The black dashed line divides the graph into its
simplest definable parts: The first half shows the rate of
reaction against temperature. The enzyme activity is increasing.
The other half shows the enzyme denaturing, yet enzyme
activity is occurring, but at a decreasing rate.
Apparatus
100g Chopped Apple (Granny Smith)
2 Beakers
10cm3 Of Pectinex
10cm3 Distilled Water - To dilute pectinase
Water Bath – To heat substances to desired temperatures
2 Measuring Cylinders
Filter Paper – To filter apple juice
Balance – To accurately measure apples
Thermometer – To measure temperature
Knife – To chop apples.
Tile – To cut apples on
Sealable container – To hold pectinase solution
Method
-
First measure 1cm3 of enzyme substance (Pectinex) in a measuring cylinder. Mix this with 1cm3 of water, also measured in a measuring cylinder, making a total of 2cm3 of 0.5mol enzyme solution.
-
Put this 2cm3 of enzyme solution into a beaker and 2cm3 of water into another beaker (this will be used as a control).
- Chop the apple.
- Take the apple and tile to the balance and put the tile down first press the ‘tare’ button on the balance and make sure it is displaying 0.00g. Then proceed to weigh 2 batches of apple weighing ten grams each.
- Set the water bath to 30 degrees Celsius.
- Place 10g apple in a beaker with the pectinase solution and 10g of apple in a beaker with the water, stir thoroughly and leave to incubate for 45 minutes in the water bath.
- Put some filter paper in a funnel and then put the funnel on a measuring cylinder. Pour all the contents on the small beaker into the funnel and leave until all the juice has gone through the paper and into the measuring cylinder.
- Record your results in a table.
- Repeat the experiment at 20, 40, 50, 60, 70 and 75 degrees Celsius, making sure controls are carried out at each temperature.
Diagram
Controlled Variables
- Use the same mass of apple.
- Same volume of pectinase
- Same concentration of pectinase
- Same amount of incubation time.
The three key variables (described above) must be controlled throughout the experiment because as they have a great effect on the efficiency of enzymes and if slightly altered could effect the pectinase. The pH and concentration of everything were kept constant throughout the experiment.
Risk Assessment
All enzymes are allergens and must be handled with care.
Pilot Experiments
Pilot tests are carried out to determine which concentration of pectinase and what mass of apple will be most suitable.
- mol solution of pectinase
0.5 mol solution of pectinase
These pilot results suggest it will be most effective to use a 0.5mol solution of pectinase with 10g of apple.
Results
Table Showing The Volume Of Juice Produced.
Table Showing Percentage Increase
Analysis
Although care was taken to obtain the greatest possible accuracy the results are not accurate enough to fully up hold the hypothesis. Between 20oC and 30oC my hypothesis is accurate as the average volume of juice produced goes up from 2.00cm3 to 3.00cm3 with the 10oC increase in temperature. However the average volume then decreases to 1.57cm3 at 40oC and then increase again to 2.37cm3 at 50oC. From this point the results seem to follow the hypothesis as the average yield of juice decreases as more of the enzyme is denatured at the higher temperatures. Overall it does show the higher the temperature the greater the volume of juice produced to a certain temperature.
For a non-enzymic chemical reaction, the general rule is: the higher the temperature, the faster the reaction: this same rule holds true for a reaction catalysed by an enzyme, but only up to a certain temperature, in my case (ignoring the anomalous 40oC result) from 50oC. Above this temperature, enzyme molecules begin to vibrate so violently that the delicate bonds that maintain tertiary and quaternary structure are broken, irreversibly changing the shape of the molecule. This has a lot in common with the collision theory too as in the gain of energy by the enzyme leads to it vibrating or gaining Kinetic Energy. This can also apply to the denaturing of the enzyme (when the enzyme gains too much kinetic energy it begins to vibrate violently and eventually when the hydrogen bonds and disulphide bridges break the enzyme becomes denatured). The enzyme can no longer function once this happens.
The optimum temperature for the effectiveness of pectinase must be between 30oC and 50oC as the most juice was produced around these temperatures although more experiments would have to be carried out around these temperatures to find the exact temperature and because the results for 40oC seem to be unreliable. All reactions that take place in the body proceed because the products have less energy than the substrates. However, most substrates require an input of energy to get the reaction going, the reaction is not spontaneous. The energy required to start the reaction is called the “activation energy”. When the substrates react, they need to form a complex called the transition state before the reaction actually occurs. This transition state has a higher energy level than either the substrate or the products. Outside the body, high temperatures often supply the energy required for a reaction. Enzymes provide an alternative way with a different transition state and lower activation energy. The rate of the reaction without any external means of providing the activation energy continues at a much faster rate with an appropriate enzyme than without it. The maximum rate that any reaction can proceed at will depend, among other things, upon the number of enzyme molecules and therefore the number of active sites available and at the optimum temperature more collision will be taking place meaning more substrate will be binding with more active sites.
The obvious anomalous result is the one for 40oC as I would have expected it to be much closer to and perhaps higher than the result for 30oC and for the optimum temperature to be near this. However more experiments will be needed to prove this as my results do not show it.
Evaluation
The error bars on the graph are a good indicator to whether the results are reliable. The error bars for 20oC, 30oC and 40oC are large and indicate a large variance in the results. The relatively small error bars from 50oC onwards show that although the results may not be precise they are consistent which would mean that they are more reliable. This means I can be fairly confident that at certain temperatures the enzyme does denature and become ineffective is valid but it is more difficult to see an optimum temperature as predicted in the hypothesis as the results for 40oC were anomalous. However it is difficult to draw conclusions from single investigations as there are always factors which can go wrong no matter how much care is taken, which can lead to misleading results and many more repeats are needed to clear all issues of validity and reliability.
The method used may have produced limitations into the usefulness of the data. Obvious changes like using more accurate equipment (balance, thermometers etc) would lead to more accurate results. Also if more time was available more experiments could be carried out especially at temperatures where anomalous results were obtained and the more results obtained the more reliable the data will tend to be. Also more time could have been allocated to incubating the enzyme at the relative temperatures allowing more time for the pectinase to break down the pectin in the cell walls. Using larger volumes would make the readings easier to read and any differences between temperatures more significant allowing more confident conclusions.
One of the largest factors in the variance of the results has to be the ripeness of the fruit. Invariably different apples will have different volumes of juice. The method tried to combat this by using the same type and mass of apple but it would be very difficult to obtain the same ripeness in all the apples used. One improvement that could be made is to make sure that the apple is only chopped right before the experiment is carried out as for some of the experiments a apple that had been cut in half the day before was used the following day, which no doubt contributed to the anomalous results.
To further extend research into the effects of temperature on pectinase there are many possible experiments.
The effect of temperature on the rate of enzyme activity could be useful to draw further conclusions on how pectinase activity is affected by temperature. Research into the optimum ph for an enzyme to operate as well as the temperature, would give those in the biotechnologies industry the ideal information for the use of enzymes in for example washing up liquid. It would be very simple to modify this experiment that looked at the effects of temperature, to look at the effects of pH. If a chosen temperature (around 40oC) was used and substrate concentration and enzyme concentration were controlled properly then the experiment could be modified to look at the optimum operating temperature for pectinase. If the optimum temperature and pH for every enzyme in living organisms then it would give a great insight into reactions within the environment around us.