The rate decreases above the optimum temperature as the enzyme become denatured. The thermal energy causes the hydrogen bonds that are holding the secondary and tertiary structure of the enzyme together to break. Therefore the active site loses its shape and the hence the substrate can not bind so the reaction is left un-catalysed.
pH level
Similar to temperature there is also an optimum pH level for enzymes where they work best in. For most enzymes the optimum pH is around pH 6-8. However some enzymes work best at extreme pH levels. For example the protease enzyme in the human stomach works best at pH 2. The pH level affects the charge of the amino acids at the active site, thereby changing the chemical nature of the amino acid which results in changes in properties of the active site. This means the substrate can no longer bind.
Enzyme concentration
As the enzyme concentration increases the rate of reaction increases proportionally as there are more active sites available for the substrates meaning less competition to find an active site. At a very high enzyme concentration the substrate concentration could become the limiting rate factor thereby preventing the rate from increasing.
Substrate concentration
The graph is this time a curve dependent upon the substrate concentration. At a low substrate concentration there are a lot of unoccupied active sites causing the rate of reaction to be low. At a higher concentration of substrate enzyme-substrate complexes allowing more collisions to take place. The increase in concentration also means that the enzyme molecules become saturated with substrate.
Pectin:
Is a group of polysaccharides occurring in the cell walls and intercellular layers of all land plants. They are extractable with hot water, dilute acid, or ammonium oxalate solutions. Pectin’s are precipitated from aqueous solution by alcohol and are commercially used for their excellent gel-forming ability.
Commercially, the primary source of pectin is the peel of citrus fruits such as lemon and lime, although orange and grapefruit may be used. A secondary source is apple pomace and sunflower heads.
Pectin is widely used in the food industry, principally in the preparation of gels. It is used as a base for jelly and as a stabilizer in some dairy products and frozen desserts, such as sherbet, and also as edible protective coatings for sausages, almonds, candied dried fruit, and soft dates.
They are synthesised in the plant's Golgi apparatus and form a matrix in which the hemicellulose polysaccharides of the plant cell are embedded. An important part of fruit walls, pectin is broken down by the enzyme pectinase to pectinic acid and finally pectic acid. During this chemical breakdown process, the fruit gets softer as the cell walls degenerate.
Structure of Pectin:
Pectinase:
Pectinase is a general term for enzymes that break down pectin, a polysaccharide substrate that is found in the cell walls of plants. One of the most studied and widely used commercial pectinases is polygalacturonase. It is useful because pectin is the jelly-like matrix which helps cement plant cells together and in which other cell wall components, such as cellulose fibrils, are embedded. Therefore pectinase enzymes are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juice from fruit, including apples. Pectinases have also been used in wine production since the 1960s.it joins together with the substrate at the active site
Like all enzymes, pectinases have an optimum temperature and pH at which they are most active. For example, a commercial pectinase might typically be activated at 45 to 55 °C and work well at a pH of 4.8 to 5. If it is hotter it will make the reaction go more quickly. However, the higher temperature will eventually denature the enzyme, ultimately destroying the enzymatic action.
Pectinase also breaks down pectin best at a pH of 4.8 to 5; too high or too low a PH would also result in the enzyme denaturing. The logical explanation of why the enzyme denatures would be that if the pH is decreased too much, there would be too many H+ ions around the protein and thus the H+ ions would be attracted to the places in the enzyme which were more negative than the enzyme, thus forming a hydrogen bond there. Also, if the pH is increased, there would be too many OH- ions and they would interact with the positive regions in the protein.
This negative or positive region could possibly be in the active site, and even if it is not it does end up disfiguring the enzyme. Due to the fact that the enzyme-substrate interaction is so specific, even the slightest deformity of the active site directly or indirectly will result in the enzyme involved not working properly. This change, if permanent, will render the enzyme useless.
After considering the previous information and from my own knowledge I have been able to formulate a hypothesis.
Hypothesis
- As the temperature gradually increases the rate of reaction will also increase with it. Therefore more juice will be produced as the temperature is being increased. This is a result of a higher number of collisions per second and a lower activation energy.
- If the temperature is increased too much the rate of reaction will come to a complete stop or it will hugely decrease. This is because the enzyme will denature as a result of the high thermal energy deforming the active site whilst breaking the weak hydrogen bonds holding the structure together.
- In accordance with my research I predict the optimum temperature for the enzymes to work would be between 45ºC – 55ºC
Pilot Experiment
The objective of the pilot experiment is vital to prevent any unexpected problems arising in our actual experiment. It is a practice to see if any improvement is needed in the method to help you to obtain more reliable and accurate results in your main experiment.
The pilot experiment can be used to calculate the following:
- The amount of fruit tissue to be used as insufficient amounts could make it difficult to measure the volume of juice thereby giving inaccurate results
- The volume of pectinase needed, as if too small an amount is used then reaction time will be very slow and vice versa if too much is used
- The range of temperatures to use, as if the temperature is too high the enzyme may denature which is a waste of the resources whilst giving inaccurate results
- The time to equilibrate at each temperature before combining the enzyme and the substrate
- The time to leave the reactants in the water bath giving enough time for the substrate molecules to turn into product
- The type of fruit to use as some may not contain enough pectin therefore producing less juice
Apparatus:
-3 x 5cm³ enzyme
-3 x 5g fruit tissue
-100 cm³ measuring cylinder
- Peeler to peel cellulose layer off the fruit
-3 test tubes to hold the enzyme and fruit together
-Syringe to obtain 10cm³ of the Pectinase
-Bunsen Burner, tripod, gauze, heat proof mat and large (250cm³) beaker to create the water bath to equilibrate the fruit and enzyme
-Cling film to prevent oxidation of fruit and evaporation of enzyme
-Filter paper and funnel to filter the juice obtained
-100g weight to put on top of the filter paper whilst juice is being extracted
-Stop clock to measure the time the mixture has been left
-Grater to obtain large surface area of fruit tissue
-Glass rod to stir the reactants together
-Electronic weighing balance to weigh fruit accurately
-Thermometer to ensure temperature is remaining correct
-Distilled water to dilute enzyme to a 1:1 concentration
-White tile
-Safety goggles to protect the eyes
-Marker pens to label the test tubes for them to be identified
Pilot Experiment Method
- Peel fruit and grate the flesh to dispose of the different tissues and obtain a large surface area.
- Measure 5g of fruit using an electronic weighing scale
- Place the fruit into a 250cm³ beaker.
- Measure 10cm³ of Pectinase enzyme using a syringe/ pipette and insert into a test tube.
- Allow these to equilibrate to the required temperature by placing the beaker containing the grated fruit and the test tube containing the 5cm³ of Pectinase enzyme into a water bath whilst starting the stop clock and time for 5 minutes.
- At the end of the 5 minutes, mix the two contents into the beaker and stir.
- Leave reactants together at the same temperature for about 5 minutes; use a thermometer to ensure the correct temperature. Label the beaker to keep it distinguishable from others.
- Keep container covered with cling film to prevent oxidation of fruit and evaporation of the enzyme.
- Whilst waiting, prepare the filter paper and funnel onto a 100cm³ measuring cylinder.
- At the end of the 5 minutes, remove the cling film.
- Filter the juice produced, into the measuring cylinder through the filter funnel and paper and put a 100g weight on top of the product.
- Leave to filter for 5 minutes and record the volume of the juice obtained into a suitable table.
- Repeat the above steps again for each temperature chosen.
- Repeat all the trials at least once as there may be mistakes in some of the results the average will ensure we get better more reliable results.
Repeat the trials at least once to ensure more accurate results. This will give us an average giving us better more reliable results
Safety
During the course of the experiment there are various hazards that must be considered and precautions must be taken:
Wearing Safety Goggles – to stop anything harmful entering the eye e.g. pectinase.
Wash apparatus with water after each use – Stops cross contamination and prevents chemicals mixing with the enzyme keeping the results most accurate.
Avoid contact with skin- if skin is in contact with the enzyme wash with plentiful water.
Avoid Spills – If the enzyme or water spills notify immediately stopping injuries from taking place
Ethical Implementation -
The removal of both the organic and chemical waste was handled in a proper manner to prevent any affect any organisms living within them e.g. the diluted enzyme was drained down a laboratory sink. Only the amount of tissue needed for the experiment was obtained avoiding any unnecessary damage to the live tissue.
Diagram:
Variables (to be kept constant)
PH level
As fore mentioned all enzyme molecules have an optimum pH at which they work best at. If pH is higher or lower than this it may lead to the denaturing of the enzyme which will mean that no results will be attained or if any are they could be misleading as the bonds holding the active site will be broken as the atoms will have a lot of energy causing them to vibrate more
Enzyme Concentration
It is important this variable remains constant because if this is increased there will be a faster reaction. This is because there will be more enzyme molecules to react with the substrate molecules. Meaning the test could be misleading as it would not show how temperature affects the rate of pectinase upon pectin. If the enzyme concentration is decreased it will slow down the reaction also making it misleading and make it difficult to conclude the affect of temperature on pectinase.
Substrate Concentration
Increasing this would also lead to a faster reaction and lowering the concentration would slow the reaction. Therefore we must keep this variable constant or we will be altering 2 variables at the same time (temperature and concentration). If it is not kept constant the results obtained would be inaccurate.
Pilot Experiment Results
From this preliminary graph we can see that the experiment was similar to what the hypothesis suggests it would be like. As the temperature increased up to the optimum the volume of juice also increased however after about 50ºC the volume of juice dropped immensely as the enzyme started denaturing.
Alterations in method:
I will now use 10g of apple and use 10 cm³ of pectinase to obtain more reliable results
I will also use a range of different temperatures to obtain the optimum temperature
I will also allow more time for filtering (10 minutes) and give 10 minutes in water bath
Ammended Method
- Peel fruit and grate the flesh to dispose of the different tissues and obtain a large surface area.
- Measure 10g of fruit using an electronic weighing scale
- Place the fruit into a 250cm³ beaker.
- Measure 10cm³ of Pectinase enzyme using a syringe/ pipette and insert into a test tube.
- Allow these to equilibrate to the required temperature by placing the beaker containing the grated fruit and the test tube containing the 10cm³ of Pectinase enzyme into a water bath whilst starting the stop clock and time for 5 minutes.
- At the end of the 5 minutes, mix the two contents into the beaker and stir.
- Leave reactants together at the same temperature for about 10 minutes; use a thermometer to ensure the correct temperature. Label the beaker to keep it distinguishable from others.
- Keep container covered with cling film to prevent oxidation of fruit and evaporation of the enzyme.
- Whilst waiting, prepare the filter paper and funnel onto a 100cm³ measuring cylinder.
- At the end of the 10 minutes, remove the cling film.
- Filter the juice produced, into the measuring cylinder through the filter funnel and paper and put a 100g weight on top of the product.
- Leave to filter for 10 minutes and record the volume of the juice obtained into a suitable table.
- Repeat the above steps again for each temperature chosen.
- Repeat all the trials at least twice as there may be mistakes in some of the results the average will ensure we get better more reliable results.
Analysis
On the next page the original results table has been enclosed
Results Table
* = recurring
Conclusion
From my graph and results I can firstly conclude that the maximum amount of juice produced was 18 cm3. This was at the temperature of 45ºC which we can with a sufficient amount of certainty say that this is the optimum temperature.
In agreement with my hypothesis the temperature up until the optimum increased with the average volume of juice. Also in accordance with my hypothesis increasing the temperature after the optimum resulted in denaturing of the enzyme meaning a decrease in the volume of juice produced.
The average increase in the amount of juice being produced between temperatures 30ºC - 45ºC increased from 13.86cm3 – 16.6cm3. Where as the average decrease in the amount of juice between temperatures 45ºC – 70ºC decreased from 16.6 cm3 – 12.3 cm3
All of the above results support my hypothesis which states “As the temperature gradually increases the rate of reaction will also increase with it. If the temperature is increased too much the rate of reaction will come to a complete stop or it will hugely decrease.” This is what the results obtained show because the molecules attained more kinetic energy making it easier for them to overcome the low activation energy. The probability of the enzyme and substrate colliding successfully heighten as the molecules move around faster.
Nevertheless after the optimum temperature had been reached the volume of juice produced decreased as the enzyme denatured. The rise in temperature increases the kinetic energy of the atoms causing them to vibrate vigorously thereby breaking the bonds which hold the structure in shape. Progressively the shape of the active site is distorted which causes a decline in the number of successful collisions.
In comparison with my pilot experiment the results were quite similar signifying the fact that the results obtained were quite accurate and reliable and they proved my hypothesis correct that the average volume of juice produced would rise as the temperature increased, until after the optimum temperature where denaturing takes place where the rate of reaction decreases again.
Evaluation
The pilot and main experiment results signify the same effects of pectinase upon pectin. In the preliminary there was insufficient mass of tissue and amount of enzyme. I increased both of these in the main experiment to obtain more accurate and reliable results. In the main experiment I used a range of different temperatures to achieve a smoother and accurate curve on my graph as there were not vast gaps between results allowing us a more calculated prediction of the results in between. These results are more reliable also because I repeated the results for each temperature 3 times. I also increased the time left to filter and the time left in the water bath from 5 minutes to 10 minutes, allowing the maximum volume of juice to be obtained in the revised experiment.
I didn’t achieve many anomalies however there were some results which were not on the path of the curve and we see an abnormal bend. There could be several reasons for this: Firstly the mass of apple may not be measured correctly. Secondly I maybe did not mange the time efficiently on the stop clocks. Thirdly it could have been the fact that the pectinase solution was not diluted correctly causing a change in concentrations at different temperatures. Perhaps the water bath temperature was not correct leading to slight errors such as the above
If I were to repeat the experiment I would use more accurate and stable water baths and accurately measure the concentration of the enzyme. On the whole I was fairly happy with my results and the general curve of the graph is similar to the prediction in the hypothesis.
