AS Biology Coursework

Enzymes and Temperature

Plan
Introduction
The purpose of this experiment is to study the effect of temperature on enzyme efficiency, measuring the amount of gas produced by a catalysed reaction at different temperatures. An enzyme is basically a biological catalyst, which is best described as a protein that assists in a biological reaction without being used up in the reaction itself. In this reaction, yeast is catalysing the break-down of hydrogen peroxide into water and oxygen:
2 H 2 O 2 2 H 2 OO2 formulaic representation

Yeast is a fungus, a member of the plant family, and originates on plants, in the air, in soil, and in and on humans and animals. It metabolises simple sugars, producing alcohol and carbon dioxide through the process of fermentation. The yeast contains an enzyme called catalyse, which is used by organisms (including humans) to catalyse the breakdown of the poisonous hydrogen peroxide (which is a by-product of respiration) into less harmful products: oxygen, water. In this reaction the independent variable is the temperature of the reaction. This is set by the participant and not a result of the experiment. The dependant variable is the amount of oxygen produced what we are actually measuring. Other variables may affect the result such as PH, enzyme concentration, and substrate concentration. The PH can be monitored using universal indicator.

Effects of extraneous variables on enzyme activity

Other variables may affect the results such as pH, enzyme and substrate concentration, and temperature.

Temperature
At lower temperatures, for instance between 5 and 10 degrees centigrade, increasing the temperature increases the rate of reaction. As the temperature is increased, the increased thermal energy results in increased kinetic energy the molecules start to move faster, and collide more often. Once the temperature reaches a certain level, individual to each enzyme: the enzyme denatures, the rate of reaction drops. In detail, the enzyme molecules lose their rigid, defined, shape and the active site is effected so the substrate molecule will no longer fit into it. If denatured, the enzyme will not automatically regain its shape.

pH level
pH measures the concentration of hydrogen ions in a solution. Since most enzymes function best over a narrow pH range, a major increase or decrease in pH level will affect the rate of reaction.

Enzyme and Substrate concentration

Equipment
To measure the amount of gas produced, I will be using a gas syringe. This is accurate and measures up to one millilitre resolution clearly. A measuring beaker is less ideal because of reduced accuracy, also a large rubber-bung would have to be found and attached to prevent the gas produced from escaping. To be exact I am going to be using a 20 cm syringe with a maximum capacity of 100ml. This range (100ml) is enough to produce a accurate graph from which a detailed conclusion can be drawn.
Rubber Tubing For the transfer of gases between beakers and measuring implements, I shall use standard rubber tubing, which is: resilient, strong, and secure. Unfortunately, this has to be forced onto specific points on the other apparatus, causing damage or weaknesses. For instance, the gas syringe may break as the rubber tubing is forced on the nib.

I shall use a measuring cylinder to create individual testtubes, each representing one of either the hydrogen peroxide or yeast solutions. To identify each test-tube, they will be labelled with the temperature, and test iteration. To heat the solutions before combining the yeast and peroxide on each iteration, I shall use a water-bath. This is because of the ability to easily control the temperature, which then remains constant. Also, the energy should randomly disperse throughout the water, preventing half of the solution from remaining relatively cold.

I will use a clamp to secure the gas syringe to prevent the force of gravity on the syringe handle modifying the result. It will be held in a horizontal fashion so that the syringe can be handled easily, although a slightly diagonal (upwards) skew could be useful in preventing the handle form falling out and possibly breaking. The solutions (enzyme, solute) concentrations will be kept constant by stirring.

Gas Syringe - 100ml range

Gas Syringe held horizontally by a Clamp

Method
Connecting equipment
The gas syringe must be attached the the rubber pipe attached to a bung which can be placed over the reaction test-tube solution. This will be produced by combining the yeast and hydrogen peroxide solutions.

Measuring liquid
Solutions must be poured slowly into measuring cylinders until the required volume is met. This is measured at the bottom of the Meniscus. To stop spillage, and increase accuracy, Pipettes can be used to transfer a small sample at a time.

Measuring from the Meniscus of the solution, using a Measuring Cylinder

Procedure for finding the volume of gas produced

For each test iteration a random or justifiable test value of yeast and hydrogen peroxide solution must be poured into a test-tube. This must be covered with a bung, preventing gas from escaping. As the reaction takes place, bubbles should be apparent, and the Gas Syringe value will start to rise. After one minute, the value on the syringe must be recorded. This is to be repeated until an ideal

solute concentration is reached.

Ideal solute concentration

To calculate the ideal solute concentration I will try concentrations at random until one produces the ideal output of gas. In this case, the ideal amount would be around 20ml so that when the temperature is increased, the amount produced will be unlikely to rise above 100ml (out of range). This is timed to prevent discrepancies caused by extra reaction time. The amount of yeast solution should be less than the amount of hydrogen peroxide, since the yeast solution cannot catalyse more molecules than it holds all the active sites are taken up. Hydrogen Peroxide /ml 10 Yeast Solution /ml 5 Oxygen produced /ml 7 Commentary This combination produced far too little oxygen, I should increase the yeast solution. Far too high production, could break syringe if heated (increased kinetic energy: more reactions). Too low. Attempt less Hydrogen Peroxide and more Yeast. Fine for the experiment.

10

10

60

10

10

20

In conclusion, I will be using 5ml of hydrogen peroxide and 4ml of yeast throughout the experiment.

The experiment
The time will be kept constant for each reaction. One minute of reaction is long enough for a valuable result, given that this is the same amount of time used to generate the optimum hydrogen and yeast solution amount. I have chosen to test temperatures from 10..60 in 10 degree increments. This will produce six independent variables to test with: 10, 20, 30, 40, 50, 60. Each temperature will be tested five times, with the average becoming the value used to plot graphs and make conclusions. The average reduces the effect of extraneous results, which may skew them unexpectedly. The water bath will be pre-heated to each temperature and the constant amount of solutions heated. The solution will be combined and capped with a rubber bung. The result, read off the gas syringe will then be measured and recorded, identified by its appropriate iteration and temperature.

Procedure/Instructions
1. The pH level of both hydrogen peroxide and yeast must be measured using universal indicator paper 2. Using a measuring cylinder, pour 5ml of Hydrogen Peroxide, and 4ml of Yeast Solution into separate Test-Tubes 3. These must be submerged into a water bath at one of the temperatures specified, and left for approximately two minutes so that the temperature inside and outside the test-tubes reach an equilibrium 4. The tubes should then be removed, and the yeast solution poured into the hydrogen peroxide, catalysing the reaction 5. Immediately after combining the solutions, fix the rubber bung to the test tube so that the test-tube contents are linked directly to the gas syringe without being exposed 6. After exactly one minute, the millilitre measurement on the side of the gas syringe must be recorded 7. The pH level of the result should be measured, after removing the rubber bung

Results
Computerised version of results table
Temperature /C 10 20 30 40 50 60 PH before Volume of Gas Produced /ml 19 39 48 41 44 35 Yeast 6 6 6 6 6 6 Hydrogen PH after Peroxide 8 8 8 8 8 8 7 7 7 7 7 7

Analysis
An enzyme is a biological catalyst. The effect of an enzyme is affected directly by four factors: PH level, temperature, enzyme concentration, substrate concentration. These are explained earlier in the planning section. My graph seems to increase steeply from 10, 20, to 30C. The graph reduces less steeply then the earlier increase between 40, 50 and 60C.

To work out the average optimum temperature, the highest point must be calculated using the graph. It is apparent that the largest volume occurred at 30C: 48ml of gas was produced. The graph appeared similar to the graph of a negative square, reaching a peak then somewhat dropping off. The graph shows that after 30C, the enzyme starts to lose efficiency. These results prove the theory connecting temperature with enzyme efficiency: the amount of gas produced increases gradually until the optimum temperature, where it suddenly trails off. Between 40 and 50C, the amount of gas produced increases. This is in contrast to the theory, which shows a gradual incline to the optimum temperature, then a steep drop in production when the enzyme becomes denatured. The actual optimum temperature may have been anywhere between 20 to 40C because my results only represent increments of 10C. For instance, maximum efficiency may have occurred at 25C then dropped to 30C, then 40C. Using the method described, it is not possible to speculate this. The more temperatures tested must give a better idea of the optimum temperature.

Evaluation
The method of measuring the solutions and gas is accurate enough for this experiment considering the scale has a smaller-than millilitre resolution. I measured this reasonable accurately, although the different solutions would have differed slightly. Using automated machines to distribute the solutions into testtubes would have been more accurate. The odd result between 40 and 50C may have been caused by human error. For instance, I think I may have left the 40C test-tube reacting for over a minute. Also, there may have been two optimum temperatures, I cannot tell using the proscribed method. This would disagree with the enzyme/temperature theory. Also, I may have placed the bung into the test-tube too slowly or some such these types of factors were not controlled. I did not make mistakes copying the temperature from the water bath's LCD (liquid crystal display) screen, however I may have read the thermometer incorrectly for the 10 and 20 set of results. This could have been a cause of error. The temperature may have reduced after being removed from the waterbath. For instance, the 60C test-tube would have cooled in the far lower room temperature. Therefore, the 60C result may have been for 50C or another value instead. To counteract this problem, the temperature could be maintained in a water bath while the reaction takes place, instead of removing the solutions. Repeats were impossible because the exact temperature could not be reproduced. However, more that one test-tube could have been used at once using samples from the solutions in the water-bath. The one minute would not have been constant for all of the temperatures, considering time waits for no-one, there will be fractional differences.