any potential heat sources such as the Bunsen burner until they have to be added to the Benedict’s reagent.

Change in pH: - Most enzymes have their own pH which they work best at, their optimum pH. This usually depends on the area of the body on the environment that the enzyme usually occurs in. Changes in pH can affect the rate of reaction as the hydrogen ions can affect the hydrogen bonding in the tertiary structure. Small changes will not denature the enzyme but extreme changes will affect the shape. The introduction of H+ (acidity) and OH- (alkalinity) ions affect the charges on the amino acids that make up the active site which changes the shape, but also if becomes flooded with these ions then it can prevent the substrate and enzyme coming together, the diagram below explains why.

The diagram shows that in the optimum pH the ratio of positive ions and negative ions is correct and so the charges of the active site and substrate are complementary and so attract each other. In low pH conditions then there are more positives charges and so the enzyme and substrate and repel each other and this is also the case in high pH as there are too many negative ions. This is the variable that I will change in my main experiment.

Enzyme concentration: - Enzymes are catalysts and so are not used up in the reactions, so being able to be used over and over again. This means only a small amount of enzyme is required to catalyse a huge number of substrate molecules. This means that providing the temperature and pH conditions are suitable, and that there is an excess of substrate, then the rate of reaction is directly proportional to the enzyme concentration. This will be kept constant in my main experiment.

Substrate concentration: - as the substrate concentration, the rate of reaction would also increase to a point. This is as the more substrate there is the more active sites are being filled so the quicker the reaction. However once all the active sites have been filled up, i.e. they are saturated, the rate of reaction cannot increase any further. Again this will be kept constant in my main experiment.

Type of enzyme: - There are different sources of the enzyme that we are using, which is amylase, and so there may be differences in the range ...

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Substrate concentration: - as the substrate concentration, the rate of reaction would also increase to a point. This is as the more substrate there is the more active sites are being filled so the quicker the reaction. However once all the active sites have been filled up, i.e. they are saturated, the rate of reaction cannot increase any further. Again this will be kept constant in my main experiment.

Type of enzyme: - There are different sources of the enzyme that we are using, which is amylase, and so there may be differences in the range of temperatures and pHs at which they react. It can be found in the mouth of humans, as well as in the pancreas or both humans and other animals. It can also be produced commercially using bacterial sources such as Bacillus licheniformis. This can be genetically engineered and the amylase extracted. In the main experiment just genetically engineered amylase will be used to make sure only one variable is being changed.

Starch:

Starch is the substrate that is being used, i.e. the substance that is being broken down. It is made up of two polysaccharide chains. One is amylose, which is made up of the monosaccharide alpha glucose in long chains. Alpha 1,4 glycosidic bonds join them. As they only have 1,4 glycosidic bonds amylose is an un-branched. This makes up around 30% of starch and the remaining 70% is made up from amylopectin, which is made up from the same alpha glucose but has 1,6 as well as 1.4 glycosidic bonds and this leads to a branched structure. The diagram below shows the structure of these two polysaccharides visually.

How is starch broken down?

Amylases hydrolyse the glycosidic bonds in starch, i.e. they add a molecule of water and so break the bonds converting starch into smaller glucose chains called dextrins or to maltose (which is the disaccharide made of two alpha glucose molecules). Different enzymes in the amylase group work in different ways and on different bonds in the starch molecule. The diagram on the following page summarises the action of 4 different amylases and gives the products that they form.

Now I have all the background knowledge that I require I can make a hypothesis based on it.

Hypothesis and Justification:

I believe that increasing or decreasing the temperature from the optimum temperature, which I predict to be ph7, the less the concentration of maltose will be. This will produce a graph that has one peak and slopes off either side, a bell shape. I believe this as the amylase that we are using is from a bacillus. Bacilli are found in a wide range of places but are most commonly found in soils, and so generally work in neutral condition, i.e. pH 7. As the conditions become more alkali or acidic then the shape of the active site of the enzyme may become altered as the bonds that hold the tertiary structure together will be broken and so the enzyme will not function properly decreasing the rate of reaction. Also the more

Preliminary Work:

To find a suitable range of pHs to test a preliminary or pilot experiment was set up. It also allowed several techniques that were to be used in the main experiment to be tested and perfected.

Firstly 3 pH starch solutions were taken (2.2, 6 and 11) that would gives us a wide range to see if they would produce results that were firstly notable but also were not too close together. These starch solution had buffer added to them so that when the enzyme was added they would be able to acclimatise to the pH conditions. Then an amylase stock solution had to be made up, this was to make sure the amylase used was standardised so eliminating variation and therefore creating more accurate results. To do this, a 1cm³ graduated pipette was used draw up 0.5cm³ of the concentrated amylase. The 1cm³ pipette was used as the amylase required was very small and so 1cm³ gave us the greatest accuracy and the smallest percentage error. The amylase sample was then poured into a 100cm³ volumetric flask so as to give the concentration that was desired. Distilled water was then used to make up the solution to the 100cm³ mark, but before the solution came to the neck of the flask it was shaken to make sure it was properly mixed. Much care was then take to fill to the 100cm³ line. When the bottom of the meniscus was level with the line no more was added, the bottom was used, as because of the nature on bonding in water it is “sticky” and so sticks to the edges of the flask and this is not an accurate measurement of the volume of water in the flask. Once this point has been reached the stopper was placed on top and the flask labelled with the concentration, which was 0.005% of the conc. amylase solution. This was chosen as the experiment was to take place over a three minute time period and so a small concentration was needed so the starch would not be broken down too quickly. From research I have found that if a higher concentration of enzyme were to be used then lower transmission values would be produced as more maltose would be formed and because of the sensitivity of the colorimeter it would be likely that anomalous results would be produced in the lower transmission region. This is as it is impossible to fully block out all of the blue light from passing through simply by increasing the severity of the precipitate but the colorimeter would be likely to read zero as some of the light may be scattered and not go straight through the solution so anomalies may be produced. So a weaker enzyme would give higher transmission values where the percentage error would be smaller.

Next 2.5cm³ of the stock amylase solution was placed into three test tubes for the three pHs that were to be tested, and to this 2.5cm³ of the conc. starch solutions containing buffer were placed into the test tubes, each test tube with a different pH solution. Both were done using two separate 5cm³ graduated pipettes, so as to give the greatest degree of accuracy and also prevent cross contamination. Simultaneously, 5cm³ of Benedict’s reagent was placed into three boiling tubes (as they were to be heated and test tubes might crack) and these were then placed into a boiling water bath for three minutes. This was to make sure the benedict’s solution was at a very high temperature, so as when the amylase/starch solution is added the heat will denature the amylase and so stop any further reaction. After the three minutes allowed for the amylase to react with the starch the contents of the three test tubes were indeed poured into the boiling Benedict’s solution, and left in there for a further eight minutes in the boiling water so the Benedict’s test for reducing sugars could take place.

The Benedict’s test- This is a test for the presence of a reducing sugar or sugars. The Benedict’s reagent is made up from an alkaline solution of copper sulphate CuSO4 that is blue in colour. When it is heated with a reducing sugar the solution becomes green, then orange before ending up as a red/brown colour depending on how much of the reducing sugar is present. This happens as the either the carbonyl/ aldehyde group are able to reduce the Cu2+ ion in the copper sulphate, to Cu1+ ions which form copper oxide which is brown in colour giving the colour changes, so the more reducing sugar that there is present the more copper oxide that is formed, so the more brown the solution.

After the eight minutes had passed the boiling tubes were removed from the water bath using tongs so as to reduce the risk of being burnt and placed into a boiling tube holder. Samples of each were then poured into a small colorimeter vessel, called a cuvette, and tested using the colorimeter. The same cuvette will be used for all readings so any imperfections in it will be constant with all results. To maintain accuracy it will be washed thoroughly with distilled water after each reading.

The Colorimeter: - This is a piece of equipment that shines light of a certain wavelength through the sample and measures the amount that is transmitted through. Different filters are used to block the light that is shone through; in this case a blue filter will be used so as to give blue light shining through the solutions, as the red precipitate will absorb this. The percentage transmission values are then taken from the dial on top of the piece of apparatus. Before any samples are put into the colorimeter it has to be calibrated, this is done by putting a sample in that is a mixture of water and Benedict’s reagent, this should have 100% transmission but if the dial does not read this the needle can be set to it using a button on the colorimeter. If there is a high transmission then this means there is les maltose so a slower rate of reaction and vice versa.

For each pH condition a percentage transmission value was taken and noted down here are the results:

This shows that from pH’s 2 to 11 there are measurable results that have a wide range of transmissions. Now 7 different pHs will be take within this range to give me a wide range of pHs that are to be tested as this is a manageable number and so will be able to be carried out in the time allowed and give a graph with a suitable number of points to draw a best fit line.

Calibration Curve:

In this experiment we are trying to see how much maltose (as well as glucose and other dextrins) that are produced but the readings that are taken are of percentage transmission not concentration of maltose. However a calibration curve can be drawn to work out these values. To do this known concentrations of maltose are needed. To get these the following procedure was followed. 5cm³ of conc. maltose is added to 50cm³ of water, using 5cm³ and 10cm³ graduated pipettes and pipette fillers to give the greatest accuracy. This gives a one in ten solution, this was down so as if drops of the maltose solution were left in the pipette then the concentration of the solution in the test tube will be closer to what is required than I f a higher concentration was used, as the percentage error will be smaller and so this improves accuracy. This solution then can be divided up using another graduated pipette into 1cm³ of maltose and 9cm³ of distilled water, 2cm³s of maltose and 8cm³ of distilled water all the way down to 9cm³ of maltose with 1cm³ of water. This gives various concentrations of maltose. Then 5cm³ samples of each concentration were taken using a 5cm³-graduated pipette, which was cleaned thoroughly after each concentration, and place into a boiling tube along with 5cm³ of Benedict’s solution. This was boiled for 8 minutes using a hot water bath and then sampled using the colorimeter twice and an average given to ensure accuracy. The table of results are shown below:

(‘-‘ Indicates that this concentration was not measured as only a certain number were needed to produce an accurate curve)

From these results a graph plotting final concentration to average transmission can be produced. This is the calibration curve and it will allow me to read off unknown values of maltose as long as I know the percentage transmission. The calibration curve itself is in the analysis section of this investigation.

Main Experiment Method (Implementing):

The method used in the main experiment is much the same as that used in the preliminary experiment, as a water bath will be set up using a tri-pod, Bunsen burner and a heat proof mat. The beaker will be the 500cm³ volume, so as to fit the 7 boiling tubes for the seven-pH values that are being used. This will be filled with water and left to boil. Once it has begun to boil a 5cm³ sample of Benedict’s solution will be placed into 7 boiling tubes using a graduated 5cm³ pipette and pipette filler. Tin foil will then be placed over the tops of the boiling tubes to stop any of the Benedict’s solution evaporating away. They will then be marked with the pHs that are being used (2.2, 4, 6, 7, 8, 9 and 11) with a marker so they can be identified when they are take out of the water bath. These will then be placed in the hot water bath all at the same time so they would boil at the same rate. At the same time 2.5cm³ samples of the stock amylase solution made in the preliminary experiment (which had been refrigerated for a short time) will be taken using a 5cm³-graduated pipette as a 1cm³ one would mean three measurements so increasing the risk of an error, and this sample placed into 7 different test tubes. These will then be marked with the different pH values. Now the different starch solutions will be taken and 2.5cm³ samples take out using another 5cm³ pipette and placed into their corresponding test tube, i.e. the one with the correct pH written on it, simultaneously so as to make sure they all have the same time to react and a stopwatch will be started. This will give us an accurate timing device to measure exactly three minutes. After these three minutes the foil over the boiling tubes will be carefully removed and the contents of the test tubes added to them making sure the pH on the side of the test tube corresponds to the pH on the side of the boiling tube. The stopwatch will then be started again after being reset and left to run for a further eight minutes. After these eight minutes are up tongs will be used to remove the test tubes so as to be safe from possible burns, they will be placed into a boiling tube holder in order of increasing pH so as to prevent confusion. Each will then be shaken carefully so as the make sure the precipitate has diffused equally through the solution so as to make sure the small samples that will be taken reflect the colour of the main solution. Three small samples of each solution will be taken to try to reduce the risk of anomalies. The colorimeter test will then be carried out as described in the preliminary experiment, to give accurate transmission readings and the results will be noted.

Here is what the results table will look.

Safety:

I must also consider the ethical implications of using amylase. If the enzyme were to be genetically engineered from extracting from animals then there may be some ethical implications, however, the enzyme used in this investigation was engineered from bacteria, by inserting a specific DNA ring into bacteria that will multiply many times over then extracting the enzyme produced. So in my view there appear to be no ethical implications.

Implementing: Here is the raw data produced from the main experiment

There were no changes made to the original method that was stated in the planning section of this investigation.

I will now use the calibration curve that was made plotting the results below, to find the unknown concentrations of maltose for each pH corresponding to the percentage transmission.

These of the results of using the calibration curve (found in the analysis section)

Analysis:

Firstly looking at the calibration curve produced, the general trend is that which was expected. That is that as the concentration of maltose increases the percentage transmission decreases, i.e. there is an in verse relationship to the results. Further more it is a curve, which again was expected because of the nature of the sensitivity of the colorimeter. There appear to be no major anomalies and indeed very few minor ones and so I believe that the evidence shown from this graph is accurate enough to use in my analysis.

The aim of this investigation was to find the relationship between changing pH and the rate at which starch is hydrolysed by way of looking at the amount of maltose produced, so I can now link the calibration curve and my raw data. From the raw data shown n the implementing section, I will take the average % transmission values and convert them to concentrations of maltose using the calibration curve that was made during the planning section of this investigation, and is found on the next page. Here are the results:

I will now plot the graph of pH against concentration of maltose and from this I will be able to look at the general trend and look how it compares to my initial hypothesis. The graph produced can be found after the calibration curve in this section. I have also added error bars to look at the variability of the results, these show the maximum and minimum concentration values for each pH. This will allow me to draw a more accurate line of best and will also allow me to comment on the reliability of results in the nest section of this investigation. Here are the maximum and minimum values of transmission for each pH and their corresponding concentration of maltose. (See page following graphs)

Firstly, I can say that there are no apparent anomalous results, this can be deduce from the fact that the error bars showing the minimum and maximum results for each pH all fall on the line of best fit, showing all the data follows the same general pattern meaning there is a high correlation.

Secondly, looking at the general shape of the graph, it appears to be bell shaped, i.e. has one main peak and slopes off either side. So from pH 2.2 to approximately pH 7.2 the concentration of maltose rises as the pH increases, i.e. has a positive trend, then from pH 7.2 to pH 11 there is a decrease in the concentration of maltose, i.e. a negative trend. However both the increase up to pH 7.2 and the decrease from it are not directly proportional as the line of best fit is not a straight one. In fact both sides are curved as the bell curve name may suggest. This shows that there is a geometric progression in the increase and decrease; this means that the rate at which they change through the different pH values is not constant. This can be displayed if we look at the negative trend of the graph by looking at the gradient at the top of the curve at that at the bottom, and simply by looking you can deduce they are different with the top section being steeper than the bottom section. This is also true for the increasing trend up to pH 7.2. Although you would expect the gradients of corresponding points on the increasing trend of the graph to be the same as those on the decreasing trend, but if you look at the working on the graph you see clearly that they are not, as the tangent gradient values are not the same. This fact will be explained in the following section, the evaluation. However in general the trend shown supports my hypothesis and can also be explained scientifically.

Amylase is a globular protein and this mean it has a tertiary structure. This structure is formed after the polypeptides (the basic units/monomers) form chain, the primary structure then twist into alpha helices and beta-pleated sheets to form a secondary structure and then in amylase a combination of the two secondary structures forming a complex 3D shape which is the tertiary structure. Different types of bonds such as hydrogen bonds and disulphide bridges hold this structure together, but the bonds that are concerned with this investigation are ionic bonds. These bonds form between oppositely charged ends of the polypeptide chains. These ends are the amino group, which in an uncharged amino acid is an NH2 group and at the other end of the amino acid is a carboxyl group, which is a COOH group in the uncharged state. However in neutral conditions this carboxyl group will dissociate, as it is a weak acid, and this form is COO- ion and an H+ ion. This gives the amino acid a negative charge and also giving a negative charge at one end of the polypeptide chain. Similarly, in certain conditions even at pH 7 the amino group will accept a proton/ H+ to form a NH3+ group, which gives this amino acid a positive charge and so this end will attract the negative end and so form an ionic bond. These bonds define the tertiary structure of the enzyme and so define the shape of the active site that has to bond with the substrate. This is a specific shape and has to be to fit with a specific substrate and this is a key principle of enzymes. However if the pH of the solution the enzyme and substrate are in increase or decrease the following happens:

COOH COO- + H+ and NH2 NH3+

So in high pH conditions, i.e. with a low hydrogen ion concentration, the COOH becomes a COO- ion, which can form new bonds so altering the shape of the structure and also the NH3+ ion becomes uncharged so breaking the ionic bond and altering the structure and the reverse processes are true in low pH, i.e. high hydrogen ion concentration. So this shows how the varying pH affects the enzyme and so it follows that the more extreme the pH conditions the less the enzyme will function, as there will be more alterations to its shape, giving the downwards trends away from the optimum pH. Also, a further affect of increasing of decreasing pH is that if the substrate and enzyme become flooded then they may end up with the same charge and so repel each other and not forming the enzyme substrate complex and so not breaking down the substrate, giving a low concentration of maltose, so all these factors add up to show why the concentration of maltose decreases away from the optimum pH. The optimum pH is also around what was expected as the bacteria the amylase was engineered from was a bacillus which are found in soils and the air all of which are neural conditions so the optimum pH of 7.2 is what was expected. It is also possible to comment that the amylase is greatly affected by slight changes in pH as the curve is steeper at the smaller changes from the optimum pH and smoothes off as the effect is not much greater in extreme pHs as the enzyme is denatured simply more quickly, showing why the graph slopes off at the bottom of the bell shape.

Evaluation:

I will now look at the reliability of the results produced and whether or not they are reliable enough for me to fully support the comments and conclusions that I drew in the previous section, the analysis. Looking the correlation of the results it appears that there are no anomalies, this is as the error bars that were drawn on the graph all fall on the bell curve that best fits the results, this shows that the results for that general trend well. However I do realise that there is a degree of variability in the results as there are results that have small standard deviations from the mean, i.e. the results are all concentrated close to the mean values such as the result for pH 11 which has a standard deviation of around 0.2 which means the values are very close to the mean. Although there are also values such as that for pH 8 where the standard deviation is high as the mean is accurate to + or – 6.1, which shows that some of the results are quite variable from the mean value, but I would still say that as they fall on the best fit lien the method that was used to obtain the results was accurate to a large extent and so the conclusions can be seen to be reliable to a large extent.

The reason that some of the results did show such a large variability could be because although many precautions were made to reduce percentage error the method used still does involve many dilutions of solutions and because of the nature of the equipment used it is possible that the dilution required at they actually made in reality may not be the same. This is even if a very small amount of liquid was left at the end of the pipette or at the bottom of the test tube when pouring the solutions then there will be inaccuracies and it is almost impossible to get all of the liquid out. So to refine my method a burette could have been used to measure volumes as this piece of equipment allows for residual liquid in the nozzle, and so the volume required is more accurately obtained. Another refine to the method may be to measure the temperature of each of the starch/amylase solutions to make sure they are all at the same temperature, as although room temperature was assumed there may have slight fluctuations in the temperature at which they were reacting as heat may have been coming from other sources such as the Bunsen burner, and enzymes can be effected by very small changes. To make sure the temperature was not affecting the rate of reaction thermometers could be used to observe the temperatures and so improve reliability. A final problem with the method itself is the fact that in the main experiment the stock amylase used had been refrigerated and so the activity of the enzyme reduced so that it would last longer, however, it is possible that insufficient time was given to allow the enzyme to acclimatise to room temperature before using it, which may have caused slight inaccuracies.

A further problem although not strictly with the method was the fact that in the main investigation a transmission value was produced that was lower than any transmission value that had been produced in the calibration curve experiment, and so the calibration curve did not extend down to this point. This meant that the curve had to be extrapolated, which means it had to be extended with out using any further points just simply following the curve on. However, there is no concrete evidence that the curve will continue in this way as it is only an estimate and that is the problem with extrapolation and means the results gained from it may be inaccurate. To rectify this, higher known concentrations of maltose could be tested using the method outlined in the calibration curve section of my plan, and so produce lower percentage transmission values that could be added to the calibration values already obtained and then plotted on the calibration curve produced in my investigation and alter if needed the curve that has been estimated past the result that I had first obtained. Then if the curve has had to be altered then the conversion of the low result will have to be reassessed and this may affect the graph plotting concentration of maltose against pH but this would improve the accuracy of the conclusions drawn from it.

However, there is a more general problem with this investigation and that is that that the results rely on random collisions of the enzyme with the substrate and so because of this randomness even with three repeats variation was inevitable so simply doing more repeats of the same solution would not give a truer average. To get this you would have to carry out the experiment again and again, possibly using the refinements highlighted above, and although the equipment was there the time this would have taken made it impracticable. This point very clearly shows that one investigation cannot be used alone to prove a set of results and shows that the conclusions and results obtained should be dealt with tentatively.

To further my investigation, aside from repeating the same experiment to give more results that would serve to either back up the conclusions found ion this investigation or undermine them, would be to focus in on the optimum value for pH. It was found that pH 7.3 was the optimum temperature, but was only an approximate as the range of values is large. To more accurately find the optimum temperature pH solutions could be made up, such as 6.8, 7, 7.2, etc. The same method used in the main experiment with the changes suggested earlier in this section could be used and the results would produced the same sort of curve but would be much broader and would allow the optimum temperature to be found more accurately. From this smaller range of results you would also be able to deduce more effectively the effect that the change in pH has on genetically engineered amylase, as you will be able to see the effect of changing the optimum pH in small increments and use these results in conjunction with the results obtained in this investigation to see how sensitive amylase is to a change in pH from its optimum pH, i.e. whether small changes in pH do not have much effect shown by the graph further investigation having broad sides or whether small pH changes are significant shown by the graph having steep sides showing greater drop in activity, and with a more accurate optimum pH the conclusions are likely to be more reliable.