It has, however, been discovered that competitors for an active site (similar in shape to the substrate) could fit even though they are larger than the substrate. This means that the substrate and active site are a little flexible. It is believed that when the enzyme and substrate form a complex, structural changes occur so that the active site moulds around the substrate (the substrate induces the active site to change shape). The reaction will take place and the product, being a different shape to the substrate, moves away from the active site. The active site then returns to its original shape. This theory is known as the induced fit theory and is illustrated in the diagram below:
Reactions take place because the products have less energy than the substrates. However, most substrates require an input of energy to get the reaction going and this is called the activation energy. Enzymes work by lowering the activation energy required to advance a reaction and therefore help increase the rate of reaction. There are, however, many factors which affect the efficiency of enzymes and therefore the rate of reaction. The key factors are temperature, pH and concentration.
Temperature has an immense affect of the efficiency of enzymes. Enzymes have a unique optimum temperature (around 40oC) which they work best in. Below the optimum temperature, the molecules involved in the reaction will be provided with less kinetic energy meaning that the numbers of collisions between enzyme and substrate will decrease, as the speed of molecular movement is decreased. Any temperatures above the optimum will cause the enzymes to be denatured - bonds holding the structure together will be broken and the active site loses its shape and will no longer work. Therefore during reactions involving enzymes, the temperature has to be greatly considered.
The pH condition in which an enzyme is made to work also has a great effect on the efficiency of the enzyme. As with temperature, enzymes have an optimum pH (usually a small range). If the pH changes much from the optimum range, the chemical nature of the amino acids can change. This may result in a change in the bonds and so the tertiary structure may break down, causing the active site to be disrupted and the enzyme to be denatured.
Concentration is another important factor affecting the efficiency of enzymes. Firstly the enzyme concentration has to be considered. At low enzyme concentration there is great competition for the active sites and therefore the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the rate of reaction will increase. However, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor. The substrate concentration has also got to be considered. At a low substrate concentration there are many active sites that are not occupied, therefore the rate of reaction is low. When more substrate molecules are added, more enzyme-substrate complexes can be formed and the rate of reaction increases. Eventually, there will be a point when increasing the substrate concentration further will have no effect as the active sites will be saturated so no more enzyme-substrate complexes can be formed.
(reference: Nelson thornes)
As proven in my preliminary experiment, the enzyme pectinase significantly increases the amount of fruit juice produced from apple pieces. This, as explained above, is due to the fact that pectinase degrades the pectin, making the cell wall of the plant softer and therefore allowing the fruit juice to flow more easily. If I did an experiment investigating the effect of pectinase on different sized apple pieces (of the same shape), I predict that the smaller the apple pieces, the greater amount of apple juice produced. This is because the smaller the apple piece, the larger the surface area which would mean more binding sites of the substrate exposed to the enzymes. If more binding site are exposed to the enzymes, there is more chance of enzyme-substrate complexes being formed (i.e. between the pectinase and pectin) and therefore the rate of reaction will increase which can be displayed by the fact that more fruit juice will be produced.
Main Experiment
The main experiment will use the combination of the preliminary experiment and the research to provide the technique and knowledge to carry out a full more thorough investigation to achieve the aim of the project.
Apparatus list:
- Whole apples (x7 of different makes)
- Knife – for cutting apples
- Pectinase enzyme
- Distilled water
- Glass stirring rods (x7) – to stir apples when in bath
- Syringes – to extract enzyme and water
- Coffee filter papers (x7) – to filter pulp for juice
- Filter funnels (x7) – to place filter paper and pulp in
-
100cm3 measuring cylinders (x7) – next size up from 10cm3
-
100cm3 beakers (x7) – for measuring weight of apples in
-
Water bath set at 40oC – to heat apples
- Stop clock (x7) – to keep accurate timing of stages
- Blender – to create apple pulp for bigger surface area
- Passim and Mortar – to crush apple before put into blender so blender can cut it better
Method:
The method below is applied to each apple but I have written it as if I’m applying it to one apple, the same method applies to all 7 apples. The medium-sized apple will be chopped into small pieces and placed in a blender to create a pulp. Half will be placed in beaker A, and the other half will be placed in beaker B (to ensure there were equal amounts in both beakers, each beaker will be weighed empty and then re-weighed with the apple pieces). The beakers will then be covered and left to stand for 30 minutes 2cm3 of diluted pectinase enzyme will be added to beaker A and 2cm3 of distilled water will be added to beaker B using syringe. The two beakers will then be incubated in a water bath set at 40oC for 20 minutes. The juice from the apple pieces in each beaker is to be filtered using coffee filter paper in funnels into separate measuring cylinders. The volume of apple juice obtained from the apple pieces in each beaker will recorded at 5 minute intervals for 40 minutes and recorded in a table. Other form of expression of data will be used.
Variables:
There a 3 key variable to this experiment which are explained in detail in the research –temperature, pH and concentration. To make sure that the experiment is fair and reliable, these key variables must be controlled throughout the experiment. Apart from the three key variables above, there are also a few more things which could be changed in the experiment that would affect my results:
- The amount of pectinase enzyme used. More enzyme would mean more active sites for the substrate to form enzyme-substrate complexes in and therefore a greater rate of reaction.
-
The time the beakers containing the apple pieces and pectinase were left in the water bath. This would have affected the experiment because a longer time in the water bath would mean more kinetic energy provided to the molecules involved in the reaction and therefore the number of collisions between enzyme and substrate will increase.
- The apparatus
- The plan method – this has been amended in comparison to the preliminary to produce more accurate results. This time I shale use filter funnels to filter the juice out with the filter paper placed inside with a cone shape, this allows the juice to flow much easier into the pinnacle point of the cone into the funnel into a measuring cylinder.
Things to note which are not in my control but will affect the results of the experiment
- Different densities of tissue (cell volumes and intercellular air-spaces)
- Different percentage composition of cell wall components (with different susceptibilities to whatever maceration process used)
- Different concentrations of sugars and acids in juice
- Storage previous to experiment and sources of where they were grown
Fair Test
The three key variables (described above) must be controlled throughout the experiment because as explained in the research, they have a great effect on the efficiency of enzymes and if slightly altered, could therefore affect the pectinase. The temperature will be controlled by putting each beaker in a water bath set at 40oC for 20 minutes before actually filtering the apple juice. The pH and concentration of everything will be kept constant throughout the experiment.
- The same amount of apple pulp will be put in each beaker and this will be controlled by weighing exact amounts of apple pieces.
- Distilled water will be used instead of tap water to ensure that no unwanted substances will interfere with the control experiment i.e. distilled water is purer.
- The volume of the diluted pectinase enzyme/distilled water used will be the same for each experiment.
- The diluted pectinase enzyme used will be the same concentration for all experiments.
- Each stop clock will be started as soon as all the apple pieces are in the filter funnels; therefore each experiment gets the same time to produce the apple juice– 40 minutes.
- The readings on the measuring cylinder will be noted down very carefully because it is quite easy to make a mistake when reading off such small scales.
- A measuring cylinder with a large scale will be used to measure out the amount of apple juice produced because it is much more accurate.
Risk assessment:
In this investigation, there are many areas where care must be taken. You have to be extremely careful when cutting the apple pieces into precise lengths with the knife as it is very sharp and could cause a serious cut. The blender should be handled very carefully, when cleaning slowly wipe blades to prevent the risk of cutting yourself, make sure it isn’t connected to the plug whilst doing this. Any activity when removing the lid should require safety goggles. Also before switching it on confirm lid is firmly on and cannot fall off. Do not wave the knife around because it could cut someone else or it could poke someone in the eye. Take care when handling the glass equipment (i.e. the beakers, measuring cylinders, funnels and glass rods) because if dropped, they will smash and sharp glass can also be very harmful. Also be careful when using the water bath as there are precautions to be taken with all electrical appliances. Care must also be taken when handling the pectinase. If in contact with the skin, wash immediately to prevent it from possibly entering the mouth. Finally, try not to spill any solution on the ground because someone could slip on it but if some liquid is spilt, wipe it up straight away
Environmental/Ethical concerns
The disposal of any pectinase enzyme or solution should have the dilution factor considered before it is poured away. Is it dilute enough for disposal? All apple content should be covered and placed in the bin for hygiene issues, i.e. spiders, insects, animals and fungi or bacteria may use the apples and this causes human hygiene and safety problems
Hypothesis:
I believe that the addition of the pectinase enzyme will lead to a greater yield of apple juice than if only water is added. This will be because enzymes speed up reactions. The pectinase will degrade the soluble pectin in the middle lamella which will in turn release the parenchyma cells and their contents. This will make the juice flow more freely resulting in a greater yield of apple juice in a set time period. Water does not act so fast on the pectin and so a smaller yield will be expected in the same time period.
H0: There is no difference between water and pectinase enzyme on the
yield of apple juice.
H1: Pectinase increases the yield of juice from apples in comparison to water.
H0(2): There is no difference in the variety of apple and the amount of juice they
contain.
H1(2): There is difference in the variety of apple and the amount of juice they
contain.
Cox
Granny Smith
Pink Lady
Braeburn
Highland fuji
Royal Gala
Golden Delicious
Anomaly
Anomaly
The graphs shows the means to be varied somewhat. This means that there was too much variation which maybe be explained by the sample size being too small, perhaps more apples and test samples are needed to produce an accurate mean to fully justify the hypothesis. More samples could also set different extremes which would get us more accuracy for the hypothesis although it would be preferred that they do no vary too much and the graph would begin to level of consistently. The main anomalies on both treated and untreated shouldn’t be ignored. They both exist on the same apple in the treated and untreated samples of that apple (Cox) which has the top yield of all the apples, being the top yield and an anomaly it has an affect of rising the average slightly producing inaccuracies.
The Graph above shows the progression of more juice being released over time. In both the Cox apple and the Pink lady we see the gradual trend of an increase in the amount produced. Between 0-5 minutes the Cox apple produces more than pink lady with it producing 8.5cm³ in the same time the Pink lady apple produced 7cm³. But apples biggest acceleration points are in the first 5 minutes. Both apples reduce after 5mins in production but continue to produce. Even at the end of the experiment after 40 minutes the line does not level off in both apples which leaves questions to be raised about potential juice that could have been excreted after this 40 minutes.
Mean =
= 13.73
=
= 10.19
Standard Deviation (s) =
sª = = 2.66
sb = = 1.88
Students T-test t =
t =
t = = 3.072
Degrees of Freedom d.f. =
= 8 + 8 – 2 = 14
Critical Value c.v. = 2.10
This value of t (3.072) is higher than the critical value (2.10) which means there is a big difference between our results. This means I can reject the null hypothesis (H0(2)) and accept there is a significant difference (p <0.05) between the yield of juice produced in different varieties of apples.
I’ll begin with the observations that were made and noted during the practical experiment:
- The pectinase used has an optimum of 60ºC and I used it at 40ºC
- Water bath varied temperatures ± 4ºC (36ºC-44ºC)
- At the end of the experiment after 40 mins of filtering all the apple samples remained moist and therefore causes potential juice which was not accounted for. This also varied in amount between apples
These observations will cause an affect to the experiment. The first and second issues of the pectinase enzymes optimum being 60ºC and I used it at 36ºC - 44ºC means that less juice than the results show was produced theoretically than if I had used it at the optimum temperature. This is an issue for further investigation and in a further experiment could be employed into the method and the contrasts made. Theoretically this would widen the gap between water and pectinase treated apples as more juice would be extracted and its effect on variety is unknown which allows more investigation. The variance due to the failure of equipment shouldn’t have many implications as all the apples equally encountered this variance so it’s standardised. The potential juice is a key issue in the evaluating of the results. This juice that was left over was defiantly varied in the amounts between each apple. This could have drastically changed the results if left long enough to filter down and alternate conclusions could be drawn as to the variety of apples and the differences in juice content. This is another suggestion for further experimenting and next time leaving the apples for longer but not allowing evaporation to occur so the total juice content can be accurately measured for a more accurate conclusion.
The results obtained, and from the statistical analysis of all the apples treated and untreated allow me to reject the null hypothesis, (H0) and accept there is a significant difference (p <0.05) between the yield of juice produced between pectinase treated apples and water treated apples. However this conclusion has its limitations. Due to time pressures I was unable to repeat the experiment and gather and average result of the juice content. Repeating it would have allowed me to gather more data and better averages for more accurate results, it is a major issue in any experiment but was unable to do it in this one for the afore mentioned reason. Also the tentative nature of results gathered from a single experiment come into play. This is because of issues mentioned previously in the plans variables section, such as: the storage of the apples previously before the experiment from where they were purchased, the chemicals used on them if grown in arid conditions perhaps. These variables allow me to assume on a different day, the same types of apples from alternate sources may produce alternate results.
To conclude this hypothesis, the results support of the hypothesis means that the addition of the pectinase enzyme leads to a greater yield of apple juice than if only water is added. This is because enzymes speed up reactions. The pectinase degraded the soluble pectin in the middle lamella which in turn released the parenchyma cells and their contents. This made the juice flow more freely resulting in a greater yield of apple juice in a set time period. Water did not act so fast on the pectin and so a smaller yield was measured in the same time period. From this I can reject the null hypothesis and that pectin has more of an effect than water. However the results are limited as they were on one day with no repetition for a mean or average, such as the pectinase optimum temperature being different to what I used and equipment problems could play there part in the results meaning they could be inaccurate.
The second hypothesis of variety was distinguished using the lowest (Pink Lady) and highest (Cox) figures and comparing them superficially and analysed more in depth statistically. This value of t (3.072) was higher than the critical value (2.10) which means there is a big difference between our results. This means I can reject the null hypothesis (H0(2)) and accept there is a significant difference (p <0.05) between the yield of juice produced in different varieties of apples. With this said it is very similar to the above in that there are issues of unreliability and inaccuracies perhaps. Afore mentioned time pressure and what repetition would have allowed me to do is a critical factor in evaluating the results and experiment. Also again the tentative nature of results gathered from a single experiment come into play. The explanations for the variable results in variety are: different densities of tissue (cell volumes and intercellular air-spaces) more air spaces would mean less juice and less air spaces would mean more juice. The different percentage composition of cell wall components (with different susceptibilities to whatever maceration process used) with less surface area from maceration then the pectin or water would have less effect, the opposite can be said if there was more surface area from better crushing or the blender. Different concentrations of sugars and acids in juice, this is what the variation is and how the pectin and water takes effect with this variation. Then finally storage previous to experiment and sources of where they were grown, this was explained above in the previous hypothesis. T
In conclusion to the second hypothesis the results when analysed statistically. This means I can reject the null hypothesis (H0(2)) and accept there is a significant difference (p <0.05) between the yield of juice produced in different varieties of apples. These differences can be mostly put down to, different densities and intercellular spaces, different percentage composition of cell wall and different susceptibilities to techniques employed and different concentrations of acids and sugars. The results gathered do however have there limitations to there reliability and how far we can go to stating firm conclusions. There is a clear variety in the difference of the apples but to what extent is this variety is what is unknown as we have little knowledge of all the apples available with my sample size being only 7 of hundreds available. Also the validation of results can be called into disrepute perhaps through the problems of sources and storage and the nature of results tending to vary from one experiment to the next as there was no repition.
In overall conclusion both variables had the null hypothesis rejected from the results gathered. There is much evidence to oppose the conclusions drawn as there are issues such as sample size which had an effect. The line graphs for the treated and untreated comparisons show a vastly varied line and no consistency. The anomaly of the Cox apple in both untreated and treated supports the notion of a sample size being too small as it has no gradual incline to it as no other apples come close. The lack of repetition is critical to analysis and the ability to create averages such as means to conclusive state any rejection or acceptance of a hypothesis. This provides a firm base for further investigation of additional evidence to support or not support the hypothesis. In a future investigation I would increase sample size, providing a wider range of results and analysing more in depth of the extent of any variation if any at all. Apples would be analysed from there source they came from and sufficient investigation to any allowances that may have to be made. To alter the experiment for better accuracy I would use the pectin enzyme at its optimum temperature, 60ºC, to judge weather or not this affects results in comparison to using it at 40ºC in this experiment. The potential juice that remained after my experiment finished after 40 minutes causes much uncertainty to how much juice each apple really contains. So a future investigation could research into this and leave the apples for a much longer time such as a day or 2 for the full or at least most of juice to be filtered down. Evaporation would have to be completely stopped as this would affect results.
