- Measure 25cm3 of wall paper paste solution into the 50cm3 beaker.
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Check that the water bath has reached 20°C. Then place the 50cm3 beaker of wall paper paste and the 25cm3 beaker of enzyme concentration into the water bath. Put thermometers into the each of the beakers and leave the solutions in the water bath until they reach 20°C.
- While leaving the solution to heat, fix the 25cm3 syringe barrel in a clamp which is attached to the retort stand, so that syringe nose is pointing down and is about 10cm above the bench.
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Place the other 50cm3 beaker under the syringe nose and in a water bath set at 20°C.
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Once heated to 20°C add the 1cm3 of 1% cellulase solution into the 50cm3 beaker of heated wall paper paste, mix with the glass rod for 30 seconds, time it on the stop watch.
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Then pour the solution into the syringe barrel, (if it does not pour use the glass rod to help transfer it) placing a finger over the end of the nose until the syringe is full. Release the finger when the stop watch reaches 1 minute and start timing. (Remember to take a 1 minute off the result at the end, as the first minute of the timing is not the time the solution has passed through the syringe. In this experiment the result recorded is the time taken for the solution to pass through the syringe barrel)
- Stop the stopwatch when all the solution has flowed through the syringe barrel and record the time taken for this to occur.
- Repeat step 3 – 10 two more time with the 1% concentration of enzyme.
- Then repeat step 3 – 10 with the other concentrations, so varying step 3 with different enzyme concentrations as shown on the table in the apparatus section. Repeat each concentration 3 times.
- So three readings of time will have been taken for this enzyme concentration and wall paste to pass through the syringe barrel. Then the average can be calculated, to give a more accurate result.
- As with all experiments a control will be needed to enable a base line result to be compared with the other result. This is the last one on the table, where the cellulase concentration is 0%.
- To plot the graph the rate needs to be calculated for each mean result this is done by doing 1/Time (1/T).
- To then draw the error bars the highest and lowest rate needs to be calculated for each enzyme concentration.
Key variables
The factors that affect enzyme activity are:
- Enzyme concentration
- Substrate concentration
- Temperature
- PH
All the factors above will need controlling other than enzyme concentration, which is the variable to be measured. The other factors need to be controlled to maintain a fair test and to get reliable results.
Temperature needs to be controlled because heat energy causes the enzyme and substrate molecules to increase random movement so collide more often. The more collisions the greater the chance that substrate will fit into the active site of an enzyme up to a certain temperature. So any variation in temperature will change the rate of reaction, so an increase in temperature will increase the rate of reaction while a decrease will have the opposite effect. In this investigation the temperature will be controlled by carrying out the experiment by using a water bath to maintain the enzyme solution at 20°C. 20°C was chosen as the temperature to maintain as fruit ripening which is cellulase main function would probably take place in the warm summer with a temperature range of 18 - 30°C.
Substrate concentration has to remain constant as it effects the rate of reaction. With a low substrate concentration all the active sites of the enzyme molecules many not be used. As the substrate concentration is increased more active sites come into use. If enzyme concentration is constant a point is reached where substrate concentration can not increase the rate of reaction because all the active sites are being used on the enzyme molecules. So in this investigation where the enzyme concentration will be varied it is important that the substrate concentration is not only constant but is high enough to always use all the active sites available at each different concentration of the enzyme. This is why a high volume of substrate will be used in relation to a low volume of enzyme. The concentration of the substrate will 2% and will be constant because the volumes of wall paper paste and water will be measured accurately.
pH is a measure of hydrogen ion concentration. The functioning of enzymes is partly the result of hydrogen bonding effecting the shape of the active site. Each enzyme has a optimum pH most working best in neutral (pH7) or slightly alkaline solution (one greater than pH7). Any change in pH can effectively denature the enzyme. This is due to the three dimensional shape of the enzyme held together by ionic and hydrogen bonds. The hydrogen ions in solution change the pH and will alter these bonds and therefore the functioning of the enzyme. So it is important that the investigation is carried at a constant pH. No specific pH tests will be carried out in this experiment but by keeping the other factors expect enzyme concentration constant, pH should also be constant.
Risk Assessment
Personal safety will be achieved by wearing protective clothes (lab coats) and glasses, to stop the enzyme, cellulase coming in contact with the clothes and eyes. Most Enzymes are not dangerous but it could be avoid having them in contact with the skin as they could cause allergic reactions. Also following safety techniques for handling chemicals e.g. enzymes such as washing hands after dealing with the enzymes, as they are water soluble and so are removed by water. When using the enzyme, ensure lid is replaced because if liquid enzyme dries up the dried enzyme could be inhaled and may provoke asthma or hayfever. Care should be taken when heating the water bath. It is recommended that the enzyme cellulase be stored at a temperature 25°C or below to maintain it declared activity for at least 3 months. Keeping it at lower temperatures (5-10°C) will increase the shelf life. Wallpaper paste contains fungicide so hands should be washed after handling it.
Pilot experiment
The pilot experiment that will be carried out will be determine whether the method that has be planned for works effectively.
Then to try to find the optimum or best substrate concentration that will give a viscosity that will flow through the syringe barrel within the time constraints (maximum 10 minutes for the substrate to flow through) and giving a recordable result. This will be tested by trying the two different concentrations 1% and 2% of the wall paper paste. With the two extremes of the enzyme concentrations of 0% and 1%. (this test will be carried at the planned temperature of 20°C).
Having decided on the optimum or best substrate concentration, the optimum temperature needs to be found for the solution of substrate and enzyme to flow through the syringe barrel again within the time constraints, but it does not want to be so fast that it can’t be recorded. To test the temperature as with testing the substrate concentration the two extremes of enzyme concentration will be used. The temperatures tested will be 20°C and 30°C which are the extremes of temperatures required for fruit ripening. I will also test at 60°C to see what happens at this temperature.
Modifications
Having carried out the pilot experiment, a few changes were made for the following reasons:
- 2% concentration substrate –
1% enzyme concentration – 3 minutes 41 second
0% enzyme concentration – left it for 30 minutes and did pass through the syringe barrel.
- 1% concentration of substrate –
1% enzyme concentration – 1 minute 20 seconds
0% enzyme concentration – left it for 30 minutes and did pass through the syringe barrel.
So having seen that the 0% enzyme did not pass through the syringe barrel at either of the substrate concentration. The substrate concentration that will be used in the experiment will remain the same as in the planned method 2%. However the enzyme concentration will be tested and if the solution has not passed through in 30 minutes the result infinity will be recorded. So unlike the other experiments being done one at a time, the 0% concentration will set up and left, this will be done three times.
Having found that the enzyme concentration of 0% does not pass through the syringe barrel in less than 30 minutes. To test the temperature the enzyme concentration used was 1%.
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20°C – 4 minutes 5 seconds
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30°C – 2 minutes 40 seconds
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60°C – 56 seconds
The temperature that was planned for this experiment to be carried out at was 20°C, however from carrying out the pilot experiment it would be better to use the temperature of 30°C. This is because the enzyme concentration being used is the highest out of the concentration, so all the other concentrations will pass through the syringe at a slower rate than this one. At 60°C the experiment occurred at too fast a time so the other concentration are also likely to occur quickly so the difference between the results would be hard to see. So this experiment will be carried out at what seems to be the optimum temperature 30°C.
Results
A table to show the time taken for wall paper paste to pass through the syringe barrel when mixed with the different concentrations of cellulase
The result infinity was recorded for any result that took longer than 30 minutes (1800 seconds) to pass through the syringe barrel.
A table to show the average time taken for wall paper paste to pass through the syringe barrel when mixed with the different concentrations of cellulase
Table to show the rate (1/T) using the average time taken for wall paper paste to pass through the syringe barrel when mixed with the different concentrations of cellulase
The rate answer has been rounded to 4 decimal places. This rate result is the figure that is plotted on the graph. The 0% concentration can not be plotted on the graph as the result has been recorded as infinity.
Table to show the highest and lowest rate (1/T) for wall paper paste to pass through the syringe barrel when mixed with the different concentrations of cellulase
To draw the error bars on the graph, the rate has to worked out for the highest and lowest valves of the time taken for the different concentrations of cellulose/wall paper paste to pass through the syringe barrel.
The highest and lowest valve rates have been rounded to 4 decimal places. The highest and lowest valves are plotted on the graph and a line is drawn between these valves and the error bars have been drawn for each concentration of cellulase.
Table to show the percentage difference in average rate taken for wall paper paste to pass through the syringe barrel when mixed with the different concentrations of cellulase
On the next page is the graph of the mean rate of each different concentration of cellulase and the error bars to show the accuracy of the results.
Analysis of Results
The results of this investigation confirm my general prediction that the rate of break down of cellulose to glucose, cellobiase and higher glucose polymers increases as the concentration of the enzyme cellulase also increases. This is because by increasing the enzyme concentration it increases the active sites available and so increases the rate of reaction by breaking down more substrate. The active site is a cavity with a precise shape in the enzyme, the substrate, is acted on by the enzyme to form the correct shape to fit into the active site. There are fewer active sites for hydrolysis to occur when the enzyme concentration is less than the number of substrate molecules.
From the table showing percentage change in rate it can be seen, that the rate increases most when the enzyme concentration is raised from 0.2% to 0.4% and is increased by 25%. However further increases in concentration of 0.2% up to 1%, results in a lower increase in rate, between 8.5% and 13% giving an average of 10%. These results show that the rate of breakdown of cellulose to sugar is greatest between 0.2 and 0.4% concentration of cellulase enzyme. The more specific prediction that as the concentration of cellulase doubles so the rate of break down of cellulose to sugar will double is not confirmed by this investigation. The rate of breakdown is not directionally proportional to the enzyme concentration as the rate does not double by doubling the enzyme concentration. This is shown by the graph as it is not a straight line.
The graph shows that the rate of breakdown of cellulose does not level off. This result is expected as a high concentration of substrate was used to ensure all the active sites available at each different concentration of the enzyme were used. In this investigation, as the substrate concentration in relation to enzyme concentration is high, time restraints and cost restricted the use of a wider range of enzyme concentrations for the 2% substrate concentration.
From the raw data collected it would appear that for each concentration of cellulase the three readings taken are within a small range and so there appears to be no anomalous results. The error bars have been plotted on the graph for each value. They show how accurate each result is for each enzyme concentration. So as enzyme concentration increases the accuracy decreases as the error bars are longer. The graph shape could be anywhere within the boundary lines of the error bar. As there is a wide range of possible graphs it is difficult to identify any anomalous results. The result at 1% concentration of cellulase is likely to be an anomalous result as it has the largest error bar range. However 0.4% concentration of cellulase could also be an anomalous result as it does not follow the general trend in graph shape.
The results of this experiment show that the rate of hydrolysis of cellulose to glucose was affected by the concentration of the enzyme cellulase. As the cellulase concentration increases the rate of hydrolysis of cellulose increases.
Evaluation
There were several limitations to the practical techniques used in this investigation. Firstly, due to cost constraints the range of enzyme concentration used was limited (0.2% -1% in interval increases of 0.2%). As only a small amount of enzyme is usually required to accelerate/ catalyse a reaction, it would increase the reliability of the experiment if smaller increases in concentration were used. By using smaller increases i.e. 0.1% instead 0.2% the greater range of enzyme concentration would have given a greater number of results to plot on the graph. This in turn would have given a more detailed graph pattern to interpret.
Secondly, time constraints on the investigation lead to limitations. At each enzyme concentration the experiment was repeated 3 times. However if this was increased to 5 times a more accurate average would be calculated and so reduce the margin of error in the results.
Another limitation of the practical technique was the difficulty in transferring 25cm3 of wall paper paste to the syringe barrel. As it was so viscous it stuck to the sides of the beaker so it was not always possible to transfer the total 25cm3 of wall paper paste to the syringe barrel each time. A modification would be to measure 25cm3 of wall paper paste directly into the syringe barrel and plug up the nozzle with blue tac then place in the water bath. This would ensure accurate measurement of substrate and reduce the heat loss that previously occurred when transferring. The enzyme would then be added to the substrate in the syringe barrel and mixed as before.
Finally, a constant temperature was difficult to maintain during the investigation, especially after the enzyme and substrate were mixed and placed in the barrel syringe. This was a major limitation as enzyme activity is very dependent on temperature, so a small variation could affect the results significantly. A modification that could be made would be to insulate the syringe barrel with a clear insulating material such as bubble wrap. This would reduce heat loss from the substrate/enzyme solution and so help to maintain a constant temperature.
There was variability in the results obtained for each concentration of enzyme. The lowest variation was at 0.2% concentration with a range of 16 seconds, while the highest range of 19 seconds was at 1% concentration. This range is confirmed by the error bars on the graph. From the pattern of the graph it is difficult to determine which results are anomalous. However the result at 0.4% concentration could be because it does not follow the pattern of the graph, as could the one at 1% concentration as it has the largest error bars.
When evaluating this investigation I do not think the results obtained were very reliable. From the results I can conclude that my general prediction that increasing enzyme concentration will result in an increase in the rate at which cellulose is broken down is correct. However the results do not confirm my specific prediction that doubling enzyme concentration leads to a doubling of the hydrolysis of cellulose. To be able to draw a firm conclusion regarding this part
of my prediction more practical evidence needs to be collected. This evidence could be obtained by using the modifications previously mentioned, namely:
- Make smaller increases in concentration of the enzyme (0.1% instead of 0.2%)
- Increase repeats of experiment at each enzyme concentration (5 instead of 3)
- Maintain a constant temperature (insulate syringe barrel to reduce heat loss)
- Ensure accurate measurement of substrate (measure substrate in syringe barrel)
This investigation could be extended to determine how the breakdown of cellulose is affected by the enzyme, cellulase, by changing the other variables that affect enzyme activity i.e. temperature, pH, and substrate concentration. So for instant temperature could be changed in this investigation instead of the enzyme concentration, which would need to be kept constant. The same experiment and method could be used with the substrate, cellulose (2% concentration, (wall paper paste)) and the enzyme, cellulase (1% concentration) being heated to a range of temperatures e.g. 20-70˚C at 10˚C intervals in thermostatically controlled water baths. At each temperature the rate at which the cellulose/cellulase solution passes through the syringe barrel would be recorded. As in this experiment the rate 1/Time would be calculated and plotted on a graph. By investigating this variable, temperature, a conclusion could be made regarding the optimum temperature of substrate and enzyme required for the hydrolysis of cellulose to glucose using the enzyme, cellulase.
Bibliography
To help me complete this individual investigation I used the following sources:
Books:
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Encyclopaedia Britannica (15th Edition) – Micropaedia Book 4 & Macropaedia book 14
- Letts Revision Guide for AS Biology by John Parker
- Understanding Biology for Advanced Level (Third Edition) by Glenn and Susan Toole
Websites:
Other Sources:
- Booklet on “Commerical Uses of Enzymes”
- Class Notes 2002/03
- Enzyme Information sheets on cellulase
